HK40066141A - Genetically engineered cells and uses thereof - Google Patents

Description

Genetically engineered cells and their uses

Cross-references

This application claims priority to Chinese patent application CN201910746355.8 filed on August 13, 2019 and international patent application PCT/CN2019/124321 filed on December 10, 2019, each of which is incorporated herein by reference in its entirety.

Technical Field

This invention relates to genetically engineered cells, and more particularly to engineered cells that are resistant to allogeneic immune systems, and further to methods for preparing said cells and the use of said cells in allogeneic transplantation.

Background Technology

Allogeneic transplant rejection is a major cause of failure in allogeneic organ or cell transplantation. While T cells play a crucial role in allogeneic transplant rejection, recent studies have shown that non-T cells, such as natural killer (NK) cells, B cells, macrophages, and mast cells, play an unexpected role in regulating graft outcomes (Li, Transplantation, 2010; 90(10):1043-1047). For example, the unique self-non-self recognition system of NK cells is highly correlated with allogeneic transplantation. In short, individual NK cells possess both stimulatory and inhibitory receptors on their cell surface and require signals from both receptors to establish NK tolerance to autologous cells. In humans, inhibitory receptors include killer cell immunoglobulin-like receptors (KIRs). In addition, NKG2A and CD94 typically form heterodimers on the cell surface and function as inhibitory receptors on both NK and T cells. The binding of “self” class I MHCs to these inhibitory receptors inhibits NK cells and prevents them from attacking “self” cells. In graft models, recipient NK cells readily recognize MHC-incompatible allogeneic cells through "missing self" recognition, as these allogeneic cells lack the self-contained class I MHC receptors necessary for binding to NK inhibitory receptors. This lack of inhibitory signaling triggers NK cell activation, including cytolytic activity and the production of potent pro-inflammatory cytokines. The same mechanism also applies to the activation of other immune cells, such as T cells, leading to allogeneic transplant rejection.

Chimeric antigen receptor T-cell (CAR-T) therapy has shown promising efficacy in cancers such as relapsed/refractory lymphoma, leukemia, and multiple myeloma. However, existing autologous CAR-T technologies require individualized cell preparation, which is limited by long production cycles, high costs, and the fact that patients often lack sufficient T cells. Therefore, universal CAR-T (UCAR-T) therapy has attracted considerable attention, in which T cells are derived from healthy donors and prepared in advance for use by any patient. However, as in any other allogeneic transplantation, the bidirectional rejection between the T-cell graft and the recipient needs to be addressed. This invention solves the problem of overcoming allogeneic transplant rejection in allogeneic organ and cell transplantation and provides related advantages.

Summary of the Invention

The present invention provides a fusion protein comprising a presenting peptide covalently linked to a β-2-microglobulin (β2M) peptide via a linker, wherein the fusion protein binds a major histocompatibility (MHC) heavy chain to form an MHC complex, the MHC complex binding an inhibitory receptor of an immune cell to inhibit the immune cell, and wherein the fusion protein (1) comprises less than 500 amino acids or (2) lacks an HLA-E heavy chain.

The fusion protein provided by this invention binds to major histocompatibility (MHC) heavy chains to form an MHC complex, which binds to inhibitory receptors of immune cells to inhibit the immune cells. In some embodiments, the inhibitory receptor of the immune cells is NKG2A. In some embodiments, the inhibitory receptor of the immune cells is selected from the group consisting of KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, KIR3DL3, and LIR1.

In some embodiments, the MHC heavy chain is a classical class I MHC heavy chain, a non-classical class I MHC heavy chain, or an MHC-like heavy chain. In some embodiments, the MHC heavy chain is a classical class I MHC heavy chain selected from the group consisting of HLA-A heavy chains, HLA-B heavy chains, and HLA-C heavy chains. In some embodiments, the MHC heavy chain is a non-classical class I MHC heavy chain selected from the group consisting of HLA-E heavy chains, HLA-F heavy chains, and HLA-G heavy chains. In some embodiments, the MHC heavy chain is an MHC-like molecular heavy chain selected from the group consisting of CD1 heavy chains, MR1 heavy chains, FcRn heavy chains, and UL18. In some embodiments, the MHC heavy chain is an HLA-E heavy chain.

In some embodiments, the present invention provides a fusion protein comprising a presenting peptide covalently linked to a β2M peptide via a linker, wherein the presenting peptide is an HLA-E restricted presenting peptide, and wherein the fusion protein (1) comprises less than 500 amino acids, or (2) lacks the HLA-E heavy chain.

In some embodiments of the fusion protein provided by this invention, the presenting peptide is derived from a virus, prokaryote, eukaryote, or mammal. In some embodiments, the presenting peptide is derived from a human.

In some embodiments of the fusion protein provided by the present invention, the presenting peptide is a signal peptide of a class I MHC molecule or a fragment thereof.

In some embodiments, the fusion protein provided by the present invention comprises a presenting peptide covalently linked to a β2M peptide via a linker, wherein the presenting peptide is a signal peptide of a class I MHC molecule or a fragment thereof, and wherein the fusion protein (1) comprises less than 500 amino acids, or (2) lacks the HLA-E heavy chain.

In some embodiments, the presenting peptide has 5-30 amino acids. In some embodiments, the presenting peptide has 7-20 amino acids. In some embodiments, the presenting peptide has 8-10 amino acids.

In some embodiments of the fusion protein provided by the present invention, the presenting peptide is a signal peptide of a class I MHC molecule or a fragment thereof, wherein the class I MHC molecule is selected from the group consisting of the heavy chains of the following: HLA-A1, HLA-A2, HLA-A*3401, HLA-A*80, HLA-B7, HLA-B*13, HLA-B15, HLA-Cw3, HLA-Cw*2, HLA-Cw*0809, HLA-Cw7, HLA-Cw*1701, HLA-G, and HLA-F.

In some embodiments, the amino acid sequence of the presenting peptide includes _{X1 X2 X3 X4 X5 X6 X7 X8} L; wherein _X1 is V or I; _X2 is T, A, M or L; X3 is A, P, K or N; _X4 is P, L or T; _X5 is R, Q or K; _X6 is T or A; _X7 is L, I, V or P; _X8 is V, L, I, F or T ₍ SEQ ID NO: 115). In some embodiments, the amino acid sequence of the presenting peptide is selected from the group consisting of SEQ ID NOs: 21-73. In some embodiments, the amino acid sequence of the presenting peptide is VMAPRTVLL (SEQ ID NO: 38).

In some embodiments, the fusion protein provided by the present invention comprises less than 500 amino acids, less than 400 amino acids, less than 300 amino acids, or less than 200 amino acids. In some embodiments, the fusion protein comprises about 120-180 amino acids.

In some embodiments, the fusion protein provided by the present invention includes the presenting peptide, the linker, and the β2M peptide from the N-terminus to the C-terminus.

In some embodiments of the fusion protein provided by this invention, the amino acid sequence of the linker includes (EAAAK)n, where n = 3, 4, or 5 (SEQ ID NO: 110), and wherein the linker has 5 to 30 amino acids. In some embodiments, the amino acid sequence of the linker includes (GGGGS)n, where n = 3, 4, or 5 (SEQ ID NO: 112), and wherein the linker has 5 to 30 amino acids. In some embodiments, the amino acid sequence of the linker is SEQ ID NO: 1 or 2.

In some embodiments of the fusion protein provided by this invention, the amino acid sequence of the β2M peptide has at least 85%, at least 90%, or at least 95% identity with SEQ ID NO:81. In some embodiments, the amino acid sequence of the β2M peptide is SEQ ID NO:81.

In some embodiments, the fusion protein provided by the present invention comprises the presenting peptide, the linker, and the β2M peptide.

In some embodiments, the amino acid sequence of the fusion protein provided by the present invention has at least 85%, at least 90%, or at least 95% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs:5 and 13-18. In some embodiments, the amino acid sequence of the fusion protein is selected from the group consisting of SEQ ID NOs:5 and 13-18.

The present invention also provides a nucleic acid encoding the fusion protein provided by the present invention.

In some embodiments, the present invention provides a nucleic acid comprising (i) a first segment encoding a fusion protein comprising a presenting peptide and a β2M peptide covalently linked by a linker, wherein the fusion protein (1) comprises fewer than 500 amino acids or (2) lacks the HLA-E heavy chain; and (ii) a second segment encoding a synthetic receptor.

In some embodiments, the synthetic receptor is selected from the group consisting of chimeric antigen receptors (CARs), T-cell receptors (TCRs), TCR receptor fusion constructs (TRuCs), T-cell antigen conjugates (TACs), antibody-TCR receptors (AbTCRs), and chimeric CD3 receptors. In some embodiments, the synthetic receptor is a CAR.

In some embodiments, the synthetic receptor includes an antigen-binding domain that specifically binds to a tumor antigen. In some embodiments, the tumor antigen is selected from the group consisting of CD19, CD20, CD22, CD30, CD123, CD138, CD33, CD70, BCMA, CS1, C-Met, IL13Ra2, EGFRvIII, CEA, Her2, GD2, MAGE, GPC3, mesothelin, PSMA, ROR1, EGFR, MUC1, and NY-ESO-1. In some embodiments, the tumor antigen is CD19 or BCMA.

In some embodiments, the synthetic receptor includes an antigen-binding domain that specifically binds to a viral antigen. In some embodiments, the viral antigen is EBV or HPV.

In some embodiments of the nucleic acid provided by the present invention, the first fragment and the second fragment are linked by a polynucleotide encoding a 2A peptide. In some embodiments, the 2A peptide is a P2A peptide, a T2A peptide, an F2A peptide, or an E2A peptide. In some embodiments, the nucleic acid provided by the present invention encodes the synthetic receptor, the 2A peptide, and the fusion protein from the N-terminus to the C-terminus.

In some embodiments of the nucleic acid provided by the present invention, the first fragment and the second fragment are linked by an IRES sequence.

The present invention also provides a vector comprising the nucleic acid disclosed herein.

In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a lentiviral vector, an adenovirus vector, or an adeno-associated virus vector.

The present invention also provides genetically engineered cells expressing the fusion protein disclosed herein.

The present invention also provides genetically engineered cells comprising the nucleic acids disclosed herein or the vectors disclosed herein.

In some embodiments, the cells provided by the present invention further express synthetic receptors.

In some embodiments, the cell provided by the present invention further includes a second nucleic acid encoding a synthetic receptor. In some embodiments, the synthetic receptor is selected from the group consisting of CAR, TCR, TruC, TAC, AbTCR, and chimeric CD3 receptors. In some embodiments, the synthetic receptor is a CAR.

In some embodiments, the synthetic receptor includes an antigen-binding domain that specifically binds to a tumor antigen. In some embodiments, the tumor antigen is selected from the group consisting of CD19, CD20, CD22, CD30, CD123, CD138, CD33, CD70, BCMA, CS1, C-Met, IL13Ra2, EGFRvIII, CEA, Her2, GD2, MAGE, GPC3, mesothelin, PSMA, ROR1, EGFR, MUC1, and NY-ESO-1. In some embodiments, the tumor antigen is CD19 or BCMA.

In some embodiments, the synthetic receptor is a CAR having an antigen-binding domain that specifically binds to CD19 or BCMA, wherein the amino acid sequence of the CAR is selected from the group consisting of SEQ ID NOs:74-80 and 136.

In some embodiments, the synthetic receptor is a TCR having an antigen-binding domain that specifically binds to NY-ESO-1, wherein the amino acid sequence of the TCR is SEQ ID NO:132.

In some embodiments, the synthetic receptor includes an antigen-binding domain that specifically binds to a viral antigen. In some embodiments, the viral antigen is EBV or HPV.

In some embodiments of the cells provided by the present invention, the fusion protein provided by the present invention forms a complex with the endogenous MHC heavy chain on the cell surface.

In some embodiments, the cells provided by the present invention lack endogenous expression of at least one gene encoding class I MHC molecules or MHC-like molecules on the cell surface. In some embodiments, the cells provided by the present invention lack endogenous expression of class I MHC molecules on the cell surface, wherein the class I MHC molecules are selected from the group consisting of HLA-A heavy chain, HLA-B heavy chain, HLA-C heavy chain, HLA-E heavy chain, HLA-F heavy chain, and HLA-G heavy chain. In some embodiments, the cells provided by the present invention lack endogenous expression of β2M on the cell surface. In some embodiments, the cells provided by the present invention lack endogenous expression of MHC-like molecules on the cell surface, wherein the MHC-like molecules are selected from the group consisting of CD1 heavy chain, MR1 heavy chain, FcRn heavy chain, and UL18.

In some embodiments, the cells provided by the present invention are immune cells. In some embodiments, the cells provided by the present invention are leukocytes. In some embodiments, the leukocytes are selected from the group consisting of T cells, NK cells, NKT cells, B cells, plasma cells, dendritic cells, neutrophils, monocytes, macrophages, and granulocytes. In some embodiments, the leukocytes are NK cells. In some embodiments, the leukocytes are T cells.

In some embodiments, at least one gene encoding a component of the TCR complex is inactivated in the T cell.

The present invention also provides pharmaceutical compositions having the cells and pharmaceutically acceptable carriers provided by the present invention.

The present invention also provides the use of the cells provided by the present invention in allogeneic transplantation.

The present invention also provides the use of the cells provided by the present invention in cancer treatment.

The present invention also provides the use of the cells provided by the present invention in the preparation of medicaments for treating cancer.

The present invention also provides the use of the cells provided by the present invention in allogeneic transplantation, wherein the cells comprise nucleic acids encoding a fusion protein, the fusion protein comprising a presenting peptide and a β2M peptide covalently linked by a linker, and wherein the fusion protein (1) comprises less than 500 amino acids or (2) lacks the HLA-E heavy chain.

The present invention also provides the use of the cells provided by the present invention in allogeneic transplantation, wherein the cells express a fusion protein comprising a presenting peptide and a β2M peptide covalently linked by a linker, and wherein the fusion protein (1) comprises less than 500 amino acids or (2) lacks the HLA-E heavy chain.

The present invention also provides a method for treating a subject who requires allogeneic transplantation, the method comprising administering an effective amount of the cells provided by the present invention to the subject.

The present invention also provides a method for treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of cells provided by the present invention.

The present invention also provides a method for treating a subject requiring allogeneic transplantation, the method comprising administering to the subject an effective amount of cells, wherein the cells comprise nucleic acids encoding a fusion protein, the fusion protein comprising a presenting peptide and a β2M peptide covalently linked by a linker, and wherein the fusion protein (1) comprises fewer than 500 amino acids, or (2) lacks the HLA-E heavy chain.

The present invention also provides a method for treating a subject requiring allogeneic transplantation, the method comprising administering to the subject an effective amount of cells, wherein the cells express a fusion protein comprising a presenting peptide and a β2M peptide covalently linked by a linker, and wherein the fusion protein (1) comprises less than 500 amino acids or (2) lacks the HLA-E heavy chain.

The present invention also provides a method for genetically engineering cells for use in allogeneic grafts, the method comprising transducing the cells with a nucleic acid encoding a fusion protein, wherein the fusion protein comprises a presenting peptide and a β2M peptide covalently linked by a linker, and wherein the fusion protein (1) comprises fewer than 500 amino acids or (2) lacks the HLA-E heavy chain.

The present invention also provides a method for genetically engineering cells for allogeneic transplantation, the method comprising transducing the cells with nucleic acids provided by the present invention.

The present invention also provides a method for genetically engineering cells for allogeneic transplantation, the method comprising transducing the cells using a vector provided by the present invention.

In some embodiments, the method provided by the present invention further comprises inactivating at least one gene encoding a class I MHC molecule or an MHC-like molecule in the cell. In some embodiments, the inactivated gene encodes a class I MHC molecule selected from the group consisting of HLA-A heavy chain, HLA-B heavy chain, HLA-C heavy chain, HLA-E heavy chain, HLA-F heavy chain, and HLA-G heavy chain. In some embodiments, the inactivated gene encodes β2M. In some embodiments, the inactivated gene encodes an MHC-like molecule selected from the group consisting of CD1 heavy chain, MR1 heavy chain, FcRn heavy chain, and UL18.

In some embodiments, the gene is inactivated by DNA splicing, DNA splicing and repair, base editing, prime editing, RNA interference, or RNA editing. In some embodiments, the gene is inactivated by DNA splicing using a rare endonuclease selected from the group consisting of RNA-directed endonucleases, TAL nucleases, homing nucleases, zinc finger nucleases, and Mega-TAL nucleases. In some embodiments, the gene is inactivated by DNA splicing and repair. In some embodiments, the gene is inactivated using a CRISPR-Cas system. In some embodiments, the CRISPR-Cas system is the CRISPR-Cas9 system.

In some embodiments of the method provided by this invention, the cells are immune cells. In some embodiments, the immune cells are selected from a group consisting of T cells, NK cells, NKT cells, B cells, plasma cells, monocytes, macrophages, dendritic cells, and granulocytes.

The present invention also provides a method for treating subjects requiring allogeneic transplantation, the method comprising: preparing genetically engineered cells according to the preparation method disclosed in the present invention; and applying the engineered cells to the subject.

Attached Figure Description

Figure 1 is a schematic diagram of the fusion protein and the expression vector. Area A of Figure 1 is a schematic diagram of an exemplary fusion protein (or "chimeric molecule" or "CM") provided by the present invention. Area B of Figure 1 is a schematic diagram of a vector having nucleic acid fragments encoding the fusion protein (CM) and a CAR.

Figure 2 illustrates the co-expression of the fusion protein (CM) and CAR in genetically engineered Jurkat T cells. Section A shows the results for untransduced cells in the control group. Section B shows the results for cells transduced with CAR19-P2A-CM virus, where CM includes an HLA-E-restricted presenting peptide. HLA-E expression at the cell surface was detected by HLA-E antibody, as indicated by the arrows. Section C shows the results for cells transduced with CAR19-P2A-CM-c, where CM includes an HLA-C-restricted presenting peptide. HLA-E expression at the cell surface was not detected by HLA-E antibody.

Figure 3 illustrates the expression of the fusion protein (CM) in naïve T cells. Section A shows the total UCAR-T-CM cells. Section B shows that approximately 80% of UCAR-T-CM cells with simultaneous TCR and B2M gene knockouts were double-negative when stained with CD3 and HLA-ABC antibodies. Section C shows the expression of the fusion protein (CM) in CD3 and HLA-ABC double-negative UCAR-T-CM cells, as indicated by positive staining against HLA-E.

Figure 4 shows that UCAR-T cells expressing the fusion protein (CM) are resistant to NK cell killing.

Figure 5 shows that UCAR-T cells expressing the fusion protein (CM) are resistant to growth inhibition by allogeneic PBMC cells and rapidly proliferate after stimulation by tumor cells.

Figure 6 shows that UCAR-T cells expressing the fusion protein (CM) effectively arrested tumor cell growth and killed tumor cells in an allogeneic PBMC environment.

Figure 7 shows the results of flow cytometry analysis, demonstrating that the fusion protein (CM) forms a complex with HLA-E heavy chain molecules on the cell membrane of β2M knockout cells.

Figures 8A-8B show the results of flow cytometry analysis, demonstrating that different presenting peptides are associated with varying expression levels of the HLA-E complex on the cell membrane. Figure 8A shows the data from the flow cytometry analysis, and Figure 8B quantifies the data shown in Figure 8A.

Figure 9 shows the flow cytometry analysis results of different fusion proteins expressed in genetically engineered naïve human T cells. The different fusion proteins (CM, CM12, CM13, CM14) contain different HLA-E-restricted presenting peptides. Cells in all groups had TRAC and β2M knocked out using CRISPR/Cas9 and expressed a CD19-targeting CAR. The control UCAR-T group did not express any fusion proteins.

Figure 10 shows that UCAR-T cells expressing different fusion proteins (CM, CM12, CM13, CM14) are resistant to growth inhibition by allogeneic PBMC cells and rapidly proliferate after being stimulated by tumor cells.

Figure 11 shows the flow cytometry analysis results of the co-expression of exogenous TCR (1G4) and fusion protein (CM) targeting NY-ESO-1 in genetically engineered Jurkat T cells. NTC: Untransduced control (no lentiviral transduction). Areas A-D: Wild-type Jurkat T cells; Areas E-P: Jurkat T cells with double knockout of TRAC and β2M, thus being CD3 and β2M double negative.

Figure 12 shows that TRAC and β2M double knockout naïve human T cells are resistant to growth inhibition of allogeneic PBMC cells and continue to proliferate after stimulation by target cells. These T cells express TCR(1G4) targeting NY-ESO-1 and the fusion protein CM.

Figure 13 shows the results of flow cytometry analysis of surface expression of HLA-ABC and CD3 in different T cell groups with or without TRAC and β2M double knockout.

Figure 14 shows the flow cytometry analysis results of BCMA CAR and fusion protein CM expression, as shown by HLA-E expression in different T cell groups with or without TRAC and β2M double knockout. Notably, BCMA CAR and CM were both positive/co-expressed only in the BCMA-CM CAR-T and BCMA-CM UCAR-T groups.

Figure 15 shows an overview of cytokine release in different T cell groups. Notably, both BCMA-CM CAR-T and BCMA-CM UCAR-T cells released large amounts of IL-2 upon stimulation by BCMA-positive tumor cells.

Figure 16 shows that BCMA-CM UCAR-T cells expressing CM are resistant to NK cells compared to UT cells without CM expression.

Detailed Implementation

Before further describing the embodiments of the present invention, it should be understood that the present invention is not limited to the specific embodiments listed herein, and it should also be understood that the terminology used in the present invention is for describing specific embodiments and is not intended to limit the present invention.

This invention relates to compositions and methods for improving the success rate of allogeneic organ transplantation or allogeneic cell transplantation (e.g., the universal CAR-T therapy). One advantage provided by the compositions and methods of this invention is overcoming the recipient's immune system's rejection of the allogeneic graft. Specifically, a fusion protein comprising a β2M polypeptide and a presenting peptide has been found to be expressed in cells used for transplantation. This fusion protein can form an MHC complex on the cell surface with endogenously expressed MHC heavy chains, allowing the MHC complex to bind to inhibitory receptors on the recipient's immune cells (e.g., NK cells and T cells) and prevent the recipient's immune cells from triggering an immune response against the allogeneic cells.

For example, it is known that allogeneic transplanted cells will be recognized and killed by the recipient's immune cells if they lack HLA-E expression on their cell surface (HLA-E is a non-classical class I MHC complex that binds to the inhibitory receptor NKG2A on immune cells (e.g., NK cells or T cells)). (Saunders et al., Immunological Reviews (2015) 267:148-166). In some embodiments, the present invention provides genetically engineered cells expressing a fusion protein having a β2M peptide and an HLA-E-restricted presenting peptide. The fusion protein can form an HLA-E complex on the cell surface with an endogenously expressed HLA-E heavy chain, which binds to the inhibitory receptor NKG2A on NK cells or T cells to inhibit NK cell or T cell activation. The compositions and methods disclosed in this invention are advantageous compared to expressing a complete MHC complex (e.g., β2M plus HLA-E heavy chain, at least 500 amino acids), at least because the fusion proteins provided by this invention can be significantly smaller (e.g., less than 300 amino acids), which makes genetic engineering more accurate and efficient.

6.1 Definition

Unless otherwise defined in this invention, the scientific and technical terms used herein shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include plural terms, and plural terms shall include singular terms. Generally, the terms and techniques used in this invention related to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and proteins, chemistry, and hybridization are well-known and commonly used in the art.

The terms “polypeptide,” “peptide,” “protein,” and their grammatical equivalents used interchangeably in this invention refer to an amino acid polymer of any length, which may be linear or branched. It may include non-natural or modified amino acids, or be broken down by non-amino acid components. Polypeptides, peptides, or proteins may also be modified, for example by disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other manipulation or modification.

The term "fusion protein" refers to a protein, peptide, or polypeptide whose amino acid sequence is derived from two or more separate proteins, peptides, or polypeptides. The fusion protein also includes an amino acid linker region derived from the amino acid portions of the separate proteins, peptides, or polypeptides. This linker region is referred to as a "linker" in this invention.

The terms “major histocompatibility complex,” “MHC complex,” and “MHC” are used interchangeably in this invention, consistent with their common usage in the art. In short, the above terms refer to a tightly linked group of genes that encode cell surface proteins essential for the adaptive immune system to recognize foreign molecules in vertebrates, thereby determining histocompatibility. The primary function of the MHC is antigen presentation, and extremely rich polymorphism has been observed. Human MHC is also known as human leukocyte antigen (“HLA”). The MHC complex is a heterodimer and consists of two “MHC proteins.” It should be understood that “MHC gene” refers to a polynucleotide encoding an MHC protein.

MHC complexes include class I MHC (or MHC-I), class II MHC (or MHC-II), and MHC-like complexes (or "MHC analogs" or "MHC homologs"). Class I MHC complexes are heterodimeric glycoproteins composed of two peptide chains linked by non-covalent bonds. One chain is a polymorphic heavy chain (i.e., the "class I MHC heavy chain," also known as the "class I MHC α chain"), and the other is a light chain (i.e., β-2-microglobulin (β2M)). Class I MHC complexes present peptide fragments of intracellular proteins to immune cells, triggering an immediate immune response to specific non-self antigens presented by the class I MHC complex. In humans, classical class I MHC (also known as MHC Ia) includes HLA-A, HLA-B, and HLA-C; and non-classical class I MHC (also known as MHC-Ib) includes HLA-E, HLA-F, and HLA-G.

MHC-like complexes, also known as MHC analogs or MHC homologs, are structurally similar to class I MHC complexes. Some MHC-like complexes also consist of non-covalently linked heavy chains (i.e., "MHC-like heavy chains") and β2M proteins. Exemplary MHC-like complexes include, but are not limited to, CD1, MR1, neonatal Fc receptor (FcRn), and UL18 derived from human cytomegalovirus (HCMV). CD1 is structurally similar to other class I MHC complexes and consists of heavy chains and β2M. CD1 presents lipid antigens and further includes CD1a, CD1b, CD1c, CD1d, and CD1e. MR1 (MHC-associated protein 1) is structurally similar to class I MHC complexes and consists of heavy chains and β2M. MR1 primarily presents small aromatic molecules that activate mucosa-associated inertial T cells (MAIT). These small aromatic molecules are typically smaller than peptides presented by class I MHC or lipids presented by CD1. Neonatal Fc receptors (FcRn) transport immunoglobulin G (IgG) across the cell layer to prolong the half-life of IgG in circulation and provide humoral immunity to newborns. Like other class I MHCs, FcRN consists of a heavy chain and β2M. UL18 is homologous to the MHC heavy chain and binds to β2M to form a complex expressed on the cell surface, enabling cells to evade NK cell-mediated cytolysis.

The term "β-2-microglobulin (β2M) peptide" refers to the full-length β-2-microglobulin protein, or a functional fragment or variant thereof. β2M is a light chain of the class I MHC complex. Inactivation of the β2M gene effectively eliminates the expression of class I MHC on the cell surface. Cells that do not express class I MHC on their surface are recognized and killed by NK cells. Human β2M has a molecular weight of 11,800 Daltons and consists of 119 amino acids (SEQ ID NO:7). The first 20 amino acids at the N-terminus form the signal peptide of human β2M, which can be cleaved. The mature form of human β2M does not contain the signal peptide and consists of 99 amino acids (SEQ ID NO:81).

Exemplary functional fragments of β2M include, for example, truncated forms: ΔN6β2M (93 amino acids) lacking the 6 N-terminal amino acids of mature β2M; and ΔN10β2M (89 amino acids) lacking the 10 N-terminal amino acids of mature β2M ((Sulatskaya et al., Int. J. Mol. Sci. (2018) 19: 2762).). Exemplary variants include, for example, the following isotypes provided on UniProt: entries F5H6I0 (101 amino acids), H0YLF3 (71 amino acids), B4E0X1 (122 amino acids), A6XMH4 (124 amino acids), A6XMH5 (92 amino acids), A6XND9 (101 amino acids), Q9UM88 (29 amino acids), Q16446 (51 amino acids), and J3KNU0 (57 amino acids).

The term "variant" is associated with a protein or polypeptide ("reference protein" or "reference polypeptide") having specific sequence characteristics, referring to a different protein or polypeptide compared to a reference protein or polypeptide, including one or more (e.g., about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) amino acid substitutions, deletions, and/or additions. Changes in the amino acid sequence can be amino acid substitutions. Changes in the amino acid sequence can be conserved amino acid substitutions. Functional fragments or functional variants of the protein or polypeptide retain the basic structural and functional properties of the reference protein or polypeptide. For example, variants of human β2M may have one or more (e.g., about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) changes in the amino acid sequence of wild-type human β2M. Variants of human β2M retain the basic structural and functional properties of human β2M. For example, like full-length human β2M, human β2M variants can also bind to MHC heavy chains to form MHC complexes that can be recognized by immune cells.

The term "presenting peptide" refers to a short peptide that can stably bind to the antigen-binding groove of the MHC to form a stable MHC complex, which can then be recognized by receptors on immune cells. Presenting peptides typically have 7-30 amino acids. In some embodiments, presenting peptides have 7-20, 7-17, 7-15, 7-12, or 8-10 amino acids.

Each MHC has a matching presenting peptide. A presenting peptide “restricted” to a specific MHC molecule means that the presenting peptide can form a stable complex with that specific MHC molecule. For example, an HLA-C-restricted presenting peptide can form a stable complex with HLA-C on the cell surface, and an HLA-E-restricted presenting peptide can form a stable complex with HLA-E on the cell surface. By selecting presenting peptides that bind stably to HLA, presenting peptides specific to a particular HLA can be identified. Presenting peptides restricted to a specific class I MHC or MHC-like complex can be covalently linked to β2M to form fusion proteins that can form stable complexes with a specific MHC heavy chain. For example, a fusion protein with an HLA-C-restricted presenting peptide covalently linked to β2M can form a stable complex with the HLA-C heavy chain on the cell surface, and a fusion protein with an HLA-E-restricted presenting peptide covalently linked to β2M can form a stable complex with the HLA-E heavy chain on the cell surface.

The term "inhibitory receptor" in the context of immune cells refers to receptor molecules expressed on the surface of immune cells that, upon binding to their ligands, can inhibit the activity of immune cells. The immune system employs various inhibitory mechanisms to control immune responses and ensure immune tolerance and homeostasis, including inhibitory pathways mediated by inhibitory receptors. For example, NK cells are regulated by autologous class I MHC-binding receptors, which exert dominant inhibition on NK cells upon binding to autologous class I MHC molecules. Class I MHC-binding inhibitory receptors are expressed not only by NK cells but also by T cell subsets, particularly CD8+ T cells. These inhibitory receptors belong to at least two families: the cytotoxic immunoglobulin-like receptor (KIR) family and the C-type lectin-like family (e.g., NKG2A/B, CD94).

The term "specific binding" refers to a peptide or molecule interacting with an epitope, protein, or target molecule more frequently, rapidly, for a longer duration, with higher affinity, or a combination of these effects compared to alternative substances (including related and unrelated proteins). The binding moiety (e.g., antibody) that specifically binds to a target molecule (e.g., antigen) can be identified by immunoassays, ELISAs, SPR (e.g., Biacore), or other techniques known to those skilled in the art. Typically, the specific reaction will be at least twice the background signal or noise, and can be more than ten times the background. The binding moiety of a specific target molecule can bind to the target molecule with a higher affinity than it has for different molecules. In some embodiments, "specific binding" means that the binding moiety binds to the molecular target at about 0.1 mM or less K_{_D}. In some embodiments, "specific binding" means that the peptide or molecule binds to the target at about 30 μM or less K_{_D}. In some embodiments, "specific binding" means that the peptide or molecule binds to the target at about 10 μM or less K_{_D}, or at about 1 μM or less K_{_D}. In some implementations, "specific binding" means that the peptide or molecule binds to the target at about 0.1 μM or less _KD , or at about 0.01 μM or less _KD , or at about 1 nM or less _KD .

The terms “polynucleotide,” “nucleic acid,” and their grammatical equivalents used interchangeably in this invention refer to nucleotide polymers of any length, including DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or analogs thereof, or any substrate that can be bound to the polymer by DNA or RNA polymerases.

The terms "identity," "percentage identity," and their grammatical equivalents refer to two or more sequences or subsequences that are identical or have a specific percentage of identical nucleotide or amino acid residues when compared and aligned (with gaps introduced where necessary) to obtain maximum correspondence, without considering any conserved amino acid substitutions as part of sequence identity. The sequence percentage can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software available for obtaining amino acid or nucleotide sequence alignments are well known in the art. These algorithms or software include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variants thereof. In some embodiments, the two polynucleotides or polypeptides provided by this invention are substantially identical, meaning that when compared and aligned using sequence comparison algorithms or by visual inspection to obtain maximum correspondence, they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity. In some embodiments, identity exists in regions of amino acid sequences of any integer value between at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues, or so. In some embodiments, identity exists in regions longer than 60-80 residues, such as at least about 80-100 residues, and in some embodiments, these sequences are substantially identical in full length to the sequence being compared, such as the coding region of a target protein or antibody. In some embodiments, identity exists in regions of nucleotide sequences of any integer value between at least about 10 bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases, or so. In some embodiments, identity exists in regions longer than 60-80 bases, such as at least about 80-1000 bases or more, and in some embodiments, these sequences are substantially identical in full length to the sequence being compared, such as the nucleotide sequence encoding a target protein.

The term "antibody" and its grammatical equivalents refer to immunoglobulin molecules that recognize and specifically bind to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or any combination thereof, through at least one antigen-binding site, wherein the antigen-binding site is typically located within the variable region of the immunoglobulin molecule. As used herein, the term includes intact polyclonal antibodies, intact monoclonal antibodies, single-domain antibodies (sdABs, e.g., camel antibodies, alpaca antibodies), single-chain Fv (scFv) antibodies, heavy-chain antibodies (HCAbs), light-chain antibodies (LCAbs), multispecific antibodies, bispecific antibodies, monospecific antibodies, monovalent antibodies, and any other modified immunoglobulin molecule containing an antigen-binding site (e.g., a dual-variable-region immunoglobulin molecule), provided that the antibody exhibits the desired biological activity. The antibodies also include, but are not limited to, mouse antibodies, camel antibodies, chimeric antibodies, humanized antibodies, and humanized antibodies. Antibodies can be any of the five major immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), respectively designated α, δ, ε, γ, and μ based on the identity of their heavy chain constant regions. Unless otherwise explicitly stated, the term "antibody" as used herein includes the "antigen-binding fragment" of a complete antibody. The term "antigen-binding fragment" as used herein refers to a portion or fragment of a complete antibody, which is the antigen-determining variable region of the complete antibody. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, linear antibodies, single-chain antibody molecules (e.g., scFv), heavy chain antibodies (HCAbs), light chain antibodies (LCAbs), disulfide-linked scFv (dsscFv), biantibodies, triantibodies, tetraantibodies, small antibodies, bivariate antibodies (DVD), single variable region antibodies (sdAb, e.g., camel antibodies, alpaca antibodies), and single variable region (VHH) of heavy chain antibodies.

The term "vector" and its grammatical equivalents refer to a carrier for carrying genetic material (e.g., a polynucleotide sequence) that can be introduced into a host cell and replicated and/or expressed within the host cell. Applicable vectors include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may include selectable sequences or markers operable for stable integration into the host cell chromosome. Furthermore, the vector may include one or more selectable marker genes and appropriate expression control sequences. The selectable marker genes may be capable of, for example, providing resistance to antibiotics or toxins, supplementing nutritional deficiencies, or providing critical nutrients absent in the culture medium. The expression control sequences may include constitutive and inducible promoters, transcription enhancers, transcription terminators, etc., known in the art. When two or more polynucleotides are to be co-expressed, both polynucleotides may be inserted, for example, in a single expression vector or in separate expression vectors. For single-vector expression, the encoded polynucleotide may be operatively linked to a common expression control sequence or to different expression control sequences, such as an inducible promoter and a constitutive promoter. The introduction of polynucleotides into the host cell can be confirmed using methods known in the art. Those skilled in the art will understand that sufficient expression of polynucleotides can produce the desired product (e.g., the fusion protein described in this invention), and will further understand that expression levels can be optimized using methods known in the art to obtain sufficient expression.

The term "operable link" and its syntactic equivalents refer to a functional link between a regulatory sequence (such as a promoter and/or enhancer) and a second nucleic acid sequence, where the regulatory sequence directs the transcription of the second nucleic acid sequence; or a link between two nucleic acid sequences to be co-expressed within the same reading frame. For example, the first and second nucleic acid sequences are operably linked when they are functionally related. Similarly, if a promoter influences the transcription or expression of a coding sequence, the promoter and coding sequence are operably linked. Typically, operably linked DNA sequences are contiguous and, in cases where two protein-coding regions need to be linked, are located within the same reading frame.

The term "exogenous" and its grammatical equivalents refer to the introduction of a molecule into a host cell. For example, this can be achieved by introducing a molecule encoding a nucleic acid into the host's genetic material (e.g., integration into the host chromosome) or into non-chromosomal genetic material (e.g., a plasmid). When referring to the expression of a nucleic acid, the term means the introduction of the nucleic acid into the cell in an expressible form.

The term "endogenous" and its grammatical equivalents refer to molecules that are naturally present in host cells. Similarly, when it comes to the expression of nucleic acids, the term refers to the expression of nucleic acids that are naturally present in the cell.

The term "genetic engineering," or its grammatical equivalents, when used to refer to cells, refers to alterations in the cellular genetic material that are not normally present in naturally occurring cells. Genetic alterations include, for example, the introduction of modifications that express nucleic acids, other additions, mutations/alterations, deletions, and/or other functional disruptions of nucleic acids. Such modifications can occur, for example, in the coding regions of genes and their functional segments. Additional modifications can occur, for example, in non-coding regulatory regions, where these modifications alter gene expression.

The terms “transduction,” “transfection,” and their grammatical equivalents refer to the process of introducing exogenous polynucleotides into host cells. “Transfected” or “transduced” cells are cells that have been transferred, transduced, or transfected with exogenous polynucleotides. These cells include the initial recipient cells and their progeny.

The gene-related terms “inactivation,” “damage,” and their grammatical equivalents refer to gene alterations that inactivate or attenuate the encoded gene product. Gene alterations can be, for example, the deletion of an entire gene, the deletion of a regulatory sequence required for transcription or translation, the deletion of a portion of a gene that produces a truncated gene product, or any of various mutational strategies that inactivate or attenuate the encoded gene product. Gene damage also includes null mutations, which refer to mutations in a gene or a region containing a gene that prevent the gene from being transcribed into RNA and/or translated into a functional gene product. Such null mutations can be produced by a variety of types of mutations, including, for example, inactivating point mutations, partial gene deletions, entire gene deletions, or chromosomal segment deletions.

The term "encoding" and its grammatical equivalents refer to the inherent properties of a specific nucleotide sequence in a polynucleotide or nucleic acid, such as a gene, cDNA, or mRNA, which serves as a template for the synthesis of other polymers and macromolecules having specific nucleotide sequences (i.e., rRNA, tRNA, and mRNA) or specific amino acid sequences in biological processes, and the resulting biological characteristics. Thus, if the transcription and translation of the mRNA corresponding to a gene produces a protein, then the gene encodes that protein. Unless otherwise stated, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are mutually degenerate or encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNA may include introns.

The term "isolated" polypeptides, peptides, proteins, antibodies, polynucleotides, carriers, cells, or compositions refers to polypeptides, peptides, proteins, antibodies, polynucleotides, carriers, cells, or compositions that are not found in nature. Isolated polypeptides, peptides, proteins, antibodies, polynucleotides, carriers, cells, or compositions include those that have been purified to a certain extent and no longer exist in forms found in nature. In some embodiments, the isolated polypeptides, peptides, proteins, antibodies, polynucleotides, carriers, cells, or compositions are substantially purified.

The term “treatment” and its grammatical equivalents relate to a disease or condition, or a subject suffering from a disease or condition, and refer to actions that suppress, eliminate, reduce, and/or improve symptoms, symptom severity, and/or the frequency of symptoms associated with the disease or disorder being treated. For example, when referring to cancer or tumor, the term “treatment” and its grammatical equivalents refer to actions that reduce the severity of cancer or tumor, or delay or slow the progression of cancer or tumor, including (a) inhibiting the growth of cancer or tumor or preventing its development, (b) causing cancer or tumor to regress, or (c) delaying, improving, or minimizing one or more symptoms associated with the presence of cancer or tumor.

The terms “administration,” “application,” and their grammatical equivalents refer to the act of delivering or resulting in the delivery of a therapeutic agent or pharmaceutical composition to a subject by the methods described in this invention or other known methods in the art. The therapeutic agent may be a compound, peptide, cell, or cell population. Administering a therapeutic agent or pharmaceutical composition involves prescribing a therapeutic or pharmaceutical composition to a subject. Typical forms of administration include oral dosage forms such as tablets, capsules, syrups, and suspensions; injectable dosage forms such as intravenous (IV), intramuscular (IM), or intraperitoneal (IP) injections; transdermal dosage forms, including creams, gels, powders, or patches; oral dosage forms; inhaled powders, sprays, suspensions, and rectal suppositories.

The terms "effective amount," "therapeutic effective amount," and their grammatical equivalents refer to an amount administered to a subject, alone or as part of a pharmaceutical composition, in a single dose or as part of a series of doses, that would produce any detectable positive effect at the time of administration on any symptom, aspect, or characteristic of a disease, disorder, or condition. Therapeutic effective amounts can be determined by measuring the relevant physiological effects. The exact amount required varies from subject to subject, depending on the subject's age, weight and general condition, the severity of the symptoms being treated, the clinician's judgment, etc. In any individual case, the appropriate "effective amount" can be determined by a person skilled in the art using routine laboratory methods.

The term "pharmaceutically acceptable carrier" refers to a material suitable for individual administration with an active pharmaceutical ingredient, which does not cause any adverse biological effects or interact with any other component of the pharmaceutical composition in a harmful manner.

The term "subject" refers to any animal (e.g., a mammal), including but not limited to humans, non-human primates, canines, felines, rodents, etc., that is an animal to receive a specific treatment. A subject can be a human. A subject can be a patient suffering from a specific disease or condition. A subject can also be a recipient of a transplant.

Scope: Throughout the invention, various aspects of the invention may be presented in the form of scope. It should be understood that the scope description is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Therefore, the scope description should be considered as having specifically disclosed all possible sub-scopes and individual numerical values within that scope. For example, a description of a scope from 1 to 6 should be considered as having disclosed sub-scopes such as from 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and individual numbers within that scope, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the scope.

This invention describes exemplary genes and peptides with reference to GenBank numbers, GI numbers, and/or SEQ ID NO. It should be understood that those skilled in the art can readily identify homologous sequences by referring to sequence sources, including but not limited to GenBank (ncbi.nlm.nih.gov/GenBank/) and EMBL (EMBL.org/).

6.2 Fusion Protein

Research has found that fusion proteins, including a presenting peptide covalently linked to a β2M peptide via a linker, can form a complex with endogenous MHC heavy chains on the cell surface when expressed in host cells. This complex can bind to inhibitory receptors on immune cells to suppress them. For example, a fusion protein containing human β2M covalently linked to an HLA-E-restricted presenting peptide can be expressed in T cells, forming an HLA-E complex with endogenous HLA-E heavy chains on the T cell surface. When genetically engineered T cells are administered as allogeneic grafts to recipients, the HLA-E complex on the T cell surface can then be recognized by the recipient's immune cells (e.g., NK cells), thereby suppressing the immune cells and protecting the T cells from potential allogeneic immune responses (e.g., NK cell-mediated cell lysis).

Therefore, the present invention provides a fusion protein comprising a presenting peptide covalently linked to a β2M peptide via a linker, wherein the fusion protein binds to an MHC heavy chain to form an MHC complex, the MHC complex binding to an inhibitory receptor of an immune cell to inhibit the immune cell. In some embodiments, the fusion protein has fewer than 500 amino acids. In some embodiments, the fusion protein has fewer than 400 amino acids. In some embodiments, the fusion protein has fewer than 300 amino acids. In some embodiments, the fusion protein has fewer than 200 amino acids. In some embodiments, the fusion protein has from about 100 amino acids to about 300 amino acids. In some embodiments, the fusion protein has from about 100 amino acids to about 200 amino acids. In some embodiments, the fusion protein has from about 120 amino acids to about 180 amino acids.

In some embodiments, the fusion protein has about 120 to about 160 amino acids. In some embodiments, the fusion protein has about 140 to about 160 amino acids. In some embodiments, the fusion protein lacks an MHC heavy chain. In some embodiments, the fusion protein lacks a class I MHC heavy chain. In some embodiments, the fusion protein lacks an MHC-like heavy chain. In some embodiments, the fusion protein lacks an HLA-A heavy chain. In some embodiments, the fusion protein lacks an HLA-B heavy chain. In some embodiments, the fusion protein lacks an HLA-C heavy chain. In some embodiments, the fusion protein lacks an HLA-E heavy chain. In some embodiments, the fusion protein lacks an HLA-F heavy chain. In some embodiments, the fusion protein lacks an HLA-G heavy chain. In some embodiments, the fusion protein lacks a CD1 heavy chain. In some embodiments, the fusion protein lacks an MR1 heavy chain. In some embodiments, the fusion protein lacks an FcRn heavy chain. In some embodiments, the fusion protein lacks UL18.

This invention provides a fusion protein comprising a presenting peptide covalently linked to a β2M peptide via a linker, wherein the fusion protein binds to an MHC heavy chain to form an MHC complex, which in turn binds to an inhibitory receptor of an immune cell to suppress the immune cell. In some embodiments, the immune cell is a T cell; and this invention provides a fusion protein that binds to an MHC heavy chain to form an MHC complex, which in turn binds to an inhibitory receptor of a T cell to suppress the T cell. In some embodiments, the immune cell is an NK cell; and this invention provides a fusion protein that binds to an MHC heavy chain to form an MHC complex, which in turn binds to an inhibitory receptor of an NK cell to suppress the NK cell.

In some embodiments, the inhibitory receptor for immune cells is NKG2A. This invention provides a fusion protein comprising a presenting peptide covalently linked to a β2M peptide via a linker, wherein the fusion protein binds to an MHC heavy chain to form an MHC complex, which binds to the NKG2A receptor on immune cells to inhibit the immune cells. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are NK cells. Human NKG2A may have an amino acid sequence corresponding to the sequence provided below in GenBank No. AAL65234.1 (accession number: AAL65234.1, GI: 18182682; see below).

1 MDNQGVIYSD LNLPPNPKRQ QRKPKGNSS ILATEQEITY AELNLQKASQ DFQGNDKTYH

61 CKDLPSAPEK LIVGILGIIC LILMASVVTI VVIPSTLIQR HNNSSLNTRT QKARHCGHCP

121 EEWITYSNSC YYIGKERRTW EESLLACTSK NSSLLSIDNE EEMKFLSIIS PSSWIGVFRN

181 SSHHPWVTMN GLAFKHEIKD SDNAELNCAV LQVNRLKSAQ CGSSIIYHCK HKL(SEQ IDNO:82)

In some embodiments, the inhibitory receptor for immune cells is a KIR receptor. The inhibitory KIR receptor may be selected from the group consisting of KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5, KIR3DL1, KIR3DL2, and KIR3DL3.

In some embodiments, the inhibitory receptor for immune cells is KIR2DL1. This invention provides a fusion protein comprising a presenting peptide covalently linked to a β2M peptide via a linker, wherein the fusion protein binds to an MHC heavy chain to form an MHC complex, which binds to the KIR2DL1 receptor on immune cells to inhibit the immune cells. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are NK cells. Human KIR2DL1 may have an amino acid sequence corresponding to the sequence provided below in GenBank No. SPC71652.1 (accession number: SPC71652.1, GI: 1373834531; see below).

In some embodiments, the inhibitory receptor for immune cells is KIR2DL2. This invention provides a fusion protein comprising a presenting peptide covalently linked to a β2M peptide via a linker, wherein the fusion protein binds to an MHC heavy chain to form an MHC complex, which binds to the KIR2DL2 receptor on immune cells to inhibit the immune cells. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are NK cells. Human KIR2DL2 may have the amino acid sequence corresponding to the sequence provided below, GenBank No. ACI49717.1 (accession number: ACI49717.1, GI: 209512829; see below).

In some embodiments, the inhibitory receptor for immune cells is KIR2DL3. This invention provides a fusion protein comprising a presenting peptide covalently linked to a β2M peptide via a linker, wherein the fusion protein binds to an MHC heavy chain to form an MHC complex, which binds to the KIR2DL3 receptor on immune cells to inhibit the immune cells. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are NK cells. Human KIR2DL3 may have the amino acid sequence corresponding to the sequence provided below, GenBank No. ADN34702.1 (accession number: ADN34702.1, GI: 307141824; see below).

In some embodiments, the inhibitory receptor for immune cells is KIR2DL4. This invention provides a fusion protein comprising a presenting peptide covalently linked to a β2M peptide via a linker, wherein the fusion protein binds to an MHC heavy chain to form an MHC complex, which binds to the KIR2DL4 receptor on immune cells to inhibit the immune cells. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are NK cells. Human KIR2DL4 may have the amino acid sequence corresponding to the sequence provided below, GenBank No. ABW73959.1 (accession number: ABW73959.1, GI: 158551989; see below).

In some embodiments, the inhibitory receptor for immune cells is KIR2DL5. This invention provides a fusion protein comprising a presenting peptide covalently linked to a β2M peptide via a linker, wherein the fusion protein binds to an MHC heavy chain to form an MHC complex, which binds to the KIR2DL5 receptor on immune cells to inhibit the immune cells. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are NK cells. Human KIR2DL5 may have an amino acid sequence corresponding to the sequence provided below in GenBank No. SHO29775.1 (accession number: SHO29775.1, GI: 1313716695; see below).

In some embodiments, the inhibitory receptor for immune cells is KIR3DL1. This invention provides a fusion protein comprising a presenting peptide covalently linked to a β2M peptide via a linker, wherein the fusion protein binds to an MHC heavy chain to form an MHC complex, which binds to the KIR3DL1 receptor on immune cells to inhibit the immune cells. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are NK cells. Human KIR3DL1 may have an amino acid sequence corresponding to the sequence provided below in GenBank No. ADM64608.1 (accession number: ADM64608.1, GI: 305690575; see below).

In some embodiments, the inhibitory receptor for immune cells is KIR3DL2. This invention provides a fusion protein comprising a presenting peptide covalently linked to a β2M peptide via a linker, wherein the fusion protein binds to an MHC heavy chain to form an MHC complex, which binds to the KIR3DL2 receptor on immune cells to inhibit the immune cells. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are NK cells. Human KIR3DL2 may have an amino acid sequence corresponding to the sequence provided below in GenBank No. CUX91181.1 (accession number: CUX91181.1, GI: 998428963; see below).

In some embodiments, the inhibitory receptor for immune cells is KIR3DL3. This invention provides a fusion protein comprising a presenting peptide covalently linked to a β2M peptide via a linker, wherein the fusion protein binds to an MHC heavy chain to form an MHC complex, which binds to the KIR3DL3 receptor on immune cells to inhibit the immune cells. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are NK cells. Human KIR3DL3 may have an amino acid sequence corresponding to the sequence provided below in GenBank No. SHO29793.1 (accession number: SHO29793.1, GI: 1313716772; see below).

In some embodiments, the inhibitory receptor for immune cells is leukocyte immunoglobulin-like receptor 1, “LIR1”. This invention provides a fusion protein comprising a presenting peptide covalently linked to a β2M peptide via a linker, wherein the fusion protein binds to an MHC heavy chain to form an MHC complex, which binds to the LIR1 receptor on immune cells to inhibit the immune cells. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are NK cells. Human LIR1 may have an amino acid sequence corresponding to the sequence provided below in GenBank No. AAG08984.1 (accession number: AAG08984.1, GI: 9954210; see below).

This invention provides a fusion protein comprising a presenting peptide covalently linked to a β2M peptide via a linker, wherein the fusion protein binds to an MHC heavy chain to form an MHC complex, which in turn binds to an inhibitory receptor on immune cells to inhibit immune cells. In some embodiments, the MHC heavy chain is a classical class I MHC heavy chain, a non-classical class I MHC heavy chain, or an MHC-like heavy chain. In some embodiments, this invention provides a fusion protein comprising a presenting peptide covalently linked to a β2M peptide via a linker, wherein the fusion protein binds to a classical class I MHC heavy chain to form an MHC complex, which in turn binds to an inhibitory receptor on immune cells to inhibit immune cells. The classical class I MHC heavy chain may be selected from the group consisting of HLA-A heavy chains, HLA-B heavy chains, and HLA-C heavy chains.

In some embodiments, the classic class I MHC heavy chain is the HLA-A heavy chain. This invention provides a fusion protein comprising an HLA-A restricted presenting peptide covalently linked to a β2M peptide via a linker, wherein the fusion protein binds to the HLA-A heavy chain to form an HLA-A complex, which binds to inhibitory receptors on immune cells to inhibit immune cells. In some embodiments, the fusion protein does not have an HLA-A heavy chain. In some embodiments, the fusion protein has fewer than 500 amino acids, fewer than 400 amino acids, fewer than 300 amino acids, or fewer than 200 amino acids. In some embodiments, the fusion protein has about 100 to about 300 amino acids, about 100 to about 200 amino acids, about 120 to about 180 amino acids, about 120 to about 160 amino acids, or about 140 to about 160 amino acids. The human HLA-A heavy chain is highly polymorphic. For example, an allelic form of the human HLA-A heavy chain has an amino acid sequence corresponding to the sequence Genbank No. BA B63400I (accession number: BAB63400I, GL:15277272).

In some embodiments, the classic class I MHC heavy chain is the HLA-B heavy chain. This invention provides a fusion protein comprising an HLA-B-restricted presenting peptide covalently linked to a β2M peptide via a linker, wherein the fusion protein binds to the HLA-B heavy chain to form an HLA-B complex, which binds to inhibitory receptors on immune cells to inhibit immune cells. In some embodiments, the fusion protein does not have an HLA-B heavy chain. In some embodiments, the fusion protein has fewer than 500 amino acids, fewer than 400 amino acids, fewer than 300 amino acids, or fewer than 200 amino acids. In some embodiments, the fusion protein has about 100 to about 300 amino acids, about 100 to about 200 amino acids, about 120 to about 180 amino acids, about 120 to about 160 amino acids, about 120 to about 160 amino acids, or about 140 to about 160 amino acids. The human HLA-B heavy chain is highly polymorphic. For example, an allelic form of the human HLA-B heavy chain has an amino acid sequence corresponding to the sequence Genbank No. AAA59682.1 (accession number: AAA59682.1, GI: 403145).

In some embodiments, the classic class I MHC heavy chain is the HLA-C heavy chain. This invention provides a fusion protein comprising an HLA-C-restricted presenting peptide covalently linked to a β2M peptide via a linker, wherein the fusion protein binds to the HLA-C heavy chain to form an HLA-C complex, which binds to inhibitory receptors on immune cells to suppress immune cells. In some embodiments, the fusion protein does not have an HLA-C heavy chain. In some embodiments, the fusion protein has fewer than 500 amino acids, fewer than 400 amino acids, fewer than 300 amino acids, or fewer than 200 amino acids. In some embodiments, the fusion protein has about 100 to about 300 amino acids, about 100 to about 200 amino acids, about 120 to about 180 amino acids, about 120 to about 160 amino acids, or about 140 to about 160 amino acids. The human HLA-C heavy chain is highly polymorphic. For example, an allelic form of the human HLA-C heavy chain has an amino acid sequence corresponding to the sequence Genbank No. BAB63310.1 (accession number: BAB63310.1, GI: 15277217).

In some embodiments, the present invention provides a fusion protein comprising a presenting peptide covalently linked to a β2M peptide via a linker, wherein the fusion protein binds to a non-classical class I MHC heavy chain to form an MHC complex, the MHC complex binding to an inhibitory receptor on immune cells to inhibit immune cells. The non-classical class I MHC heavy chain may be selected from the group consisting of HLA-E heavy chains, HLA-F heavy chains, and HLA-G heavy chains.

In some embodiments, the non-classical class I MHC heavy chain is the HLA-E heavy chain. This invention provides a fusion protein comprising an HLA-E-restricted presenting peptide covalently linked to a β2M peptide via a linker, wherein the fusion protein binds to the HLA-E heavy chain to form an HLA-E complex, which binds to inhibitory receptors on immune cells to inhibit immune cells. In some embodiments, the fusion protein does not have an HLA-E heavy chain. In some embodiments, the fusion protein has fewer than 500 amino acids, fewer than 400 amino acids, fewer than 300 amino acids, or fewer than 200 amino acids. In some embodiments, the fusion protein has about 100 to about 300 amino acids, about 100 to about 200 amino acids, about 120 to about 180 amino acids, about 120 to about 160 amino acids, about 120 to about 160 amino acids, or about 140 to about 160 amino acids. The human HLA-E heavy chain is polymorphic. For example, an allelic form of the human HLA-E heavy chain has an amino acid sequence corresponding to the sequence Genbank No. ARB08449.1 (accession number: ARB08449.1, GI: 1168024009).

In some embodiments, the non-classical class I MHC heavy chain is the HLA-F heavy chain. This invention provides a fusion protein comprising an HLA-F restricted presenting peptide covalently linked to a β2M peptide via a linker, wherein the fusion protein binds to the HLA-F heavy chain to form an HLA-F complex, which binds to inhibitory receptors on immune cells to inhibit immune cells. In some embodiments, the fusion protein does not have an HLA-F heavy chain. In some embodiments, the fusion protein has fewer than 500 amino acids, fewer than 400 amino acids, fewer than 300 amino acids, or fewer than 200 amino acids. In some embodiments, the fusion protein has about 100 to about 300 amino acids, about 100 to about 200 amino acids, about 120 to about 180 amino acids, about 120 to about 160 amino acids, or about 140 to about 160 amino acids. The human HLA-F heavy chain is polymorphic. For example, an allelic form of the human HLA-F heavy chain has an amino acid sequence corresponding to the sequence Genbank No. BAB63337.1 (accession number: BAB63337.1, GI: 15277244).

In some embodiments, the non-classical class I MHC heavy chain is the HLA-G heavy chain. This invention provides a fusion protein comprising an HLA-G-restricted presenting peptide covalently linked to a β2M peptide via a linker, wherein the fusion protein binds to the HLA-G heavy chain to form an HLA-G complex, which binds to an inhibitory receptor on immune cells to inhibit immune cells. In some embodiments, the fusion protein does not have an HLA-G heavy chain. In some embodiments, the fusion protein has fewer than 500 amino acids, fewer than 400 amino acids, fewer than 300 amino acids, or fewer than 200 amino acids. In some embodiments, the fusion protein has about 100 to about 300 amino acids, about 100 to about 200 amino acids, about 120 to about 180 amino acids, about 120 to about 160 amino acids, or about 140 to about 160 amino acids. The human HLA-G heavy chain is highly polymorphic. For example, an allelic form of the human HLA-G heavy chain has an amino acid sequence corresponding to the sequence Genbank No. BAB63336.1 (accession number: BAB63336.1, GI: 15277243).

In some embodiments, the present invention provides a fusion protein comprising a presenting peptide covalently linked to a β2M peptide via a linker, wherein the fusion protein binds to an MHC-like heavy chain to form an MHC-like complex, the MHC-like complex binding to an inhibitory receptor of an immune cell to inhibit the immune cell. The MHC-like complex may be selected from the group consisting of CD1, MR1, FcRN, and UL18.

In some embodiments, the MHC-like heavy chain is the CD1 heavy chain. The present invention provides a fusion protein comprising a CD1-restricted presenting molecule covalently linked to a β2M peptide via a linker, wherein the fusion protein binds to the CD1 heavy chain to form a CD1 complex, the CD1 complex binding to an inhibitory receptor on an immune cell to inhibit the immune cell. In some embodiments, the fusion protein does not have a CD1 heavy chain. In some embodiments, the fusion protein has fewer than 500 amino acids, fewer than 400 amino acids, fewer than 300 amino acids, or fewer than 200 amino acids. In some embodiments, the fusion protein has about 100 to about 300 amino acids, about 100 to about 200 amino acids, about 120 to about 180 amino acids, about 120 to about 160 amino acids, or about 140 to about 160 amino acids. In some embodiments, the MHC-like heavy chain comprises CD1a, CD1b, CD1c, CD1d, and CD1e heavy chains. The human CD1a heavy chain corresponds to the amino acid sequence of NCBI reference sequence NP_001754.2 (accession number: NP_001754.2, GI: 110618224). The human CD1b heavy chain corresponds to the amino acid sequence of NCBI reference sequence XP_011508421.1 (accession number: XP_011508421.1, GI: 767910806). The human CD1c heavy chain corresponds to the amino acid sequence of NCBI reference sequence XP_005245636.1 (accession number: XP_005245636.1, GI: 530365569). Human CD1d heavy chain can have an amino acid sequence corresponding to the NCBI reference sequence: NP_001358692.1 (accession number: NP_001358692.1, GI: 1707918971; precursor). Human CD1e heavy chain can have an amino acid sequence corresponding to the sequence Genbank No. AAI31694.1 (accession number: AAI31694.1, GI: 124297103).

In some embodiments, the MHC-like heavy chain is the MR1 heavy chain. The human MR1 heavy chain may have an amino acid sequence corresponding to the sequence in Genbank No. AAH12485.1. This invention provides a fusion protein comprising an MR1-restricted presenting molecule covalently linked to a β2M peptide via a linker, wherein the fusion protein binds to the MR1 heavy chain to form an MR1 complex, which binds to an inhibitory receptor on immune cells to inhibit immune cells. In some embodiments, the fusion protein does not have an MR1 heavy chain. In some embodiments, the fusion protein has fewer than 500 amino acids, fewer than 400 amino acids, fewer than 300 amino acids, or fewer than 200 amino acids. In some embodiments, the fusion protein has about 100 to about 300 amino acids, about 100 to about 200 amino acids, about 120 to about 180 amino acids, about 120 to about 160 amino acids, or about 140 to about 160 amino acids. Human MR1 heavy chain can have an amino acid sequence that corresponds to the sequence Genbank No. CAB77667.1 (accession number: CAB77667.1, GI: 7271191).

In some embodiments, the MHC-like heavy chain is an FcRn heavy chain. The present invention provides a fusion protein comprising an FcRn-restricted presentation molecule covalently linked to a β2M peptide via a linker, wherein the fusion protein binds to the FcRn heavy chain to form an FcRn complex, the FcRn complex binding to an inhibitory receptor on an immune cell to inhibit the immune cell. In some embodiments, the fusion protein does not have an FcRn heavy chain. In some embodiments, the fusion protein has fewer than 500 amino acids, fewer than 400 amino acids, fewer than 300 amino acids, or fewer than 200 amino acids. In some embodiments, the fusion protein has about 100 to about 300 amino acids, about 100 to about 200 amino acids, about 120 to about 180 amino acids, about 120 to about 160 amino acids, or about 140 to about 160 amino acids. Human FcRn heavy chains can have an amino acid sequence corresponding to the sequence UniProtKB/Swiss-Prot: P55899.1 (accession number: P55899.1; GI: 2497331).

In some embodiments, the MHC-like heavy chain is UL18. The present invention provides a fusion protein comprising a UL18-restricted presenting molecule covalently linked to a β2M peptide via a linker, wherein the fusion protein binds to the UL18 heavy chain to form a UL18 complex, the UL18 complex binding to an inhibitory receptor on an immune cell to inhibit the immune cell. In some embodiments, the fusion protein does not have a UL18 polypeptide. In some embodiments, the fusion protein has fewer than 500 amino acids, fewer than 400 amino acids, fewer than 300 amino acids, or fewer than 200 amino acids. In some embodiments, the fusion protein has about 100 to about 300 amino acids, about 100 to about 200 amino acids, about 120 to about 180 amino acids, about 120 to about 160 amino acids, or about 140 to about 160 amino acids. Human UL18 can have an amino acid sequence corresponding to the sequence Genbank No. AJY57863.1 (accession number: AJY57863.1, GI: 777959438).

This invention provides a fusion protein comprising a presenting peptide covalently linked to a β2M peptide via a linker. The presenting peptide may be derived from a virus, prokaryote, eukaryote, or mammal. In some embodiments, the presenting peptide is derived from a virus. In some embodiments, the presenting peptide is derived from a prokaryote. In some embodiments, the presenting peptide is derived from a eukaryote. In some embodiments, the presenting peptide is derived from a mammal. In some embodiments, the presenting peptide is derived from a human. The presenting peptide may have 5-30 amino acids, 5-25 amino acids, 5-20 amino acids, 7-20 amino acids, 7-18 amino acids, 7-15 amino acids, 8-12 amino acids, or 8-10 amino acids. In some embodiments, the presenting peptide has 5-30 amino acids. In some embodiments, the presenting peptide has 5-25 amino acids. In some embodiments, the presenting peptide has 5-20 amino acids. In some embodiments, the presenting peptide has 7-20 amino acids. In some embodiments, the presenting peptide has 7-18 amino acids. In some embodiments, the presenting peptide has 7-15 amino acids. In some embodiments, the presenting peptide has 8-12 amino acids. In some embodiments, the presenting peptide has 8-10 amino acids.

The presenting peptide may be a signal peptide of a class I MHC molecule or a fragment thereof. In some embodiments, the presenting peptide is a fragment of a signal peptide of a class I MHC molecule. In some embodiments, the present invention provides a fusion protein comprising a presenting peptide covalently linked to a β2M peptide via a linker, wherein the presenting peptide is a signal peptide of a class I MHC molecule or a fragment thereof. In some embodiments, the fusion protein does not have an MHC heavy chain. In some embodiments, the fusion protein does not have an HLA-E heavy chain. In some embodiments, the fusion protein does not have an HLA-A heavy chain, HLA-B heavy chain, HLA-C heavy chain, HLA-E heavy chain, HLA-F heavy chain, or HLA-G heavy chain. In some embodiments, the fusion protein has fewer than 500 amino acids, fewer than 400 amino acids, fewer than 300 amino acids, or fewer than 200 amino acids. In some embodiments, the fusion protein has about 100 to about 300 amino acids, about 100 to about 200 amino acids, about 120 to about 180 amino acids, about 120 to about 160 amino acids, or about 140 to about 160 amino acids.

The signal peptide of class I MHC molecules is a peptide located at the N-terminus of the class I MHC heavy chain. It guides newly synthesized class I MHC heavy chain proteins to the endoplasmic reticulum, allowing mature heavy chain molecules, β2M, and the presenting peptide to form a complex on the cell surface. This polypeptide is then cleaved within the mature protein. Class I MHC signal peptides typically have 13–80 amino acids. Some class I MHC signal peptides have 20–50 amino acids.

In some embodiments, the presenting peptide is a signal peptide of a class I MHC molecule or a fragment thereof. The class I MHC molecule may be selected from the group consisting of HLA-A1, HLA-A2, HLA-A*3401, HLA-A*80, HLA-B7, HLA-B*13, HLA-B15, HLA-Cw3, HLA-Cw*2, HLA-Cw*0809, HLA-Cw7, HLA-Cw*1701, HLA-G, and HLA-F. In some embodiments, the presenting peptide is a signal peptide of HLA-A1 or a fragment thereof. In some embodiments, the presenting peptide is a signal peptide of HLA-A2 or a fragment thereof. In some embodiments, the presenting peptide is a signal peptide of HLA-A*3401 or a fragment thereof. In some embodiments, the presenting peptide is a signal peptide of HLA-A*80 or a fragment thereof. In some embodiments, the presenting peptide is a signal peptide of HLA-B7 or a fragment thereof. In some embodiments, the presenting peptide is the signal peptide of HLA-B*13 or a fragment thereof. In some embodiments, the presenting peptide is the signal peptide of HLA-B15 or a fragment thereof. In some embodiments, the presenting peptide is the signal peptide of HLA-Cw3 or a fragment thereof. In some embodiments, the presenting peptide is the signal peptide of HLA-Cw*2 or a fragment thereof. In some embodiments, the presenting peptide is the signal peptide of HLA-Cw*0809 or a fragment thereof. In some embodiments, the presenting peptide is the signal peptide of HLA-Cw7 or a fragment thereof. In some embodiments, the presenting peptide is the signal peptide of HLA-Cw*1701 or a fragment thereof. In some embodiments, the presenting peptide is the signal peptide of HLA-G or a fragment thereof. In some embodiments, the presenting peptide is the signal peptide of HLA-F or a fragment thereof. In some embodiments, these exemplary class I MHC molecule signal peptides or fragments thereof are HLA-E-restricted presenting peptides. The presenting peptide is a signal peptide of a class I MHC molecule or a fragment thereof, and may have 5-30 amino acids. In some embodiments, the presenting peptide is a signal peptide of a class I MHC molecule or a fragment thereof, and may have 5-25 amino acids, 5-20 amino acids, 7-18 amino acids, 7-15 amino acids, 8-12 amino acids, or 8-10 amino acids. In some embodiments, the presenting peptide is a signal peptide of a class I MHC molecule or a fragment thereof, and may have 8-10 amino acids.

In some embodiments, the presenting polypeptide is a signal peptide of a class I MHC molecule or a fragment thereof, and has the following amino acid sequence: _{X1 X2 X3 X4 X5 X6 X7 X8} L; wherein, _X1 is V or I; _X2 is T, A, M or L; _X3 is A, P, K or N; _X4 is P, L or T; _X5 is R, Q or K; _X6 is T or A; _X7 is L, I, V or P; _X8 is V, L, I, F or T (SEQ ID NO:115).

In some embodiments, the presenting peptide is a signal peptide of a class I MHC molecule or a fragment thereof, and the amino acid sequence of the presenting peptide is selected from (1) YLLPRRGPRL (SEQ ID NO: 21); (2) ALALVRMLI (SEQ ID NO: 22); (3) AISPRTLNA (SEQ ID NO: 23); (4) QMRPVSRVL (SEQ ID NO: 24); (5) SQQPYLQLQ (SEQ ID NO: 25); (6) VTAPRTL LL(SEQ ID NO:26); (7)VTAPRTVLL(SEQ ID NO:27); (8)VTAPRTLVL(SEQ ID NO:28); (9)VMAPQALLL(SEQ ID NO:29 ); (10) VMAPRALLL (SEQ ID NO: 30); (11) VMAPRTLTL (SEQ ID NO: 31); (12) VMAPRTLFL (SEQ ID NO: 32); (13) VMAPR TLVL(SEQ ID NO:33); (14)VMAPRTLIL(SEQ ID NO:34); (15)IMAPRTLVL(SEQ ID NO:35); (16)VMAPRTLLL(SEQ ID NO: 36); (17) VMPPRTLLL (SEQ ID NO: 37); (18) VMAPRTVLL (SEQ ID NO: 38); (19) VMAPRSLLL (SEQ ID NO: 39); (20) VMAPRSLIL(SEQ ID NO:40); (21)VMTPRTLVL(SEQ ID NO:41); (22)VMAPRILIL(SEQ ID NO:42); (23)AMAPRTLIL(S EQ ID NO:43); (24)VIAPRTLVL(SEQ ID NO:44); (25)VMAPQSLLL(SEQ ID NO:45); (26)VMAPRTFVL(SEQ ID NO:46 ); (27) VMTPRTLIL (SEQ ID NO: 47); (28) VTAPRTLIL (SEQ ID NO: 48); (29) VMAPWTLLL (SEQ ID NO: 49); (30) VMVPRS LIL(SEQ ID NO:50); (31)AMAPTLVL(SEQ ID NO:51); (32)VIAPRTLIL(SEQ ID NO:52); (33)VIAPRTLLL(SEQ ID NO:53); (34)VLAPRTLIL(SEQ ID NO:54); (35)VMALRTLIL(SEQ ID NO:55); (36)VMAPRGLIL(SEQ ID NO:56); (37)V MAPRNLIL(SEQ ID NO:57); (38)VMAPRTLFV(SEQ ID NO:58); (39)VMAPRTLLM(SEQ ID NO:59); (40)VMAPRTLVM(SEQ ID NO:60); (41) VMAPRTSLL (SEQ ID NO:61); (42) VMAPRTSVL (SEQ ID NO:62); (43) VMAPWTLIL (SEQ ID NO:63); ( 44) VMAPWTLVL (SEQ ID NO: 64); (45) VMDPRTLLL (SEQ ID NO: 65); (46) VMGPRTLIL (SEQ ID NO: 66); (47) VMGPRTLL The group consisting of (48) VMVPQTLIL (SEQ ID NO: 68); (49) VMVPRTLLL (SEQ ID NO: 69); (50) VVAPRTLIL (SEQ ID NO: 70); (51) VVAPRTLLL (SEQ ID NO: 71); (52) VMVPRTLIL (SEQ ID NO: 72); and (53) VMATRTLLL (SEQ ID NO: 73). These exemplary presenting peptides are HLA-E restricted presenting peptides.

In some embodiments, the amino acid sequence of the presenting peptide is (1) YLLPRRGPRL (SEQ ID NO: 21). In some embodiments, the amino acid sequence of the presenting peptide is (2) ALALVRMLI (SEQ ID NO: 22). In some embodiments, the amino acid sequence of the presenting peptide is (3) AISPRTLNA (SEQ ID NO: 23). In some embodiments, the amino acid sequence of the presenting peptide is (4) QMRPVSRVL (SEQ ID NO: 24). In some embodiments, the amino acid sequence of the presenting peptide is (5) SQQPYLQLQ (SEQ ID NO: 25). In some embodiments, the amino acid sequence of the presenting peptide is (6) VTAPRTLLL (SEQ ID NO: 26). In some embodiments, the amino acid sequence of the presenting peptide is (7) VTAPRTVLL (SEQ ID NO: 27). In some embodiments, the amino acid sequence of the presenting peptide is (8) VTAPRTLVL (SEQ ID NO: 28). In some embodiments, the amino acid sequence of the presenting peptide is (9) VMAPQALLL (SEQ ID NO: 29). In some embodiments, the amino acid sequence of the presenting peptide is (10) VMAPRALLL (SEQ ID NO: 30). In some embodiments, the amino acid sequence of the presenting peptide is (11) VMAPRTLTL (SEQ ID NO: 31). In some embodiments, the amino acid sequence of the presenting peptide is (12) VMAPRTLFL (SEQ ID NO: 32). In some embodiments, the amino acid sequence of the presenting peptide is (13) VMAPRTLVL (SEQ ID NO: 33). In some embodiments, the amino acid sequence of the presenting peptide is (14) VMAPRTLIL (SEQ ID NO: 34). In some embodiments, the amino acid sequence of the presenting peptide is (15) IMAPRTLVL (SEQ ID NO: 35). In some embodiments, the amino acid sequence of the presenting peptide is (16) VMAPRTLLL (SEQ ID NO: 36). In some embodiments, the amino acid sequence of the presenting peptide is (17) VMPPRTLLL (SEQ ID NO: 37). In some embodiments, the amino acid sequence of the presenting peptide is (18) VMAPRTVLL (SEQ ID NO: 38). In some embodiments, the amino acid sequence of the presenting peptide is (19) VMAPRSLLL (SEQ ID NO: 39). In some embodiments, the amino acid sequence of the presenting peptide is (20) VMAPRSLIL (SEQ ID NO: 40). In some embodiments, the amino acid sequence of the presenting peptide is (21) VMTPRTLVL (SEQ ID NO: 41). In some embodiments, the amino acid sequence of the presenting peptide is (22) VMAPRILIL (SEQ ID NO: 42). In some embodiments, the amino acid sequence of the presenting peptide is (23) AMAPRTLIL (SEQ ID NO: 43). In some embodiments, the amino acid sequence of the presenting peptide is (24) VIAPRTLVL (SEQ ID NO: 44). In some embodiments, the amino acid sequence of the presenting peptide is (25) VMAPQSLLL (SEQ ID NO: 45). In some embodiments, the amino acid sequence of the presenting peptide is (26) VMAPRTFVL (SEQ ID NO: 46). In some embodiments, the amino acid sequence of the presenting peptide is (27) VMTPRTLIL (SEQ ID NO: 47). In some embodiments, the amino acid sequence of the presenting peptide is (28) VTAPRTLIL (SEQ ID NO: 48). In some embodiments, the amino acid sequence of the presenting peptide is (29) VMAPWTLLL (SEQ ID NO: 49). In some embodiments, the amino acid sequence of the presenting peptide is (30) VMVPRSLIL (SEQ ID NO: 50). In some embodiments, the amino acid sequence of the presenting peptide is (31) AMAPRTLVL (SEQ ID NO: 51). In some embodiments, the amino acid sequence of the presenting peptide is (32) VIAPRTLIL (SEQ ID NO: 52). In some embodiments, the amino acid sequence of the presenting peptide is (33) VIAPRTLLL (SEQ ID NO: 53). In some embodiments, the amino acid sequence of the presenting peptide is (34) VLAPRTLIL (SEQ ID NO: 54). In some embodiments, the amino acid sequence of the presenting peptide is (35) VMALRTLIL (SEQ ID NO: 55). In some embodiments, the amino acid sequence of the presenting peptide is (36) VMAPRGLIL (SEQ ID NO: 56). In some embodiments, the amino acid sequence of the presenting peptide is (37) VMAPRNLIL (SEQ ID NO: 57). In some embodiments, the amino acid sequence of the presenting peptide is (38) VMAPRTLFV (SEQ ID NO: 58). In some embodiments, the amino acid sequence of the presenting peptide is (39) VMAPRTLLM (SEQ ID NO: 59). In some embodiments, the amino acid sequence of the presenting peptide is (40) VMAPRTLVM (SEQ ID NO: 60). In some embodiments, the amino acid sequence of the presenting peptide is (41) VMAPRTSLL (SEQ ID NO: 61). In some embodiments, the amino acid sequence of the presenting peptide is (42) VMAPRTSVL (SEQ ID NO: 62). In some embodiments, the amino acid sequence of the presenting peptide is (43) VMAPWTLIL (SEQ ID NO: 63). In some embodiments, the amino acid sequence of the presenting peptide is (44) VMAPWTLVL (SEQ ID NO: 64). In some embodiments, the amino acid sequence of the presenting peptide is (45) VMDPRTLLL (SEQ ID NO: 65). In some embodiments, the amino acid sequence of the presenting peptide is (46) VMGPRTLIL (SEQ ID NO: 66). In some embodiments, the amino acid sequence of the presenting peptide is (47) VMGPRTLLL (SEQ ID NO: 67). In some embodiments, the amino acid sequence of the presenting peptide is (48) VMVPQTLIL (SEQ ID NO: 68). In some embodiments, the amino acid sequence of the presenting peptide is (49) VMVPRTLLL (SEQ ID NO: 69). In some embodiments, the amino acid sequence of the presenting peptide is (50) VVAPRTLIL (SEQ ID NO: 70). In some embodiments, the amino acid sequence of the presenting peptide is (51) VVAPRTLLL (SEQ ID NO: 71). In some embodiments, the amino acid sequence of the presenting peptide is (52) VMVPRTLIL (SEQ ID NO: 72). In some embodiments, the amino acid sequence of the presenting peptide is (53) VMATRTLLL (SEQ ID NO: 73).

Other HLA-E-restricted presenting peptides are also described in this art. See, for example, United States Patent Application 20190314445; Celik et al., Immunogenetics 2016.68:29-41; Hannoun et al. Immunology Letters 2018.202:65-72; et al. Cell Rep 2018.14(8):1967-1976; et al. Front Immunol 2018.9:2410. All of these references are incorporated herein by reference in their entirety. Similarly, presenting peptides restricted to HLA-A, HLA-B, HLA-C, HLA-F, or HLA-G are also well known in the art. See, for example, Gfeller et al.Front Immunol 2018.9:1716; Luo et al.Bioinform BiolInsights 2015.9(Suppl 3):21-9; You et al.PATTERN RECOGNITION IN BIOINFORMATICS2007.pp 337-3 48.Lecture Notes in Computer Science,vol 4774.Springer,Berlin,Heidelberg;Purcell et al.Nat Protoc 2019.14(6):1687-1707;Rock et al.TrendsImmunol 2016.37(11):724- 737; Celik et al. Immunogenetics 2018.70(8):485–494; DiMarco et al.J Immunol 2017.1 99(8):2639-2651; Dulberger et al.Immunity 2017.46(6):1018–1029.e7; Ho et al.Immuno genetics 2019.71(5-6):353-360; Sim etal. Immunity 2017.46(6):972-974; Rammensee et al. al.Annu Rev Immunol 1993.11:213-44; Schellens et al.PLoS One 2015.10(9):e0136417. All of these references are incorporated herein by reference in their entirety.

In some embodiments, the presenting peptide may also be derived from viral proteins, such as CMV, EBV, HIV, etc. In some embodiments, the presenting peptide is a fragment of CMV. In some embodiments, the presenting peptide is a fragment of EBV. In some embodiments, the presenting peptide is a fragment of HIV.

The fusion protein provided by this invention has a presenting peptide covalently linked to a β2M peptide via a linker. In some embodiments, the fusion protein provided by this invention has a presenting peptide, a linker, and a β2M peptide from the N-terminus to the C-terminus. In some embodiments, the fusion protein provided by this invention has a β2M peptide, a linker, and a presenting peptide from the N-terminus to the C-terminus. In some embodiments, the fusion protein disclosed by this invention further includes a membrane localization signal peptide at its N-terminus, which guides the fusion protein to localize to the cell membrane and is subsequently cleaved from the fusion protein. The length of the membrane localization signal peptide can be between about 20 and 80 amino acids. An exemplary membrane localization signal peptide may have the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4. In some embodiments, the fusion protein provided by this invention consists of a presenting peptide, a linker, and a β2M peptide from the N-terminus to the C-terminus. In some embodiments, the fusion protein provided by this invention consists of a β2M peptide, a linker, and a presenting peptide from the N-terminus to the C-terminus.

The fusion protein provided by this invention has a presenting peptide covalently linked to a β2M peptide via a linker. The linker connects the presenting peptide and the β2M peptide of the fusion protein and provides sufficient flexibility for the fusion protein to form a stable complex with the MHC heavy chain. The linker provided by this invention may have about 5 to 50 amino acids and includes at least one of the following five amino acids (Gly, Ala, Pro, Val, and Leu) and at least one of the following eight amino acids (Ser, Thr, Glu, Lys, Asn, Gln, Asp, and Arg). In some embodiments, the linker provided by this invention has 5 to 30 amino acids.

In some embodiments, the amino acid sequence of the linker is (EAAAK)n, where n = 1, 2, 3, 4, or 5 (SEQ ID NO: 109). In some embodiments, the amino acid sequence of the linker is (EAAAK)n, where n = 3, 4, or 5 (SEQ ID NO: 110). In some embodiments, the amino acid sequence of the linker is (GGGGS)n, where n = 1, 2, 3, 4, or 5 (SEQ ID NO: 111). In some embodiments, the amino acid sequence of the linker is (GGGGS)n, where n = 3, 4, or 5 (SEQ ID NO: 112).

In some embodiments, the amino acid sequence of the linker is (G)nS, where n = 1, 2, 3, 4, or 5 (SEQ ID NO: 113). In some embodiments, the amino acid sequence of the linker is an m-series combination of (G)nS, where n = 1, 2, 3, 4, or 5; and m = 1, 2, 3, 4, or 5 (SEQ ID NO: 122). For example, the amino acid sequence of the linker may be (G4S)(G2S)(GS)(G3S), i.e., GGGGSGGSGSGGGS (SEQ ID NO: 114).

In some embodiments, the amino acid sequence of the linker is (GGGGS)3 or (G4S)3, namely GGGGSGGGGSGGGGS (SEQ ID NO:1). In some embodiments, the amino acid sequence of the linker is (G4S)4, namely GGGGSGGGGSGGGGSGGGS (SEQ ID NO:2).

The fusion protein provided by this invention has a presenting peptide covalently linked to a β2M peptide via a linker. In some embodiments, the β2M peptide is a wild-type β2M protein. In some embodiments, the β2M peptide is wild-type human β2M (SEQ ID NO:7). In some embodiments, the β2M peptide is a functional fragment of the wild-type β2M protein. In some embodiments, the β2M peptide is a functional variant of the wild-type β2M protein. In some embodiments, the β2M peptide is a functional fragment of wild-type human β2M. In some embodiments, the β2M peptide is a functional variant of wild-type human β2M. In some embodiments, the β2M peptide is a functional variant of the wild-type β2M protein, and its amino acid sequence has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO:7. In some embodiments, the amino acid sequence of the β2M peptide has at least 85% identity with SEQ ID NO:7. In some embodiments, the amino acid sequence of the β2M peptide has at least 90% identity with SEQ ID NO:7. In some embodiments, the amino acid sequence of the β2M peptide has at least 95% identity with SEQ ID NO:7. In some embodiments, the amino acid sequence of the β2M peptide has at least 98% identity with SEQ ID NO:7. In some embodiments, the amino acid sequence of the β2M peptide has at least 99% identity with SEQ ID NO:7. In some embodiments, the amino acid sequence of the β2M peptide is SEQ ID NO:7.

In some embodiments, the β2M peptide is the mature form of human β2M (SEQ ID NO: 81), and the fusion protein provided by the present invention has a presenting peptide covalently linked to the mature human β2M peptide via a linker. In some embodiments, the β2M peptide is a functional fragment of the mature form of human β2M protein. In some embodiments, the β2M peptide is a functional variant of the mature form of human β2M protein. In some embodiments, the β2M peptide is a functional variant of the mature form of human β2M protein, having an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO: 81. In some embodiments, the amino acid sequence of the β2M peptide has at least 85% identity with SEQ ID NO: 81. In some embodiments, the amino acid sequence of the β2M peptide has at least 90% identity with SEQ ID NO: 81. In some embodiments, the amino acid sequence of the β2M peptide has at least 95% identity with SEQ ID NO: 81. In some embodiments, the amino acid sequence of the β2M peptide has at least 98% identity with SEQ ID NO:81. In some embodiments, the amino acid sequence of the β2M peptide has at least 99% identity with SEQ ID NO:81. In some embodiments, the amino acid sequence of the β2M peptide is SEQ ID NO:81.

In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs:5 and 13-18. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:5 and 13-18. In some embodiments, the fusion protein does not have an MHC heavy chain. In some embodiments, the fusion protein does not have an HLA-E heavy chain. In some embodiments, the fusion protein does not have an HLA-A heavy chain, HLA-B heavy chain, HLA-C heavy chain, HLA-E heavy chain, HLA-F heavy chain, or HLA-G heavy chain. In some embodiments, the fusion protein has fewer than 500 amino acids, fewer than 400 amino acids, fewer than 300 amino acids, or fewer than 200 amino acids. In some embodiments, the fusion protein has about 100 to about 300 amino acids, about 100 to about 200 amino acids, about 120 to about 180 amino acids, about 120 to about 160 amino acids, or about 140 to about 160 amino acids. In some embodiments, the fusion protein has about 120 to about 180 amino acids.

In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises an amino acid sequence having at least 85% identity with amino acids 21-143 of SEQ ID NO:5 or SEQ ID NO:5. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises an amino acid sequence having at least 90% identity with amino acids 21-143 of SEQ ID NO:5 or SEQ ID NO:5. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises an amino acid sequence having at least 95% identity with amino acids 21-143 of SEQ ID NO:5 or SEQ ID NO:5. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises an amino acid sequence having at least 98% identity with amino acids 21-143 of SEQ ID NO:5 or SEQ ID NO:5. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises an amino acid sequence having at least 99% identity with amino acids 21-143 of SEQ ID NO:5 or SEQ ID NO:5. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention includes SEQ ID NO:5. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention includes amino acids 21-143 of SEQ ID NO:5.

In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises an amino acid sequence having at least 85% identity with amino acids 21-143 of SEQ ID NO:13. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises an amino acid sequence having at least 90% identity with amino acids 21-143 of SEQ ID NO:13. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises an amino acid sequence having at least 95% identity with amino acids 21-143 of SEQ ID NO:13. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises an amino acid sequence having at least 98% identity with amino acids 21-143 of SEQ ID NO:13. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises an amino acid sequence having at least 99% identity with amino acids 21-143 of SEQ ID NO:13. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises amino acids 21-143 of SEQ ID NO:13. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises amino acids 21-143 of SEQ ID NO:13.

In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises an amino acid sequence having at least 85% identity with amino acids 21-143 of SEQ ID NO:14. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises an amino acid sequence having at least 90% identity with amino acids 21-143 of SEQ ID NO:14. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises an amino acid sequence having at least 95% identity with amino acids 21-143 of SEQ ID NO:14. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises an amino acid sequence having at least 98% identity with amino acids 21-143 of SEQ ID NO:14. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises an amino acid sequence having at least 99% identity with amino acids 21-143 of SEQ ID NO:14. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises amino acids 21-143 of SEQ ID NO:14.

In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises an amino acid sequence having at least 85% identity with amino acids 21-143 of SEQ ID NO:15 or SEQ ID NO:15. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises an amino acid sequence having at least 90% identity with amino acids 21-143 of SEQ ID NO:15 or SEQ ID NO:15. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises an amino acid sequence having at least 95% identity with amino acids 21-143 of SEQ ID NO:15 or SEQ ID NO:15. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises an amino acid sequence having at least 98% identity with amino acids 21-143 of SEQ ID NO:15 or SEQ ID NO:15. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises an amino acid sequence having at least 99% identity with amino acids 21-143 of SEQ ID NO:15 or SEQ ID NO:15. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises amino acids 21-143 of SEQ ID NO:15.

In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises an amino acid sequence having at least 85% identity with amino acids 21-143 of SEQ ID NO:16 or SEQ ID NO:16. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises an amino acid sequence having at least 90% identity with amino acids 21-143 of SEQ ID NO:16 or SEQ ID NO:16. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises an amino acid sequence having at least 95% identity with amino acids 21-143 of SEQ ID NO:16 or SEQ ID NO:16. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises an amino acid sequence having at least 98% identity with amino acids 21-143 of SEQ ID NO:16 or SEQ ID NO:16. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises an amino acid sequence having at least 99% identity with amino acids 21-143 of SEQ ID NO:16 or SEQ ID NO:16. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises amino acids 21-143 of SEQ ID NO:16.

In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises an amino acid sequence having at least 85% identity with amino acids 21-143 of SEQ ID NO:17 or SEQ ID NO:17. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises an amino acid sequence having at least 90% identity with amino acids 21-143 of SEQ ID NO:17 or SEQ ID NO:17. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises an amino acid sequence having at least 95% identity with amino acids 21-143 of SEQ ID NO:17 or SEQ ID NO:17. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises an amino acid sequence having at least 98% identity with amino acids 21-143 of SEQ ID NO:17 or SEQ ID NO:17. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises an amino acid sequence having at least 99% identity with amino acids 21-143 of SEQ ID NO:17 or SEQ ID NO:17. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises amino acids 21-143 of SEQ ID NO:17.

In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises an amino acid sequence having at least 85% identity with amino acids 21-144 of SEQ ID NO:18 or SEQ ID NO:18. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises an amino acid sequence having at least 90% identity with amino acids 21-144 of SEQ ID NO:18 or SEQ ID NO:18. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises an amino acid sequence having at least 95% identity with amino acids 21-144 of SEQ ID NO:18 or SEQ ID NO:18. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises an amino acid sequence having at least 98% identity with amino acids 21-144 of SEQ ID NO:18 or SEQ ID NO:18. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises an amino acid sequence having at least 99% identity with amino acids 21-144 of SEQ ID NO:18 or SEQ ID NO:18. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises amino acids 21-144 of SEQ ID NO:18.

In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs:5 and 13-18. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:5 and 13-18.

In some embodiments, the amino acid sequence of the fusion protein provided by the present invention has at least 85% identity with amino acids 21-143 of SEQ ID NO:5. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention has at least 90% identity with amino acids 21-143 of SEQ ID NO:5. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention has at least 95% identity with amino acids 21-143 of SEQ ID NO:5. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention has at least 98% identity with amino acids 21-143 of SEQ ID NO:5. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention has at least 99% identity with amino acids 21-143 of SEQ ID NO:5. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention is composed of SEQ ID NO:5. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention consists of amino acids 21-143 of SEQ ID NO:5.

In some embodiments, the amino acid sequence of the fusion protein provided by the present invention has at least 85% identity with amino acids 21-143 of SEQ ID NO:13. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention has at least 90% identity with amino acids 21-143 of SEQ ID NO:13. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention has at least 95% identity with amino acids 21-143 of SEQ ID NO:13. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention has at least 98% identity with amino acids 21-143 of SEQ ID NO:13. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention has at least 99% identity with amino acids 21-143 of SEQ ID NO:13. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention consists of SEQ ID NO:13. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention consists of amino acids 21-143 of SEQ ID NO:13.

In some embodiments, the amino acid sequence of the fusion protein provided by the present invention has at least 85% identity with amino acids 21-143 of SEQ ID NO:14. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention has at least 90% identity with amino acids 21-143 of SEQ ID NO:14. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention has at least 95% identity with amino acids 21-143 of SEQ ID NO:14. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention has at least 98% identity with amino acids 21-143 of SEQ ID NO:14. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention has at least 99% identity with amino acids 21-143 of SEQ ID NO:14. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention consists of SEQ ID NO:14. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention consists of amino acids 21-143 of SEQ ID NO:14.

In some embodiments, the amino acid sequence of the fusion protein provided by the present invention has at least 85% identity with amino acids 21-143 of SEQ ID NO:15. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention has at least 90% identity with amino acids 21-143 of SEQ ID NO:15. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention has at least 95% identity with amino acids 21-143 of SEQ ID NO:15. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention has at least 98% identity with amino acids 21-143 of SEQ ID NO:15. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention has at least 99% identity with amino acids 21-143 of SEQ ID NO:15. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention consists of SEQ ID NO:15. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention consists of amino acids 21-143 of SEQ ID NO:15.

In some embodiments, the amino acid sequence of the fusion protein provided by the present invention has at least 85% identity with amino acids 21-143 of SEQ ID NO:16 or SEQ ID NO:16. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention has at least 90% identity with amino acids 21-143 of SEQ ID NO:16 or SEQ ID NO:16. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention has at least 95% identity with amino acids 21-143 of SEQ ID NO:16 or SEQ ID NO:16. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention has at least 98% identity with amino acids 21-143 of SEQ ID NO:16 or SEQ ID NO:16. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention has at least 99% identity with amino acids 21-143 of SEQ ID NO:16 or SEQ ID NO:16. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention consists of SEQ ID NO:16. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention consists of amino acids 21-143 of SEQ ID NO:16.

In some embodiments, the amino acid sequence of the fusion protein provided by the present invention has at least 85% identity with amino acids 21-143 of SEQ ID NO:17 or SEQ ID NO:17. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention has at least 90% identity with amino acids 21-143 of SEQ ID NO:17 or SEQ ID NO:16. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention has at least 95% identity with amino acids 21-143 of SEQ ID NO:17 or SEQ ID NO:16. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention has at least 98% identity with amino acids 21-143 of SEQ ID NO:17 or SEQ ID NO:16. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention has at least 99% identity with amino acids 21-143 of SEQ ID NO:17 or SEQ ID NO:16. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention consists of SEQ ID NO:17. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention consists of amino acids 21-143 of SEQ ID NO:17.

In some embodiments, the amino acid sequence of the fusion protein provided by the present invention has at least 85% identity with amino acids 21-144 of SEQ ID NO:18 or SEQ ID NO:18. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention has at least 90% identity with amino acids 21-144 of SEQ ID NO:18 or SEQ ID NO:18. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention has at least 95% identity with amino acids 21-144 of SEQ ID NO:18 or SEQ ID NO:18. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention has at least 98% identity with amino acids 21-144 of SEQ ID NO:18 or SEQ ID NO:18. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention has at least 99% identity with amino acids 21-144 of SEQ ID NO:18 or SEQ ID NO:18. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention consists of SEQ ID NO:18. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention consists of amino acids 21-144 of SEQ ID NO:18.

The fusion proteins described in this invention can be prepared by any method known in the art, including chemical synthesis and recombinant expression techniques. Unless otherwise stated, the practice of this invention employs techniques from molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and other conventional techniques within this technical field. These techniques are described in the references cited in this invention and are fully explained therein. See, for example, Maniatis et al. (1982) Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory Press; Sambrook et al. (1989), Molecular Cloning: A Laboratory Manual , Second Edition, Cold Spring Harbor Laboratory Press; Sambrook et al. (2001) MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., Current Protocols in Molecular Biology , John Wiley&Sons (1987and annual updates); Current Protocols in Immunology , John Wiley&Sons (1987and annual updates) Gait (ed.) (1984) Oligonucleotides Synthesis: A Practical Approach , IRL Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach ,IRL Press; Birren et al. (eds.) (1999) Genome Analysis: A Laboratory Manual , Cold Spring Harbor Laboratory Press; Borrebaeck (ed.) (1995) Antibody Engineering , Second Edition, Oxford University Press; Lo (ed.) (2006) Antibody Engineering: Methods and Protocols (Methods in Molecular Biology) ; Vol. 248, Humana Press, Inc.; Each of these works is incorporated herein by reference in its entirety.

The fusion proteins described in this invention can be prepared and isolated using methods known in the art. Peptides can be synthesized wholly or partially using chemical methods (see, for example, Caruthers (1980). Nucleic Acids Res. Symp. Ser. 215; Horn (1980); and Banga, AK, Therapeutic Peptides and Proteins, Formulation, Processing and Delivery Systems (1995) Technomic Publishing Co., Lancaster, PA). Peptide synthesis can be performed using various solid-phase techniques (see, for example, Roberge Science 269:202 (1995); Merrifield, Methods. Enzymol. 289:3 (1997)) and can be automated, for example, using an ABI 431A peptide synthesizer (Perkin Elmer) according to the manufacturer's instructions. Peptides can also be synthesized using combinatorial methods. Artificially synthesized residues and peptides can be synthesized using a variety of procedures and methods known in the art (see, for example, Organic Syntheses Collective Volumes, Gilman, et al. (Eds) John Wiley & Sons, Inc., NY). Modified peptides can be produced by chemical modification methods (see, for example, Belousov, Nucleic Acids Res. 25:3440 (1997); Frenkel, Free Radic. Biol. Med. 19:373 (1995); and Blommers, Biochemistry 33:7886 (1994)). Peptide sequence variations, derivatives, substitutions, and modifications can also be performed using methods such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR-based mutagenesis. Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res. 10:6487 (1987)), cassette mutagenesis (Wells et al., Gene 34:315 (1985)), restriction selection mutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA 317:415 (1986)) and other techniques can be performed on cloned DNA to produce the peptide sequences, variants, fusions and chimeras of the present invention, as well as their variants, derivatives, substitutions and modifications.

In some embodiments, the recombinant expression vector is used to amplify and express nucleic acids encoding the fusion proteins described in this invention. For example, the recombinant expression vector may be a reproducible DNA construct comprising a synthetic or cDNA-derived DNA fragment encoding a fusion protein operatively linked to a suitable transcriptional and/or translational regulatory element derived from a mammalian, microbial, viral, or insect gene. In some embodiments, a viral vector is used. DNA regions are operatively linked when they are functionally interconnected. For example, if a promoter controls transcription of a sequence, it is operatively linked to a coding sequence; or if the location of a ribosome-binding site allows translation, it is operatively linked to a coding sequence. In some embodiments, the structural element intended for use in a yeast expression system includes a leader sequence that enables the host cell to secrete the translated protein extracellularly. In some embodiments, where no leader or transport sequence is available for the expression of the recombinant protein, the polypeptide may include an N-terminal methionine residue.

A variety of expression host/vector combinations can be used. Expression vectors useful for eukaryotic hosts include, for example, vectors containing expression control sequences from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Expression vectors useful for bacterial hosts include known bacterial plasmids, such as plasmids from *E. coli*, including pCR1, pBR322, pMB9, and their derivatives, as well as plasmids with a broader host range, such as M13 and other filamentous single-stranded DNA bacteriophages.

In some embodiments, the fusion protein of the present invention is expressed by one or more vectors. Suitable host cells for expressing the fusion protein include prokaryotic cells, yeast cells, insect cells, or higher eukaryotic cells under the control of a suitable promoter. Suitable cloning and expression vectors for bacterial, fungal, yeast, and mammalian cell hosts, as well as methods for protein preparation, are well known in the art.

Examples of suitable mammalian host cell lines include, but are not limited to, COS-7 (derived from monkey kidney), L-929 (derived from mouse fibroblasts), C127 (derived from mouse mammary tumors), 3T3 (derived from mouse fibroblasts), CHO (derived from Chinese hamster ovaries), HeLa (derived from human cervical cancer), BHK (derived from hamster kidney fibroblasts), HEK-293 (derived from human embryonic kidney) cell lines and their variants. Mammalian expression vectors may include non-transcriptional elements (e.g., origin of replication), suitable promoters and enhancers linked to the gene to be expressed, and other 5' or 3' flanking non-transcriptional and 5' or 3' untranslated sequences (e.g., necessary ribosome binding sites, polyadenylation sites, splicing donor and acceptor sites, and transcription termination sequences). Expression of recombinant proteins in insect cell culture systems (such as baculoviruses) also provides a powerful method for producing proteins that are correctly folded and have biological functions. Baculovirus systems for producing heterologous proteins in insect cells are well known to those skilled in the art.

6.3 Synthetic receptors

In some embodiments, the fusion protein provided by the present invention can be co-expressed with a synthetic receptor in a cell. In some embodiments, the fusion protein provided by the present invention can bind to a synthetic receptor. As used in this invention, the term "synthetic receptor" refers to an engineered cell surface protein or protein complex comprising (1) a target-binding domain that specifically binds to a target molecule and (2) a functional domain that activates an engineered signal transduction pathway in the cell. The target-binding domain includes an extracellular domain. The functional domain includes an intracellular domain. The synthetic receptor also contains a transmembrane sequence. The synthetic receptor is a protein complex that may include a protein expressed by an exogenous nucleic acid, or at least one exogenously expressed protein and at least one endogenously expressed protein. In some embodiments, the engineered cell may be an immune cell, such as a T cell, natural killer (NK) cell, B cell, macrophage, etc., and the functional domain may directly or indirectly activate the immune cell. The synthetic receptor can be selected from the group consisting of chimeric antigen receptors (“CAR”), T-cell receptors (“TCR”), TCR receptor fusion constructs (“TRuC”), T-cell antigen conjugates (“TAC”), antibody-TCR receptors (“AbTCR”), and chimeric CD3ε receptors. In some embodiments, the synthetic receptor is a CAR. In some embodiments, the synthetic receptor is a TCR. In some embodiments, the synthetic receptor is a TruC.

In some embodiments, the synthetic receptor is a TAC. In some embodiments, the synthetic receptor is an AbTCR. In some embodiments, the synthetic receptor is a chimeric CD3 receptor.

6.3.1 Target-binding domain

The synthetic receptor provided by this invention includes a target-binding domain. A target-binding domain is an extracellular domain capable of binding a target molecule. In some embodiments, the target molecule is an antigen on a target tissue. In some embodiments, the target molecule is a viral antigen. In some embodiments, the target molecule is a cancer antigen. In some embodiments, the target-binding domain of the synthetic receptor provided by this invention may also be an epitope that can be recognized by antibodies (e.g., bispecific or multispecific antibodies) that can bind to target molecules on a target tissue, such as cancer antigens.

Such antigen-binding domains are typically derived from antibodies. In one embodiment, the antigen-binding domain may be an scFv or Fab, or any suitable antigen-binding fragment of the antibody (see Sadelain et al., Cancer Discov. 3:388-398 (2013)). In some embodiments, the extracellular antigen-binding domain is an scFv. Many antibodies or antigen-binding domains derived from antibodies that bind to cancer antigens are known in the art. Alternatively, such antibodies or antigen-binding domains can be prepared using conventional methods. Methods for preparing antibodies are well known in the art, including methods for preparing monoclonal antibodies or screening libraries to obtain antigen-binding peptides (including screening human Fabs libraries) (Winter and Harris, Immunol. Today 14:243-246 (1993); Ward et al., Nature 341:544-546 (1989); Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1988); Hilyard et al., Protein Engineering: A practical approach (IRL Press 1992); Borrabeck, Antibody Engineering, 2nd ed. (Oxford University Press 1995); Huse et al., Science 246:1275-1281 (1989)).

Depending on the requirements, the antigen-binding domain derived from the antibody can be human, humanized, chimeric, CDR-transplanted, etc. For example, if a mouse monoclonal antibody is the source antibody for generating the antigen-binding domain, such antibodies can be humanized by transplanting the CDRs of the mouse antibody onto a human frame (see Borrabeck, ibid., 1995), which can be beneficial for administering synthetic receptors to human subjects. In a preferred embodiment, the antigen-binding domain is scFv. The generation of scFvs is well known in the art (see, for example, Huston, et al., Proc. Nat. Acad. Sci. USA 85:5879-5883 (1988); Ahmad et al., Clin. Dev. Immunol. 2012: ID980250 (2012); U.S. Patent Nos. 5,091,513,5,132,405 and 4,956,778; and U.S. Patent Publication Nos. 20050196754 and 20050196754).

Regarding obtaining cancer antigen-binding activity, those skilled in the art can use any well-known method for generating and screening antibodies that bind to the desired antigen to obtain suitable cancer antigen-binding activity, such as antibodies comprising scFvs that bind to cancer antigens, which are particularly useful in the synthetic receptors disclosed in this invention. In addition, many cancer antigen antibodies, especially monoclonal antibodies, are commercially available and can also be used as a source of cancer antigen-binding activity (such as scFvs) to generate synthetic receptors.

In some embodiments, the target-binding domain may be an antibody fragment, a derivative thereof, or a mimic, wherein these fragments, derivatives, and mimics possess the necessary binding affinity for the target molecule. Such antibody fragments or derivatives typically contain at least the _VH and _VL domains of the antibody under test to preserve the binding properties of the antibody under test. As used in this invention, an antibody fragment refers to a molecule other than a complete antibody, comprising a portion of the antibody and a typical antigen-binding site. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain antibody molecules (e.g., scFv), disulfide-linked scFv (dsscFv), biantibodies, triantibodies, tetraantibodies, microantibodies, bivariate domain antibodies (DVD), monovariate domain antibodies (e.g., camel antibodies, alpaca antibodies), monovariate domain (VHH) of heavy chain antibodies, nanobodies, and multispecific antibodies formed from antibody fragments. In some embodiments, the target-binding domain is Fab. In some embodiments, the target-binding domain is scFv. In some embodiments, the target-binding domain comprises a monovariate domain antibody.

In some implementations, the target-binding domain includes antibody mimics. Antibody mimics can be molecules that specifically bind to antigens like antibodies, but are structurally independent of antibodies. Antibody mimics are typically artificial peptides with a molar mass of about 2 to 20 kDa. Nucleic acids and small molecules are sometimes also considered antibody mimics. Antibody mimics known in the art include adnectins, affibodies, affiliins, affimers, affitins, alphabodies, antiicalins, aptamers, avimers, bicyclic peptides, Centyrin (J&J), DARPins, Fynomers, Knottins, Kunitz domain peptides, monobodies, and nanoCLAMP.

In some implementations, the target-binding domain includes an antibody-like scaffold (e.g., Owens, Nature Biotechnology 35:602 603 (2017); Simeon and Chen, Protein Cell 9(1):3-14 (2018)).

In addition to using antibody-derived antigen-binding domains, the target-binding domains of synthetic receptors can include ligand or extracellular ligand-binding domains of the receptor (see Sadelain et al., Cancer Discoy. 3:388-398 (2013); Sharpe et al., Dis Model Mech. 8:337-350 (2015)). In this case, the ligand or extracellular ligand-binding domain of the receptor provides the synthetic receptor with the ability to target cells expressing the synthetic receptor to the corresponding receptor or ligand. The selection of the ligand or extracellular ligand-binding domain allows cells expressing the synthetic receptor to target cancer cells or tumors (see Sadelain et al., Cancer Discoy. 3:388-398 (2013); Sharpe et al., Dis. Model Mech. 8:337-350 (2015), and the references cited therein). In one embodiment, a ligand or extracellular ligand binding domain is selected to bind to a cancer antigen that is a corresponding receptor or ligand (see, Sadelain et al., CancerDiscov.3:388-398 (2013)).

Additionally, in some embodiments, the target-binding domain of the synthetic receptor may include a chimeric autoantibody receptor (CAAR), which is an autoantigen that can be recognized by the B cell receptor (BCR) (see Ellebrecht et al. Science 353 (6295), 179-184 (2016)).

For synthetic receptors targeting cancer antigens, the antigen-binding domain of the synthetic receptor is selected to bind to antigens expressed in cancer cells. These cancer antigens may be uniquely expressed in cancer cells, or they may be overexpressed in cancer cells relative to non-cancer cells or tissues. The selection of cancer antigens that bind to synthetic receptors makes them more targeted to cells expressing the synthetic receptors than to non-cancer cells or tissues.

In some embodiments, the synthetic receptor includes an antigen-binding domain that specifically binds to a viral antigen. In some embodiments, the viral antigen is EBV. In some embodiments, the viral antigen is HPV. It should be understood that the synthetic receptor disclosed in this invention can target these or other viral antigens.

Cancer antigens can be tumor antigens. Any suitable cancer antigen can be selected based on the type of cancer exhibited by the subject to be treated (cancer patient). It should be understood that the selected cancer antigen is expressed in a manner capable of binding to synthetic receptors. Typically, cancer antigens targeted by cells expressing synthetic receptors are expressed on the cell surface of cancer cells. However, it should be understood that any cancer antigen that can bind to synthetic receptors is suitable for targeting cells expressing synthetic receptors to cancer cells.

Suitable antigens include, but are not limited to, B-cell maturation antigen (BCMA), mesothelin (MSLN), prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD70, CD74, CD123, CD133, CD138, CD33, IL3Ra2, CS1, C-Met, and epithelial glycoprotein 2 (EGP). 2) Epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-α and β (FRα and β), ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER-2/ERB2), epidermal growth factor receptor (EGFR), epidermal growth factor receptor vIII (EGFRvIII), ERB3, ERB4, human telomerase reverse transcriptase (hTERT), interleukin-13 receptor α-2 subunit (IL-13Rα2), κ- Light chain, kinase insertion domain receptor (KDR), Lewis A (CA19.9), Lewis Y (LeY), L1 cell adhesion molecule (LlCAM), melanoma-associated antigen 1 (melanoma antigen family A1, MAGE-A1), mucin 16 (Muc-16), mucin 1 (Muc-1), NKG2D ligand, cancer-testis antigen NY-ESO-1, oncoemulsification antigen (h5T4), tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), nephroblastoma protein (WT-1), type 1 tyrosine protein kinase transmembrane receptor The receptors include ROR1, B7-H3 (CD276), B7-H6 (Nkp30), chondroitin sulfate proteoglycan-4 (CSPG4), DNAX helper molecule (DNAM-1), Ephrin type A receptor 2 (EpHA2), fibroblast-associated protein (FAP), Gp100/HLA-A2, Glypican3 (GPC3), HA-1H, HERK-V, IL-11Rα, latent membrane protein 1 (LMP1), MAG3, neural cell adhesion molecule (N-CAM/CD56), NY-ESO-1, and Trail receptor (TRAIL R). It should be understood that these or other cancer antigens can be used for targeting the synthetic receptors disclosed in this invention.

In some embodiments, the fusion protein disclosed in this invention binds to a synthetic receptor targeting a tumor antigen, wherein the tumor antigen is selected from the group consisting of CD19, CD20, CD22, CD30, CD123, CD138, CD33, CD70, BCMA, CS1, C-Met, IL13Ra2, EGFRvIII, CEA, Her2, GD2, MAGE, GPC3, mesothelin, PSMA, ROR1, EGFR, MUC1, and NY-ESO-1. In some embodiments, the synthetic receptor provided by this invention includes a target-binding domain that binds to CD19. In some embodiments, the synthetic receptor provided by this invention includes a target-binding domain that binds to CD20. In some embodiments, the synthetic receptor provided by this invention includes a target-binding domain that binds to CD22. In some embodiments, the synthetic receptor provided by this invention includes a target-binding domain that binds to CD30. In some embodiments, the synthetic receptor provided by this invention includes a target-binding domain that binds to CD123. In some embodiments, the synthetic receptor provided by the present invention includes a target-binding domain for binding CD138. In some embodiments, the synthetic receptor provided by the present invention includes a target-binding domain for binding CD33. In some embodiments, the synthetic receptor provided by the present invention includes a target-binding domain for binding CD70. In some embodiments, the synthetic receptor provided by the present invention includes a target-binding domain for binding BCMA. In some embodiments, the synthetic receptor provided by the present invention includes a target-binding domain for binding CS1. In some embodiments, the synthetic receptor provided by the present invention includes a target-binding domain for binding C-Met. In some embodiments, the synthetic receptor provided by the present invention includes a target-binding domain for binding IL13Ra2. In some embodiments, the synthetic receptor provided by the present invention includes a target-binding domain for binding EGFRvIII. In some embodiments, the synthetic receptor provided by the present invention includes a target-binding domain for binding CEA. In some embodiments, the synthetic receptor provided by the present invention includes a target-binding domain for binding Her2. In some embodiments, the synthetic receptor provided by the present invention includes a target-binding domain for binding GD2. In some embodiments, the synthetic receptor provided by the present invention includes a target-binding domain for binding MAGE. In some embodiments, the synthetic receptor provided by the present invention includes a target-binding domain that binds to GPC3. In some embodiments, the synthetic receptor provided by the present invention includes a target-binding domain that binds to mesothelin. In some embodiments, the synthetic receptor provided by the present invention includes a target-binding domain that binds to PSMA. In some embodiments, the synthetic receptor provided by the present invention includes a target-binding domain that binds to ROR1. In some embodiments, the synthetic receptor provided by the present invention includes a target-binding domain that binds to EGFR. In some embodiments, the synthetic receptor provided by the present invention includes a target-binding domain that binds to MUC1. In some embodiments, the synthetic receptor provided by the present invention includes a target-binding domain that binds to NY-ESO-1.

6.3.2 CARs

In some embodiments, the synthetic receptor is a CAR, and the fusion protein provided by the present invention can be co-expressed with the CAR in cells. In some embodiments, the fusion protein provided by the present invention can bind to a CAR. A CAR is a synthetic receptor that retargets immune cells (e.g., T cells) to tumor surface antigens (Sadelain et al., Nat. Rev. Cancer. 3(1):35-45 (2003); Sadelain et al., Cancer Discovery 3(4):388-398 (2013)). CARs are engineered receptors that provide antigen binding and immune cell activation. CARs can be used to specifically transplant antibodies (e.g., monoclonal antibodies) onto immune cells (e.g., T cells, NK cells, or macrophages). First-generation receptors link antibody-derived tumor-binding elements responsible for antigen recognition, such as scFv, to a CD3zeta or Fc receptor signaling domain, thereby triggering T cell activation. The emergence of second-generation CARs combining activation and co-stimulatory signal transduction domains has yielded encouraging results in patients with chemotherapy-refractory B-cell malignancies (Brentjens et al., Science Translational Medicine 5(177):177ra38(2013); Brentjens et al., Blood 118(18):4817-4828(2011); Davila et al., Science Translational Medicine 6(224):224ra25(2014); Grupp et al., N. Engl. J. Med. 368(16):1509-1518(2013); Kalos et al., Science Translational Medicine 3(95):95ra73(2011)). The extracellular antigen-binding domain of CARs is typically derived from monoclonal antibodies (mAbs) or from receptors or their ligands. Antigen binding of CARs triggers phosphorylation of immune receptor tyrosine activation motifs (ITAMs) within the intracellular domain, initiating a signal cascade amplification required for cell lysis induction, cytokine secretion, and proliferation.

In some embodiments, the fusion protein provided by the present invention can bind to a CAR having an antigen-binding domain that binds to a cancer antigen. In some embodiments, the CAR can be a "first-generation" CAR, a "second-generation" CAR, or a "third-generation" CAR (see, for example, Sadelain et al., Cancer Discov. 3(4):388-398 (2013); Jensen et al., Immunol. Rev. 257:127-133 (2014); Sharpe et al., Dis. Model Mech. 8(4):337-350 (2015); Brentjens et al., Clin. Cancer Res. 13:5426-5435). 2007); Gade et al., CancerRes.65:9080-9088(2005); Maher et al., Nat.Biotechnol.20:70-75(2002); Kershaw etal.,J.Immunol.173 :2143-2150(2004); Sadelain et al., Curr.Opin.Immunol.21(2):215-223(2009); Hollyman et al., J.Immunother.32:169-180(2009)).

First-generation CARs typically consist of an extracellular antigen-binding domain, such as a single-chain variable region fragment (scFv), with the extracellular antigen-binding domain fused to a transmembrane domain, which in turn fuses to a cytoplasmic/intracellular domain of the T-cell receptor chain. First-generation CARs also typically possess an intracellular domain derived from the CD3δ-chain, the primary signaling pathway for endogenous T-cell receptors (TCRs). First-generation CARs can provide de novo antigen recognition and induce activation of both CD4 ⁺ and CD8 ⁺ T cells via the CD3δ-chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation. "Second-generation" CARs include a cancer antigen-binding domain fused with an intracellular signaling domain and a co-stimulatory domain. The intracellular signaling domain activates immune effector cells (e.g., T cells), while the co-stimulatory domain is designed to enhance the potency and persistence of immune effector cells (e.g., T cells) (Sadelain et al., Cancer Discov. 3:388-398 (2013)). Therefore, CAR design can combine antigen recognition and signal transduction, two functions physiologically performed by two separate complexes (the TCR heterodimer and the CD3 complex). "Second-generation" CARs include intracellular domains derived from various co-stimulatory molecules, such as CD28, 4-1BB, ICOS, OX40, etc., located in the cytoplasmic tail of the CAR to provide additional signaling to the cell. "Second-generation" CARs provide both co-stimulation (e.g., via the CD28 or 4-1BB domain) and activation (e.g., via the CD3δ signaling domain). Studies have shown that "second-generation" CARs can enhance the antitumor activity of T cells. "Third-generation" CARs offer multiple co-stimulatory mechanisms (e.g., by simultaneously containing CD28 and 4-1BB domains) and activation mechanisms (e.g., by containing a CD3δ activation domain).

As described above, CAR also includes a signal transduction domain that functions in CAR-expressing immune cells. Such a signal transduction domain can, for example, be derived from CDδ or Fc receptor γ (see, Sadelain et al., Cancer Discov. 3:388-398 (2013)). Generally, the signal transduction domain will induce persistent, transport, and/or effector functions in transduced immune effector cells (e.g., T cells) (Sharpe et al., Dis. Model Mech. 8:337-350 (2015); Finney et al., J. Immunol. 161:2791-2797 (1998); Krause et al., J. Exp. Med. 188:619-626 (1998)). In the case of CDδ or Fc receptor γ, the signal transduction domain corresponds to an intracellular domain of the corresponding polypeptide, or corresponds to an intracellular domain fragment sufficient to perform signal transduction. An exemplary signal conduction structure domain is described in more detail below.

CD3δ. In a non-limiting embodiment, the CAR may include a signal transduction domain derived from a CD3δ polypeptide, such as a signal transduction domain derived from an intracellular domain of CD3δ, which can activate or stimulate immune cells, such as T cells. CD3δ contains three immune receptor tyrosine activation motifs (ITAMs) and delivers an activation signal to cells, such as lymphoid cells, like T cells, upon antigen binding. The CD3δ polypeptide may have an amino acid sequence or fragment thereof corresponding to the sequence provided below (GenBank No. NP_932170 (NP_932170.1, GI:37595565; see below)). In one embodiment, the CD3δ polypeptide has an amino acid sequence of amino acids 52 to 164 of the CD3δ polypeptide sequence provided below, or a fragment thereof sufficient for signal transduction activity. An exemplary CAR has an intracellular domain comprising a CD3δ polypeptide containing amino acids 52 to 164 of the CD3δ polypeptide sequence provided below. Another exemplary CAR has an intracellular domain comprising a CD3δ polypeptide containing amino acids 52 to 164 of the CD3δ polypeptide provided below. Yet another exemplary CAR has an intracellular domain comprising a CD3δ polypeptide containing amino acids 52 to 164 of the CD3δ polypeptide provided below. See GenBank NP_932170 for reference to the domains within CD3δ, such as the signal peptide of amino acids 1-21; the extracellular domain of amino acids 22-30; the transmembrane domain of amino acids 31-51; and the intracellular domain of amino acids 52-164.

1 MKWKALFTAA ILQAQLPITE AQSFGLLDPK LCYLLDGILF IYGVILTALF LRVKFSRSAD

61APAYQQGQNQ LYNELNLGRR EEYDVLDKRR GRDPEMGGKP QRRKNPQEGL YNELQKDKMA

121 EAYSEIGMKG ERRRGKGHDG LYQGLSTATK DTYDALHMQA LPPR(SEQ ID NO:92)

It should be understood that "CD3δ nucleic acid" refers to a polynucleotide encoding a CD3δ polypeptide. In one embodiment, the CD3δ nucleic acid encoding a CD3δ polypeptide (including exemplary CARs Mz, M28z, or MBBz) in the intracellular domain of a CAR comprises the nucleotide sequence described below.

AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTA CAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA(SEQ ID NO:93)

In some non-limiting embodiments, the intracellular domain of the CAR may further include at least one co-stimulatory signaling domain. In some embodiments, the intracellular domain of the CAR may include two co-stimulatory signaling domains. Such co-stimulatory signaling domains can increase the activation of immune cells. The co-stimulatory signaling domains may be derived from the CD28 peptide, 4-1BB peptide, OX40 peptide, ICOS peptide, DAP10 peptide, 2B4 peptide, CD27 peptide, CD30 peptide, CD40 peptide, etc. CARs including intracellular domains comprising co-stimulatory signaling regions comprising 4-1BB, ICOS, or DAP-10 have been previously described (see U.S. 7,446,190, which is incorporated herein by reference, and which also describes representative sequences of 4-1BB, ICOS, and DAP-10). In some embodiments, the intracellular domain of the CAR may include a co-stimulatory signal transduction region comprising two co-stimulatory molecules, such as CD28 and 4-IBB (see, Sadelain et al., Cancer Discov. 3:388-398 (2013)), or CD28 and OX40, or other combinations of co-stimulatory ligands disclosed herein.

CD28. Differentiation cluster 28 (CD28) is a protein expressed on T cells that provides co-stimulatory signals for T cell activation and survival. CD28 is a receptor for CD80 (B7.1) and CD86 (B7.2) proteins. In one embodiment, the CAR may include a co-stimulatory signaling domain derived from CD28. For example, as disclosed in this invention, the CAR may include at least a portion of the intracellular/cytoplasmic domain of CD28, such as an intracellular/cytoplasmic domain that can be used as a co-stimulatory signaling domain. The CD28 polypeptide may have an amino acid sequence or fragment thereof corresponding to the sequence provided below in GenBank No. P10747 (P10747.1, GI:115973) or NP_006130 (NP_006130.1, GI:5453611). If desired, CD28 sequences outside the intracellular domain may be included in the CAR of this invention. For example, the CAR may include a transmembrane portion of the CD28 polypeptide. In one embodiment, the CAR may have an amino acid sequence comprising an intracellular domain of CD28 corresponding to amino acids 180 to 220 or a fragment thereof. In another embodiment, the CAR may have an amino acid sequence comprising a transmembrane domain of CD28 corresponding to amino acids 153 to 179 or a fragment thereof. An exemplary CAR may include a costimulatory signaling domain corresponding to the intracellular domain of CD28. An exemplary CAR may include a transmembrane domain derived from CD28. Thus, an exemplary CAR may include two domains from CD28: a costimulatory signaling domain and a transmembrane domain. In one embodiment, the CAR has an amino acid sequence comprising a transmembrane domain and an intracellular domain of CD28, and the CAR comprises amino acids 153 to 220 of CD28. In another embodiment, the CAR comprises amino acids 117 to 220 of CD28. Another exemplary CAR having a transmembrane domain and an intracellular domain of CD28 is P28z. In one embodiment, the CAR may include a transmembrane domain derived from a CD28 polypeptide comprising amino acids 153 to 179 of the CD28 polypeptide provided below. See GenBank NP_006130 for reference to domains within CD28, such as a signal peptide of amino acids 1-18; an extracellular domain of amino acids 19-152; a transmembrane domain of amino acids 153-179; and an intracellular domain of amino acids 180-220. It will be understood that, if desired, CD28 sequences shorter or longer than a specific descriptive domain may be included in the CAR.

It should be understood that "CD28 nucleic acid" refers to a polynucleotide encoding a CD28 polypeptide. In one embodiment, the CD28 nucleic acid encoding a CD28 polypeptide comprising transmembrane domains and intracellular domains (e.g., co-stimulatory signal transduction regions) includes the nucleotide sequence described below.

ATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCT AGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC(SEQ ID NO:95)

4-1BB. 4-1BB, also known as member 9 of the tumor necrosis factor receptor superfamily, can act as a ligand for tumor necrosis factor (TNF) and has stimulatory activity. In one embodiment, the CAR may include a co-stimulatory signaling domain derived from 4-1BB. The 4-1BB polypeptide may have an amino acid sequence or fragment thereof corresponding to the sequence provided below, GenBank No. P41273 (P41273.1, GI:728739) or NP_001552 (NP_001552.2, GI:5730095). In one embodiment, the CAR may have a co-stimulatory domain comprising a 4-1BB intracellular domain corresponding to amino acids 214 to 255 or a fragment thereof. In another embodiment, the CAR may have a 4-1BB transmembrane domain corresponding to amino acids 187 to 213 or a fragment thereof. An exemplary CAR is MBBz, which has an intracellular domain comprising a 4-1BB polypeptide (e.g., amino acids 214 to 255 of NP_001552, SEQ ID NO:96). See GenBank NP_001552 for reference to the domains within the 4-1BB, such as a signal peptide of amino acids 1-17; an extracellular domain of amino acids 18-186; a transmembrane domain of amino acids 187-213; and an intracellular domain of amino acids 214-255. It is understood that 4-1BB sequences shorter or longer than a specific descriptive domain may be included in the CAR if desired. It is also understood that “4-1BB nucleic acid” refers to a polynucleotide encoding a 4-1BB polypeptide.

1MGNSCYNIVA TLLLVLNFER TRSLQDPCSN CPAGTFCDNN RNQICSPCPP NSFSSAGGQR

61 TCDICRQCKG VFRTRKECSS TSNAECDCTP GFHCLGAGCS MCEQDCKQGQ ELTKKGCKDC

121 CFGTFNDQKR GICRPWTNCS LDGKSVLVNG TKERDVVCGP SPADLSPGAS SVTPPAPARE

181 PGHSPQIISF FLALTSTALL FLLFFLTLRF SVVKRGRKKL LYIFKQPFMR PVQTTQEEDG

241 CSCRFPEEEE GGCEL(NP_001552; SEQ ID NO:96)

OX40. OX40, also known as a precursor of tumor necrosis factor receptor superfamily member 4 or CD134, is a member of the TNFR-receptor superfamily. In one embodiment, the CAR may include a co-stimulatory signaling domain derived from OX40. The OX40 polypeptide may have an amino acid sequence or fragment thereof corresponding to the sequence provided below, GenBank No. P43489 (P43489.1, GI:1171933) or NP_003318 (NP_003318.1, GI:4507579). In one embodiment, the CAR may have a co-stimulatory domain comprising an intracellular domain of OX40 corresponding to amino acids 236 to 277 or a fragment thereof. In another embodiment, the CAR may have an amino acid sequence comprising an OX40 transmembrane domain corresponding to amino acids 215 to 235 or a fragment thereof. See GenBank NP_003318 for reference to the domains within OX40, such as the signal peptide at amino acids 1-28; the extracellular domain at amino acids 29-214; the transmembrane domain at amino acids 215-235; and the intracellular domain at amino acids 236-277. It should be understood that, if desired, OX40 sequences shorter or longer than a specific descriptive domain may be included in the CAR. It should also be understood that “OX40 nucleic acid” refers to a polynucleotide encoding the OX40 polypeptide.

ICOS. Inducible T cell co-stimulatory molecule precursor (ICOS), also known as CD278, is a CD28 superfamily co-stimulatory receptor expressed on activated T cells. In one embodiment, the CAR may include a co-stimulatory signaling domain derived from ICOS. The ICOS peptide may have an amino acid sequence or a fragment thereof corresponding to the sequence provided below (GenBank No. NP_036224 (NP_036224.1, GI:15029518)). In one embodiment, the CAR may have a co-stimulatory domain comprising an intracellular ICOS domain corresponding to amino acids 162 to 199 of ICOS. In another embodiment, the CAR may have an amino acid sequence comprising an ICOS transmembrane domain corresponding to amino acids 141 to 161 of ICOS or a fragment thereof. See GenBank NP_036224 for reference to the domains within ICOS, such as the signal peptide of amino acids 1-20; the extracellular domain of amino acids 21-140; the transmembrane domain of amino acids 141-161; and the intracellular domain of amino acids 162-199. It is understood that, if desired, ICOS sequences shorter or longer than a specific descriptive domain may be included in the CAR. It is also understood that “ICOS nucleic acid” refers to a polynucleotide encoding an ICOS polypeptide.

DAP10. DAP10, also known as a hematopoietic cell signal transducer, is a signal transduction subunit closely related to the receptor family in hematopoietic cells. In one embodiment, the CAR may include a co-stimulatory domain derived from DAP10. The DAP10 polypeptide may have an amino acid sequence or fragment thereof corresponding to the sequence provided in GenBank No. NP_055081.1 (GI:15826850). In one embodiment, the CAR may have a co-stimulatory domain comprising an intracellular domain of DAP10 corresponding to amino acids 70 to 93 or a fragment thereof. In another embodiment, the CAR may have a transmembrane domain of DAP10 corresponding to amino acids 49 to 69 or a fragment thereof. See GenBank NP_055081.1 for reference to domains within DAP10, such as a signal peptide of amino acids 1-19; an extracellular domain of amino acids 20-48; a transmembrane domain of amino acids 49-69; and an intracellular domain of amino acids 70-93. It is understood that, if necessary, DAP10 sequences shorter or longer than a specific descriptive domain can be included in the CAR. It is also understood that "DAP10 nucleic acid" refers to a polynucleotide encoding the DAP10 polypeptide.

1 MIHLGHILFL LLLPVAAAQT TPGERSSLPA FYPGTSGSCS GCGSLSLPLL AGLVAADAVA

61 SLLIVGAVFL CARPRRSPAQ EDGKVYINMP GRG(SEQ ID NO:99)

CD27 : CD27 (TNFRSF7) is a transmembrane receptor expressed on human CD8+ and CD4+ T cell subsets, NKT cells, NK cell subsets, and hematopoietic progenitor cells, and induced in Foxp3+CD4T cells and B cell subsets. Previous studies have found that CD27 can actively provide co-stimulatory signals in vivo, enhancing human T cell survival and antitumor activity. (See Song and Powell; Oncoimmunology 1, no. 4 (2012): 547-549). The CD27 peptide may have an amino acid sequence or fragment thereof corresponding to the sequence UniProtKB/Swiss-Prot No.: P26842.2 (GI: 269849546) provided below.

In one embodiment, the CAR may have a co-stimulatory domain comprising an intracellular domain of CD27 or a fragment thereof. In another embodiment, the CAR may have a transmembrane domain of CD27 or a fragment thereof. It is understood that, if desired, CD27 sequences shorter or longer than a specific descriptive domain may be included in the CAR. It is also understood that CD27 nucleic acid refers to a polynucleotide encoding a CD27 polypeptide.

CD30 : CD30 and its ligand (CD30L) belong to the tumor necrosis factor receptor (TNFR) and tumor necrosis factor (TNF) superfamily, respectively. CD30 behaves similarly to Ox40 in many ways and can enhance TCR-induced proliferation and cytokine production. (Goronzy and Weyand, Arthritis Research & Therapy 10, no. S1 (2008): S3.) In one embodiment, the CAR may include a co-stimulatory domain derived from CD30. The CD30 peptide may have an amino acid sequence or a fragment thereof corresponding to the sequence provided below in GenBank No.:AAA51947.1 (GI:180096).

In one embodiment, the CAR may have a co-stimulatory domain comprising an intracellular domain of CD30 or a fragment thereof. In another embodiment, the CAR may have a transmembrane domain of CD30 or a fragment thereof. It should be understood that, if desired, CD30 sequences shorter or longer than a specific descriptive domain may be included in the CAR. It should also be understood that CD30 nucleic acid refers to a polynucleotide encoding a CD30 polypeptide.

CD40 : CD40 and its ligands CD40L or CD154 were first identified as contributing to T-cell-dependent B-cell activation. This pathway is now considered a mechanism for activating APCs and enhancing their potential to activate T cells. CD154-mediated CD40 stimulation provides an important feedback mechanism for the initial co-stimulatory pathway of CD28-CD80/CD86. (Goronzy and Weyand, Arthritis Research & Therapy 10, no. S1 (2008): S3.). In one embodiment, the CAR may include a co-stimulatory domain derived from CD40. The CD40 peptide may have an amino acid sequence or a fragment thereof corresponding to the sequence UniProtKB/Swiss-Prot No.: P25942.1 (GI: 269849546) provided below.

In one embodiment, the CAR may have a co-stimulatory domain comprising an intracellular domain of CD40 or a fragment thereof. In another embodiment, the CAR may have a transmembrane domain of CD40 or a fragment thereof. It should be understood that, if desired, CD40 sequences shorter or longer than a specific descriptive domain may be included in the CAR. It should also be understood that CD40 nucleic acid refers to a polynucleotide encoding a CD40 polypeptide.

The extracellular domain of a CAR can be fused with a lead peptide or signal peptide that guides nascent proteins into the endoplasmic reticulum and subsequently to the cell surface. It should be understood that once a polypeptide containing a signal peptide is expressed on the cell surface, the signal peptide is typically removed by proteolysis during polypeptide processing and translocation from the endoplasmic reticulum to the cell surface. Therefore, polypeptides, such as CARs, are typically expressed on the cell surface as mature proteins lacking a signal peptide, while precursor forms of polypeptides may include the signal peptide. The signal peptide or lead peptide may be essential if the CAR is to be glycosylated and/or anchored to the cell membrane. A signal sequence or lead sequence is a peptide sequence typically present at the N-terminus of newly synthesized proteins, guiding them into the secretory pathway. This signal peptide is covalently linked as a fusion protein to the N-terminus of the extracellular antigen-binding domain of the CAR. In one embodiment, the signal peptide comprises a CD8 polypeptide comprising the amino acid MALPVTLPLALLLHAARP (SEQ ID NO: 116). It should be understood that the use of the CD8 signal peptide is exemplary. As is well known in the art, any suitable signal peptide can be used to guide CAR expression on the cell surface of immune cells (see Gierasch Biochem. 28:923-930 (1989); von Heijne, J. Mol. Biol. 184(1):99–105 (1985)). Particularly useful signal peptides can be derived from cell surface proteins naturally expressed in the immune cells provided by this invention, including any of the signal peptides disclosed in this invention. Therefore, any suitable signal peptide can be used to guide CAR expression on the cell surface of the immune cells provided by this invention.

In some non-limiting embodiments, the extracellular antigen-binding domain of the CAR may include a linker sequence or peptide linker connecting the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. In one non-limiting example, the linker comprises amino acids having the sequence shown in GGGGSGGGGSGGGGS (SEQ ID NO:1).

In some non-limiting embodiments, the CAR may also include spacers or sequences that interconnect the domains of the CAR. For example, spacers may be included between a signal peptide and an antigen-binding domain, between an antigen-binding domain and a transmembrane domain, between a transmembrane domain and an intracellular domain, and/or between domains within an intracellular domain, such as between a stimulatory domain and a co-stimulatory domain. The spacers may be flexible enough to allow various domains to interact with other peptides, for example, allowing the antigen-binding domain to have directional flexibility to facilitate antigen recognition. The spacers may be, for example, a hinge region from IgG, a CH2CH3 (constant) region of an immunoglobulin, and/or a portion of CD3 (differentiation cluster 3), or some other sequence suitable as a spacer.

The transmembrane domain of a CAR typically includes a hydrophobic α-helix spanning at least a portion of the membrane. Different transmembrane domains result in different receptor stability. Upon antigen recognition, the receptor aggregates and transduces a signal into the cell. In one embodiment, the transmembrane domain of the CAR provided by the present invention may be derived from another polypeptide naturally expressed in immune cells. In one embodiment, the CAR has a transmembrane domain derived from CD8, CD28, CD3δ, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, BTLA, or other polypeptides expressed in immune cells. Optionally, the transmembrane domain may be derived from a polypeptide not naturally expressed in immune cells, provided that the transmembrane domain is capable of transducing a signal from the antigen bound to the CAR to an intracellular signal transduction domain and/or a co-stimulatory domain. It should be understood that the polypeptide portion including the transmembrane domain may, as needed, include additional sequences from said polypeptide, for example, additional sequences adjacent to the N-terminus or C-terminus of the transmembrane domain, or other regions of said polypeptide.

CD8. Differentiation cluster 8 (CD8) is a transmembrane glycoprotein that acts as a co-receptor for the T-cell receptor (TCR). CD8 binds to the major histocompatibility complex (MHC) molecule and is specific for class I MHC proteins. In one embodiment, the CAR may include a transmembrane domain derived from CD8. The CD8 polypeptide may have an amino acid sequence or a fragment thereof corresponding to the sequence provided below in GenBank No.:NP_001139345.1 (GI:225007536). In one embodiment, the CAR may have an amino acid sequence containing a CD8 transmembrane domain corresponding to amino acids 183 to 203 or a fragment thereof. In one embodiment, an exemplary CAR has a transmembrane domain derived from a CD8 polypeptide. In a non-limiting embodiment, the CAR may include a transmembrane domain derived from a CD8 polypeptide containing amino acids 183 to 203. Furthermore, the CAR may include a hinge domain comprising amino acids 137-182 of the CD8 polypeptide provided below. In another embodiment, the CAR may comprise amino acids 137-203 of the CD8 polypeptide provided below. In another embodiment, the CAR may comprise amino acids 137 to 209 of the CD8 polypeptide provided below. See GenBank NP_001139345.1 for reference to domains within CD8, such as a signal peptide of amino acids 1-21; an extracellular domain of amino acids 22-182; a transmembrane domain of amino acids 183-203; and an intracellular domain of amino acids 204-235. It should be understood that, if desired, additional CD8 sequences beyond the transmembrane domain of amino acids 183-203 may be included in the CAR. It should further be understood that, if desired, CD8 sequences shorter or longer than a specific descriptive domain may be included in the CAR. It should also be understood that “CD8 nucleic acid” refers to a polynucleotide encoding a CD8 polypeptide.

CD4. Differentiation cluster 4 (CD4), also known as T cell surface glycoprotein CD4, is a glycoprotein present on the surface of immune cells such as helper T cells, monocytes, macrophages, and dendritic cells. In one embodiment, the CAR may include a transmembrane domain derived from CD4. CD4 exists in multiple isoforms. It is understood that any isoform can be selected to achieve the desired function. Exemplary isoforms include isoform 1 (NP_000607.1, GI:10835167), isoform 2 (NP_001181943.1, GI:303522479), isoform 3 (NP_001181944.1, GI:303522485; or NP_001181945.1, GI:303522491; or NP_001181946.1, GI:303522569), etc. An exemplary isoform sequence, namely isoform 1, is provided below. In one embodiment, the CAR may have an amino acid sequence comprising a CD4 transmembrane domain corresponding to amino acids 397 to 418 or a fragment thereof. See GenBank NP_000607.1 for reference to domains within CD4, such as a signal peptide of amino acids 1-25; an extracellular domain of amino acids 26-396; a transmembrane domain of amino acids 397-418; and an intracellular domain of amino acids 419-458. It should be understood that, if desired, additional CD4 sequences beyond the transmembrane domain of amino acids 397 to 418 may be included in the CAR. It should further be understood that, if desired, CD4 sequences shorter or longer than a specific descriptive domain may be included in the CAR. It should also be understood that “CD4 nucleic acid” refers to a polynucleotide encoding a CD4 polypeptide.

In addition to cytotoxic T cells, regulatory T cells (Tregs) can be effectively engineered to have predetermined antigen specificity by transfecting viral vectors encoding specific CARs (CAR-Tregs). CAR-Tregs engineered in a non-MHC-restricted manner have the advantage of wide-ranging applications, especially in transplantation and autoimmunity (Zhang et al. (2018) Front. Immunol. 9:2359).

Besides T cells, CARs can also be engineered into other types of immune cells, such as NK cells, macrophages, or B cells. In some embodiments, the engineered cells are NK cells. In some embodiments, the synthetic receptor is a CAR that retargets NK cells to tumor surface antigens (see, for example, Hu et al. Acta Pharmacol Sin 39, 167-176 (2018)). CAR-NK cells can use first-generation CAR constructs containing CD3δ as an intracellular signal transduction domain or second-generation CAR constructs that co-express a second signal transduction domain (e.g., CD28, 4-1BB) with CD3δ. Generally, second-generation CARs are more active in NK cells than first-generation CARs. In some embodiments, the CAR construct is based on NK cell activation characteristics. For example, DNAX-activator protein 12 (DAP12) is known to activate NK cell signaling.

DAP12. DAP12 is present in myeloid cells, such as macrophages and granulocytes, in which, for example, DAP12 is associated with trigger receptors expressed on myeloid cell members (TREMs) and MDL1 (myeloid DAP12-associated lectin 1/CLEC5A), both of which are involved in inflammatory responses against pathogens such as viruses and bacteria. In lymphoid cells, DAP12 is expressed in NK cells and is associated with activating receptors such as the C-type lectin receptor NKG2C, the natural cytotoxic receptor NKp44, short-tailed KIR3DS1, and KIR2DS1/2/5, respectively. In particular, NKG2C is a major activating NK cell receptor used to control CMV infection in humans and mice. Studies have found that CARs containing DAP12, after crosslinking with their Ag, generate sufficient activation signals in NK cells. et al., J Immunol 194:3201-12 (2015). In one embodiment, the CAR may include a co-stimulatory domain derived from DAP12. The DAP10 polypeptide may have an amino acid sequence or a fragment thereof corresponding to the sequence provided below in GenBank No. AAD09437.1 (GI:2905996). In one embodiment, the CAR may have a signal transduction domain comprising an intracellular domain of DAP12 or a fragment thereof. In another embodiment, the CAR may have a transmembrane domain of DAP12 or a fragment thereof. It should be understood that “DAP12 nucleic acid” refers to a polynucleotide encoding the DAP12 polypeptide.

1 MGGLEPCSRL LLLPLLLAVS GLRPVQAQAQ SDCSCSTVSP GVLAGIVMGD LVLTVLIALA

61 VYFLGRLVPR GRGAAEAATR KQRITETESP YQELQGQRSD VYSDLNTQRP YYK(SEQ IDNO:118)

Since the receptor NKG2D is important for NK cell activity, and NKG2D ligands are commonly overexpressed in various tumors, a unique CAR structure containing NKG2D has been developed. NKG2D serves as the extracellular domain and is linked to signal transduction proteins (e.g., DAP10 and CD3δ). This CAR structure can be co-expressed or coupled with the fusion protein disclosed in this invention in engineered cells. These CAR-expressing NK cells exhibit enhanced cytotoxicity against multiple tumor subtypes, with the best response observed in ALL, osteosarcoma, prostate cancer, and rhabdomyosarcoma (Chang et al., Cancer Res 2013; 73:1777-86). The human NKG2D polypeptide may have an amino acid sequence or fragment thereof corresponding to the sequence provided below in Genbank No. CAA04925.1 (GI: 2980865). It should be understood that "NKG2D nucleic acid" refers to the polynucleotide encoding the NKG2D polypeptide.

In some implementations, CARs can be engineered into macrophages. In some implementations, the engineered cells are macrophages. For example, a family of phagocytic chimeric antigen receptors (CAR-Ps) has been generated that direct macrophages to phagocytose specific targets, including cancer cells (Morrissey et al., eLife 2018; 7:e36688). Specifically, cytoplasmic domains derived from MEGF10 and FcRV can trigger phagocytosis independently of their native extracellular domains, and CAR-Ps can drive specific phagocytosis of antigen-coated synthetic particles and whole human cancer cells. Adding a tandem PI3K recruitment domain can further enhance cancer cell phagocytosis (Morrissey et al., eLife 2018; 7:e36688).

MEGF10, also known as EMARDD or SR-F3, is a membrane receptor involved in the phagocytic activity of macrophages and apoptotic astrocytes. The intracellular domain of Megf10 contains immunoreceptor tyrosine activation motifs (ITAMs) phosphorylated by Src family kinases. In one embodiment, the CAR may include a transmembrane domain derived from MEGF10. In one embodiment, the CAR may include a co-stimulatory domain derived from MEGF10. The MEGF10 polypeptide may have an amino acid sequence or fragment thereof corresponding to the sequence provided below in Genbank No. AAH20198.1 (GI: 18044366). In one embodiment, the CAR may have a co-stimulatory domain comprising an intracellular domain of MEGF10 or a fragment thereof. In another embodiment, the CAR may have a transmembrane domain of MEGF10 or a fragment thereof. It should be understood that “MEGF10 nucleic acid” refers to a polynucleotide encoding a MEGF10 polypeptide.

The activation type of FcRγ IgG receptors (FcγRs) forms a multimeric complex comprising an Fc receptor γ chain (FCRγ) containing an intracellular tyrosine activation motif (ITAM), the activation of which triggers reactive oxygen species bursts, cytokine release, phagocytosis, antibody-dependent cell-mediated cytotoxicity, and degranulation. In one embodiment, the CAR may include a transmembrane domain derived from FCRγ. In one embodiment, the CAR may include a co-stimulatory domain derived from FCRγ. The FcRγ polypeptide may have an amino acid sequence or fragment thereof corresponding to the NCBI reference sequence provided below: NP_004097.1 (GI:4758344). In one embodiment, the CAR may have a co-stimulatory domain comprising an intracellular FCRγ domain or a fragment thereof. In another embodiment, the CAR may have a transmembrane domain of FCRγ or a fragment thereof. It should be understood that “FCRγ nucleic acid” refers to a polynucleotide encoding an FCRγ polypeptide.

1 MIPAVVLLLL LLVEQAAALG EPQLCYILDA ILFLYGIVLT LLYCRLKIQV RKAAITSYEK

61 SDGVYTGLST RNQETYETLK HEKPPQ(SEQ ID NO:121)

The CAR provided by this invention may include the target-binding domain as disclosed above. In some embodiments, the fusion protein disclosed in this invention may be co-expressed with a CAR targeting a tumor antigen selected from the group consisting of CD19, CD20, CD22, CD30, CD123, CD138, CD33, CD70, BCMA, CS1, C-Met, IL13Ra2, EGFRvIII, CEA, Her2, GD2, MAGE, GPC3, mesothelin, PSMA, ROR1, EGFR, MUC1, and NY-ESO-1 in cells. In some embodiments, the fusion protein disclosed in this invention is conjugated to a CAR targeting a tumor antigen, wherein the tumor antigen is selected from the group consisting of CD19, CD20, CD22, CD30, CD123, CD138, CD33, CD70, BCMA, CS1, C-Met, IL13Ra2, EGFRvIII, CEA, Her2, GD2, MAGE, GPC3, mesothelin, PSMA, ROR1, EGFR, MUC1, and NY-ESO-1. In some embodiments, the fusion protein provided by this invention can be co-expressed or conjugated to a CD19-targeting CAR (CAR19). In some embodiments, the fusion protein provided by this invention can be conjugated to a CAR. In some embodiments, the fusion protein provided by this invention can be conjugated to a CD19-binding CAR (CAR19). In some embodiments, the amino acid sequence of CAR19 is SEQ ID NO:74, which may be encoded by, for example, the nucleic acid sequence of SEQ ID NO:20. In some embodiments, the amino acid sequence of CAR19 is SEQ ID NO:75. In some embodiments, the amino acid sequence of CAR19 is SEQ ID NO:76. In some embodiments, the synthetic receptor provided by the present invention comprises a target-binding domain that binds to BCMA. In some embodiments, the amino acid sequence of the BCMA-binding CAR is SEQ ID NO:77. In some embodiments, the amino acid sequence of the BCMA-binding CAR is SEQ ID NO:78. In some embodiments, the amino acid sequence of the BCMA-binding CAR is SEQ ID NO:79. In some embodiments, the amino acid sequence of the BCMA-binding CAR is SEQ ID NO:80. In some embodiments, the amino acid sequence of the BCMA-binding CAR is SEQ ID NO:136, which may be encoded by, for example, a nucleic acid sequence of SEQ ID NO:137.

6.3.3 TCRs

The fusion protein provided by this invention can be co-expressed with or coupled to a synthetic receptor in cells. In some embodiments, the synthetic receptor includes a TCR, and the fusion protein provided by this invention can be co-expressed with or coupled to a TCR in cells. In some embodiments, the synthetic receptor can bind to and activate an endogenous TCR, and the fusion protein provided by this invention can be co-expressed with or coupled to a synthetic receptor capable of binding to and activating an endogenous TCR.

T cell receptors (TCRs) are antigen-specific molecules responsible for recognizing antigenic peptides that appear on the surface of antigen-presenting cells (APCs) or any nucleated cell in the context of MHC products. This system endows T cells with the potential to recognize an entire array of intracellular antigens expressed by the cell, including viral proteins, which are processed into short peptides, bound to intracellular MHC molecules, and delivered to the surface as peptide-MHC complexes. This system allows exogenous proteins (e.g., variant cancer antigens or viral proteins) or aberrantly expressed proteins to serve as targets for T cells (e.g., Davis and Bjorkman (1988) Nature, 334, 395-402; Davis et al. (1998) Annu Rev Immunol, 16, 523-544).

The interaction between TCRs and peptide-MHC complexes can drive T cells into different activation states, depending on the binding affinity (or dissociation rate). The TCR recognition process allows T cells to distinguish normal, healthy cells from cells transformed by viruses or malignancies by providing a diverse library of TCRs, in which there are likely one or more TCRs whose binding affinity to exogenous peptides bound to MHC molecules is higher than the threshold for stimulating T cell activity (Manning and Kranz (1999) Immunology Today, 20, 417-422).

Wild-type TCRs isolated from human or mouse T cell clones and identified in vitro have been shown to have relatively low binding affinity ( _KD = 1-300 μM) (Davis et al. (1998) Annu Rev Immunol, 16, 523-544). This is partly because T cells developing in the thymus undergo negative selection (tolerance induction) of their own peptide-MHC ligands, thus eliminating T cells with excessive affinity (Starr et al. (2003) Annu Rev Immunol, 21, 139-76). To compensate for these relatively low affinityes, T cells have evolved a co-receptor system in which cell surface molecules CD4 and CD8 bind to MHC molecules (class II and class I, respectively) and co-mediate signal transduction activity with TCRs. CD8 is particularly effective in this process, allowing TCRs with very low affinity (e.g., _KD = 300 μM) to mediate strong antigen-specific activity.

Directed evolution can be used to generate TCRs with higher affinity for specific peptide-MHC complexes. Methods that can be used include yeast display (Holler et al. (2003) Nat Immunol, 4, 55-62; Holler et al. (2000) Proc Natl Acad Sci U S A, 97, 5387-92), phage display (Li et al. (2005) Nat Biotechnol, 23, 349-54), and T cell display (Chervin et al. (2008) J Immunol Methods, 339, 175-84). All three methods involve modifying or altering normal, low-affinity TCRs that exhibit wild-type TCRs to increase affinity for homologous peptide-MHC complexes (T cell-specific primitive antigens).

Thus, in some embodiments, the synthetic receptor includes a TCR, and the fusion protein provided by the present invention can be co-expressed with the TCR in cells. In some embodiments, the fusion protein provided by the present invention can be coupled to a TCR. In some embodiments, the TCR includes an alpha (α) chain and a beta (β) chain (encoded by TRAC and TRBC, respectively). Human TRAC may have an amino acid sequence corresponding to UniProtKB/Swiss-Prot No.: P01848.2 (accession number: P01848.2, GI: 1431906459). Human TRBC may have an amino acid sequence corresponding to the GenBank sequence ALC78509.1 (accession number: ALC78509.1, GI: 924924895). In some embodiments, the TCR includes a gamma (γ) chain and a delta (δ) chain (encoded by TRGC and TRDC, respectively). Human TRGCs can have an amino acid sequence corresponding to UniProtKB/Swiss-Prot: P0CF51.1 (accession number: P0CF51.1, GI: 294863156) or to UniProtKB/Swiss-Prot: P03986.2 (accession number: P03986.2, GI: 1531253869). Human TRDCs can have an amino acid sequence corresponding to UniProtKB/Swiss-Prot: B7Z8K6.2 (accession number: B7Z8K6.2, GI: 294863191). The extracellular region of the αβ chain (or γδ chain) is responsible for antigen recognition and binding. Antigen binding stimulates downstream signal transduction via a polyCD3 complex, which binds to the intracellular domain of the αβ (or γδ) chain in three dimers (εγ, εδ, δδ).

The TCRs provided by this invention can bind to specific antigens through genetic engineering. In some embodiments, the fusion protein disclosed in this invention can be co-expressed with a TCR targeting a tumor antigen in the target cell. In some embodiments, the fusion protein disclosed in this invention can be coupled to a TCR targeting a tumor antigen. In some embodiments, the tumor antigen is selected from the group consisting of CD19, CD20, CD22, CD30, CD123, CD138, CD33, CD70, BCMA, CS1, C-Met, IL13Ra2, EGFRvIII, CEA, Her2, GD2, MAGE, GPC3, mesothelin, PSMA, ROR1, EGFR, MUC1, and NY-ESO-1.

In some embodiments, the fusion protein provided by the present invention can be coupled to a TCR. In some embodiments, the fusion protein provided by the present invention can be coupled to a TCR that binds to NY-ESO-1. In some embodiments, the amino acid sequence of the TCR that binds to NY-ESO-1 is SEQ ID NO:132, which can be encoded by a nucleic acid sequence such as SEQ ID NO:133.

In some embodiments, the synthetic receptor does not include the αβ (or γδ) chain of the TCR, but can bind to and activate the endogenous TCR complex. For example, in some embodiments, the synthetic receptor may be a TCR receptor fusion construct (“TRuC”) (Baeuerle et al., Nature Communications 10:2087(2019)), a T-cell antigen conjugate (“TAC”) (Helsen et al., Nature communications 9(1):3049(2018)), an antibody TCR receptor (“AbTCR”) (Xuet et al., Cell Discovery(2018)4:62), or a chimeric CD3 receptor (WO2016/054520).

In some embodiments, the synthetic receptor is TRuC (Baeuerle et al., Nature Communications 10:2087 (2019)). In some embodiments, the fusion protein disclosed in this invention can be co-expressed with TRuC. In some embodiments, the fusion protein disclosed in this invention can be conjugated with TRuC. TRuCs include an antibody-based binding domain fused to a T cell receptor (TCR) subunit and can be efficiently reprogrammed to recognize tumor surface antigens. Unlike CARs, TRuCs become a functional component of the TCR complex. TRuC-T cells have shown potent antitumor activity in both liquid and solid tumor xenograft models. To generate TRuCs, the target-binding domain disclosed in this invention (e.g., scFV or single-domain antibodies) can be fused to the N-terminus of human TCRα, TCRβ, CD3γ, CD3δ, or CD3ε via a linker sequence, thereby providing novel target specificity and HLA-A-independent cell lysis potential for TCRs and engineered cells. In some embodiments, the target-binding domain is fused to the N-terminus of full-length human TCRα (α-TRuC). In some embodiments, the target-binding domain is fused to the N-terminus of full-length human TCRβ (β-TRuC). In some embodiments, the target-binding domain is fused to the N-terminus of full-length human CD3γ (γ-TRuC). In some embodiments, the target-binding domain is fused to the N-terminus of full-length human CD3δ (δ-TRuC). In some embodiments, the target-binding domain is fused to the N-terminus of full-length human CD3ε (ε-TRuC).

In some embodiments, the synthetic receptor is TAC (Helsen et al., Nature communications 9(1):3049(2018)). In some embodiments, the fusion protein disclosed in this invention can be co-expressed with TAC. In some embodiments, the fusion protein disclosed in this invention can be coupled with TAC. TAC is a chimeric receptor that designates an initial TCR via a CD3-binding domain, triggering the aggregation and activation of endogenous TCRs upon binding to tumor antigens, and inducing a more efficient antitumor response and reducing toxicity compared to previous generation CARs. TAC is designed to mimic the TCR-CD3:co-receptor complex and has three components: (1) an antigen-binding domain, (2) a TCR recruitment domain, and (3) a co-receptor domain (hinge, transmembrane, and cytoplasmic region). The TAC receptor redirects the TCR-CD3 complex to a selected antigen using interchangeable antigen-binding moieties, recruits the TCR-CD3 complex using scFv, and integrates co-receptor properties by including a CD4 hinge, a TM region, and a cytoplasmic tail. The antigen-binding domain can be any target-binding domain disclosed in this invention. In some embodiments, the antigen-binding domain is anti-HER2 DARPin. In some embodiments, the antigen-binding domain is anti-CD19 scFV. In some embodiments, the TCR recruitment domain is a CD3ε targeting domain. In some embodiments, the CD3ε targeting domain is selected from the group consisting of huUCHT1, UCHT1, F6K, L2K, and OKT3. In some embodiments, the co-receptor domain is a CD4 co-receptor domain. In some embodiments, the co-receptor domain is a CD8α co-receptor domain.

In some embodiments, the synthetic receptor is an AbTCR, which associates antibody antigen-binding with the endogenous TCR activation pathway (Xu et al., Cell Discovery (2018) 4:62). In some embodiments, the fusion protein disclosed in this invention can be co-expressed with an AbTCR. In some embodiments, the fusion protein disclosed in this invention can be coupled to an AbTCR. To generate an AbTCR, the Fab domain of the antibody binds to the γ and δ chains of the TCR as effector domains. Specifically, in the AbTCR receptor, the heavy chain domain of the Fab fragment is fused to a portion of the δ chain of the γδTCR. However, the light chain domain of the Fab fragment is fused to a portion of the γ chain of the γδTCR. Similar to the exogenously expressed TCR in TCR-T cells, the AbTCR binds to the endogenous CD3 complex to initiate T cell activation. However, because the γδTCR chain does not bind to the TCR in αβT cells, the AbTCR avoids the mismatch problem of conventional αβTCR-based synthetic receptors. Furthermore, by employing Fab as the antigen-binding domain, AbTCRs can target peptide MHC complexes or cell surface antigens using either TCR mimic antibodies or conventional antibodies, respectively. AbTCR-T cells can trigger the production of antigen-specific cytokines, degranulation, and the killing of cancer cells.

In some embodiments, the synthetic receptor is a chimeric CD3 receptor (WO2016/054520). In some embodiments, the fusion protein disclosed in this invention can be co-expressed with the chimeric CD3 receptor. In some embodiments, the fusion protein disclosed in this invention can be coupled to the chimeric CD3 receptor. The chimeric CD3 receptor includes an extracellular domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular domain contains a CD3 epitope. Therefore, cells expressing the chimeric CD3 receptor can be brought into close proximity to cancer cells expressing the cancer antigen by using a bispecific antibody that binds to CD3 and a cancer antigen. In some embodiments, the CD3 epitope originates from the CD3ε extracellular domain. The intracellular signaling protein is a co-stimulatory domain, which may include a 4-1BB signaling domain, a CD28 signaling domain, an ICOS signaling domain, an OX40 signaling domain, a CD27 signaling domain, a CD30 signaling domain, a CD150 signaling domain, a DAP-10 signaling domain, an NKG2D signaling domain, or a portion thereof or a combination thereof. The intracellular domain may further include the T cell receptor delta chain. The chimeric CD3 receptor may further include a transmembrane domain. The transmembrane domain may be a CD8 transmembrane domain, a CD28 transmembrane domain, an FcR transmembrane domain, or a CD3ε transmembrane domain. (W02016/054520).

6.4 Nucleic Acid

The present invention also provides nucleic acids encoding the fusion proteins disclosed herein. In some embodiments, the present invention provides nucleic acids encoding fusion proteins comprising a presenting peptide covalently linked to a β2M peptide via a linker, wherein the fusion protein binds to an MHC heavy chain to form an MHC complex, the MHC complex binding to an inhibitory receptor of an immune cell to inhibit the immune cell.

In some embodiments, the present invention provides a nucleic acid encoding a fusion protein, said fusion protein comprising an HLA-E-restricted presenting peptide covalently linked to a β2M peptide via a linker. In some embodiments, the present invention provides a nucleic acid encoding a fusion protein comprising a presenting peptide covalently linked to a β2M peptide via a linker, wherein said presenting peptide is a signal peptide of a class I MHC molecule or a fragment thereof. In some embodiments, the fusion protein does not have an MHC heavy chain. In some embodiments, the fusion protein does not have an HLA-E heavy chain. In some embodiments, the fusion protein does not have an HLA-A heavy chain, HLA-B heavy chain, HLA-C heavy chain, HLA-E heavy chain, HLA-F heavy chain, or HLA-G heavy chain. In some embodiments, the fusion protein has fewer than 500 amino acids, fewer than 400 amino acids, fewer than 300 amino acids, or fewer than 200 amino acids. In some embodiments, the fusion protein has about 100 to about 300 amino acids, about 100 to about 200 amino acids, about 120 to about 180 amino acids, about 120 to about 160 amino acids, or about 140 to about 160 amino acids. Various embodiments of fusion proteins encoded by nucleic acids provided by the present invention are provided in Section 6.2 above.

In some embodiments, the nucleic acid provided by the present invention comprises (i) a first fragment encoding a fusion protein, said fusion protein comprising a presenting peptide and a β2M peptide covalently linked by a linker, and (ii) a second fragment encoding a synthetic receptor. The synthetic receptor can be any synthetic receptor disclosed in this invention. In some embodiments, the synthetic receptor can be selected from the group consisting of chimeric antigen receptors (CARs), T-cell receptors (TCRs), TCR receptor fusion constructs (TRuCs), T-cell antigen conjugates (TACs), antibody-TCR receptors (AbTCRs), and chimeric CD3 receptors.

In some embodiments, the synthetic receptor is a CAR, and the nucleic acid provided by the present invention comprises a first fragment encoding a fusion protein as disclosed herein, the fusion protein comprising a presenting peptide and a β2M peptide covalently linked by a linker, and a second fragment encoding a CAR, wherein the second fragment is operatively linked to the first fragment. The CAR may be any CAR disclosed herein or known in the art. In some embodiments, the CAR includes an antigen-binding domain that specifically binds to tumor antigens.

In some embodiments, the synthetic receptor is a TCR, and the nucleic acid provided by the present invention comprises a first fragment encoding a fusion protein as disclosed herein, said fusion protein comprising a presenting peptide and a β2M peptide covalently linked by a linker, and a second fragment encoding a TCR, wherein said second fragment is operatively linked to the first fragment. The TCR may be any TCR disclosed herein or known in the art. In some embodiments, the TCR includes an antigen-binding domain that specifically binds to tumor antigens.

In some embodiments, the synthetic receptor is TRuc, and the nucleic acid provided by the present invention comprises a first fragment encoding a fusion protein as disclosed herein, the fusion protein comprising a presenting peptide and a β2M peptide covalently linked by a linker, and a second fragment encoding TRuc, wherein the second fragment is operatively linked to the first fragment. In some embodiments, TRuc includes an antigen-binding domain that specifically binds to tumor antigens.

In some embodiments, the synthetic receptor is a TAC, and the nucleic acid provided by the present invention comprises a first fragment encoding a fusion protein as disclosed herein, the fusion protein comprising a presenting peptide and a β2M peptide covalently linked by a linker, and a second fragment encoding a TAC, wherein the second fragment is operatively linked to the first fragment. In some embodiments, the TAC includes an antigen-binding domain that specifically binds to tumor antigens.

In some embodiments, the synthetic receptor is AbTCR, and the nucleic acid provided by the present invention comprises a first fragment encoding a fusion protein as disclosed herein, the fusion protein comprising a presenting peptide and a β2M peptide covalently linked by a linker, and a second fragment encoding AbTCR, wherein the second fragment is operatively linked to the first fragment. In some embodiments, AbTCR comprises an antigen-binding domain that specifically binds to tumor antigens.

In some embodiments, the synthetic receptor is a chimeric CD3 receptor, and the nucleic acid provided by the present invention includes a first fragment encoding a fusion protein as disclosed herein, said fusion protein including a presenting peptide and a β2M peptide covalently linked by a linker, and a second fragment encoding a chimeric CD3 receptor, wherein said second fragment is operatively linked to the first fragment. In some embodiments, the chimeric CD3 receptor includes an antigen-binding domain that specifically binds to tumor antigens.

In some embodiments, the nucleic acid provided by the present invention comprises a first fragment encoding a fusion protein and a second fragment encoding a synthetic receptor, wherein the fusion protein comprises a presenting peptide and a β2M peptide, and the first and second fragments are linked by a polynucleotide encoding a self-cleaving peptide. In some embodiments, the first and second fragments are linked by a polynucleotide encoding a 2A peptide. 2A peptides, also known as 2A self-cleaving peptides, comprise a class of peptides 18-22 amino acids long and can be used for bicistronic or polycistronic expression of protein sequences (see, Szymczak et al., Expert Opin. Biol. Therapy 5(5):627-638(2005); Liu et al.(2017). Scientific Reports 7(1):2193). 2A peptides include, for example, P2A peptides, T2A peptides, F2A peptides, or E2A peptides. An exemplary P2A sequence is (GSG)ATNFSLLKQAGDVEENPGP (SEQ ID NO: 105). An exemplary T2A sequence is (GSG)EGRGSLLTCGDVEENPGP (SEQ ID NO: 106). An exemplary E2A sequence is (GSG)QCTNYALLKLAGDVESNPGP (SEQ ID NO: 107). An exemplary F2A sequence is (GSG)VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 108). The N-terminal sequence “GSG” (Gly-Ser-Gly) of the 2A peptide is optional.

In some embodiments, the nucleic acid provided by the present invention includes a first fragment encoding a fusion protein and a second fragment encoding a synthetic receptor, wherein the fusion protein includes a presenting peptide and a β2M peptide, the first fragment and the second fragment are linked by a polynucleotide encoding a 2A peptide, and the nucleic acid encodes the synthetic receptor, the 2A linker, and the fusion protein from the N-terminus to the C-terminus. In some embodiments, the nucleic acid provided by the present invention encodes the fusion protein, the 2A linker, and the synthetic receptor from the N-terminus to the C-terminus.

In some embodiments, the nucleic acid provided by the present invention comprises a first fragment encoding a fusion protein and a second fragment encoding a CAR, wherein the fusion protein comprises a presenting peptide and a β2M peptide, the first fragment and the second fragment are linked by a polynucleotide encoding a 2A peptide, and the nucleic acid encodes the CAR, the 2A linker, and the fusion protein from the N-terminus to the C-terminus. In some embodiments, the nucleic acid provided by the present invention encodes the fusion protein, the 2A linker, and the CAR from the N-terminus to the C-terminus.

In some embodiments, the nucleic acid provided by the present invention comprises a first segment encoding a fusion protein and a second segment encoding a synthetic receptor, wherein the fusion protein comprises a presenting peptide and a β2M peptide, and the first and second segments are linked by an IRES sequence. An “internal ribosome entry site” or “IRES” refers to an element that facilitates direct entry of an internal ribosome into a cistron (protein-coding region) start codon (e.g., ATG), thereby leading to cap-independent translation of the gene. See, for example, Jackson et al., 1990. Trends Biochem Sci 15(12):477-83) and Jackson and Kaminski. 1995. RNA 1(10):985-1000. Examples of IRES commonly used by those skilled in the art are contained in U.S. Pat. No. 6,692,736. Examples of "IRES" known in the art also include, but are not limited to, IRES obtained from picomaviruses (Jackson et al., 1990). In some embodiments, the nucleic acid provided by the present invention has a first fragment, an IRES sequence, and a second fragment from the 5' end to the 3' end. In some embodiments, the nucleic acid provided by the present invention has a second fragment, an IRES fragment, and a first fragment from the 5' end to the 3' end.

In some embodiments, the nucleic acid provided by the present invention encodes a fusion protein whose amino acid sequence has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs:5 and 13-18. In some embodiments, the amino acid sequence of the fusion protein provided by the present invention comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:5 and 13-18. The present invention also provides a nucleic acid for hybridization with a nucleic acid encoding an amino acid sequence selected from the group consisting of SEQ ID NOs:5 and 13-18. In some embodiments, hybridization is performed under highly stringent conditions known to those skilled in the art.

In some embodiments, the nucleic acid sequence provided by the present invention has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO: 6 or 12. In some embodiments, the nucleic acid sequence provided by the present invention is SEQ ID NO: 6 or 12.

In some embodiments, the nucleic acid sequence provided by the present invention has at least 85% identity with SEQ ID NO:6. In some embodiments, the nucleic acid sequence provided by the present invention has at least 90% identity with SEQ ID NO:6. In some embodiments, the nucleic acid sequence provided by the present invention has at least 95% identity with SEQ ID NO:6. In some embodiments, the nucleic acid sequence provided by the present invention has at least 98% identity with SEQ ID NO:6. In some embodiments, the nucleic acid sequence provided by the present invention has at least 99% identity with SEQ ID NO:6. In some embodiments, the nucleic acid sequence provided by the present invention is SEQ ID NO:6.

In some embodiments, the nucleic acid sequence provided by the present invention has at least 85% identity with SEQ ID NO:12. In some embodiments, the nucleic acid sequence provided by the present invention has at least 90% identity with SEQ ID NO:12. In some embodiments, the nucleic acid sequence provided by the present invention has at least 95% identity with SEQ ID NO:12. In some embodiments, the nucleic acid sequence provided by the present invention has at least 98% identity with SEQ ID NO:12. In some embodiments, the nucleic acid sequence provided by the present invention has at least 99% identity with SEQ ID NO:12. In some embodiments, the nucleic acid sequence provided by the present invention is SEQ ID NO:12.

The present invention also contemplates variants of the specific nucleic acids provided by the present invention. Variants may contain alterations in coding regions, non-coding regions, or both. In some embodiments, nucleic acid variants contain alterations that produce silent substitutions, additions, or deletions, but do not alter the properties or activity of the encoded polypeptide. In some embodiments, nucleic acid variants include silent substitutions that result in no change to the amino acid sequence of the polypeptide (due to codon degeneracy). Nucleic acid variants are generated for various reasons, such as optimizing codon expression for a specific host (e.g., changing codons in human mRNA to codons suitable for a particular bacterial host, such as *E. coli*). In some embodiments, nucleic acid variants include at least one silent mutation in a non-coding or coding region of the sequence.

In some embodiments, nucleic acid variants are prepared to modulate or alter the expression (or expression level) of the encoding nucleic acid. In some embodiments, nucleic acid variants are prepared to increase the expression of the encoding polypeptide. In some embodiments, nucleic acid variants are prepared to decrease the expression of the encoding polypeptide. In some embodiments, the nucleic acid variant increases the expression of the encoding polypeptide compared to the parental polynucleotide. In some embodiments, the nucleic acid variant decreases the expression of the encoding polypeptide compared to the parental nucleic acid.

If desired, the nucleic acid provided by this invention can be codon-optimized to improve the expression efficiency of the fusion protein in a given cell. Codon optimization can be used to achieve higher levels of expression in a given cell. Factors involved in different stages of protein expression include codon adaptability, mRNA structure, and various cis-elements in transcription and translation. Any suitable codon optimization methods or techniques known to those skilled in the art can be used to modify the nucleic acid provided by this invention. These codon optimization methods are well-known and include commercially available codon optimization services, such as OptimumGene ^™ (GenScript; Piscataway, NJ), Encor optimization (EnCor Biotechnology; Gainseville FL), Blue Heron (Blue Heron Biotech; Bothell, WA), etc. Optionally, multiple codon optimizations can be performed based on different algorithms, and the optimization results can be mixed to generate codons encoding peptides to obtain optimized nucleic acids.

In some embodiments, the nucleic acid is isolated. In some embodiments, the nucleic acid is substantially purified.

The present invention also provides vectors comprising the nucleic acids provided by the present invention. The vector may be an expression vector. The vector may be a viral vector. In one embodiment, the vector is a retroviral vector, such as a gamma retroviral vector, for introducing the nucleic acids of the present invention into target cells. The vector may be a lentiviral vector. The vector may be an adenovirus vector. The vector may be an adeno-associated virus vector. In some embodiments, the vector and construct may optionally be designed to contain a reporter gene.

The nucleic acids provided by this invention can be prepared, manipulated, and/or expressed using any of the various well-established techniques known and available in the art. Examples of vectors include plasmids, autonomously replicating sequences, and transposon elements. Typical transposon systems such as Sleeping Beauty and PiggyBac can be used, which can be stably integrated into the genome (e.g., Ivics et al., Cell, 91(4):501-510 (1997); et al., (2007) Nucleic Acids Research. 35(12):e87). Other exemplary vectors include, but are not limited to, plasmids, bacteriophages, granules, artificial chromosomes (such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC)), bacteriophages (such as λ phage or M13 phage), and animal viruses. Examples of animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and multivacuolar papillomaviruses (e.g., SV40). Examples of expression vectors include pClneo (Promega) for expression in mammalian cells; and pLenti4/V5-DEST ^™ , pLenti6/V5-DEST ^™ , and pLenti6.2/V5-GW/lacZ (Invitrogen) for lentivirus-mediated gene transduction and expression in mammalian cells. In certain embodiments, the coding sequence of the fusion protein disclosed in this invention can be ligated into such expression vectors for expression in mammalian cells.

In certain embodiments, the vector is an episomal vector or a vector maintained outside the chromosome. As used herein, the term "episomal" refers to a vector capable of replication without integration into the host's chromosomal DNA and without gradual loss from dividing host cells; it also means that the vector replicates outside the chromosome or in a free form. The vector is modified to contain a sequence encoding the origin of DNA replication or "ori" from lymphotrophic herpesvirus or gamma herpesvirus, adenovirus, SV40, bovine papillomavirus, or yeast, particularly the origin of replication of lymphotrophic herpesvirus or gamma herpesvirus corresponding to oriP of EBV. In some embodiments, the lymphotrophic herpesvirus may be Epstein-Barr virus II (EBV), Kaposi's sarcoma herpesvirus (KSHV), squirrel monkey herpesvirus (HS), or Marek's disease virus (MDV). Epstein-Barr virus II (EBV) and Kaposi's sarcoma herpesvirus (KSHV) are also examples of gamma herpesviruses. Typically, the host cell includes a viral replication transactivator protein that activates replication.

The "expression control sequences," "control elements," or "regulatory sequences" present in expression vectors refer to the vector's untranslated regions (such as the origin of replication, selector, promoter, enhancer, translation initiation signal (Shine Dalgarno or Kozak sequence) introns, polyadenylated sequences, and 5' and 3' untranslated regions) that interact with host cell proteins to enable transcription and translation. These elements vary in strength and specificity. Depending on the vector system and host used, any number of suitable transcriptional and translational elements can be used, including ubiquitous promoters and inducible promoters.

Illustrative and general expression control sequences that can be used in this invention include, but are not limited to, cytomegalovirus (CMV) immediate early promoters, viral simian virus (SV40) promoters (e.g., early or late), Moloney mouse leukemia virus (MoMLV) LTR promoters, Raul's sarcoma virus (RSV) LTR, herpes simplex virus (HSV) (thymidine kinase) promoters, H5, P7.5, and P11 promoters from vaccinia virus, elongation factor 1-α (EF1a) promoters, early growth response factor 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), eukaryotic... Cellular translation initiation factor 4A1 (EIF4A1), heat shock protein 70kDa 5 (HSPA5), heat shock protein 90kDa β-member 1 (HSP90B1), heat shock protein 70kDa (HSP70), β-kinin (β-KIN), human ROSA 26 gene locus (Irions et al., Nature Biotechnology 25, 1477-1482 (2007)), ubiquitin C promoter (UBC), phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer/chicken β-actin (CAG) promoter and β-actin promoter.

Illustrative examples of inducible promoters/systems include, but are not limited to, steroid-inducible promoters (e.g., promoters of genes encoding glucocorticoids or estrogen receptors (inducible by treatment with the corresponding hormone)), metallothionein promoters (inducible by treatment with various heavy metals), MX-1 promoters (inducible by interferon), the mifepristone-regulated “gene switch” system (Sirin et al., 2003, Gene, 323:67), cumate-induced gene switches (WO 2002/088346), tetracycline-dependent regulatory systems, and so on.

6.5 Genetically Engineered Cells

This invention provides genetically engineered cells expressing the fusion protein disclosed herein. This invention also provides genetically engineered cells comprising the nucleic acids disclosed herein. In some embodiments, this invention further provides genetically engineered cells comprising the vector disclosed herein.

In some embodiments, the present invention provides genetically engineered cells expressing a fusion protein, said fusion protein comprising a presenting peptide covalently linked to a β2M peptide via a linker, wherein the fusion protein binds to an MHC heavy chain to form an MHC complex, the MHC complex binding to an inhibitory receptor of an immune cell to inhibit the immune cell. In some embodiments, the present invention provides genetically engineered cells expressing a fusion protein comprising a presenting peptide covalently linked to a β2M peptide via a linker, wherein the presenting peptide is a signal peptide of a class I MHC molecule or a fragment thereof.

In some embodiments, the present invention provides genetically engineered cells comprising nucleic acids encoding a fusion protein, said fusion protein comprising a presenting peptide covalently linked to a β2M peptide via a linker, wherein said fusion protein binds to an MHC heavy chain to form an MHC complex, said MHC complex binding to an inhibitory receptor of an immune cell to inhibit the immune cell. In some embodiments, the present invention provides genetically engineered cells comprising nucleic acids encoding a fusion protein, said fusion protein comprising a presenting peptide covalently linked to a β2M peptide via a linker, wherein said presenting peptide is a signal peptide of a class I MHC molecule or a fragment thereof.

In some embodiments, the fusion protein has fewer than 500 amino acids. In some embodiments, the fusion protein has fewer than 400, fewer than 300, or fewer than 200 amino acids. In some embodiments, the fusion protein has about 100 to about 200 amino acids, about 120 to about 180 amino acids, about 120 to about 160 amino acids, or about 140 to about 160 amino acids. In some embodiments, the fusion protein lacks the MHC heavy chain. In some embodiments, the fusion protein lacks the HLA-A heavy chain, HLA-B heavy chain, HLA-C heavy chain, HLA-E heavy chain, HLA-F heavy chain, or HLA-G heavy chain. In some embodiments, the fusion protein lacks the HLA-E heavy chain. Various embodiments of the fusion protein have been provided in the foregoing sections.

The cells disclosed in this invention are genetically engineered to express a fusion protein, said fusion protein comprising a presenting peptide covalently linked to a β2M peptide via a linker, wherein the fusion protein binds to an MHC heavy chain to form a stable MHC complex on the cell surface. In some embodiments, the fusion protein binds to an endogenously expressed MHC heavy chain to form a stable MHC complex on the cell surface. In some embodiments, the MHC complex may bind to inhibitory receptors on immune cells to inhibit immune cells. Therefore, this invention also provides a stable MHC complex comprising the fusion protein disclosed herein and an MHC heavy chain. The MHC heavy chain may be endogenously expressed.

In some embodiments, the present invention provides a stable HLA-A complex comprising the fusion protein disclosed herein and an HLA-A heavy chain, wherein the fusion protein comprises an HLA-A-restricted presenting peptide covalently linked to a β2M peptide via a linker. In some embodiments, the present invention provides a stable HLA-B complex comprising the fusion protein disclosed herein and an HLA-B heavy chain, wherein the fusion protein comprises an HLA-B-restricted presenting peptide covalently linked to a β2M peptide via a linker. In some embodiments, the present invention provides a stable HLA-C complex comprising the fusion protein disclosed herein and an HLA-C heavy chain, wherein the fusion protein comprises an HLA-C-restricted presenting peptide covalently linked to a β2M peptide via a linker.

In some embodiments, the present invention provides a stable HLA-E complex comprising the fusion protein disclosed herein and an HLA-E heavy chain, wherein the fusion protein comprises an HLA-E-restricted presenting peptide covalently linked to a β2M peptide via a linker. In some embodiments, the present invention provides a stable HLA-F complex comprising the fusion protein disclosed herein and an HLA-F heavy chain, wherein the fusion protein comprises an HLA-F-restricted presenting peptide covalently linked to a β2M peptide via a linker. In some embodiments, the present invention provides a stable HLA-G complex comprising the fusion protein disclosed herein and an HLA-G heavy chain, wherein the fusion protein comprises an HLA-G-restricted presenting peptide covalently linked to a β2M peptide via a linker.

In some embodiments, the present invention provides a stable CD1 complex comprising the fusion protein disclosed herein and a CD1 heavy chain, wherein the fusion protein comprises a CD1-restricted presenting molecule covalently linked to a β2M peptide via a linker. In some embodiments, the present invention provides a stable MR1 complex comprising the fusion protein disclosed herein and an MR1 heavy chain, wherein the fusion protein comprises an MR1-restricted presenting molecule covalently linked to a β2M peptide via a linker. In some embodiments, the present invention provides a stable FcRn complex comprising the fusion protein disclosed herein and an FcRn heavy chain, wherein the fusion protein comprises an FcRn-restricted presenting molecule covalently linked to a β2M peptide via a linker. In some embodiments, the present invention provides a stable UL18 complex comprising the fusion protein disclosed herein and a UL18 protein, wherein the fusion protein comprises a UL18-restricted presenting peptide covalently linked to a β2M peptide via a linker.

The formation of the MHC complex can be detected by any method disclosed in this invention or known in the art. For example, the co-localization of the fusion protein disclosed in this invention and the MHC heavy chain in the MHC complex can be detected by β2M co-immunoprecipitation (Lee et al., J Immunol 1998; 160:4951-60). As another example, the interaction between the MHC heavy chain and the fusion protein disclosed in this invention in the MHC complex can be detected by surface plasmon resonance (SPR) technology (Goodridge et al., Immunol. 2010; 184(11):6199-6208). Furthermore, the fusion protein and the MHC heavy chain can be labeled with different fluorescent markers, and the co-localization of the MHC heavy chain and the fusion protein disclosed in this invention in the MHC complex can be detected by microscopy or flow cytometry. Those skilled in the art will understand that a variety of assays for detecting protein-protein interactions can be used to detect MHC complexes, including, for example, pull-down assays, cross-linking, label transfer, far-western blot analysis, protein affinity purification, two-hybrid assays (yeast, mammals), fluorescence resonance energy transfer (FRET), gel migration assays, etc. (see, for example, Berggard et al. Proteomics 2007, 7, 2833-2842; Phizicky et al. (1995) Microbiol Rev. 59: 94-123.; Golemis E (2002) Protein-protein interactions: A molecular cloning manual. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press. p ix, 682.).

In some embodiments, the genetically engineered cells disclosed in this invention further express synthetic receptors, said synthetic receptors comprising (1) a target-binding domain capable of specifically binding to a target molecule, and (2) a functional domain capable of activating signal transduction pathways in the engineered cells. The synthetic receptor may be CAR, TCR, TruC, TAC, AbTCR, or a chimeric CD3 receptor. In some embodiments, the genetically engineered cells disclosed in this invention further express CAR. In some embodiments, the genetically engineered cells disclosed in this invention further express TCR. In some embodiments, the genetically engineered cells disclosed in this invention further express TruC. In some embodiments, the genetically engineered cells disclosed in this invention further express TAC. In some embodiments, the genetically engineered cells disclosed in this invention further express AbTCR. In some embodiments, the genetically engineered cells disclosed in this invention further express a chimeric CD3 receptor.

In some embodiments, the synthetic receptor is a CAR. The CAR can be any CAR disclosed in this invention or known in the art. In some embodiments, the CAR includes an antigen-binding domain that specifically binds to tumor antigens. Therefore, in some embodiments, this invention also provides genetically engineered cells expressing the fusion protein and CAR disclosed in this invention. In some embodiments, the genetically engineered cells provided by this invention include nucleic acids comprising a first fragment encoding the fusion protein and a second fragment encoding the CAR. The first and second fragments of the nucleic acid can be linked by a polynucleotide encoding a 2A peptide. The first and second fragments of the nucleic acid can also be linked to an IRES sequence. In some embodiments, the genetically engineered cells provided by this invention include a nucleic acid encoding the fusion protein and a second nucleic acid encoding the CAR, wherein the fusion protein includes a presenting peptide and a β2M peptide covalently linked by a linker.

In some embodiments, the synthetic receptor is a TCR. The TCR can be any TCR disclosed in this invention or known in the art. In some embodiments, the TCR includes an antigen-binding domain that specifically binds to tumor antigens. Therefore, in some embodiments, genetically engineered cells expressing the fusion protein disclosed in this invention and the TCR are also provided. In some embodiments, the genetically engineered cells provided by this invention include nucleic acids comprising a first fragment encoding the fusion protein and a second fragment encoding the TCR. The first and second fragments of the nucleic acid can be linked by a polynucleotide encoding a 2A peptide. The first and second fragments of the nucleic acid can also be linked to an IRES sequence. In some embodiments, the genetically engineered cells provided by this invention include a nucleic acid encoding the fusion protein and a second nucleic acid encoding the TCR, wherein the fusion protein includes a presenting peptide and a β2M peptide covalently linked by a linker.

In some embodiments, the synthetic receptor is TruC. TruC can be any TruC disclosed in this invention or known in the art. In some embodiments, TruC includes an antigen-binding domain that specifically binds to tumor antigens. Therefore, in some embodiments, this invention also provides genetically engineered cells expressing the fusion protein disclosed in this invention and TruC. In some embodiments, the genetically engineered cells provided by this invention comprise nucleic acids including a first fragment encoding the fusion protein and a second fragment encoding TruC. The first and second fragments of the nucleic acid can be linked by a polynucleotide encoding a 2A peptide. The first and second fragments of the nucleic acid can also be linked to an IRES sequence. In some embodiments, the genetically engineered cells provided by this invention comprise nucleic acids encoding the fusion protein and a second nucleic acid encoding TruC, wherein the fusion protein includes a presenting peptide and a β2M peptide covalently linked by a linker.

In some embodiments, the synthetic receptor is a TAC. The TAC can be any TAC disclosed in this invention or known in the art. In some embodiments, the TAC includes an antigen-binding domain that specifically binds to tumor antigens. Therefore, in some embodiments, this invention also provides genetically engineered cells expressing the fusion protein and TAC disclosed in this invention. In some embodiments, the genetically engineered cells provided by this invention comprise nucleic acids including a first fragment encoding the fusion protein and a second fragment encoding the TAC. The first and second fragments of the nucleic acid can be linked by a polynucleotide encoding a 2A peptide. The first and second fragments of the nucleic acid can also be linked to an IRES sequence. In some embodiments, the genetically engineered cells provided by this invention comprise nucleic acids encoding the fusion protein and a second nucleic acid encoding the TAC, wherein the fusion protein includes a presenting peptide and a β2M peptide covalently linked by a linker.

In some embodiments, the synthetic receptor is an AbTCR. The AbTCR can be any AbTCR disclosed in this invention or known in the art. In some embodiments, the AbTCR includes an antigen-binding domain that specifically binds to tumor antigens. Therefore, in some embodiments, this invention also provides genetically engineered cells expressing the fusion protein disclosed in this invention and an AbTCR. In some embodiments, the genetically engineered cells provided by this invention comprise nucleic acids including a first fragment encoding the fusion protein and a second fragment encoding the AbTCR. The first and second fragments of the nucleic acid can be linked by a polynucleotide encoding a 2A peptide. The first and second fragments of the nucleic acid can also be linked to an IRES sequence. In some embodiments, the genetically engineered cells provided by this invention comprise nucleic acids encoding the fusion protein and a second nucleic acid encoding the AbTCR, wherein the fusion protein includes a presenting peptide and a β2M peptide covalently linked by a linker.

In some embodiments, the synthetic receptor is a chimeric CD3 receptor. The chimeric CD3 receptor can be any chimeric CD3 receptor disclosed herein or known in the art. In some embodiments, the chimeric CD3 receptor includes an antigen-binding domain that specifically binds to tumor antigens. Therefore, in some embodiments, the present invention also provides genetically engineered cells expressing the fusion protein disclosed herein and the chimeric CD3 receptor. In some embodiments, the genetically engineered cells provided by the present invention comprise nucleic acids including a first fragment encoding the fusion protein and a second fragment encoding the chimeric CD3 receptor. The first and second fragments of the nucleic acid can be linked by a polynucleotide encoding a 2A peptide. The first and second fragments of the nucleic acid can also be linked to an IRES sequence. In some embodiments, the genetically engineered cells provided by the present invention comprise nucleic acids encoding the fusion protein and a second nucleic acid encoding the chimeric CD3 receptor, wherein the fusion protein includes a presenting peptide and a β2M peptide covalently linked by a linker.

In some embodiments, the cells provided by this invention are further modified to reduce the endogenous expression of class I MHC molecules or MHC-like molecules on the cell surface. Inhibiting the expression of class I MHC molecules and/or MHC-like molecules on the donor cell surface can reduce or eliminate allogeneic antigen presentation on the donor cells and help them evade recognition and clearance by host T cells, thus improving the compatibility of allogeneic grafts. For example, in the context of CAR-T therapy, it has been suggested that removing class I MHC molecules from donor T cells can eliminate the presentation of “non-self” antigens by allogeneic donor cells, thereby protecting them from the host immune system, especially CD8+ T cells (WO2015136001).

In some embodiments, the cells provided by this invention are modified to reduce the endogenous expression of class I MHC molecules or MHC-like molecules. In some embodiments, the cells disclosed in this invention lack the endogenous expression of class I MHC molecules or MHC-like molecules on the cell surface. In some embodiments, the cells disclosed in this invention lack the endogenous expression of class I MHC molecules on the cell surface. In some embodiments, the cells disclosed in this invention lack the endogenous expression of classical class I MHC molecules on the cell surface. In some embodiments, the cells disclosed in this invention lack the endogenous expression of non-classical class I MHC molecules on the cell surface. In some embodiments, the cells disclosed in this invention lack the endogenous expression of MHC-like molecules on the cell surface. In some embodiments, the cells disclosed in this invention lack the endogenous expression of HLA-A heavy chain, HLA-B heavy chain, HLA-C heavy chain, HLA-E heavy chain, HLA-F heavy chain, HLA-G heavy chain, or any combination thereof on the cell surface. In some embodiments, the cells disclosed in this invention lack the endogenous expression of HLA-A heavy chain on the cell surface. In some embodiments, the cells disclosed in this invention lack endogenous expression of HLA-B heavy chain on the cell surface. In some embodiments, the cells disclosed in this invention lack endogenous expression of HLA-C heavy chain on the cell surface. In some embodiments, the cells disclosed in this invention lack endogenous expression of HLA-E heavy chain on the cell surface. In some embodiments, the cells disclosed in this invention lack endogenous expression of HLA-F heavy chain on the cell surface. In some embodiments, the cells disclosed in this invention lack endogenous expression of HLA-G heavy chain on the cell surface. In some embodiments, the cells disclosed in this invention lack endogenous expression of HLA-A heavy chain, HLA-B heavy chain, and HLA-C heavy chain on the cell surface. In some embodiments, the cells disclosed in this invention lack endogenous expression of HLA-E heavy chain, HLA-F heavy chain, and HLA-G heavy chain on the cell surface. In some embodiments, the cells disclosed in this invention lack endogenous expression of HLA-A heavy chain, HLA-B heavy chain, HLA-C heavy chain, HLA-E heavy chain, HLA-F heavy chain, and HLA-G heavy chain on the cell surface. In some embodiments, the cells disclosed in this invention lack endogenous expression of at least MHC-like molecules on the cell surface. In some embodiments, the cells disclosed in this invention lack endogenous expression of CD1 heavy chain, MR1 heavy chain, FcRN heavy chain, UL18, or any combination thereof on the cell surface. In some embodiments, the cells disclosed in this invention lack endogenous expression of CD1 heavy chain on the cell surface. In some embodiments, the cells disclosed in this invention lack endogenous expression of MR1 heavy chain on the cell surface. In some embodiments, the cells disclosed in this invention lack endogenous expression of FcRn heavy chain on the cell surface. In some embodiments, the cells disclosed in this invention lack endogenous expression of UL18 on the cell surface. In some embodiments, the cells disclosed in this invention lack endogenous expression of CD1 heavy chain, MR1 heavy chain, FcRn heavy chain and UL18 on the cell surface.

In some embodiments, the cells disclosed in this invention lack endogenous expression of β2M on their cell surface. Since β2M is an essential structural component of various MHC complexes with different heavy chains, inhibiting β2M expression can reduce or eliminate class I MHC molecules and some MHC-like molecules on the engineered cell surface. In some embodiments, the cells disclosed in this invention are genetically engineered to (1) express the fusion protein disclosed in this invention, said fusion protein comprising a presenting peptide covalently linked to a β2M peptide via a linker, and (2) lack endogenous expression of β2M. The fusion protein can be any fusion protein disclosed in this invention. In some embodiments, the fusion protein comprises an HLA-E-restricted presenting peptide and forms a complex with an endogenous HLA-E heavy chain, thereby being recognized by inhibitory receptors of immune cells (e.g., NKG2A receptors on NK cells).

When endogenous expression of β2M is eliminated, the formation of the MHC complex can be detected by the co-localization of the fusion protein and the heavy chain on the cell surface. The MHC complex comprises the fusion protein disclosed in this invention and an endogenous MHC heavy chain. The fusion protein can be detected by an anti-β2M antibody, and the endogenous MHC heavy chain can be detected by an antibody that recognizes the heavy chain. For example, antibody clone W6/32 can be used to detect HLA-ABC; antibody clone 3D12 can be used to detect HLA-E; and antibody clones can be used to detect β2M.

The lack of endogenous expression of class I MHC molecules or MHC-like molecules on the cell surface, as disclosed in this invention, refers to a significantly reduced level of endogenous expression of these molecules at the cell surface compared to unengineered cells. In some embodiments, the endogenous expression of molecules at the cell surface is reduced by at least 50% compared to unengineered cells. In some embodiments, the endogenous expression of molecules at the cell surface is reduced by at least 60% compared to unengineered cells. In some embodiments, the endogenous expression of molecules at the cell surface is reduced by at least 70% compared to unengineered cells. In some embodiments, the endogenous expression of molecules at the cell surface is reduced by at least 80% compared to unengineered cells. In some embodiments, the endogenous expression of molecules at the cell surface is reduced by at least 90% compared to unengineered cells. In some embodiments, the endogenous expression of molecules at the cell surface is reduced by at least 95% compared to unengineered cells. In some embodiments, the endogenous expression of molecules at the cell surface is undetectable. For example, in some embodiments, the cell membrane portion of the cells disclosed in this invention cannot be detected by Western blotting or by immunofluorescence on the cell membrane. In some embodiments, the cells provided by this invention are modified to delete (or knock out) the gene encoding the class I MHC molecule or MHC-like molecule.

In some embodiments, the present invention provides a genetically engineered cell population in which at least 50% of the cells lack endogenous expression of class I MHC molecules or MHC-like molecules on the cell surface. In some embodiments, the present invention provides a genetically engineered cell population in which at least 60% of the cells lack endogenous expression of class I MHC molecules or MHC-like molecules on the cell surface. In some embodiments, the present invention provides a genetically engineered cell population in which at least 75% of the cells lack endogenous expression of class I MHC molecules or MHC-like molecules on the cell surface. In some embodiments, the present invention provides a genetically engineered cell population in which at least 80% of the cells lack endogenous expression of class I MHC molecules or MHC-like molecules on the cell surface. In some embodiments, the present invention provides a genetically engineered cell population in which at least 85% of the cells lack endogenous expression of class I MHC molecules or MHC-like molecules on the cell surface. In some embodiments, the present invention provides a genetically engineered cell population in which at least 90% of the cells lack endogenous expression of class I MHC molecules or MHC-like molecules on the cell surface. In some embodiments, the present invention provides a genetically engineered cell population wherein at least 95% of the cells lack endogenous expression of class I MHC molecules or MHC-like molecules on the cell surface.

The genetically engineered cells provided by this invention can be of any type. In some embodiments, the cells are suitable for transplantation. In some embodiments, the cells are suitable for allogeneic transplantation. In some embodiments, the cells are selected from the group consisting of stem cells, pluripotent cells, progenitor cells, hematopoietic stem cells and/or progenitor cells, CD34+ mobilized peripheral blood cells, CD34+ umbilical cord blood cells, CD34+ bone marrow cells, hepatocytes, somatic cells, immune cells, and non-transformed cells. In some embodiments, the cells are stem cells. In some embodiments, the cells are pluripotent cells. In some embodiments, the cells are progenitor cells. In some embodiments, the cells are hematopoietic stem cells and/or progenitor cells. In some embodiments, the cells are CD34+ mobilized peripheral blood cells. In some embodiments, the cells are CD34+ umbilical cord blood cells. In some embodiments, the cells are CD34+ bone marrow cells. In some embodiments, the cells are hepatocytes. In some embodiments, the cells are somatic cells. In some embodiments, the cells are non-transformed cells.

In some embodiments, the cells provided by this invention are stem cells. The cells can be somatic cells. The cells can be non-pluripotent cells. The cells can be incompletely or partially pluripotent stem cells. The cells can be pluripotent cells. The cells can be oligopotent cells. The cells can be unipotent cells. The cells can be terminally differentiated cells. Pluripotent cells suitable for a particular embodiment include, but are not limited to, natural stem cells, embryonic stem cells, or induced pluripotent cells (iPSCs). This invention also provides mixed cell populations combining any of the above-described cells. For example, the cell populations provided by this invention can include reprogrammed cells, including pluripotent cells, partially pluripotent cells, and non-pluripotent cells, such as fully differentiated cells.

In some embodiments, the cells provided by the present invention are adult stem/progenitor cells. In some embodiments, the cells provided by the present invention are neonatal stem/progenitor cells. In some embodiments, the cells are selected from the group consisting of mesodermal stem/progenitor cells, endoderm stem/progenitor cells, and ectoderm stem/progenitor cells. In some embodiments, the cells are mesodermal stem/progenitor cells. In some embodiments, the cells are endoderm stem/progenitor cells. In some embodiments, the cells are ectoderm stem/progenitor cells.

Illustrative examples of mesodermal stem/progenitor cells include, but are not limited to: mesodermal stem/progenitor cells, endothelial stem/progenitor cells, bone marrow stem/progenitor cells, umbilical cord stem/progenitor cells, adipose tissue-derived stem/progenitor cells, hematopoietic stem/progenitor cells (HSCs), mesenchymal stem/progenitor cells, muscle stem/progenitor cells, kidney stem/progenitor cells, osteoblast stem/progenitor cells, chondrocyte stem/progenitor cells, etc. In some embodiments, the cell is a mesodermal stem/progenitor cell. In some embodiments, the cell is an endothelial stem/progenitor cell. In some embodiments, the cell is a bone marrow stem/progenitor cell. In some embodiments, the cell is an umbilical cord stem/progenitor cell. In some embodiments, the cell is adipose tissue-derived stem/progenitor cells. In some embodiments, the cell is a hematopoietic stem/progenitor cell (HSC). In some embodiments, the cell is a mesenchymal stem/progenitor cell. In some embodiments, the cell is a muscle stem/progenitor cell. In some embodiments, the cells are renal stem/progenitor cells. In some embodiments, the cells are osteoblast stem/progenitor cells. In some embodiments, the cells are chondrocyte stem/progenitor cells.

Illustrative examples of ectodermal stem/progenitor cells include, but are not limited to, neural stem/progenitor cells, retinal stem/progenitor cells, and skin stem/progenitor cells. In some embodiments, the cells are neural stem/progenitor cells. In some embodiments, the cells are retinal stem/progenitor cells. In some embodiments, the cells are skin stem/progenitor cells.

Illustrative examples of endoderm stem cells or progenitor cells include, but are not limited to, liver stem/progenitor cells, pancreatic stem/progenitor cells, epithelial stem/progenitor cells, etc. In some embodiments, the cells are endoderm stem/progenitor cells. In some embodiments, the cells are liver stem/progenitor cells. In some embodiments, the cells are pancreatic stem/progenitor cells. In some embodiments, the cells are epithelial stem/progenitor cells.

In some embodiments, the cells provided by this invention are selected from the group consisting of pancreatic islet cells, CNS cells, PNS cells, cardiomyocytes, skeletal muscle cells, smooth muscle cells, hematopoietic cells, osteocytes, hepatocytes, adipocytes, kidney cells, lung cells, chondrocytes, skin cells, follicular cells, vascular cells, epithelial cells, immune cells, and endothelial cells. In some embodiments, the cells are pancreatic islet cells. In some embodiments, the cells are CNS cells. In some embodiments, the cells are PNS cells. In some embodiments, the cells are cardiomyocytes. In some embodiments, the cells are skeletal muscle cells. In some embodiments, the cells are smooth muscle cells. In some embodiments, the cells are hematopoietic cells. In some embodiments, the cells are osteocytes. In some embodiments, the cells are hepatocytes. In some embodiments, the cells are adipocytes. In some embodiments, the cells are kidney cells. In some embodiments, the cells are lung cells. In some embodiments, the cells are chondrocytes. In some embodiments, the cells are skin cells. In some embodiments, the cells are follicular cells. In some embodiments, the cells are vascular cells. In some embodiments, the cells are epithelial cells. In some implementations, the cells are endothelial cells.

In some embodiments, the cells provided by this invention are immune cells. The cells may be leukocytes. Leukocytes are nucleated cells that develop from hematopoietic stem cells and play an important role in the hematopoietic and lymphatic systems. Leukocytes include myeloid cells, lymphoid cells, granulocytes (such as neutrophils, eosinophils, and basophils), lymphocytes (such as T cells, B cells, NK cells, and NKT cells), plasma cells, dendritic cells, monocytes, and cells differentiated from them (such as macrophages). In some embodiments, the cells provided by this invention are leukocytes. In some embodiments, the cells provided by this invention are myeloid cells. In some embodiments, the cells provided by this invention are lymphoid cells. In some embodiments, the cells provided by this invention are granulocytes. In some embodiments, the cells provided by this invention are neutrophils. In some embodiments, the cells provided by this invention are eosinophils. In some embodiments, the cells provided by this invention are basophils. In some embodiments, the cells provided by this invention are T cells. In some embodiments, the cells provided by this invention are B cells. In some embodiments, the cells provided by the present invention are plasma cells. In some embodiments, the cells provided by the present invention are NK cells. In some embodiments, the cells provided by the present invention are NKT cells. In some embodiments, the cells provided by the present invention are dendritic cells. In some embodiments, the cells provided by the present invention are monocytes. In some embodiments, the cells provided by the present invention are macrophages.

In some embodiments, the cells provided by the present invention are tumor-infiltrating lymphocytes (TILs).

In some embodiments, the present invention provides the cell populations disclosed herein. The cell population can be a homogeneous cell population. The cell population can be a heterogeneous cell population. In some embodiments, the cell population can be a heterogeneous cell population comprising any combination of the cells disclosed herein.

The T cells provided by this invention can be used for allogeneic transplantation. For example, some CAR-T cells disclosed in this invention can be universal CAR-T cells and can be used for allogeneic transplantation in cancer treatment. In some embodiments, this invention provides genetically engineered T cells expressing the fusion protein disclosed in this invention. In some embodiments, this invention provides genetically engineered T cells comprising the nucleic acids disclosed in this invention. In some embodiments, this invention provides CAR-T cells.

In some embodiments, the genetically engineered cells provided by this invention are universal lymphocytes, i.e., lymphocytes applicable to allogeneic transplantation that do not produce or produce a controllable GvHD response. GvHD (graft-versus-host disease) is a medical complication following tissue transplantation with immune-active cells, in which immune-active cells in the graft tissue recognize the recipient's healthy tissue as a foreign body and initiate an immune response against the recipient's healthy tissue. GvHD can be clinically controlled with pharmacological intervention and is not life-threatening; however, severe GvHD can be life-threatening. GvHD is a particular concern for allogeneic CAR-T therapy because allogeneic T cells can not only recognize cancer but also recognize the recipient's non-cancerous tissue as non-self and attack such non-cancerous tissue.

The T cell receptor (TCR) is a transmembrane octamer on T cells, composed of a TCR dimer and various subunits of CD3. The TCR is responsible for antigen recognition by T cells, which is essential for T cell activation. The components of the TCR receptor include the α and β subunits of the TCR, and the δ, ε, γ, δ, or ε subunits of CD3. Removal of functional TCR components from donor T cells prevents donor T cells from attacking host cells. Therefore, in some embodiments, the T cells provided by the present invention also lack expression of components of the TCR complex. In some embodiments, at least one gene encoding a component of the TCR is inactivated in the T cells provided by the present invention. In some embodiments, a gene encoding the α subunit of the TCR (e.g., TRAC) is inactivated in the T cells provided by the present invention. Human TRAC may have an amino acid sequence corresponding to UniProtKB/Swiss-Prot No.: P01848.2 (accession number: P01848.2, GI: 1431906459). In some embodiments, the gene encoding the β subunit of the TCR (e.g., TRBC) is inactivated in the T cells provided by this invention. Human TRBC may have an amino acid sequence corresponding to the GenBank sequence ALC78509.1 (accession number: ALC78509.1, GI: 924924895).

In some embodiments, the gene encoding the δ subunit of CD3 is inactivated in the T cells provided by this invention. The δ subunit of human CD3 may have an amino acid sequence corresponding to the NCBI reference sequence NP_000723.1 (accession number: NP_000723.1, GI: 4502669; isomer A) or NCBI reference sequence NP_001035741.1 (accession number: NP_001035741.1, GI: 98985801; isomer B). In some embodiments, the gene encoding the ε subunit of CD3 is inactivated in the T cells provided by this invention. The ε subunit of human CD3 may have an amino acid sequence corresponding to the sequence UniProtKB/Swiss-Prot: P07766.2 (accession number: P07766.2, GI: 1345708). In some embodiments, the gene encoding the γ subunit of CD3 is inactivated in the T cells provided by this invention. The γ subunit of human CD3 may have an amino acid sequence corresponding to the NCBI reference sequence NP_000064.1 (accession number: NP_000064.1, GI: 4557429) provided below. In some embodiments, the gene encoding the δ subunit of CD3 is inactivated in the T cells provided by this invention. The δ subunit of human CD3 may have an amino acid sequence corresponding to the sequence UniProtKB/Swiss-Prot: P20963.1 (accession number: P20963.1, GI: 23830999) provided below. In some embodiments, the gene encoding the ε subunit of CD3 is inactivated in the T cells provided by this invention. The ε subunit of human CD3 may have an amino acid sequence corresponding to the sequence UniProt Identifier P20963-2 provided below.

For illustrative purposes, in some embodiments, the present invention provides genetically engineered T cells expressing a fusion protein comprising a presenting peptide covalently linked to a β2M peptide via a linker, wherein the T cells further express a CAR, and an endogenous gene encoding β2M and the α subunit of the TCR is inactivated in the T cells. The fusion protein may be any fusion protein disclosed in the present invention. The CAR may be any CAR disclosed in the present invention or known in the art. In some embodiments, the fusion protein comprises human β2M covalently linked to a presenting peptide via a (G4S)3 linker, wherein the presenting peptide is an 8-10 amino acid fragment of an HLA-E-restricted signal peptide that is a class I MHC molecule. In some embodiments, the CAR comprises an antigen-binding domain that specifically binds to a tumor antigen (e.g., CD19).

In some embodiments, the cells provided by the present invention are genetically engineered T cells expressing a fusion protein having an amino acid sequence selected from the group consisting of SEQ ID NOs:5 and 13-18. In some embodiments, the T cells further express a CAR having an antigen-binding domain that specifically binds to CD19 or BCMA. In some embodiments, the amino acid sequence of the CD19-targeting CAR is selected from the group consisting of SEQ ID NOs:74-76. In some embodiments, the amino acid sequence of the BCMA-targeting CAR is selected from the group consisting of SEQ ID NOs:77-80 and 136. In some embodiments, the T cells further express a TCR having an antigen-binding domain that specifically binds to NY-ESO-1. In some embodiments, the NY-ESO-1-targeting TCR has the amino acid sequence of SEQ ID NO:132. In some embodiments, the T cells comprise nucleic acids containing a first fragment encoding the fusion protein and a second fragment encoding the CAR or TCR. In some embodiments, the T cell includes a nucleic acid encoding a fusion protein and a CAR, the fusion protein having an amino acid sequence selected from the group consisting of SEQ ID NOs:5 and 13-18, and the CAR having an amino acid sequence selected from the group consisting of SEQ ID NOs:74-80 and 136. In some embodiments, the T cell includes a nucleic acid encoding a fusion protein and a TCR, the fusion protein having an amino acid sequence selected from the group consisting of SEQ ID NOs:5 and 13-18, and the TCR having an amino acid sequence of SEQ ID NO:132. In some embodiments, the T cell includes a nucleic acid encoding an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with an amino acid sequence selected from the group consisting of SEQ ID NOs:11, 124, 126, 128, 130, 134, and 138. In some embodiments, the T cell includes a nucleic acid encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 124, 126, 128, 130, 134, and 138. In some embodiments, endogenous genes encoding β2M and the α subunit of the TCR are inactivated in the T cell. In some embodiments, endogenous genes encoding β2M and the α subunit of the TCR are knocked out in the T cell.

In some embodiments, the T cell comprises a nucleic acid encoding an amino acid sequence having at least 85% identity with SEQ ID NO:11. In some embodiments, the T cell comprises a nucleic acid encoding an amino acid sequence having at least 90% identity with SEQ ID NO:11. In some embodiments, the T cell comprises a nucleic acid encoding an amino acid sequence having at least 95% identity with SEQ ID NO:11. In some embodiments, the T cell comprises a nucleic acid encoding an amino acid sequence having at least 98% identity with SEQ ID NO:11. In some embodiments, the T cell comprises a nucleic acid encoding an amino acid sequence having at least 99% identity with SEQ ID NO:11. In some embodiments, the T cell comprises a nucleic acid encoding the amino acid sequence of SEQ ID NO:11. In some embodiments, the T cell comprises a nucleic acid having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO:12. In some embodiments, the T cell comprises a nucleic acid having the nucleotide sequence of SEQ ID NO:12. In some embodiments, endogenous genes encoding the α subunits of β2M and TCR are inactivated in the T cell. In some embodiments, endogenous genes encoding the α subunits of β2M and TCR are knocked out in the T cell.

In some embodiments, the T cell includes a nucleic acid encoding an amino acid sequence having at least 85% identity with SEQ ID NO:124. In some embodiments, the T cell includes a nucleic acid encoding an amino acid sequence having at least 90% identity with SEQ ID NO:124. In some embodiments, the T cell includes a nucleic acid encoding an amino acid sequence having at least 95% identity with SEQ ID NO:124. In some embodiments, the T cell includes a nucleic acid encoding an amino acid sequence having at least 98% identity with SEQ ID NO:124. In some embodiments, the T cell includes a nucleic acid encoding an amino acid sequence having at least 99% identity with SEQ ID NO:124. In some embodiments, the T cell includes a nucleic acid encoding the amino acid sequence of SEQ ID NO:124. In some embodiments, the T cell includes a nucleic acid having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO:125. In some embodiments, the T cell comprises a nucleic acid having the nucleotide sequence of SEQ ID NO:125. In some embodiments, endogenous genes encoding the α subunits of β2M and TCR are inactivated in the T cell. In some embodiments, endogenous genes encoding the α subunits of β2M and TCR are knocked out in the T cell.

In some embodiments, the T cell includes a nucleic acid encoding an amino acid sequence having at least 85% identity with SEQ ID NO:126. In some embodiments, the T cell includes a nucleic acid encoding an amino acid sequence having at least 90% identity with SEQ ID NO:126. In some embodiments, the T cell includes a nucleic acid encoding an amino acid sequence having at least 95% identity with SEQ ID NO:126. In some embodiments, the T cell includes a nucleic acid encoding an amino acid sequence having at least 98% identity with SEQ ID NO:126. In some embodiments, the T cell includes a nucleic acid encoding an amino acid sequence having at least 99% identity with SEQ ID NO:126. In some embodiments, the T cell includes a nucleic acid encoding the amino acid sequence of SEQ ID NO:126. In some embodiments, the T cell includes a nucleic acid having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO:127. In some embodiments, the T cell comprises a nucleic acid having the nucleotide sequence of SEQ ID NO:127. In some embodiments, endogenous genes encoding the α subunits of β2M and TCR are inactivated in the T cell. In some embodiments, endogenous genes encoding the α subunits of β2M and TCR are knocked out in the T cell.

In some embodiments, the T cell includes a nucleic acid encoding an amino acid sequence having at least 85% identity with SEQ ID NO:128. In some embodiments, the T cell includes a nucleic acid encoding an amino acid sequence having at least 90% identity with SEQ ID NO:128. In some embodiments, the T cell includes a nucleic acid encoding an amino acid sequence having at least 95% identity with SEQ ID NO:128. In some embodiments, the T cell includes a nucleic acid encoding an amino acid sequence having at least 98% identity with SEQ ID NO:128. In some embodiments, the T cell includes a nucleic acid encoding an amino acid sequence having at least 99% identity with SEQ ID NO:128. In some embodiments, the T cell includes a nucleic acid encoding the amino acid sequence of SEQ ID NO:128. In some embodiments, the T cell includes a nucleic acid having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO:129. In some embodiments, the T cell comprises a nucleic acid having the nucleotide sequence of SEQ ID NO:129. In some embodiments, endogenous genes encoding the α subunits of β2M and TCR are inactivated in the T cell. In some embodiments, endogenous genes encoding the α subunits of β2M and TCR are knocked out in the T cell.

In some embodiments, the T cell comprises a nucleic acid encoding an amino acid sequence having at least 85% identity with SEQ ID NO:130. In some embodiments, the T cell comprises a nucleic acid encoding an amino acid sequence having at least 90% identity with SEQ ID NO:130. In some embodiments, the T cell comprises a nucleic acid encoding an amino acid sequence having at least 95% identity with SEQ ID NO:130. In some embodiments, the T cell comprises a nucleic acid encoding an amino acid sequence having at least 98% identity with SEQ ID NO:130. In some embodiments, the T cell comprises a nucleic acid encoding an amino acid sequence having at least 99% identity with SEQ ID NO:130. In some embodiments, the T cell comprises a nucleic acid encoding the amino acid sequence of SEQ ID NO:130. In some embodiments, the T cell comprises a nucleic acid having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO:131. In some embodiments, the T cell comprises a nucleic acid having the nucleotide sequence of SEQ ID NO:131. In some embodiments, endogenous genes encoding the α subunits of β2M and TCR are inactivated in the T cell. In some embodiments, endogenous genes encoding the α subunits of β2M and TCR are knocked out in the T cell.

In some embodiments, the T cell includes a nucleic acid encoding an amino acid sequence having at least 85% identity with SEQ ID NO:134. In some embodiments, the T cell includes a nucleic acid encoding an amino acid sequence having at least 90% identity with SEQ ID NO:134. In some embodiments, the T cell includes a nucleic acid encoding an amino acid sequence having at least 95% identity with SEQ ID NO:134. In some embodiments, the T cell includes a nucleic acid encoding an amino acid sequence having at least 98% identity with SEQ ID NO:134. In some embodiments, the T cell includes a nucleic acid encoding an amino acid sequence having at least 99% identity with SEQ ID NO:134. In some embodiments, the T cell includes a nucleic acid encoding the amino acid sequence of SEQ ID NO:134. In some embodiments, the T cell includes a nucleic acid having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO:135. In some embodiments, the T cell comprises a nucleic acid having the nucleotide sequence of SEQ ID NO:135. In some embodiments, endogenous genes encoding the α subunits of β2M and TCR are inactivated in the T cell. In some embodiments, endogenous genes encoding the α subunits of β2M and TCR are knocked out in the T cell.

In some embodiments, the T cell comprises a nucleic acid encoding an amino acid sequence having at least 85% identity with SEQ ID NO:138. In some embodiments, the T cell comprises a nucleic acid encoding an amino acid sequence having at least 90% identity with SEQ ID NO:138. In some embodiments, the T cell comprises a nucleic acid encoding an amino acid sequence having at least 95% identity with SEQ ID NO:138. In some embodiments, the T cell comprises a nucleic acid encoding an amino acid sequence having at least 98% identity with SEQ ID NO:138. In some embodiments, the T cell comprises a nucleic acid encoding an amino acid sequence having at least 99% identity with SEQ ID NO:138. In some embodiments, the T cell comprises a nucleic acid encoding the amino acid sequence of SEQ ID NO:138. In some embodiments, the T cell comprises a nucleic acid having at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO:139. In some embodiments, the T cell comprises a nucleic acid having the nucleotide sequence of SEQ ID NO:139. In some embodiments, endogenous genes encoding the α subunits of β2M and TCR are inactivated in the T cell. In some embodiments, endogenous genes encoding the α subunits of β2M and TCR are knocked out in the T cell.

6.6 Pharmaceutical Compositions

The present invention also provides pharmaceutical compositions comprising the cells disclosed herein. The pharmaceutical compositions comprise an effective amount of the cells disclosed herein and a pharmaceutically acceptable carrier. The cells disclosed herein and the compositions comprising the cells can be conveniently provided in the form of sterile liquid formulations, for example, typically isotonic aqueous solutions containing a cell suspension, or optionally emulsions, dispersions, etc., which are typically buffered to a specified pH. The compositions may include carriers, such as water, saline, phosphate-buffered saline, etc., which are suitable for the integrity and viability of the cells and facilitate administration of the cell composition.

The cells disclosed in this invention can be incorporated into a suitable solvent and other components in varying amounts to prepare a sterile injectable solution, as needed. Such compositions may contain pharmaceutically acceptable carriers, diluents, or excipients (e.g., sterile water, physiological saline, glucose, dextrose, etc.) suitable for use with the cell composition and for administration to a subject (e.g., a human). Suitable biological buffers for the cell composition are well known in the art. Any mediators, diluents, or additives used are compatible with maintaining the integrity and viability of the cells disclosed in this invention.

The composition is typically isotonic, meaning it has the same osmotic pressure as blood and tears. The desired isotonicity of the cell compositions provided by this invention can be achieved using sodium chloride or other pharmaceutically acceptable reagents (e.g., dextran, boric acid, sodium tartrate, or other inorganic or organic solutes). Sodium chloride is particularly preferred for buffer solutions containing sodium ions. A particularly useful buffer solution is saline, such as physiological saline. Those skilled in the art will recognize that the components of the composition should be chemically inert and will not affect the viability or efficacy of the cells disclosed in this invention, and are suitable for administration to a subject (e.g., a human). Those skilled in the art can readily determine the amount of cells and optional additives, mediators, and/or carriers in the composition to be administered in the methods of this invention.

The cells disclosed in this invention can be administered in any physiologically acceptable medium. Suitable dosages are described in this invention. Cell populations comprising the cells disclosed in this invention may include purified cell populations. As described in this invention, those skilled in the art can readily determine the percentage of cells in a cell population using various well-known methods. The purity range of cell populations comprising the genetically modified cells provided by this invention can be from about 20% to about 25%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 55% to about 60%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%; about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%. In some embodiments, the purity of the cell population including the genetically modified cells provided by the present invention can range from about 20% to about 30%, about 20% to about 50%, about 20% to about 80%, about 20% to about 100%, about 50% to about 80%, or about 50% to about 100%. Those skilled in the art can readily adjust the dosage; for example, a decrease in purity may require an increase in dosage.

The present invention also provides a kit for preparing the cells disclosed herein. In one embodiment, the kit includes one or more vectors for generating genetically engineered cells (such as T cells) expressing the fusion protein disclosed herein for allogeneic transplantation. The kit can be used to generate genetically engineered cells from non-autologous cells for administration to a compatible subject. In another embodiment, the kit may include the cells disclosed herein, such as non-autologous cells, for administration to a subject. In a particular embodiment, the kit includes the cells disclosed herein in one or more containers.

6.7 Preparation method

This invention provides a method for genetically engineering cells for allogeneic transplantation, including transducing cells with nucleic acids disclosed in this invention. This invention also provides a method for genetically engineering cells for allogeneic transplantation, including transducing cells with a vector comprising nucleic acids disclosed in this invention. In some embodiments, the nucleic acid encodes a fusion protein disclosed in this invention. In some embodiments, the method provided by this invention includes transducing cells with nucleic acids encoding a fusion protein, wherein the fusion protein comprises a presenting peptide and a β2M peptide covalently linked by a linker.

In some embodiments, the present invention provides a method for genetically engineering cells for allogeneic transplantation, comprising using nucleic acid transduced cells encoding a fusion protein, the fusion protein comprising a presenting peptide covalently linked to a β2M peptide via a linker, wherein the fusion protein binds to an MHC heavy chain to form an MHC complex, the MHC complex binding to an inhibitory receptor of an immune cell to inhibit the immune cell. In some embodiments, the present invention provides a method for genetically engineering cells for allogeneic transplantation, comprising using nucleic acid transduced cells encoding a fusion protein, the fusion protein comprising an HLA-E-restricted presenting peptide covalently linked to a β2M peptide via a linker. In some embodiments, the present invention provides a method for genetically engineering cells for allogeneic transplantation, comprising using nucleic acid transduced cells encoding a fusion protein, the fusion protein comprising a presenting peptide covalently linked to a β2M peptide via a linker, wherein the presenting peptide is a signal peptide of a class I MHC molecule or a fragment thereof. In some embodiments, the fusion protein does not have an MHC heavy chain. In some embodiments, the fusion protein does not have an HLA-E heavy chain. In some embodiments, the fusion protein does not have an HLA-A heavy chain, HLA-B heavy chain, HLA-C heavy chain, HLA-E heavy chain, HLA-F heavy chain, or HLA-G heavy chain. In some embodiments, the fusion protein has fewer than 500 amino acids, fewer than 400 amino acids, fewer than 300 amino acids, or fewer than 200 amino acids. In some embodiments, the fusion protein has about 100 to about 300 amino acids, about 100 to about 200 amino acids, about 120 to about 180 amino acids, about 120 to about 160 amino acids, or about 140 to about 160 amino acids. Various embodiments of fusion proteins encoded by nucleic acids provided by the present invention are provided in Section 6.2 above.

The cells disclosed in this invention (e.g., stem cells or immune cells) can be placed under conditions known in the art that are conducive to maintaining or expanding the cells. (De Libero, T Cell Protocols, Vol. 514 of Methods in Molecular Biology, Humana Press, Totowa NJ (2009); Parente-pereira et al., J. Biol. Methods 1(2)e7 (doi 10.14440/jbm.2014.30) (2014); Movassagh et al., Hum. Gene Ther.II:1189-1200(2000); Rettig et al., Mol.Ther.8:29-41(2003); Agarwalet al., J.Virol.72:3720-3728(1998); Pollok et al., Hum.Gene Ther.10:2221-2236(1999); Quinn et al. al.,Hum.Gene Ther. 9:1457-1467 (1998); see also commercially available methods, such as Dynabeads ^™ Human T-cell activator products, Thermo Fisher Scientific, Waltham, MA). The cells disclosed in this invention (e.g., stem cells or immune cells) may optionally be expanded before or after in vitro genetic engineering. Cell expansion is particularly useful for increasing the number of cells administered to a subject. Such cell expansion methods are well known in the art (see, for example, Kaiser et al., Cancer Gene Therapy 22:72-78 (2015); Wolfl et al., Nat. Protocols 9:950-966 (2014)). Furthermore, cells may optionally be cryopreserved after expansion of isolated and/or genetically engineered cells (see Kaiser et al., ibid., 2015). The method of cryopreservation of cells is well known in the art (see, for example, Freshney, Culture of Animal Cells: A Manual of Basic Techniques, 4th ed., Wiley-Liss, New York (2000); Harrison and Rae, General Techniques of Cell Culture, Cambridge University Press (1997)).

To generate cells recombinantly expressing the fusion protein disclosed in this invention, one or more nucleic acids encoding the fusion protein are introduced into target cells using a suitable expression vector. Target cells (e.g., stem cells or immune cells) are transduced with one or more nucleic acids, or a synthetic receptor and fusion protein, wherein the nucleic acid encodes the fusion protein. In the case of expressing a synthetic receptor and fusion protein, the synthetic receptor and fusion protein may encode the nucleic acid on separate vectors or on the same vector, as needed. For example, the nucleic acid encoding the synthetic receptor or fusion protein disclosed in this invention can be cloned into a suitable vector, such as a retroviral vector, and introduced into target cells using well-known molecular biology techniques (Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999)). Any vector suitable for expression in the cells of this invention, particularly human immune cells or stem cells, can be used. The vector contains suitable expression elements such as promoters providing expression of the encoded nucleic acid in target cells. In the case of retroviral vectors, cells can be optionally activated to improve transduction efficiency (see Parente-Pereira et al., J. Biol. Methods 1(2)e7(doi 10.14440/jbm.2014.30)(2014); Movassagh et al., Hum. Gene Ther. 11:1189-1200(2000); Rettig et al., Mol. Ther. 8:29-41(2003); Agarwal et al., J. Virol. 72:3720-3728(1998); Pollok et al., Hum. Gene Ther. 10:2221-2236(1999); Quinn et al., Hum. Gene Ther. 9:1457-1467 (1998); Additionally, commercially available methods are available, such as Dynabeads ^™ Human T-cell activator products (Thermo Fisher Scientific, Waltham, MA).

In one implementation, the vector is a retroviral vector, such as a gamma retrovirus or lentiviral vector, used to introduce the fusion protein and/or synthetic receptor into target cells. Retroviral vectors are commonly used for transduction to genetically modify cells to express the fusion protein and/or synthetic receptor. However, it should be understood that any suitable viral vector or non-viral delivery system may be used. Combinations of retroviral vectors and appropriate packaging cell lines are also suitable, where the capsid protein will function to infect human cells. Several cell lines that produce facultative viruses are known, including, but not limited to, PA12 (Miller et al., Mol. Cell. Biol. 5:431-437 (1985)); PA317 (Miller et al., Mol. Cell. Biol. 6:2895-2902 (1986)); and CRIP (Danos et al., Proc. Natl. Acad. Sci. USA 85:6460-6464 (1988)). Non-amphotropic particles are also applicable, for example, with VSVG, RD114 or GALV envelopes and any other pseudotyped particles known in the art (Relander et al., Mol. Therap. 11:452-459 (2005)). Possible transduction methods also include direct co-culture of cells with secretory cells (e.g., Bregni et al., Blood 80:1418-1422 (1992)), or culture alone with viral supernatant or concentrated vector stock solution (with or without appropriate growth factors and polycations) (see, for example, Xu et al., Exp. Hemat. 22:223-230 (1994); Hughes et al. J. Clin. Invest. 89:1817-1824 (1992)).

Typically, the chosen vectors exhibit high infection efficiency and stable integration and expression (see, for example, Cayouette et al., Human Gene Therapy 8:423-430 (1997); Kido et al., Current Eye Research 15:833-844 (1996); Bloomer et al., J.Virol.71:6641-6649 (1997); Naldini et al., Science 272:263-267 (1996); and Miyoshi et al., Proc.Natl.Acad.Sci.U.S.A.94:10319-10323 (1997)). Other viral vectors that may be used include, for example, adenoviruses, lentiviruses and adeno-associated virus vectors, vaccinia virus, bovine papillomavirus-derived vectors or herpesviruses, such as Epstein-Barr virus (see, for example, Miller, Hum. Gene Ther. 1(1):5-14 (1990); Friedman, Science 244:1275-1281 (1989); Eglitis et al., BioTechniques 6:608-614 (1988); Tolstoshev et al., Current Opin. Biotechnol. 1:55-61 (1990); Sharp, Lance t 337:1277-1278(1991); Cornetta et al., Prog.Nucleic AcidRes.Mol.Biol.36:311-322(1989); Anderson, Science 226:401-409(1984); Moen, BloodCells 17: 407-416(1991); Miller et al., Biotechnology 7:980-990(1989); Le Gal LaSalle et al., Science 259:988-990(1993); and Johnson, Chest 107:77S-83S (1995)). Retroviral vectors have been well developed and have been used clinically (Rosenberg et al., N. Engl. J. Med. 323: 370 (1990); Anderson et al., U.S. Pat. No. 5, 399, 346).

Particularly useful vectors for expressing the fusion proteins and/or synthetic receptors disclosed in this invention include vectors already used in human gene therapy. In one non-limiting embodiment, the vector is a retroviral vector. Expression in T cells or other immune effector cells (including engineered T cells) using retroviral vectors has been described (see, Schollerret et al., Sci. Transl. Med. 4: 132-153 (2012); Parente-Pereira et al., J. Biol. Methods 1(2): e7(1-9) (2014); Lamers et al., Blood 117(1): 72-82 (2011); Reviere et al., Proc. Natl. Acad. Sci. USA 92: 6733-6737 (1995)). In one embodiment, the vector is an SGF retroviral vector, such as an SGFγ-retroviral vector, which is a retroviral vector based on Moloney murine leukemia. SGF vectors have been described previously (see, for example, Wang et al., Gene Therapy 15:1454-1459 (2008)).

The vectors used in this invention employ suitable promoters for expression in specific host cells. The promoters can be inducible or constitutive promoters. In one specific embodiment, the promoter of the expression vector provides expression in stem cells (e.g., hematopoietic stem cells). In another specific embodiment, the promoter of the expression vector provides expression in immune effector cells (e.g., T cells). Non-viral vectors can also be used, provided the vector contains expression elements suitable for expression in target cells. Some vectors, such as retroviral vectors, can integrate into the host genome. If needed, techniques such as nucleases, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), regularly clustered short palindromic repeats (CRISPRs), homologous recombination, non-homologous end joining, micro-homologous end joining, and homologous end joining can be used to achieve site-directed integration (Gersbach et al., Nucl. Acids Res. 39: 7868-7878 (2011); Vasileva et al. Cell Death Dis. 6: e1831. (Jul 23 2015); Sontheimer, Hum. Gene Ther. 26(7): 413-424 (2015); Yao et al. Cell Research volume 27, 801-814 (2017)).

Vectors and constructs are optionally designed to contain a reporter protein. For example, a vector may be designed to express a reporter protein that can be used to recognize cells containing the vector or a polynucleotide provided on the vector (e.g., a polynucleotide integrated into a host chromosome). In one embodiment, the reporter protein may be expressed with a fusion protein or a synthetic receptor as a bicistronic or polycistronic expression construct. Exemplary reporter proteins include, but are not limited to, fluorescent proteins such as mCherry, green fluorescent protein (GFP), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, and mKalamal), cyan fluorescent proteins (e.g., ECFP, Cerulean, and CyPet), and yellow fluorescent proteins (e.g., YFP, Citrine, Venus, and YPet).

Conventional molecular biology techniques can be used to determine the transduction efficiency of the fusion protein or synthetic receptor disclosed in this invention. If a marker (e.g., a fluorescent protein) is included in the construct, gene transfer efficiency can be monitored by FACS analysis to quantify the proportion of transduced (e.g., GFP ⁺ ) immune effector cells (such as T cells), and/or by quantitative PCR. Using well-established co-culture systems (Gade et al., Cancer Res. 65:9080-9088 (2005); Gong et al., Neoplasia 1:123-127 (1999); Latouche et al., Nat. Biotechnol. 18:405-409 (2000)), it is possible to determine whether cancer antigen-expressing fibroblasts (AAPCs) (compared to controls) induce the release of cytokines from transduced immune effector cells (e.g., CAR-expressing T cells (IL-2, IL-4, IL-10, IFN-γ, TNF-α, and GM-CSF cell supernatant LUMINEX (Austin TX) assay), T cell proliferation (by carboxyfluorescein succinimide (CFSE) labeling), and T cell survival (by Annexin V staining)). The effects of CD80 and/or 4-1BBL on T cell survival, proliferation, and efficacy can be evaluated. T cells can be exposed to repeated stimulation by cancer antigen-positive target cells, and it can be determined whether T cell proliferation and cytokine responses remain similar or diminish with repeated stimulation. Cancer antigen CAR constructs can be compared side-by-side under equivalent experimental conditions. Cytotoxicity assays with various E:T ratios can be performed using chromium release assays. In addition to providing nucleic acids encoding the fusion proteins and/or synthetic receptors disclosed in this invention in vectors for expression in immune cells, nucleic acids encoding fusion proteins and/or synthetic receptors can also be provided in other types of vectors more suitable for genetic manipulation, such as various constructs for expression in bacterial cells (e.g., *E. coli*). Such vectors can be any well-known expression vector, including commercially available expression vectors (see Sambrook et al., *Molecular Cloning: A Laboratory Manual*, Third Ed., Cold Spring Harbor Laboratory, New York (2001); and Ausubel et al., *Current Protocols in Molecular Biology*, John Wiley and Sons, Baltimore, MD (1999)).

This invention also provides a method for genetically engineering cells for allogeneic transplantation, comprising inactivating genes. In some embodiments, the method of this invention comprises inactivating at least one gene in immune cells encoding class I MHC molecules or MHC-like molecules. In some embodiments, class I MHC molecules are selected from the group consisting of HLA-A heavy chain, HLA-B heavy chain, HLA-C heavy chain, HLA-E heavy chain, HLA-F heavy chain, and HLA-G heavy chain. In some embodiments, the method of this invention comprises inactivating a gene encoding β2M. In some embodiments, the method of this invention comprises inactivating a gene encoding an MHC-like molecule selected from the group consisting of CD1 heavy chain, MR1 heavy chain, FcRN heavy chain, and UL18.

Gene inactivation can be performed using any method disclosed in this invention or known in the art. In some embodiments, gene inactivation is performed via DNA splicing and repair, base editing, RNA interference, or RNA editing. In some embodiments, gene inactivation is performed via DNA splicing using a rare endonuclease selected from RNA-directed endonucleases, TAL nucleases, homing endonucleases, zinc finger nucleases, and Mega-TAL nucleases. In some embodiments, gene inactivation is performed via DNA splicing and repair. In some embodiments, DNA splicing and repair is performed using a CRISPR-Cas system. The CRISPR-Cas system is the CRISPR-Cas9 system.

Base editing

In some embodiments, the method provided by the present invention includes inactivating a gene through base editing. Precise site-specific changes and transformations of adenine (A), guanine (G), cytosine (C), thymine (T), or uracil (U) at certain specific sites in a nucleic acid (DNA or RNA) result in a change of one base to another at that specific site.

Guided Editor

In some embodiments, the method provided by this invention includes gene inactivation via guided editing. Guided editing is a "find and replace" genome editing technique that mediates site-specific insertions, deletions, and all possible base conversions. Furthermore, it can combine different types of editing. For example, the guided editing process may include the following steps: First, a modified pegRNA (prime editing guide RNA) binds to a guide editor protein, which both specifies the target site and contains the desired edit. This guide editor protein consists of a Cas9 nickase fused to a reverse transcriptase. The Cas9 nickase portion of the protein is guided by the pegRNA to the DNA target site. After Cas9 cleavage, the reverse transcriptase domain uses the pegRNA to reverse transcribe the template to be edited, directly polymerizing DNA onto the nicked target DNA strand. The edited DNA strand replaces the original DNA strand, creating a heteroduplex containing one edited strand and one unedited strand. Finally, the editor guides the disassembly of the heterogeneous double strand to facilitate the copying of the previous edits onto the unedited strand, thus completing the process (Anzalone et al. Nature (2019) doi:10.1038/s41586-019-1711-4).

RNA interference

In some embodiments, the method provided by the present invention includes inactivating a gene through RNA interference. RNA interference (RNAi) can be used to reduce the expression of a target gene or to silence a target gene. In one embodiment, for example, by expressing a double-stranded RNA or antisense RNA homologous to the target gene mRNA, the degradation (or interference with its stability) of the target gene mRNA or the inhibition of the translation efficiency of the target gene mRNA can be specifically induced.

DNA cutting and repair

In some embodiments, the method provided by this invention includes inactivating a gene through DNA cleavage and repair. DNA cleavage refers to the breaking of one or both strands of DNA at a target sequence site in double-stranded DNA. DNA cleavage can be performed using, for example, ribonucleases, FokI endonucleases, Cas proteins, etc. After DNA is cleaved, the cell repairs the cleavage site, for example, through non-homologous end joining (NHEJ) or homologous recombination (HR). The sequence near the cleavage site can be altered, and errors such as insertion and deletion mutations may occur. If the cleavage site is in an exon or regulatory region of a gene, the expression of the gene may be reduced or eliminated.

Zinc finger nucleases (ZFNs)

In some embodiments, the method provided by this invention includes gene inactivation using zinc finger nucleases (ZFNs). Zinc finger nucleases consist of a DNA recognition domain and a nonspecific endonuclease. The DNA recognition domain consists of a series of tandemly linked Cys2-His2 zinc finger proteins, and each zinc finger unit contains approximately 30 amino acids for specific DNA binding. The nonspecific endonuclease is a FokI endonuclease, which forms a dimer to cleave DNA.

TAL nuclease (TALEN)

In some embodiments, the method provided by this invention includes inactivating a gene by using TALEN. TALEN is a transcription activator-like effector nuclease. TALEN proteins are core components of DNA-binding domains and are typically composed of multiple tandem basic repeat units. These designed and assembled series of units can specifically recognize DNA sequences and cleave specific DNA sequences by coupling with the FokI endonuclease.

CRISPR/Cas system

In some embodiments, the method provided by the present invention includes inactivating a gene by using a CRISPR/Cas system. The CRISPR/Cas system is a nuclease system consisting of regularly clustered interspaced short palindromic repeats (CRISPR) and a CRISPR-binding protein (i.e., Cas protein), capable of cleaving almost any genomic sequence in eukaryotic cells adjacent to a prototypical spacer motif (PAM) (Cong et al. Science 2013.339:819-823). The term "CRISPR/Cas system" is used collectively to refer to transcripts involving CRISPR-associated ("Cas") genes, as well as other elements involved in their expression or directing their activity, including sequences encoding Cas genes, tracr (trans-activated CRISPR) sequences (e.g., tracrRNA or active tracrRNA), tracr pairing sequences (in the context of an endogenous CRISPR system, covering "direct repeats" and processed partial direct repeats), guide sequences, or other sequences from CRISPR sites and transcripts. Generally, a CRISPR system is characterized by elements that promote the formation of the CRISPR complex at the site of the target sequence (also known as the prespacer in endogenous CRISPR systems).

The formation of a CRISPR complex (including a guide sequence that hybridizes to the target sequence and is complexed with one or more Cas proteins) results in the cleavage of one or both strands of the target sequence or close to the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs). A tracr sequence (which may include all or part of the wild-type tracr sequence, or consist of all or part of the wild-type tracr sequence (e.g., about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85 or more nucleotides of the wild-type tracr sequence) may also form part of the CRISPR complex, for example, by hybridization along at least a portion of the tracr sequence to all or part of the tracr pairing sequence, wherein the tracr pairing sequence is operatively linked to the guide sequence. In some embodiments, the tracr sequence and the tracr pairing sequence are sufficiently complementary to hybridize and participate in the formation of the CRISPR complex. In some embodiments, when optimal alignment is performed, the tracr sequence has at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence complementarity along the length of the tracr pairing sequence. In some embodiments, one or more vectors driving the expression of one or more elements of the CRISPR system are introduced into a host cell such that the expression of elements of the CRISPR system guides the formation of the CRISPR complex at one or more target sites.

Generally, the tracr pairing sequence and the tracr sequence have sufficient complementarity to promote the formation of the CRISPR complex at the target sequence, wherein the CRISPR complex includes the tracr pairing sequence that hybridizes with the tracr sequence. Typically, complementarity refers to the optimal alignment of the tracr pairing sequence and the tracr sequence along the shorter of the two sequences. Optimal alignment can be determined by any suitable alignment algorithm, and the effects of secondary structures, such as self-complementarity within the tracr sequence or the tracr pairing sequence, can be further considered. At the time of optimal alignment, the complementarity of the tracr sequence and the tracr pairing sequence along the shorter of the two sequences is approximately or greater than approximately 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher. The tracr sequence has about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50 or more nucleotides in the sequence.

Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csxl2), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csxl5, Csf1, Csf2, Csf3, Csf4 homologs or modified forms thereof. In some embodiments, the Cas protein is the Cas9 protein.

Cas9, also known as Csn1, is a giant protein involved in crRNA biosynthesis and the disruption of invading DNA. Cas9 has been described in various bacterial species, such as *Streptococcus thermophilus*, *Listeria innocua* (Gasiunas, Barrangou et al. 2012; Jinek, Chylinski et al. 2012), and *S. pyogenes* (Deltcheva, Chylinski et al. 2011). The giant CaS9 protein (over 1200 amino acids) contains two predicted nuclease domains: an HNH (McrA-like) nuclease domain located in the middle of the protein and a split RuvC-like nuclease domain (RNAase H-sheet) (Makarova, Grishin et al. (2006)). Cas9 variants can be non-naturally occurring Cas9 endonucleases obtained through protein engineering or random mutation. For example, the Cas9 variant according to the invention can be obtained by mutations such as deletion, insertion, or substitution of at least one residue in the amino acid sequence of the Streptococcus pyogenes Cas9 endonuclease (COG3513). In some embodiments, the Cas9 protein is Streptococcus pneumoniae, Streptococcus pyogenes, or Streptococcus thermophilus Cas9, and may contain mutant Cas9 derived from these organisms, or variants that link other amino acid sequences to Cas9, for example, variants that link FokI enzyme to Cas9. These Cas9s are known; for example, the amino acid sequence of the Streptococcus pyogenes Cas9 protein can be found in the Swissprot database, accession number Q99ZW2; the amino acid sequence of the Neisseria meningitidis Cas9 protein can be found in the Uniprot database, accession number A1IQ68; the amino acid sequence of the S. thermophilus Cas9 protein can be found in the Uniprot database, accession number Q03LF7; and the amino acid sequence of the Staphylococcus aureus Casg protein can be found in the Uniprot database, accession number J7RUA5.

It is readily conceivable that combinations and arrangements of various methods described in this invention or other methods known in the art can be used to prepare the genetically engineered cells disclosed in this invention.

6.8 Treatment methods

The genetically engineered cells provided by this invention can be used for allogeneic transplantation and are unlikely to be rejected by the recipient's immune system, at least because the fusion protein expressed on the cells can form an MHC complex that binds to inhibitory receptors on the recipient's immune cells to prevent their activation for allogeneic transplantation.

In some embodiments, the present invention provides the use of genetically engineered cells in allogeneic transplantation, wherein the cells express a fusion protein comprising a presenting peptide and a β2M peptide covalently linked by a linker. The present invention also provides a method of providing allogeneic cell transplantation to a subject in need, comprising administering a therapeutically effective amount of genetically engineered cells to the subject, wherein the cells express a fusion protein comprising a presenting peptide and a β2M peptide covalently linked by a linker. The genetically engineered cells may be any genetically engineered cells disclosed in Part 6.5 of the present invention. The fusion protein may be any fusion protein disclosed in Part 6.2 of the present invention.

In some embodiments, the present invention provides the use of genetically engineered cells in allogeneic transplantation, wherein the cells comprise the nucleic acids disclosed in the present invention. The present invention also provides a method for providing allogeneic cell transplantation in a subject in need, the method comprising administering a therapeutically effective amount of genetically engineered cells to the subject, wherein the cells comprise the nucleic acids disclosed in the present invention.

In some implementations, the subjects receiving treatment are human. In other implementations, the subjects are individuals who require allogeneic transplantation.

Fusion proteins, nucleic acids, cells, and methods for their production have been disclosed in detail in the foregoing sections. For illustrative purposes, the present invention provides the use of genetically engineered cells in allogeneic transplantation, wherein the cells comprise nucleic acids encoding a fusion protein comprising an HLA-E-restricted presenting peptide covalently linked to human β2M via a (G4S)3 linker. The fusion protein can bind to endogenously expressed HLA-E heavy chains to form an HLA-E complex on the surface of genetically engineered cells, which can bind to inhibitory receptors (e.g., NKG2A) on immune cells (e.g., NK cells) to inhibit their potential cytotoxic activity against the genetically engineered cells.

Therefore, the present invention provides a method for administering a therapeutically effective amount of the genetically engineered cells disclosed herein to a subject in need of such cells as an allogeneic graft. The present invention also provides the use of the genetically engineered cells disclosed herein for the preparation of allogeneic grafts. Allogeneic grafts comprising the cells provided by the present invention are less likely to be rejected by the recipient, at least because the fusion protein expressed in the cells provides a mechanism to suppress a potential allogeneic immune response. In some embodiments, the present invention provides a method for providing an allogeneic graft to a subject in need by administering a therapeutically effective amount of the cells provided by the present invention to the subject. In some embodiments, the present invention provides a method for reducing the likelihood that the cells administered to the subject will trigger an allogeneic immune response in the subject. In some embodiments, the present invention provides a method for reducing the allogeneic response in the subject. In some embodiments, the method provided by the present invention reduces the occurrence of allogeneic transplant rejection.

The method of administering cells disclosed in this invention can be applied to any situation where such cells are desired. In some embodiments, the subject requiring cell administration suffers from a condition. For example, the subject may suffer from a condition characterized by a reduction in the function or number of specific cells, and therefore it is desirable to administer functional cells obtained from a healthy or normal individual (where the specific cells function normally) and to administer a sufficient number of healthy cells to the subject to restore the function provided by these cells (e.g., hormone-producing cells with reduced cell number or function, immune cells with reduced cell number or function, etc.). In such cases, the healthy cells can be modified as disclosed in this invention to reduce the likelihood of host rejection of the healthy cells. In some embodiments, the condition is a genetic condition. In some embodiments, the condition is an infectious condition. In some embodiments, the condition is HIV infection or AIDS. In some embodiments, the condition is cancer.

The methods disclosed in this invention are particularly helpful in regenerative medicine. For example, patients requiring stem cell transplantation can benefit from the genetically engineered cells disclosed in this invention. In some embodiments, this invention provides a method for providing allogeneic stem cells to a subject in need by administering a therapeutically effective amount of the stem cells provided by this invention to the subject. This invention also provides the use of the genetically engineered stem cells disclosed in this invention for the preparation of stem cell transplants. In some embodiments, the genetically engineered stem cells are differentiated into a desired cell type prior to administration to a subject in need. For illustrative purposes, individuals who may benefit from these cell therapies include patients suffering from spinal cord injury, type 1 diabetes, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Alzheimer's disease, heart disease, stroke, burns, cancer, and osteoarthritis. In some embodiments, this invention provides a method for treating a subject who may benefit from allogeneic cell therapy by administering a therapeutically effective amount of the cells provided by this invention to the subject. In some embodiments, the subject may suffer from spinal cord injury, type 1 diabetes, Parkinson's disease, ALS, Alzheimer's disease, heart disease, stroke, burns, cancer, or osteoarthritis.

This invention provides a method for using cells (e.g., T cells) expressing the fusion protein described herein in allogeneic transplantation. In some embodiments, the genetically engineered cells can be expanded prior to administration to a recipient. The cells can be administered as a population of cells expressing the fusion protein described herein. Optionally, the cells to be administered can be purified or enriched. In some embodiments, the recipient requires organ transplantation, and allogeneic organ grafts can be developed using the genetically engineered stem cells disclosed herein. Therefore, this invention also provides a method for providing allogeneic organ grafts by culturing the genetically engineered stem cells disclosed herein under appropriate conditions that allow for organ development.

In some embodiments, the present invention provides a method for providing hematopoietic stem cells and/or progenitor cells to a subject in need by administering a therapeutically effective amount of the genetically engineered cells provided by the present invention, wherein said cells are hematopoietic stem cells and/or progenitor cells. The subject may require a bone marrow graft, and the present invention also provides the use of the genetically engineered cells disclosed herein for the preparation of bone marrow grafts. The genetically engineered hematopoietic stem cells and/or progenitor cells may be CD34+ mobilized peripheral blood cells, CD34+ umbilical cord blood cells, or CD34+ bone marrow cells.

In some embodiments, the present invention provides a method for treating cancer by administering a therapeutically effective amount of the cells provided by the present invention to a subject in need. The present invention also provides the use of the genetically engineered cells disclosed herein in cancer treatment. The present invention further provides the use of the genetically engineered cells disclosed herein in the preparation of medicaments for treating cancer. The cells may be immune cells. Cells may be leukocytes, including, for example, myeloid cells, lymphoid cells, granulocytes (such as neutrophils, eosinophils, basophils), lymphocytes (such as T cells, B cells, NK cells, NKT cells), plasma cells, dendritic cells, monocytes, or cells differentiated therefrom (e.g., macrophages). In some embodiments, the present invention provides a method for treating cancer by administering a therapeutically effective amount of the genetically engineered immune cells provided by the present invention to a subject in need. In some embodiments, the present invention provides a method for treating cancer by administering a therapeutically effective amount of the genetically engineered T cells provided by the present invention to a subject in need. In some embodiments, the present invention provides a method for treating cancer by administering a therapeutically effective amount of the genetically engineered NK cells provided by the present invention to a subject in need. In some embodiments, the present invention provides a method for treating cancer by administering a therapeutically effective amount of the genetically engineered macrophages provided by the present invention to a subject in need.

In some embodiments, the present invention also provides the use of the genetically engineered immune cells disclosed herein in the preparation of medicaments for cancer treatment. In another embodiment, the method may comprise administering cancer antigen-specific immune cells to a subject in need, wherein the cells recombinantly express the fusion protein disclosed herein and a synthetic receptor comprising an antigen-binding domain specifically binding to the cancer antigen. The cancer antigen may be any cancer antigen disclosed herein or known in the art. In some embodiments, the cancer antigen is selected from the group consisting of CD19, CD20, CD22, CD30, CD123, CD138, CD33, CD70, BCMA, CS1, C-Met, IL13Ra2, EGFRvIII, CEA, Her2, GD2, MAGE, GPC3, mesothelin, PSMA, ROR1, EGFR, MUC1, and NY-ESO-1. In some embodiments, the synthetic receptor is CAR, TCR, TruC, TAC, AbTCR, or a chimeric CD3 receptor.

In some embodiments, the present invention also provides the use of the genetically engineered T cells disclosed herein in the preparation of medicaments for cancer treatment, wherein the genetically engineered T cells express synthetic receptors. In some embodiments, the genetically engineered T cells express CARs. For example, the present invention provides universal CAR-Ts that can be used for cancer treatment. In some embodiments, the genetically engineered T cells express TCRs. In some embodiments, the genetically engineered T cells express TruCs. In some embodiments, the genetically engineered T cells express TACs. In some embodiments, the genetically engineered T cells express AbTCRs. In some embodiments, the genetically engineered T cells express chimeric CD3 receptors.

In some embodiments, the immune cells provided by the present invention can be used for allogeneic transplantation. Immune cells can be leukocytes, including, for example, myeloid cells, lymphoid cells, granulocytes (such as neutrophils, eosinophils, and basophils), lymphocytes (such as T cells, B cells, NK cells, and NKT cells), plasma cells, dendritic cells, monocytes, or cells differentiated therefrom (e.g., macrophages). In some embodiments, the present invention provides a method for treating a subject's cancer by administering a therapeutically effective amount of the T cells provided by the present invention to a subject in need. In some embodiments, the present invention provides a method for treating a subject's cancer by administering a therapeutically effective amount of the CAR-T cells provided by the present invention to a subject in need. The present invention also provides the use of the genetically engineered T cells disclosed herein in cancer treatment. The present invention further provides the use of the genetically engineered T cells disclosed herein in the preparation of medicaments for cancer treatment.

For example, the method can be used to treat cancer or reduce tumor burden in a subject. In one embodiment, the method provided by the present invention is used to treat cancer. It should be understood that the method of treating cancer can include any effect that improves cancer-related signs or symptoms. Such signs or symptoms include, but are not limited to, reducing tumor burden, including inhibiting tumor growth, slowing tumor growth rate, reducing tumor size, reducing tumor number, eliminating tumor, all of which can be measured using conventional tumor imaging techniques well known in the art. Other cancer-related signs or symptoms include, but are not limited to, fatigue, pain, weight loss, and other signs or symptoms associated with various cancers. In a non-limiting example, the method of the present invention can reduce tumor burden. Therefore, administration of the cells of the present invention can reduce the number of tumor cells in a subject, reduce tumor size, and/or eradicate tumors.

In the method provided by this invention, immune cells are administered to a subject requiring cancer treatment. The subject may be a mammal, specifically a human. Preferably, the subject is a human. Suitable subjects for treatment include those with clinically measurable tumors (including “advanced disease” or “high tumor burden”). Clinically measurable tumors are tumors that can be detected based on a tumor mass, for example, by palpation, CAT scan, ultrasound, breast imaging, X-ray, etc. Positive biochemical or histopathological markers may also be used to identify these populations. A pharmaceutical composition comprising the cells provided by this invention is administered to the subject to elicit an anticancer response to alleviate the subject's symptoms. Reduction of the tumor mass in subjects with tumors may occur, but any clinical improvement constitutes a benefit. Clinical improvement includes a reduction in the risk or rate of tumor progression or a reduction in pathological consequences.

Another suitable group of subjects may be those with a history of cancer but who have responded to an alternative treatment modality. Previous treatment may include, but is not limited to, surgical resection, radiation therapy, and conventional chemotherapy. Therefore, these individuals do not have clinically measurable tumors. However, they are suspected of having a risk of disease progression, either near the original tumor site or through metastasis. This group can be further subdivided into high-risk and low-risk individuals. The subdivision is based on characteristics observed before or after the initial treatment. These characteristics are known in the clinical field and are appropriately defined for different types of cancer. A typical characteristic of the high-risk subgroup is tumor invasion of adjacent tissues or the presence of lymph node metastasis. Optionally, the cells provided by this invention may be administered for prophylactic treatment to prevent cancer in subjects suspected of having a predisposition to cancer, for example, based on family history and/or genetic testing.

Subjects may have advanced disease, in which case treatment goals may include mitigating or reversing disease progression and/or reducing side effects. Subjects may have a history of previously treated disease, in which case treatment goals may include reducing or delaying the risk of recurrence. Furthermore, the cell therapy provided by this invention can be used to treat refractory or recurrent malignancies.

The cells provided by this invention are administered to subjects requiring cancer treatment, such as human subjects. The cancer may involve a solid tumor or a hematologic malignancy that does not involve a solid tumor. Cancers treated using the cells of this invention include cancers that typically respond to immunotherapy. Exemplary types of cancer include, but are not limited to, carcinoma, sarcoma, leukemia, lymphoma, multiple myeloma, melanoma, brain and spinal cord tumors, germ cell tumors, neuroendocrine tumors, carcinoid tumors, etc. The cancer may be a solid tumor or a hematologic malignancy that does not form a solid tumor. In the case of a solid tumor, the tumor may be a primary tumor or a metastatic tumor.

Examples of other tumors or cancers that can be treated using the methods provided in this invention include bone cancer, colorectal cancer, liver cancer, skin cancer, head and neck cancer, melanoma (malignant melanoma of the skin or eye), kidney cancer (e.g., clear cell carcinoma), pharyngeal cancer, prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), blood cancers (e.g., leukemia, lymphoma, and myeloma), uterine cancer, rectal cancer, anal cancer, bladder cancer, brain cancer, stomach cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, and leukemia (e.g., acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, acute...). Myeloid leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease, non-Hodgkin's disease, Waldenstrom's macroglobulinemia), small intestine cancer, endocrine system cancers, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumors, lymphocytic lymphoma. Lymphoma, renal cell carcinoma or ureteral cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal cord axis tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers (including asbestos-induced cancers), heavy chain diseases, and solid tumors (e.g., sarcomas and carcinomas), such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovial sarcoma, mesothelioma, Ewing's sarcoma ( Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

In one embodiment, the method provided by the present invention is used to treat cancers selected from the following: malignant pleural diseases, mesothelioma, lung cancer (e.g., non-small cell lung cancer), pancreatic cancer, ovarian cancer, breast cancer (e.g., metastatic breast cancer, metastatic triple-negative breast cancer), colon cancer, pleural tumors, glioblastoma, esophageal cancer, gastric cancer, and synovial sarcoma. The present invention provides a therapy particularly suitable for treating solid tumors, such as malignant pleural diseases, mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumors, glioblastoma, esophageal cancer, gastric cancer, and synovial sarcoma. Solid tumors can be primary tumors or metastatic tumors.

For therapeutic purposes, the amount administered is the effective amount that produces the desired effect. An effective amount, or therapeutically effective amount, is an amount sufficient to provide a beneficial or desired clinical outcome after treatment. An effective amount can be provided in a single administration or in a series of administrations (one or more doses). An effective amount can be provided by rapid infusion (bolus) or by continuous perfusion. For therapeutic purposes, an effective amount is an amount sufficient to alleviate, improve, stabilize, reverse, or slow disease progression, or otherwise reduce the pathological consequences of the disease. An effective amount can be determined by a physician for a specific subject. Several factors are typically considered when determining the appropriate dose to achieve an effective amount. These factors include the subject's age, sex, and weight; the condition being treated; the severity of the condition; and the form and effective concentration of the cells of the present invention being administered.

The cells provided by this invention can typically be administered at a dose of cells per kilogram (cell/kg) based on the body weight of the subject to which the cells are administered. Typically, the cell dose is between about ^10⁴ and ^10¹⁰ cells/kg body weight, for example, about ^10⁵ to about ^10⁹ , about ^10⁵ to about ^10⁸ , about ^10⁵ to about ^10⁷ , or about ^10⁵ to ^10⁶ , depending on the route and site of administration. Generally, in cases of systemic administration, a higher dose is used than for regional administration, where the immune cells of this invention are administered to the tumor region. Exemplary dose ranges include, but are not limited to, 1× ^10⁴ to 1× ^10⁸ , 2× ^10⁴ to 1× ^10⁸ , 3× ^10⁴ to 1× ^10⁸ , 4× ^10⁴ to 1× ^10⁸ , 5× ^10⁴ to 1× ^10⁸ , 6× ^10⁴ to 1× ^10⁸ , 7× ^10⁴ to 1× ^10⁸ , 8× ^10⁴ to 1× ^10⁸ , 9× ^10⁴ to 1× ^10⁸ , 1× ^10⁵ to 1× ^10⁸ , for example, 1× ^10⁵ to 9× ^10⁷ , 1× ^10⁵ to 8× ^10⁷ , 1× ^10⁵ to 7× ^10⁷ , 1× ^10⁵ to 6× ^10⁷ , 1× ^10⁵ to 5× ^10⁷ , 1× ^10⁵ to 4×10⁸. ^7. 1× ^10⁵ to 3×10⁵ ^{; 7.} 1× ^10⁵ to 2×10⁵ ^{; 7.} 1×10⁵ to 1×10⁵ ^{; 7. 1} × ^10⁵ to 9×10⁶ ^{; 6.} 1× ^10⁵ to 8×10⁶ ^{; 6.} 1× ^10⁵ to 7×10⁶ ^; 6. 1× ^10⁵ to 6×10⁶ ^{; 6.} 1× ^10⁵ to 5×10⁶ ^{; 6.} 1× ^10⁵ to 4×10⁶ ^{; 6.} 1× ^10⁵ to 3×10⁶; ^6. 1× ^10⁵ to 2×10⁶ ^{; 6.} 1× ^10⁵ to 1×10⁶ ^{; 6.} 2× ^10⁵ to 9×10⁷ ^{; 7.} 2× ^10⁵ to 8× ^10⁷ ; 7. 2× ^10⁵ to 7×10⁷; ^7. 2× ^10⁵ to 6× ^10⁷ Dosage ranges include 2× ^10⁵ to 5× ^10⁷ , 2× ^10⁵ to 4× ^10⁷ , 2× ^10⁵ to 3× ^10⁷ , 2× ^10⁵ to 2× ^10⁷ , 2× ^10⁵ to 1× ^10⁷ , 2× ^10⁵ to 9× ^10⁶ , 2×10⁵ to 8× ^10⁶ , 2× ^10⁵ to 7× ^10⁶ , 2× ^10⁵ to 6×10⁶, 2× ^10⁵ to 5× ^10⁶ , 2× ^10⁵ to 4× ^10⁶ , and 3× ^10⁵ to 3× ^10⁶ cells ^/ kg. These dosage ranges are particularly useful for ^regional administration. In certain implementations, the drug is administered at a dose of 1× ^10⁵ to 1× ^10⁸ , such as 1× ^10⁵ to 1× ^10⁷ , 1× ^10⁵ to 1× ^10⁶ , 1× ^10⁶ to 1× ^10⁸ , 1× ^10⁶ to 1× ^10⁷ , 1× ^10⁷ to 1× ^10⁸ , or 1× ^10⁵ to 5× ^10⁶ . Specifically, it is administered at a dose of 1× ^10⁵ to 3× ^10⁶ or 3× ^10⁵ to 3× ^10⁶ cells/kg for regional administration, such as intrapleural administration. Exemplary dosage ranges may also include, but are not limited to, 5 × ^10⁵ to 1 × ^10⁸ , such as 6 × ^10⁵ to 1 × ^10⁸ , 7 × ^10⁵ to 1 × ^10⁸ , 8 × ^10⁵ to 1 × ^10⁸ , 9 × ^10⁵ to 1 × ^10⁸ , 1 × ^10⁶ to 1 × ^10⁸ , 1 × ^10⁶ to 9 × ^10⁷ , 1 × ^10⁶ to 8 × ^10⁷ , 1 × ^10⁶ to 7 × ^10⁷ , 1 × ^10⁶ to 6 × ^10⁷ , 1 × ^10⁶ to 5 × ^10⁷ , 1 × ^10⁶ to 4 × ^10⁷ , 1 × ^10⁶ to 3 × ^10⁷ cells/kg, etc. Such dosages are particularly useful for systemic administration. In a particular implementation, cells are provided at a dose of 1× ^10⁶ to 3× ^10⁷ cells/kg for systemic administration. Exemplary cell doses include, but are not limited to, 1× ^10⁴ , 2× ^10⁴ , 3× ^10⁴ , 4× ^10⁴ , 5× ^10⁴ , 6× ^10⁴ , 7×10⁴, 8× ^10⁴ , 9× ^10⁴ , 1× ^10⁵ , 2× ^10⁵ , 3× ^10⁵ , 4× ^10⁵ , 5× ^10⁵ , ⁶ × ^10⁵ , 7× ^10⁵ , 8× ^10⁵ , 9× ^10⁵ , 1× ^10⁶ , 2× ^10⁶ , 3× ^10⁶ , 4× ^10⁶ , 5× ^10⁶ , 6× ^10⁶ , 7× ^10⁶ , 8× ^10⁶ , 9× ^10⁶ , 1× ^{10⁷, 2×10⁷ ,} and 3× ^10⁷. Doses of 4× ^10⁷ , 5× ^10⁷ , 6× ^10⁷ , 7×10⁷, 8× ^10⁷ , 9× ^10⁷ , 1× ^10⁸ , 2× ^10⁸ , 3× ^10⁸ , 4× ^10⁸ , 5× ^10⁸ , 6× ^10⁸ , 7× ^10⁸ , 8× ^10⁸ , 9× ^10⁸ , and 1× ^10⁹ cells/kg are available. Furthermore, the dosage can be adjusted to consider whether a ^single dose or ^multiple doses are used. As mentioned above, accurately determining the effective dose can be based on individual factors for each subject, including ^their body type, age, sex, weight, and the specific condition of the subject. Those skilled in the art can readily determine the dosage based on the disclosure of this invention and existing knowledge in the art.

The cells provided by this invention can be administered by any method known in the art, including but not limited to intrapleural administration, intravenous administration, subcutaneous administration, intratumoral administration, intrathecal administration, intrathecal administration, intrapleural administration, intraperitoneal administration, intracranial administration, and direct thymic administration. In one embodiment, the cells provided by this invention can be locally delivered to a tumor using well-known methods, including but not limited to hepatic or aortic pumps; perfusion of the extremities, lungs, or liver; in the portal vein; via venous shunt; in cavities or blood vessels near the tumor, etc. In another embodiment, the cells provided by this invention can be administered systemically. In a preferred embodiment, the cells are administered locally at the tumor site. The cells can also be administered intratumorally, for example, by direct injection of cells at the tumor site and/or into the tumor vascular system. For example, in the case of malignant pleural diseases, mesothelioma, or lung cancer, intrapleural administration is preferred (see, Adusumilli et al., Science Translational Medicine 6(261):261ra151(2014)). Those skilled in the art can select an appropriate route of administration based on the type and/or location of the tumor to be treated. The cells can be introduced via injection or catheter. In one embodiment, intrapleural administration is performed on the desired subject, for example, using an intrapleural catheter. Optionally, the subject may be given an amplification/differentiation agent before, during, or after cell administration to increase in vivo cell generation provided by the present invention.

The cell proliferation provided by this invention is typically performed in vitro prior to administration to a subject and may be desirable in vivo after administration to a subject (see Kaiser et al., Cancer Gene Therapy 22:72-78 (2015)). Cell proliferation should be accompanied by cell survival to allow for cell (e.g., T cells) expansion and persistence.

The methods provided by this invention can further include additional treatments used before, during, or after treatment with the cells provided by this invention. Therefore, the cell therapy methods provided by this invention can be used in conjunction with other standard cancer care and/or therapies that are compatible with the administration of the cells provided by this invention.

This invention uses specific language to describe numerous embodiments. The invention also specifically includes embodiments that completely or partially exclude particular subjects, such as substances or materials, method steps and conditions, schemes, procedures, measurements, or analyses. Therefore, even if the invention is not generally expressed as something not included in the invention, aspects not expressly included in the invention are still disclosed herein.

This invention describes specific embodiments of the invention, including the best mode known to the inventors for carrying out the invention. Variations of the disclosed embodiments will become apparent to those skilled in the art upon reading the foregoing description, and it is expected that those skilled in the art will appropriately employ such variations. Therefore, the invention is intended to be practiced in a manner different from that specifically described herein, and the invention includes all modifications and equivalents to the subject matter mentioned in the appended claims, where permitted by applicable law. Furthermore, unless otherwise stated or clearly contradicted by the context, the invention covers any combination of all possible variations of the foregoing elements.

All publications, patent applications, registry numbers, and other references cited in this specification are incorporated herein by reference in their entirety, to the extent that each individual publication or patent application is specifically and individually incorporated into the invention by reference. Publications provided for discussion of the invention are used only for their publication prior to the filing date of this application. References or identifications of any references in the description of some embodiments of the invention should not be construed as an admission that such reference is available as prior art. Furthermore, the publication dates provided may differ from the actual publication dates and require independent verification.

Several embodiments of the invention have been described. However, it should be understood that various modifications can be made without departing from the spirit and scope of the invention. Therefore, the experimental description is intended to illustrate, but not limit, the scope of the invention as described in the claims.

7. Example

7.1 Experiment Overview

Allogeneic transplant rejection is a major cause of failure in allogeneic organ or cell transplantation. NK cell-mediated allogeneic responses, including cell lysis of transplanted cells, are a significant factor in this rejection (Li, Transplantation. 2010; 90(10):1043-1047). Universal CAR-T therapy has the potential to overcome the drawbacks of autologous transplantation, but it also faces the challenge of allogeneic rejection. The studies described below illustrate the preparation of genetically engineered cells disclosed in this invention, which express fusion proteins including a presenting peptide covalently linked to human β2M. Some of the cells described in this invention are T cells co-expressing CARs that specifically recognize tumor antigens. As described in detail below, the cells provided by this invention exhibit better survival and enhanced cell proliferation in an allogeneic setting. Specifically, the cells provided by this invention exhibit resistance to NK cell-mediated cell lysis. In addition, CAR-T cells expressing the fusion proteins provided by this invention have also been shown to have significantly enhanced activity in inhibiting tumor cell growth and promoting tumor cell death in an allogeneic setting.

The abbreviations have the following meanings: "h" refers to hour, "min" refers to minute, "s" refers to second, "ms" refers to millisecond, "d" refers to day, "μL" refers to microliter, "mL" refers to milliliter, "L" refers to liter, "bp" refers to base pair, "mM" refers to millimoles per liter, and "μM" refers to micromoles per liter.

7.2 Materials and Methods

Typically, methods for preparing engineered cells include the following steps: (i) expressing the fusion protein disclosed in this invention on the cell surface; said fusion protein comprising: (a) a presenting peptide; (b) a β2M peptide; and (c) a linker sequence covalently linking (a) and (b) above. The method may optionally further include: (ii) reducing the expression of endogenous class I MHC molecules on the cell surface. In some embodiments, this involves mutating or knocking out a gene encoding class I MHC molecules. The order of steps (i) and (ii) above may be interchanged.

Lentiviral preparation : A three-plasmid system can be used, comprising: a lentiviral targeting expression plasmid (Addgene ID: #12252), in which the GFP expression cassette is replaced with various expression cassettes to express CAR and fusion proteins; and packaging helper plasmids psPAX2 (Addgene ID: #12260) and pMD2.G (Addgene ID: #12259). Viral packaging can be performed in HEK293T cells (Shanghai Institute of Cell Research, Chinese Academy of Sciences). The preparation procedure is as follows: thaw HEK293T cells from frozen working cells, culture them in 10 cm culture dishes in DMEM medium (+10% FBS + 1% P/S) (Cellgro10-013-CMR), and change the medium after 2 days of culture. Once fusion occurs, the cells can be passaged (e.g., re-seeding cells from one complete plate into 5 plates), and cells from the fourth passage can be transfected with the plasmid. PEI can be used as a transfection reagent (e.g., a PEI to plasmid mass ratio of 2:1). A mixture of plasmid and PEI is added to Opti-MEM medium (Gibco, cat#31985-070), and then the mixture is added to HEK293T cells at the 4th passage. Six hours after transfection, the original medium is replaced with fresh medium containing 2% FBS, and the cells are cultured continuously for 72 hours. The supernatant from the HEK293T cells is then collected. The collected viral supernatant is concentrated by ultracentrifugation (e.g., at 82200g at 2°C to 8°C for 2 hours), and then the concentrated virus is sterilized by filtration through a 0.22 μm filter membrane and resuspended for use.

Vector construction : The target gene (e.g., encoding a single CAR or encoding a CAR and the fusion protein disclosed in this invention) is cloned into a lentiviral vector. The lentiviral vector includes a long terminal repeat (LTR) 5' LTR and a truncated 3' LTR, RRE, rev response element (cPPT), central termination sequence (CTS), and WHV post-transcriptional regulatory element (WPRE). The fusion protein can be constitutively expressed by the EF-1a (elongation factor-1a) promoter and cloned into the lentiviral vector by BmHI and SalI restriction enzyme digestion.

Naïve T cell activation : Naïve T cells can be derived from peripheral blood (PBMCs) of healthy human volunteers. The culture medium used can be complete medium, namely ImmunoCult ^™ -XF T cell expansion medium (Stem Cell Technology, cat#10981) + 300 IU/ml IL2 (Cayan, cat#HEILP-0201c). T cells are activated using Dynabeads (Thermo, cat#11141D) at a Dynabeads to cell ratio of 3:1. T cells show significant aggregation and enlargement 24 hours after activation.

Lentiviral transduction of T cells : 24 to 72 hours after initial T cell activation, the cells aggregate and enlarge, entering the mitotic phase for lentiviral transduction. The ratio of viral transduction units to cell numbers can be 10:1, i.e., MOI = 10. Protein expression can be detected 48 hours after transduction.

Gene editing : Four days after T cell activation, or two days after T cell transduction with a virus, cells were collected and washed three times with electroporation buffer (Thermo, resuspension buffer T Cat#MPK 1096 from the kit), Opti-MEM (Gibco, cat#31985-070), etc. Cells were resuspended in electroporation buffer, and the cell density was adjusted to 1× ^10⁸ /ml. The required sgRNA (total 300 to 400 ng) and 1 μg Cas9 protein were homogenized in vitro and incubated at room temperature for 10 minutes, then the mixture was added to the resuspended cells. The total volume of cells used for electroporation could be 10 μL, and electroporation could be performed using a Neon electroporation system (Thermo, Cat#MPK1096). The pulse voltage, pulse duration, and pulse number could be 1200 to 1600 V, 10 ms, and 3 pulses. Feasible sgRNA sequences can be designed based on predictive analysis from relevant websites; the Cas9 protein can be derived from the Alt-Rs.p.Cas9 nuclease 3NLS protein (IDT, Cat#1074181) of IDT DNA Technology Company.

UCAR-T cells expressing fusion proteins : T cell activation, viral transduction, and gene editing methods are as described above. An exemplary procedure is as follows: initial PBMC activation is performed on day 0; viral transduction (for fusion protein expression) is performed on day 2; gene editing (TRAC and β2M knockout) is performed on day 4, and the expression of each protein is detected by flow cytometry on days 6 and 7.

Materials : Exemplary antibodies and cell dyes used: anti-CD3-APC (BD, 555335), anti-CD3-FITC (BD, 555916), anti-β2M-PE (BD, 551337), anti-β2M-FITC (BD, 551338), anti-HLA-ABC-APC (R&D, FAB7098A), anti-HLA-E-PE (Biolegend, 342604), anti-HLA-E-APC (Biolegend, 342606), anti-mouse FMC63 (Shanghai Xingwan, R19PB-100) and dyes eFluor ^™ 670 (eBioscience, 65-0840-90), anti-HLA-ABC PE-Cy7 (Invitrogen, 25-9983-42), and anti-αβ-TCR PE (Biolegend, 306708).

7.3 Preparation of Universal CAR-T Cells Expressing Fusion Proteins (“UCAR-T-CM”)

Construction of CAR19-P2A-CM, CAR19-P2A-CM-C, and CAR19 lentiviral targeted expression plasmids :

Lentiviral targeted expression plasmids with a single CAR19 (i.e., a CAR including an antigen-binding domain that specifically binds to the tumor antigen CD19) or lentiviral targeted expression plasmids containing both CAR19 and a fusion protein (or “chimeric molecule”, “CM”) were constructed. The fusion protein (CM) used herein has the following components from N-terminus to C-terminus: 20-a.a. signal peptide (SEQ ID NO:3), presenting peptide, linker sequence GGGGSGGGGSGGGGS (SEQ ID NO:1), and β2M mature form peptide (SEQ ID NO:81). CAR19 and CM are linked by a sequence encoding a P2A peptide (ATNFSLLKQAGDVEENPGP; SEQ ID NO:105), a self-cleaving peptide typically used to co-express two or more independent peptides from the same vector. After co-expression from the same plasmid, the self-cleaving of the P2A peptide allows the two linked peptides to separate and function independently. For CAR19-P2A-CM, the fusion protein (CM) shown in SEQ ID NO:5 includes the HLA-E-restricted presenting peptide (VMAPRTVLL; SEQ ID NO:38). The nucleic acid sequence encoding CAR19-P2A-CM is SEQ ID NO:12. CAR19-P2A-CM-c is identical to CAR19-P2A-CM in all respects except that the fusion protein (CM-c) includes the HLA-C-restricted presenting peptide IIDKSGSTV (SEQ ID NO:117). The nucleic acid sequence encoding CAR19-P2A-CM-c is SEQ ID NO:19.

The viral expression plasmid (Addgene ID: #12252) was digested with BamHI/Sall ahd and used as the backbone. The CAR19 sequence (SEQ ID NO: 20), CAR19-P2A-CM sequence (SEQ ID NO: 12), or CAR19-P2A-CM-c sequence (SEQ ID NO: 19) were cloned into the backbone to form CAR19-P2A-CM, CAR19-P2A-CM-c, and CAR19 expression plasmids, respectively. As shown in Figure 1, the plasmid containing the nucleotide sequence encoding CAR19-P2A-CM (SEQ ID NO: 12) can be operatively inserted into the digested viral expression backbone.

Lentiviral preparation : A three-plasmid system was used, consisting of the following: a lentiviral targeting expression plasmid constructed using the above method, and packaging helper plasmids psPAX2 (Addgene ID: #12260) and pMD2.G (Addgene ID: #12259). Viral packaging was performed in HEK293T cells (Shanghai Institute of Cell Research, Chinese Academy of Sciences). The preparation process was as follows: HEK293T cells from frozen working cells were thawed and cultured in 10 cm culture dishes in DMEM medium (+10% FBS + 1% P/S) (Cellgro 10-013-CMR), and the medium was changed after 2 days of culture. When confluence was reached, the cells were passaged (cells from one complete plate were seeded onto 5 new plates), and the cells from the 4th passage were transfected with the plasmid. Polyethyleneimine (PEI) was used as the transfection reagent, with a PEI to plasmid mass ratio of 2:1. The mixture of the three plasmids and PEI was then added to Opti MEM medium (Gibco, cat#31985-070), and the resulting solution was added to HEK293T cells at the fourth passage. Six hours after transfection, the original medium was replaced with fresh medium containing 2% FBS, and the cells were cultured continuously for 72 hours. The supernatant of the cell culture was then collected. The virus in the supernatant was collected by ultracentrifugation (at 82200g at 4°C to 8°C for 2 hours), and the concentrated virus was sterilized by filtration through a 0.22μm filter membrane and resuspended for use.

Nascent T cell activation : Nascent T cells were derived from peripheral blood (PBMCs) of healthy human volunteers. Complete culture medium, ImmunoCult ^™ -XF T cell expansion medium (Stem Cell Technology, cat#10981) + 300 IU/ml IL2 (Cayan, cat#HEILP-0201c), was used. T cells were activated using Dynabeads (Thermo, cat#11 141D) at a Dynabeads to cell ratio of 3:1. T cells showed significant aggregation and enlargement 24 hours after activation.

Lentiviral gene transduction: 3 × ^10⁵ naïve T cells activated for 48 hours, or unactivated Jurkat cells (Shanghai Institute of Cell Research, Chinese Academy of Sciences), were seeded in 24-well culture plates. The prepared lentivirus was added to these cells at a ratio of 10:10 (MOI = 10, the ratio of viral transduction units to cell numbers), and culture medium was added to bring the volume to 500 μL (the same medium used to activate the naïve T cells). After 24 hours, culture medium was added again to bring the volume to 1 mL to promote cell growth.

Candidate sgRNAs : sgRNAs for editing the B2M gene and the TRAC gene were designed and screened. sgRNA (SEQ ID NO:8; SEQ ID NO:9) was used to edit the B2M gene, and sgRNA (SEQ ID NO:10) was used to edit the TRAC gene.

Cas9 protein : Utilizes the Alt-R spCas9 nuclease 3NLS protein from IDT DNA Technologies.

Electroporation : Forty-eight hours after transducing naïve T cells or Jurkat cells with lentivirus, cells were collected and washed three times with electroporation buffer (Thermo, resuspension buffer T Cat#MPK 1096 from the kit) or Opti MEM (Gibco, cat#31985-070), and then resuspended in electroporation buffer. The cell density was adjusted to 1× ^10⁸ /ml. β2M sgRNA and TRAC sgRNA (150 ng each) and 1 μg Cas9 protein were homogenized in vitro and incubated at room temperature for 10 min. The mixture was then added to the resuspended cells for electroporation. The total volume used for electroporation was 10 μL, and the Neon electroporation system was used. The pulse voltage, pulse duration, and pulse number were as follows: 1200 V and 10 ms, 3 times.

Measurement of viral transduction efficiency and gene editing efficiency : The efficiency of genetic engineering was measured 3 days after electroporation. A small number of cells were washed once with 1 ml PBS (Gibco, cat#C10010500BT). The cells were resuspended in 100 μL PBS, and 3 μL of primary antibody against FMC63 (for CAR19 detection) was added. The mixture was incubated at 4°C for 30 minutes. Then, 1 ml PBS was added to the mixture, and after homogenization, it was centrifuged at 350 g for 3 minutes. The cells were collected and the supernatant was discarded. 0.5 μL of secondary antibody (primary and secondary antibodies were from an anti-mouse FMC63 kit, Shanghai Xingwan, R19PB-100) and 3 μL of anti-HLA-E-APC antibody were added to the cells sequentially, and after homogenization, the mixture was incubated at 4°C for 30 minutes. 1 ml PBS was added, and after homogenization, it was centrifuged at 350 g for 3 minutes. Cells were collected and the supernatant discarded. 200 μL of PBS was added to resuspend the cells, and they were analyzed by flow cytometry. As shown in Figure 2, all Jurkat T cells had TRAC and β2M knocked out using CRISPR/Cas9 gene editing as described above. When these cells were transduced with the CAR19-P2A-CM virus (Figure 2, Panel B), the fusion protein with the HLA-E-specific presenting polypeptide (CM) was efficiently expressed in cells that expressed CAR on the surface (cells in the upper right quadrant), because the fusion protein and CAR were operatively linked together. CAR-positive cells in Panel B also showed positive staining for HLA-E heavy chains, indicating that CM binds to HLA-E heavy chains to form a complex on the cell membrane, which is due to the lack of HLA-E heavy chains in CM. Meanwhile, in cells transduced with CAR19-P2A-CM-C virus and with TRAC and β2M knocked out, only CAR expression was detected on the cell surface, while HLA-E heavy chain expression was not detected. This indicates that the fusion protein expressing HLA-C restriction presenting peptide (CM-c) failed to form a complex with endogenous HLA-E heavy chain (Figure 2, area C).

To measure the gene editing efficiency in naïve T cells, a small number of cells were taken, washed once with 1 ml of PBS, and resuspended in 100 μL of PBS. 3 μL of anti-HLA-E-PE antibody, 3 μL of anti-HLA-ABC antibody, and 3 μL of anti-CD3-FITC antibody were added. As shown in Figure 3, section B, after lentiviral transduction and gene editing as described above, approximately 80% of the naïve T cells transduced with CAR19-P2A-CM virus and possessing TRAC and β2M double knockout (i.e., UCAR-T-CM cells) were double-negative for both CD3 and HLA-ABC. These double-negative cells also highly expressed a fusion protein containing the HLA-E-restricted presenting peptide (CM), as evidenced by the detection of CM-bound HLA-E at the cell membrane (Figure 3, section C). The detection of anti-HLA-E antibodies in CD3 and HLA-ABC double-negative cells revealed that 50% of HLA-E-positive cells were positive, indicating the expression of the fusion protein. Since these cells had endogenous β2M knocked out, the HLA-E heavy chain must bind to the recombinant CM presented at the cell surface and be detected by anti-HLA-E antibodies. Therefore, these results demonstrate that β2M gene editing effectively removed the presentation of the endogenous HLA-ABC complex on the cell membrane, and TRAC gene editing effectively removed the presentation of the TCR-CD3 complex on the cell membrane, resulting in efficient expression of the fusion protein and its formation of a stable complex with the HLA-E heavy chain on the cell surface.

7.4 UCAR-T cells expressing the fusion protein (CM) are resistant to allogeneic NK cells.

Flow cytometry was used to measure NK92 cell-mediated cell lysis (human NK cell line) .

ImmunoCult ^™ (STEMCELL) was used as the culture medium described above. NK92 cells (Shanghai Institute of Cell Research, Chinese Academy of Sciences) were used as effector cells. Three (3) types of target cells were used, all derived from naïve T cells derived from peripheral blood of the healthy human volunteers described above, including: 1. CAR-T cells, which were naïve T cells transduced with CAR19 virus; 2. UCAR-T cells, which were naïve T cells transduced with CAR19 virus and with the TRAC and B2M genes knocked out; and 3. UCAR-T-CM cells, which were naïve T cells transduced with CAR19-P2A-CM virus and with the TRAC and B2M genes knocked out. The final concentration of all target cells was 5 × ^10⁴ /mL.

In each group, NK92 cells (effect cells) and target cells were co-cultured at a 1:1 ratio (cell number ratio). Target cells were also cultured separately as a control in each group. Cells were seeded in 96-well plates, supplemented with 100 μL of culture medium, and co-cultured at 37°C for 24 hours. Prior to co-culture, NK92 cells were stained with eFluor ^™ 670 to distinguish them from target cells. After 48 hours of co-culture, the number of target cells in the CAR-T cell group, UCAR-T cell group, or UCAR-T-CM cell group was measured by flow cytometry to detect changes in CAR-positive cells. CAR-positive cells were stained using an anti-mouse FMC63 kit, as described above. The killing rate of NK92 cells against target cells was calculated as follows: (number of CAR-positive cells in the co-culture group) / (number of CAR-positive cells in the target cell-only group). As shown in Figure 4, UCAR-T-CM cells from two volunteers (donor 1# and donor 2#) showed significant resistance to NK cell-mediated cell lysis ( ^** p<0.05, ^*** p<0.01).

7.5 UCAR-T cells expressing the fusion protein (CM) rapidly expanded in the presence of allogeneic PBMCs.

Co-culture of UCAR-T cells and allogeneic PBMC cells:

This study further simulated the in vivo environment. Because the human body has a complex system that includes many different types of immune cells in addition to NK cells, initial allogeneic PBMCs were used to accurately simulate the environment of the allogeneic immune system in the human body. To measure the ability of genetically engineered cells to survive and grow in an allogeneic PBMC environment, this study co-cultured three types of cells: UCAR-T cells or UCAR-T-CM cells as effector cells, Raji tumor cells (Shanghai Institute of Cell Research, Chinese Academy of Sciences) as target cells, and allogeneic PBMCs from peripheral blood of healthy human volunteers as a mimicry of the allogeneic immune environment. Initial PBMCs and T cells used to prepare UCAR-T or UCAR-T-CM were obtained from different donors to elicit an allogeneic response. Raji tumor cells were CD19 positive; therefore, CAR19-containing T cells were activated by the CD19 antigen on Raji cells and began to proliferate.

Cells were co-cultured at the following ratio: CAR-positive cells in UCAR-T cells (or UCAR-T-CM cells): Raji tumor cells: allogeneic PBMCs at a ratio of 1:5:20 (cell number ratio). 5 × ^10⁴ /ml CAR-positive cells from UCAR-T cells (or UCAR-T-CM cells) were seeded into 24-well culture plates. Appropriate numbers of Raji tumor cells and allogeneic PBMCs were calculated based on the above ratio (i.e., 2.5 × ^10⁵ /ml Raji tumor cells and 1 × ^10⁶ /ml allogeneic PBMCs) and seeded accordingly. Cells were cultured at 37°C, and the number of CAR-positive cells in different groups was recorded at different time points. The number of CAR-positive cells at different time points was divided by the initial number of CAR-positive cells on day 0 to obtain the fold change in cell expansion (Figure 5). The same CAR-positive cell staining method was used (using the anti-mouse FMC63 kit).

Prior to co-culture, allogeneic PBMCs and Raji cells were stained with eFluor ^™ 670 to distinguish them from UCAR-T cells or UCAR-T-CM cells. For cell staining, the cell density was adjusted to 1 × ^10⁷ /ml, eFluor ^™ e670 was added to a final concentration of 10 μM, the mixture was incubated in the dark at room temperature for 5 minutes, and washed three times with culture medium before use.

Figure 5 shows that in the presence of allogeneic PBMCs, UCAR-T cells cannot proliferate well upon stimulation by CD19-positive Raji cells, while UCAR-T-CM cells can proliferate rapidly. These results indicate that, upon stimulation by cancer cells, allogeneic immune cells in PBMCs inhibit the growth of UCAR-T cells but not the growth of UCAR-T-CM cells, thus further demonstrating that genetically engineered CAR-T cells expressing the fusion protein (CM) can overcome the inhibition and/or rejection by allogeneic immune cells and survive and grow in an allogeneic environment.

7.6 UCAR-T cells expressing the fusion protein (CM) efficiently induced tumor cell death in the presence of allogeneic PBMCs.

The experimental procedure was the same as described in section 7.5 above, but different cells were measured. In this experiment, the number of Raji cells in different groups was measured at different time points, rather than the number of CAR-positive cells. Raji cells were stained with dye e670 before co-culture, and could be distinguished from UCAR-T or UCAR-T-CM cells using the APC fluorescence channel in flow cytometry. Flow cytometry analysis clearly distinguished Raji cells from PBMC cells because Raji cells are tumor cells and have significantly different morphology and size compared to PBMC cells, and also because the continuous proliferation of tumor cells during co-culture led to a continuous decrease in the fluorescence of dye e670. The fold change of Raji cells at different time points was calculated by dividing the number of Raji cells at different time points by the initial number of Raji cells on day 0. As shown in Figure 6, the number of tumor cells in the Raji-only culture group increased over time, and UCAR-T cells showed weaker inhibition of tumor cells in the presence of allogeneic PBMCs—tumor cells continued to increase. In contrast, the growth of tumor cells co-cultured with UCAR-T-CM cells was significantly inhibited, and these tumor cells were gradually eliminated. These results confirm the findings described in section 7.5 above and demonstrate that UCAR-T-CM cells, which can rapidly proliferate when stimulated by tumor cells, also possess tumor-killing activity.

7.7 Detection of the complex formed by the fusion protein (CM) and HLA-E heavy chain on the cell membrane of β2M-deficient cells

Following the experimental procedures described above, Jurkat T cells were used in this study. Flow cytometry was used to detect the complex formed on the cell membrane containing the fusion protein (CM) and endogenous HLA-E heavy chain molecules (Figure 7). Jurkat cells in each group were stained with an anti-mouse FMC63 kit to detect CAR, and stained with anti-β2M-FITC and anti-HLA-E-APC, respectively, to detect β2M and HLA-E. A small amount of cells was taken, washed once with 1 ml PBS (Gibco, catalog #C10010500BT), and resuspended in 100 μL PBS. 3 μL of primary anti-FMC63 antibody (for CAR19 detection) was added to the cell mixture, and the mixture was incubated at 4°C for 30 minutes. Then, 1 ml PBS was added and the mixture was thoroughly mixed, and centrifuged at 350 g for 3 minutes. The cells were collected, and the supernatant was discarded. Add 0.5 μL of secondary antibody (primary and secondary antibodies were obtained from an anti-mouse FMC63 kit, Shanghai Xingwan, R19PB-100), 3 μL of anti-β2M-FITC antibody, and 3 μL of anti-HLA-E-APC antibody sequentially, mix thoroughly, and incubate at 4°C for 30 minutes. Add 1 ml of PBS and mix thoroughly, then centrifuge at 350 g for 3 minutes. Collect the cells and discard the supernatant. Resuspend the cells in 200 μL of PBS and load them into a flow cytometer for analysis.

Figures 7A-H show flow cytometry data from Jurkat cells transduced with CAR19-P2A-CM virus and in which the endogenous TRAC and B2M genes were knocked out. Figures 7I-L show flow cytometry data from the control group, where the endogenous TRAC and B2M genes of Jurkat cells were knocked out but not transduced with CAR19-P2A-CM virus.

Figure 7, section B, shows HLA-E positivity in cells expressing only CAR. Because the nucleic acids encoding CAR and the fusion protein (CM) are cloned to be operatively linked to the same viral vector (via the nucleotide sequence encoding the P2A peptide), cells expressing CAR also express CM. Therefore, cells expressing only CM are HLA-E positive. Figure 7, section C, shows co-expression of CAR and fusion protein (CM). Because CM includes β2M, double-positive cells for both CAR and β2M can be observed. Figure 7, section D, shows co-localization of β2M and HLA-E. Since most cells lack endogenous expression of β2M, anti-β2M antibodies effectively detect CM containing recombinant β2M. The fact that HLA-E can only be detected on the cell surface of cells expressing CM indicates that recombinantly expressed CM forms a complex with the endogenous HLA-E heavy chain on the cell surface.

Figure 7, section F, shows that the CAR-positive cell population R2 almost entirely comprises β2M and HLA-E double-positive cells, while section H shows that β2M and HLA-E double-positive cells were not detected in CAR-negative cells. These results further demonstrate that β2M signaling is generated by CM molecules co-expressed with CAR, and that CM (containing recombinant β2M and HLA-E restriction peptides) forms a complex with the HLA-E heavy chain in β2M and HLA-E double-positive cells. Membrane presentation of the HLA-E heavy chain requires β2M expression. When CM is not expressed (in CAR-negative cells), HLA-E is undetectable on the cell surface of β2M-knockout Jurkat cells. In those β2M-knockout Jurkat cells, when CM is expressed (in CAR-localized cells), CM and the HLA-E heavy chain form a complex to restore HLA-E heavy chain presentation on the cell membrane, making the HLA-E heavy chain molecules detectable on the cell surface.

Figure 7, sections I-L, shows that CAR or HLA-E was not detected on the cell surface in the control group cells. A small number of β2M-positive cells represent cells where β2M was not completely knocked out by gene editing.

7.8 The expression of different presenting peptides is associated with different expression of HLA-E molecules on the cell membrane.

Presenting peptides influence the binding between the fusion protein (CM) and the HLA-E heavy chain, thereby affecting the expression of the HLA-E complex on the cell membrane. Eighteen different presenting peptides (P1-P18; SEQ ID NOs:21-38) were selected and cloned into a lentiviral expression vector as GFP-P2A-CMx (x represents different peptide segment numbers P1-P18). The fusion protein CMx used here has the following components from the N-terminus to the C-terminus: 20-a.a. signal peptide (SEQ ID NO:3), presenting peptide x selected from P1-P18 (SEQ ID NOs:21-38), linker sequence GGGGSGGGGSGGGGS (SEQ ID NO:1), and β2M mature form peptide (SEQ ID NO:81). The lentiviral targeted expression plasmid was constructed as described above, and GFP-P2A-CMx was cloned into a lentiviral vector using BamHI/Sall. The virus was transduced into Jurkat cells with β2M knocked out. Lentiviral preparation and the method for virus production are described above.

The expression of the HLA-E complex on the cell membrane was detected using an anti-HLA-E-APC antibody (Biolegend, 342606) and by measuring GFP, which indicates lentiviral transduction efficiency. As shown in Figure 8A (quantified in Figure 8B), the expression of different presenting peptides was correlated with different expression levels of the HLA-E complex on the cell membrane (NTC, negative control; untransduced group).

7.9 Multiple fusion proteins were efficiently expressed in UCAR-T cells, and in the presence of allogeneic PBMCs, the expansion of UCAR-T cells increased with the expression of fusion proteins.

CJP-P2A-CM, CJP-P2A-CM12, CJP-P2A-CM13, CJP-P2A-CM14 and CJP Lentiviral Targeted Expression Plasmid construction and preparation of UCAR-T cells with or without fusion proteins.

The procedures for constructing lentiviral targeted expression plasmids, preparing UCAR-T cells expressing different fusion proteins, and measuring cell surface expression using flow cytometry are the same as described in sections 7.2 and 7.3 above. CJP is an anti-CD19 CAR (SEQ ID NO: 75). The fusion proteins are the same as those described in section 7.8 above. The amino acid sequences of these constructs are summarized below. The nucleotide sequences can be found in the sequence listing.

Flow cytometry analysis of CD3 and HLA-ABC expression on cell surfaces demonstrated efficient knockout of the TRAC and B2M genes (Fig. 9, regions B, E, H, K, N). All groups had >85% CD3 and HLA-ABC double-negative cells. HLA-E and CAR were co-detected on the cell surfaces of UCAR-T-CM, UCAR-T-CM12, UCAR-T-CM13, and UCAR-T-CM14 cells. Each of these cells contained different presenting peptides as shown in the table above (Fig. 9, regions C, F, I, L, O, analysis of gated CD3 and HLA-ABC double-negative cells). The results showed that, compared with the control UCAR-T group which did not have any fusion protein, all of these fusion proteins with different HLA-E restriction peptides could restore the surface expression of the HLA-E complex in TRAC and β2M double knockout T cells, but could not restore the surface expression of the HLA-ABC complex.

UCAR- with different fusion proteins (CM, CM12, CM13, CM14) in the presence of allogeneic PBMCs. T cell expansion

The experimental procedure was the same as described in section 7.5 above. Figures 10A-10C show that, in the presence of allogeneic PBMCs, UCAR-T cells without fusion protein expression did not proliferate well over time upon stimulation by CD19-positive Raji cells. In contrast, UCAR-T-CM, UCAR-T-CM12, UCAR-T-CM13, and UCAR-T-CM14 cells proliferated rapidly even in the presence of allogeneic PBMCs. Different PBMC cell populations containing PBMCs were used in parallel experiments (Figure 10). These results indicate that immune cells in allogeneic PBMCs inhibited the growth of UCAR-T cells but not the growth of UCAR-T-CM/CM12/CM13/CM14 cells, thus further demonstrating that genetically engineered T cells expressing the various forms of fusion proteins disclosed in this invention can overcome the inhibition and/or rejection by allogeneic immune cells and survive and grow in an allogeneic environment.

7.10 Exogenous TCRs targeting NY-ESO-1 and fusion proteins were co-expressed in genetically engineered cells.

A lentiviral targeting expression plasmid containing an exogenous TCR (1G4, SEQ ID NO: 132) targeting NY-ESO-1 and a fusion protein CM (SEQ ID NO: 5) was constructed, and cells were prepared using this lentiviral vector and CRISPR/Cas9 gene editing. The procedure for constructing the lentiviral plasmid with 1G4 and CM linked by the P2A peptide was the same as described in sections 7.2 and 7.3 above. The lentiviral plasmid backbone (Addgene ID: #12252) was digested with BamHI/SalI. The construct 1G4-P2A-CM had the nucleotide sequence of SEQ ID NO: 135 and the amino acid sequence of SEQ ID NO: 134.

As described in sections 7.2 and 7.3 above, cells were prepared using lentiviral transduction and CRISPR/Cas9 gene editing, and the cells were analyzed by flow cytometry after immunofluorescence staining. For flow cytometry analysis, a small number of cells were taken and washed once with 1 ml of PBS (Gibco, cat#C10010500BT). The cells were resuspended in 100 μL of PBS, and 3 μL of each of the following antibodies was added: anti-CD3-FITC, anti-αβ-TCR PE, anti-HLA-ABC PE-Cy7, and anti-HLA-E-APC. After incubating at 4°C for 30 min, 1 ml of PBS was added to the mixture, and the mixture was thoroughly mixed and centrifuged at 350 g for 3 min. Cell clumps were collected and the supernatant was discarded. The cells were then resuspended in 200 μL of PBS and analyzed by flow cytometry.

The surface expression of different molecules on the resulting cells is shown in Figure 11: Areas A-D show the results from wild-type Jurkat T cells; Areas E-P show the results from Jurkat T cells with double knockout (dKO) of TRAC and β2M, thus being CD3 and β2M double negative. In Areas E-P of Figure 11: Areas E-H show the results from Jurkat T cells with TRAC and B2M dKO without lentiviral transduction; Areas I-L show the results from Jurkat T cells with TRAC and B2M dKO transduced with a lentiviral vector carrying 1G4 TCR (SEQ ID NO: 132); Areas M-P show the results from Jurkat T cells with TRAC and B2M dKO transduced with the lentiviral vector 1G4-P2A-CM (1G4 TCR and fusion protein CM linked by the P2A peptide; SEQ ID NO: 134).

As shown in Figure 11, wild-type Jurkat T cells showed positivity for CD3, αβ-TCR, HLA-ABC, and HLA-E staining (A-D). TRAC and β2M knockout Jurkat T cells had 91.8% CD3 and αβ-TCR double-negative cells (F, bottom left), and HLA-ABC and HLA-E were undetectable on the cell surface due to the lack of β2M (G, H). Double knockout Jurkat T cells transduced with 1G4 TCR showed 42.8% CD3 and αβ-TCR double-positive cells (J, top right), indicating that the expression of this exogenous TCR (i.e., 1G4) restored the surface presentation of the CD3/TCR complex; however, HLA-ABC and HLA-E remained negative in this group (K, L). Double knockout Jurkat T cells transduced with 1G4-P2A-CM showed 40.2% CD3 and αβ-TCR double-positive cells (Figure N, top right), which is comparable to the group expressing 1G4 alone (Figure J, top right); expression of the fusion protein CM restored the surface presentation of the HLA-E complex (Figure P, top right, 36.3% HLA-E and αβ-TCR double-positive), but did not restore the surface presentation of the HLA-ABC complex (Figure O).

7.11 Expansion of genetically engineered TCR-T cells targeting NY-ESO-1 with or without fusion proteins in the presence of allogeneic PBMCs.

Preparation of genetically engineered TCR-T cells targeting NY-ESO-1 with or without the fusion protein CM

The procedures for constructing lentiviral targeted expression plasmids, activating naïve T cells, gene editing, lentiviral-mediated gene transduction, and measuring cell surface expression using flow cytometry were the same as described in sections 7.2, 7.3, and 7.10 above. The lentivirus used was the same as described in section 7.10 above. Briefly, naïve T cells were derived from peripheral blood (PBMCs) of healthy human volunteers. T cells were activated using Dynabeads (Thermo, cat#11141D) at a Dynabeads-to-cell ratio of 3:1. 72 hours after activation, T cells were collected and washed three times with electroporation buffer (Thermo, resuspension buffer T in the kit, cat#MPK1096) or Opti-MEM (Gibco, cat#31985-070), and then resuspended in electroporation buffer (without Dynabeads). The cell density was adjusted to 1 × ^10⁸ /ml. To prepare β2M/TCR dual knockout T cells, β2M sgRNA and TRAC sgRNA (150 ng each) and 1 μg Cas9 protein were homogenized in vitro and incubated at room temperature for 10 minutes. The mixture was then added to the resuspended cells for electroporation. The total volume used for electroporation was 10 μL, and a Neon electroporation system was used. The pulse voltage, pulse duration, and pulse number were as follows: 1200 V and 10 ms, 3 times. 24 hours after electroporation, 3 × ^10⁵ initial T cells were seeded in 24-well plates. The prepared lentivirus was added to the cells, with the lentivirus quantity being 10 times the cell quantity (MOI = 10, "multiple of infection," the ratio of viral transduction units to cell quantity), and the culture medium was replenished to bring the volume to 500 μL (the same medium used to activate the initial T cells). For 1G4 cells, transduction was performed using lentivirus carrying 1G4; for 1G4-P2A-CM cells, transduction was performed using lentivirus carrying 1G4-P2A-CM; after 24 hours, culture medium was added to bring the volume to 1 mL to promote cell growth.

Co-culture of genetically engineered TCR-T cells with allogeneic PBMC cells

The experimental procedure was essentially the same as that described in section 7.5 above. In Figure 12, 1G4 cells were naïve human T cells with both TRAC and β2M knocked out, and 1G4 cells expressed 1G4 TCR (SEQ ID NO:132); 1G4-P2A-CM cells were naïve human T cells with both TRAC and β2M knocked out, and 1G4-P2A-CM cells co-expressed 1G4-TCR linked by the P2A peptide and the fusion protein CM (SEQ ID NO:134). To measure the survival and growth of genetically engineered TCR-T cells (with or without CM) in an allogeneic PBMC environment, this study co-cultured three cell types: 1G4 cells or 1G4-P2A-CM cells as effector cells; K562 cells expressing a single-chain trimer (HLA-A0201 heavy chain, NY-ESO-1-derived peptide 'SLLMWITQC' (SEQ ID NO:140), and β2M) as target cells, named K562-A02-NY-ESO-1; and allogeneic PBMCs from peripheral blood of healthy human volunteers as a mimicry of the allogeneic immune environment. Initial PBMCs and T cells used to prepare TCR-T or TCR-T-CM were obtained from different donors to elicit an allogeneic response. Cells were co-cultured at the following ratio: TCR-positive cells from 1G4 or 1G4-P2A-CM cells : K562-A02-NY-ESO-1 tumor cells : allogeneic PBMCs = 1:5:20 (cell number ratio). Allogeneic PBMCs and K562-A02-NY-ESO-1 cells were stained with eFluor ^™ 670 before co-culturing to distinguish them from 1G4 or 1G4-P2A-CM cells. Cells were cultured at 37°C, and the number of HLA-ABC negative and TCR-positive cells in different groups was recorded at different time points.

Figure 12 shows that 1G4 cells could not sustainably expand upon stimulation by K562-A02-NY-ESO-1 cells in the presence of allogeneic PBMCs. Following a short period (3 days) of expansion, the number of 1G4 cells decreased rapidly, while 1G4-P2A-CM cells were able to sustainably expand throughout the entire co-culture period (7 days). These results indicate that immune cells in allogeneic PBMCs inhibited the growth of 1G4 cells but did not suppress the growth of 1G4-P2A-CM cells, suggesting that the fusion protein CM helps TCR-T cells overcome growth inhibition and/or rejection by allogeneic immune cells and contributes to survival and growth in an allogeneic environment.

7.12 BCMA UCAR-T cells expressing the fusion protein are resistant to allogeneic NK cells.

Preparation of T cells expressing BCMA CAR and fusion protein (CM)

The procedures described in sections 7.2 and 7.3 above were used to prepare the lentiviral constructs and genetically engineered T cells used in this study. First, a nucleic acid encoding a BCMA CAR sequence (SEQ ID NO: 136) was synthesized by linking a nucleic acid sequence encoding the fusion protein CM (SEQ ID NO: 5) to P2A, and this nucleic acid was cloned into a lentiviral vector named pLenti-BCMA-CM (BCMA CAR-CM: SEQ ID NO: 138). CAR-T cells (without β2M/TRAC double knockout) and UCAR-T cells (with β2M/TRAC double knockout) were activated and transduced using pLenti-BCMA-CM. The resulting T cells were named BCMA-CM CAR-T and BCMA-CM UCAR-T, respectively. Control T cells (without β2M/TRAC double knockout) and UT cells (with β2M/TRAC double knockout), neither of which underwent lentiviral transduction, were used as control cells.

Following T cell expansion, the expression levels of BCMA CAR and the fusion protein CM were assessed using fluorescently labeled antibody staining and flow cytometry analysis, following the procedures described in Section 7.3 above. As shown in Figure 13, HLA-ABC and CD3 expression were eliminated in UT and BCMA-CM UCAR-T cells. As shown in Figure 14, BCMA CAR was stably expressed in all groups except control T cells and UT cells. Based on HLA-E expression, the fusion protein CM was expressed only in BCMA-CM CAR-T and BCMA-CM UCAR-T cells, with CM expression rates of 29.6% and 27.5%, respectively.

IFN-γ release from BCMA-CM CAR-T and BCMA-CM UCAR-T cells

Target cells (MMIS.Luc cells and K562.BCMA.Luc cells) were co-cultured with effector cells (control T, UT, BCMA-CM CAR-T, and BCMA-CM UCAR-T cells) at a 1:1 ratio for 20 hours. The supernatant from the co-culture was collected to assess interferon-γ (IFN-γ) levels using a commercially available ELISA kit (Thermo Fisher Scientific; catalog number: 88-7316). Measurements were performed according to the kit instructions. As shown in Figure 15, BCMA-CM CAR-T and BCMA-CM UCAR-T cells released high levels of IFN-γ when co-cultured with BCMA-expressing target cells.

BCMA UCAR-T cells expressing the fusion protein are resistant to allogeneic NK cells.

NK-92 cytotoxicity assays were performed according to the procedures described in Section 7.4 above. Before flow cytometry analysis, different groups of T cells (control T, UT, BCMA-CM CAR-T, and BCMA-CM UCAR-T cells) were co-cultured with NK-92 cells for 20 hours. As shown in Figure 16, BCMA UCAR-T-CM cells exhibited significantly higher levels of resistance to NK cell-mediated cytotoxicity compared to UT cells without CM expression, which reproduced previous results (Note: cell lysis rate was normalized relative to control T cells; ***p<0.01).

8. Electronically submitted sequence list reference

This application incorporates by reference a sequence list that has been submitted electronically as an ASCII text file. The sequence list is named "102A001WO02_SEQLIST.TXT", created on August 7, 2020, and has a size of 210,210 bytes.

sequence list

<110> Suzhou Kerui Gene Biotechnology Co., Ltd.

<120> Genetically engineered cells and their uses

<130> 102A001WO02

<150> CN201910746355.8

<151> 2019-08-13

<150> PCT/CN2019/124321

<151> 2019-12-10

<160> 140

<170> PatentIn Version 3.5

<210> 1

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Connector GGGGS

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Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 2

<211> 20

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<213> Artificial Sequence

<220>

<223> Connector GGGGS

<400> 2

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

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Gly Gly Gly Ser

20

<210> 3

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<213> Artificial Sequence

<220>

<223> Signal peptide B2M

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1 5 10 15

Gly Leu Glu Ala

20

<210> 4

<211> 21

<212> PRT

<213> Artificial Sequence

<220>

<223> Signal peptide CD8

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His Ala Ala Arg Pro

20

<210> 5

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<212> PRT

<213> Artificial Sequence

<220>

<223> B7-B2M protein sequence

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Gly Leu Glu Ala Val Met Ala Pro Arg Thr Val Leu Leu Gly Gly Gly

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Glu Phe Thr Pro Thr Glu Lys Asp Glu Tyr Ala Cys Arg Val Asn His

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Val Thr Leu Ser Gln Pro Lys Ile Val Lys Trp Asp Arg Asp Met

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<212> DNA

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<223> DNA coding sequence of SEQ ID NO. 5

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ggtggcggct ccatccagcg tacgccaaag attcaggttt actcacgtca tccagcagag 180

aatggaaagt caaatttcct gaattgctat gtgtctgggt ttcatccatc cgacattgaa 240

gttgacttac tgaagaatgg agagaatt gaaaaagtgg agcattcaga cttgtctttc 300

agcaaggact ggtctttcta tctcttgtac tacactgaat tcaccccccac tgaaaaagat 360

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1 5 10 15

Gly Leu Glu Ala Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg

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Gly Phe His Pro Ser Asp Ile Glu Val Asp Leu Leu Lys Asn Gly Glu

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Arg Ile Glu Lys Val Glu His Ser Asp Leu Ser Phe Ser Lys Asp Trp

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Ser Phe Tyr Leu Leu Tyr Tyr Thr Glu Phe Thr Pro Thr Glu Lys Asp

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Glu Tyr Ala Cys Arg Val Asn His Val Thr Leu Ser Gln Pro Lys Ile

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Val Lys Trp Asp Arg Asp Met

115

<210> 8

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<210> 10

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<213> Artificial Sequence

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gagucucuca gcugguacag uuuuagagcu agaaauagca aguuaaaaua aggcuagucc 60

guaucaacu ugaaaaagug gcaccgaguc ggugcuuuu 99

<210> 11

<211> 659

<212> PRT

<213> Artificial Sequence

<220>

<223> CAR19-P2A-B7-B2M protein

<400> 11

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu

20 25 30

Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr

50 55 60

Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro

65 70 75 80

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile

85 90 95

Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

130 135 140

Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser

145 150 155 160

Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Tyr Asn Ser Ala Leu Lys Ser

195 200 205

Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys

210 215 220

Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

245 250 255

Thr Ser Val Thr Val Ser Ser Thr Arg Thr Thr Thr Thr Pro Ala Pro Arg

260 265 270

Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg

275 280 285

Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly

290 295 300

Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr

305 310 315 320

Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg

325 330 335

Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro

340 345 350

Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu

355 360 365

Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala

370 375 380

Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu

385 390 395 400

Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly

405 410 415

Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu

420 425 430

Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser

435 440 445

Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly

450 455 460

Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu

465 470 475 480

His Met Gln Ala Leu Pro Pro Arg His Met Arg Arg Lys Arg Gly Ser

485 490 495

Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu

500 505 510

Asn Pro Gly Pro Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala Leu

515 520 525

Leu Ser Leu Ser Gly Leu Glu Ala Val Met Ala Pro Arg Thr Val Leu

530 535 540

Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

545 550 555 560

Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg His Pro Ala Glu

565 570 575

Asn Gly Lys Ser Asn Phe Leu Asn Cys Tyr Val Ser Gly Phe His Pro

580 585 590

Ser Asp Ile Glu Val Asp Leu Leu Lys Asn Gly Glu Arg Ile Glu Lys

595 600 605

Val Glu His Ser Asp Leu Ser Phe Ser Lys Asp Trp Ser Phe Tyr Leu

610 615 620

Leu Tyr Tyr Thr Glu Phe Thr Pro Thr Glu Lys Asp Glu Tyr Ala Cys

625 630 635 640

Arg Val Asn His Val Thr Leu Ser Gln Pro Lys Ile Val Lys Trp Asp

645 650 655

Arg Asp Met

<210> 12

<211> 1980

<212> DNA

<213> Artificial Sequence

<220>

<223> The coding sequence of the CAR19-P2A-B7-B2M protein

<400> 12

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggacatcc agatgacaca gactacatcc tccctgtctg cctctctggg agacagagtc 120

accatcagtt gcagggcaag tcaggacatt agtaaatatt taaattggta tcagcagaaa 180

ccagatggaa ctgttaaact cctgatctac catacatcaa gattacactc aggagtccca 240

tcaaggttca gtggcagtgg gtctggaaca gattattctc tcaccattag caacctggag 300

caagaagata ttgccactta cttttgccaa cagggtaata cgcttccgta cacgttcgga 360

ggggggacca agctggagat cacaggtggc ggtggctcgg gcggtggtgg gtcgggtggc 420

ggcggatctg aggtgaaact gcaggagtca ggacctggcc tggtggcgcc ctcacagagc 480

ctgtccgtca catgcactgt ctcaggggtc tcattacccg actatggtgt aagctggatt 540

cgccagcctc cacgaaaggg tctggagtgg ctgggagtaa tatggggtag tgaaaccaca 600

tactataatt cagctctcaa atccagactg accatcatca aggacaactc caagagccaa 660

gttttcttaa aaatgaacag tctgcaaact gatgacacag ccatttacta ctgtgccaaa 720

cattattact acggtggtag ctatgctatg gactactggg gccaaggaac ctcagtcacc 780

gtctcctcaa cgcgtaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 840

gcgtcgcagc ccctgtccct gcgcccag gcgtgccggc cagcggcggg gggcgcagtg 900

cacacgaggg ggctggactt cgcctgtgat atctacatct gggcgccctt ggccgggact 960

tgtggggtcc ttctcctgtc actggttatc accctttact gcaaacgggg cagaaagaaa 1020

ctcctgtata tattcaaaca accattttatg agaccagtac aaactactca agaggaagat 1080

ggctgtagct gccgatttcc agaagaagaa gaaggaggat gtgaactgag agtgaagttc 1140

agcaggagcg cagacgcccc cgcgtaccag cagggccaga accagctcta taacgagctc 1200

aatctaggac gaagagagga gtacgatgtt ttggacaaga gacgtggccg ggaccctgag 1260

atggggggaa agccgagaag gaagaaccct caggaaggcc tgtacaatga actgcagaaa 1320

gataagatgg cggaggccta cagtgagatt gggatgaaag gcgagcgccg gaggggcaag 1380

gggcacgatg gcctttacca gggtctcagt acagccacca aggacacccta cgacgccctt 1440

cacatgcagg ccctgccccc tcgccatatg cgccgcaagc gcggctccgg tgccacgaac 1500

ttctctctgt taaagcaagc aggagacgtg gaagaaaacc ccggtcctat gtctcgctcc 1560

gtggcattgg ctgtgctcgc gctactctct ctttctggtc tcgaagctgt tatggctccg 1620

cggactgtgc tgttaggtgg tggcggttcc ggtggtggcg gttctggtgg tggcggctcc 1680

atccagcgta cgccaaagat tcaggtttac tcacgtcatc cagcagagaa tggaaagtca 1740

aatttcctga attgctatgt gtctgggttt catccatccg acattgaagt tgacttactg 1800

aagaatggag agagaattga aaaagtggag cattcagact tgtctttcag caaggactgg 1860

tctttctatc tcttgtacta cactgaattc accccactg aaaaagatga gtatgcctgc 1920

cgtgtgaacc atgtgacttt gtcacagccc aagatagtta agtggggatcg cgacatgtaa 1980

<210> 13

<211> 143

<212> PRT

<213> Artificial Sequence

<220>

<223> Fusion protein

<400> 13

Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu Ser

1 5 10 15

Gly Leu Glu Ala Val Met Ala Pro Arg Thr Leu Ile Leu Gly Gly Gly

20 25 30

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Gln Arg Thr

35 40 45

Pro Lys Ile Gln Val Tyr Ser Arg His Pro Ala Glu Asn Gly Lys Ser

50 55 60

Asn Phe Leu Asn Cys Tyr Val Ser Gly Phe His Pro Ser Asp Ile Glu

65 70 75 80

Val Asp Leu Leu Lys Asn Gly Glu Arg Ile Glu Lys Val Glu His Ser

85 90 95

Asp Leu Ser Phe Ser Lys Asp Trp Ser Phe Tyr Leu Leu Tyr Tyr Thr

100 105 110

Glu Phe Thr Pro Thr Glu Lys Asp Glu Tyr Ala Cys Arg Val Asn His

115 120 125

Val Thr Leu Ser Gln Pro Lys Ile Val Lys Trp Asp Arg Asp Met

130 135 140

<210> 14

<211> 143

<212> PRT

<213> Artificial Sequence

<220>

<223> Fusion protein

<400> 14

Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu Ser

1 5 10 15

Gly Leu Glu Ala Val Met Ala Pro Arg Thr Leu Val Leu Gly Gly Gly

20 25 30

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Gln Arg Thr

35 40 45

Pro Lys Ile Gln Val Tyr Ser Arg His Pro Ala Glu Asn Gly Lys Ser

50 55 60

Asn Phe Leu Asn Cys Tyr Val Ser Gly Phe His Pro Ser Asp Ile Glu

65 70 75 80

Val Asp Leu Leu Lys Asn Gly Glu Arg Ile Glu Lys Val Glu His Ser

85 90 95

Asp Leu Ser Phe Ser Lys Asp Trp Ser Phe Tyr Leu Leu Tyr Tyr Thr

100 105 110

Glu Phe Thr Pro Thr Glu Lys Asp Glu Tyr Ala Cys Arg Val Asn His

115 120 125

Val Thr Leu Ser Gln Pro Lys Ile Val Lys Trp Asp Arg Asp Met

130 135 140

<210> 15

<211> 143

<212> PRT

<213> Artificial Sequence

<220>

<223> Fusion protein

<400> 15

Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu Ser

1 5 10 15

Gly Leu Glu Ala Val Met Ala Pro Arg Thr Leu Phe Leu Gly Gly Gly

20 25 30

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Gln Arg Thr

35 40 45

Pro Lys Ile Gln Val Tyr Ser Arg His Pro Ala Glu Asn Gly Lys Ser

50 55 60

Asn Phe Leu Asn Cys Tyr Val Ser Gly Phe His Pro Ser Asp Ile Glu

65 70 75 80

Val Asp Leu Leu Lys Asn Gly Glu Arg Ile Glu Lys Val Glu His Ser

85 90 95

Asp Leu Ser Phe Ser Lys Asp Trp Ser Phe Tyr Leu Leu Tyr Tyr Thr

100 105 110

Glu Phe Thr Pro Thr Glu Lys Asp Glu Tyr Ala Cys Arg Val Asn His

115 120 125

Val Thr Leu Ser Gln Pro Lys Ile Val Lys Trp Asp Arg Asp Met

130 135 140

<210> 16

<211> 143

<212> PRT

<213> Artificial Sequence

<220>

<223> Fusion protein

<400> 16

Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu Ser

1 5 10 15

Gly Leu Glu Ala Val Met Ala Pro Arg Thr Leu Leu Leu Gly Gly Gly

20 25 30

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Gln Arg Thr

35 40 45

Pro Lys Ile Gln Val Tyr Ser Arg His Pro Ala Glu Asn Gly Lys Ser

50 55 60

Asn Phe Leu Asn Cys Tyr Val Ser Gly Phe His Pro Ser Asp Ile Glu

65 70 75 80

Val Asp Leu Leu Lys Asn Gly Glu Arg Ile Glu Lys Val Glu His Ser

85 90 95

Asp Leu Ser Phe Ser Lys Asp Trp Ser Phe Tyr Leu Leu Tyr Tyr Thr

100 105 110

Glu Phe Thr Pro Thr Glu Lys Asp Glu Tyr Ala Cys Arg Val Asn His

115 120 125

Val Thr Leu Ser Gln Pro Lys Ile Val Lys Trp Asp Arg Asp Met

130 135 140

<210> 17

<211> 143

<212> PRT

<213> Artificial Sequence

<220>

<223> Fusion protein

<400> 17

Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu Ser

1 5 10 15

Gly Leu Glu Ala Val Thr Ala Pro Arg Thr Val Leu Leu Gly Gly Gly

20 25 30

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Gln Arg Thr

35 40 45

Pro Lys Ile Gln Val Tyr Ser Arg His Pro Ala Glu Asn Gly Lys Ser

50 55 60

Asn Phe Leu Asn Cys Tyr Val Ser Gly Phe His Pro Ser Asp Ile Glu

65 70 75 80

Val Asp Leu Leu Lys Asn Gly Glu Arg Ile Glu Lys Val Glu His Ser

85 90 95

Asp Leu Ser Phe Ser Lys Asp Trp Ser Phe Tyr Leu Leu Tyr Tyr Thr

100 105 110

Glu Phe Thr Pro Thr Glu Lys Asp Glu Tyr Ala Cys Arg Val Asn His

115 120 125

Val Thr Leu Ser Gln Pro Lys Ile Val Lys Trp Asp Arg Asp Met

130 135 140

<210> 18

<211> 144

<212> PRT

<213> Artificial Sequence

<220>

<223> Fusion protein

<400> 18

Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu Ser

1 5 10 15

Gly Leu Glu Ala Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Gly

20 25 30

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Gln Arg

35 40 45

Thr Pro Lys Ile Gln Val Tyr Ser Arg His Pro Ala Glu Asn Gly Lys

50 55 60

Ser Asn Phe Leu Asn Cys Tyr Val Ser Gly Phe His Pro Ser Asp Ile

65 70 75 80

Glu Val Asp Leu Leu Lys Asn Gly Glu Arg Ile Glu Lys Val Glu His

85 90 95

Ser Asp Leu Ser Phe Ser Lys Asp Trp Ser Phe Tyr Leu Leu Tyr Tyr

100 105 110

Thr Glu Phe Thr Pro Thr Glu Lys Asp Glu Tyr Ala Cys Arg Val Asn

115 120 125

His Val Thr Leu Ser Gln Pro Lys Ile Val Lys Trp Asp Arg Asp Met

130 135 140

<210> 19

<211> 1980

<212> DNA

<213> Artificial Sequence

<220>

<223> DNA encoding the CAR19-P2A-CM-c protein

<400> 19

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggacatcc agatgacaca gactacatcc tccctgtctg cctctctggg agacagagtc 120

accatcagtt gcagggcaag tcaggacatt agtaaatatt taaattggta tcagcagaaa 180

ccagatggaa ctgttaaact cctgatctac catacatcaa gattacactc aggagtccca 240

tcaaggttca gtggcagtgg gtctggaaca gattattctc tcaccattag caacctggag 300

caagaagata ttgccactta cttttgccaa cagggtaata cgcttccgta cacgttcgga 360

ggggggacca agctggagat cacaggtggc ggtggctcgg gcggtggtgg gtcgggtggc 420

ggcggatctg aggtgaaact gcaggagtca ggacctggcc tggtggcgcc ctcacagagc 480

ctgtccgtca catgcactgt ctcaggggtc tcattacccg actatggtgt aagctggatt 540

cgccagcctc cacgaaaggg tctggagtgg ctgggagtaa tatggggtag tgaaaccaca 600

tactataatt cagctctcaa atccagactg accatcatca aggacaactc caagagccaa 660

gttttcttaa aaatgaacag tctgcaaact gatgacacag ccatttacta ctgtgccaaa 720

cattattact acggtggtag ctatgctatg gactactggg gccaaggaac ctcagtcacc 780

gtctcctcaa cgcgtaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 840

gcgtcgcagc ccctgtccct gcgcccag gcgtgccggc cagcggcggg gggcgcagtg 900

cacacgaggg ggctggactt cgcctgtgat atctacatct gggcgccctt ggccgggact 960

tgtggggtcc ttctcctgtc actggttatc accctttact gcaaacgggg cagaaagaaa 1020

ctcctgtata tattcaaaca accattttatg agaccagtac aaactactca agaggaagat 1080

ggctgtagct gccgatttcc agaagaagaa gaaggaggat gtgaactgag agtgaagttc 1140

agcaggagcg cagacgcccc cgcgtaccag cagggccaga accagctcta taacgagctc 1200

aatctaggac gaagagagga gtacgatgtt ttggacaaga gacgtggccg ggaccctgag 1260

atggggggaa agccgagaag gaagaaccct caggaaggcc tgtacaatga actgcagaaa 1320

gataagatgg cggaggccta cagtgagatt gggatgaaag gcgagcgccg gaggggcaag 1380

gggcacgatg gcctttacca gggtctcagt acagccacca aggacacccta cgacgccctt 1440

cacatgcagg ccctgccccc tcgccatatg cgccgcaagc gcggctccgg tgccacgaac 1500

ttctctctgt taaagcaagc aggagacgtg gaagaaaacc ccggtcctat gtctcgctcc 1560

gtggcattgg ctgtgctcgc gctactctct ctttctggtc tcgaagctat cattgacaag 1620

agcggctcca ccgtgggtgg tggcggttcc ggtggtggcg gttctggtgg tggcggctcc 1680

atccagcgta cgccaaagat tcaggtttac tcacgtcatc cagcagagaa tggaaagtca 1740

aatttcctga attgctatgt gtctgggttt catccatccg acattgaagt tgacttactg 1800

aagaatggag agagaattga aaaagtggag cattcagact tgtctttcag caaggactgg 1860

tctttctatc tcttgtacta cactgaattc accccactg aaaaagatga gtatgcctgc 1920

cgtgtgaacc atgtgacttt gtcacagccc aagatagtta agtggggatcg cgacatgtaa 1980

<210> 20

<211> 1467

<212> DNA

<213> Artificial Sequence

<220>

<223> DNA encoding the CAR19 protein

<400> 20

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggacatcc agatgacaca gactacatcc tccctgtctg cctctctggg agacagagtc 120

accatcagtt gcagggcaag tcaggacatt agtaaatatt taaattggta tcagcagaaa 180

ccagatggaa ctgttaaact cctgatctac catacatcaa gattacactc aggagtccca 240

tcaaggttca gtggcagtgg gtctggaaca gattattctc tcaccattag caacctggag 300

caagaagata ttgccactta cttttgccaa cagggtaata cgcttccgta cacgttcgga 360

ggggggacca agctggagat cacaggtggc ggtggctcgg gcggtggtgg gtcgggtggc 420

ggcggatctg aggtgaaact gcaggagtca ggacctggcc tggtggcgcc ctcacagagc 480

ctgtccgtca catgcactgt ctcaggggtc tcattacccg actatggtgt aagctggatt 540

cgccagcctc cacgaaaggg tctggagtgg ctgggagtaa tatggggtag tgaaaccaca 600

tactataatt cagctctcaa atccagactg accatcatca aggacaactc caagagccaa 660

gttttcttaa aaatgaacag tctgcaaact gatgacacag ccatttacta ctgtgccaaa 720

cattattact acggtggtag ctatgctatg gactactggg gccaaggaac ctcagtcacc 780

gtctcctcaa cgcgtaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 840

gcgtcgcagc ccctgtccct gcgcccag gcgtgccggc cagcggcggg gggcgcagtg 900

cacacgaggg ggctggactt cgcctgtgat atctacatct gggcgccctt ggccgggact 960

tgtggggtcc ttctcctgtc actggttatc accctttact gcaaacgggg cagaaagaaa 1020

ctcctgtata tattcaaaca accattttatg agaccagtac aaactactca agaggaagat 1080

ggctgtagct gccgatttcc agaagaagaa gaaggaggat gtgaactgag agtgaagttc 1140

agcaggagcg cagacgcccc cgcgtaccag cagggccaga accagctcta taacgagctc 1200

aatctaggac gaagagagga gtacgatgtt ttggacaaga gacgtggccg ggaccctgag 1260

atggggggaa agccgagaag gaagaaccct caggaaggcc tgtacaatga actgcagaaa 1320

gataagatgg cggaggccta cagtgagatt gggatgaaag gcgagcgccg gaggggcaag 1380

gggcacgatg gcctttacca gggtctcagt acagccacca aggacacccta cgacgccctt 1440

cacatgcagg ccctgccccc tcgctaa 1467

<210> 21

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> P1 – Exemplary Presenting Peptide

<400> 21

Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu

1 5 10

<210> 22

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P2 - Exemplary Presenting Peptide

<400> 22

Ala Leu Ala Leu Val Arg Met Leu Ile

1 5

<210> 23

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P3 - Exemplary Presenting Peptide

<400> 23

Ala Ile Ser Pro Arg Thr Leu Asn Ala

1 5

<210> 24

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P4 - Exemplary Presenting Peptide

<400> 24

Gln Met Arg Pro Val Ser Arg Val Leu

1 5

<210> 25

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P5 - Exemplary Presenting Peptide

<400> 25

Ser Gln Gln Pro Tyr Leu Gln Leu Gln

1 5

<210> 26

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P6 - Exemplary Presenting Peptide

<400> 26

Val Thr Ala Pro Arg Thr Leu Leu Leu

1 5

<210> 27

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P7 - Exemplary Presenting Peptide

<400> 27

Val Thr Ala Pro Arg Thr Val Leu Leu

1 5

<210> 28

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P8 - Exemplary Presenting Peptide

<400> 28

Val Thr Ala Pro Arg Thr Leu Val Leu

1 5

<210> 29

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P9 - Exemplary Presenting Peptide

<400> 29

Val Met Ala Pro Gln Ala Leu Leu Leu

1 5

<210> 30

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P10 - Exemplary Presenting Peptide

<400> 30

Val Met Ala Pro Arg Ala Leu Leu Leu

1 5

<210> 31

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P11 - Exemplary Presenting Peptide

<400> 31

Val Met Ala Pro Arg Thr Leu Thr Leu

1 5

<210> 32

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P12 - Exemplary Presenting Peptide

<400> 32

Val Met Ala Pro Arg Thr Leu Phe Leu

1 5

<210> 33

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P13 - Exemplary Presenting Peptide

<400> 33

Val Met Ala Pro Arg Thr Leu Val Leu

1 5

<210> 34

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P14 - Exemplary Presenting Peptide

<400> 34

Val Met Ala Pro Arg Thr Leu Ile Leu

1 5

<210> 35

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P15 - Exemplary Presenting Peptide

<400> 35

Ile Met Ala Pro Arg Thr Leu Val Leu

1 5

<210> 36

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P16 - Exemplary Presenting Peptide

<400> 36

Val Met Ala Pro Arg Thr Leu Leu Leu

1 5

<210> 37

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P17 - Exemplary Presenting Peptide

<400> 37

Val Met Pro Pro Arg Thr Leu Leu Leu

1 5

<210> 38

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P18 - Exemplary Presenting Peptide

<400> 38

Val Met Ala Pro Arg Thr Val Leu Leu

1 5

<210> 39

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P19 - Exemplary Presenting Peptide

<400> 39

Val Met Ala Pro Arg Ser Leu Leu Leu

1 5

<210> 40

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P20 - Exemplary Presenting Peptide

<400> 40

Val Met Ala Pro Arg Ser Leu Ile Leu

1 5

<210> 41

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P21 - Exemplary Presenting Peptide

<400> 41

Val Met Thr Pro Arg Thr Leu Val Leu

1 5

<210> 42

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P22 - Exemplary Presenting Peptide

<400> 42

Val Met Ala Pro Arg Ile Leu Ile Leu

1 5

<210> 43

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P23 - Exemplary Presenting Peptide

<400> 43

Ala Met Ala Pro Arg Thr Leu Ile Leu

1 5

<210> 44

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P24 - Exemplary Presenting Peptide

<400> 44

Val Ile Ala Pro Arg Thr Leu Val Leu

1 5

<210> 45

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P25 - Exemplary Presenting Peptide

<400> 45

Val Met Ala Pro Gln Ser Leu Leu Leu

1 5

<210> 46

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P26 - Exemplary Presenting Peptide

<400> 46

Val Met Ala Pro Arg Thr Phe Val Leu

1 5

<210> 47

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P27 - Exemplary Presenting Peptide

<400> 47

Val Met Thr Pro Arg Thr Leu Ile Leu

1 5

<210> 48

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P28 - Exemplary Presenting Peptide

<400> 48

Val Thr Ala Pro Arg Thr Leu Ile Leu

1 5

<210> 49

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P29 - Exemplary Presenting Peptide

<400> 49

Val Met Ala Pro Trp Thr Leu Leu Leu

1 5

<210> 50

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P30 - Exemplary Presenting Peptide

<400> 50

Val Met Val Pro Arg Ser Leu Ile Leu

1 5

<210> 51

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P31 - Exemplary Presenting Peptide

<400> 51

Ala Met Ala Pro Arg Thr Leu Val Leu

1 5

<210> 52

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P32 - Exemplary Presenting Peptide

<400> 52

Val Ile Ala Pro Arg Thr Leu Ile Leu

1 5

<210> 53

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P33 - Exemplary Presenting Peptide

<400> 53

Val Ile Ala Pro Arg Thr Leu Leu Leu

1 5

<210> 54

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P34 - Exemplary Presenting Peptide

<400> 54

Val Leu Ala Pro Arg Thr Leu Ile Leu

1 5

<210> 55

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P35 - Exemplary Presenting Peptide

<400> 55

Val Met Ala Leu Arg Thr Leu Ile Leu

1 5

<210> 56

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P36 - Exemplary Presenting Peptide

<400> 56

Val Met Ala Pro Arg Gly Leu Ile Leu

1 5

<210> 57

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P37 - Exemplary Presenting Peptide

<400> 57

Val Met Ala Pro Arg Asn Leu Ile Leu

1 5

<210> 58

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P38 - Exemplary Presenting Peptide

<400> 58

Val Met Ala Pro Arg Thr Leu Phe Val

1 5

<210> 59

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P39 - Exemplary Presenting Peptide

<400> 59

Val Met Ala Pro Arg Thr Leu Leu Met

1 5

<210> 60

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P40 - Exemplary Presenting Peptide

<400> 60

Val Met Ala Pro Arg Thr Leu Val Met

1 5

<210> 61

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P41 - Exemplary Presenting Peptide

<400> 61

Val Met Ala Pro Arg Thr Ser Leu Leu

1 5

<210> 62

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P42 - Exemplary Presenting Peptide

<400> 62

Val Met Ala Pro Arg Thr Ser Val Leu

1 5

<210> 63

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P43 - Exemplary Presenting Peptide

<400> 63

Val Met Ala Pro Trp Thr Leu Ile Leu

1 5

<210> 64

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P44 - Exemplary Presenting Peptide

<400> 64

Val Met Ala Pro Trp Thr Leu Val Leu

1 5

<210> 65

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P45 - Exemplary Presenting Peptide

<400> 65

Val Met Asp Pro Arg Thr Leu Leu Leu

1 5

<210> 66

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P46 - Exemplary Presenting Peptide

<400> 66

Val Met Gly Pro Arg Thr Leu Ile Leu

1 5

<210> 67

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P47 - Exemplary Presenting Peptide

<400> 67

Val Met Gly Pro Arg Thr Leu Leu Leu

1 5

<210> 68

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P48 - Exemplary Presenting Peptide

<400> 68

Val Met Val Pro Gln Thr Leu Ile Leu

1 5

<210> 69

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P49 - Exemplary Presenting Peptide

<400> 69

Val Met Val Pro Arg Thr Leu Leu Leu

1 5

<210> 70

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P50 - Exemplary Presenting Peptide

<400> 70

Val Val Ala Pro Arg Thr Leu Ile Leu

1 5

<210> 71

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P51 - Exemplary Presenting Peptide

<400> 71

Val Val Ala Pro Arg Thr Leu Leu Leu

1 5

<210> 72

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P52 - Exemplary Presenting Peptide

<400> 72

Val Met Val Pro Arg Thr Leu Ile Leu

1 5

<210> 73

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> P53 - Exemplary Presenting Peptide

<400> 73

Val Met Ala Thr Arg Thr Leu Leu Leu

1 5

<210> 74

<211> 488

<212> PRT

<213> Artificial Sequence

<220>

<223> Anti-CD19 CAR

<400> 74

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu

20 25 30

Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr

50 55 60

Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro

65 70 75 80

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile

85 90 95

Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

130 135 140

Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser

145 150 155 160

Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Tyr Asn Ser Ala Leu Lys Ser

195 200 205

Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys

210 215 220

Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

245 250 255

Thr Ser Val Thr Val Ser Ser Thr Arg Thr Thr Thr Thr Pro Ala Pro Arg

260 265 270

Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg

275 280 285

Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly

290 295 300

Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr

305 310 315 320

Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg

325 330 335

Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro

340 345 350

Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu

355 360 365

Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala

370 375 380

Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu

385 390 395 400

Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly

405 410 415

Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu

420 425 430

Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser

435 440 445

Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly

450 455 460

Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu

465 470 475 480

His Met Gln Ala Leu Pro Pro Arg

485

<210> 75

<211> 486

<212> PRT

<213> Artificial Sequence

<220>

<223> Anti-CD19 CAR

<400> 75

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu

20 25 30

Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr

50 55 60

Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro

65 70 75 80

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile

85 90 95

Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

130 135 140

Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser

145 150 155 160

Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Tyr Asn Ser Ala Leu Lys Ser

195 200 205

Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys

210 215 220

Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

245 250 255

Thr Ser Val Thr Val Ser Ser Thr Thr Thr Thr Pro Ala Pro Arg Pro Pro

260 265 270

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

275 280 285

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

290 295 300

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg

325 330 335

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln

340 345 350

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu

355 360 365

Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

370 375 380

Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

385 390 395 400

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

405 410 415

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

420 425 430

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

435 440 445

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

450 455 460

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

465 470 475 480

Gln Ala Leu Pro Pro Arg

485

<210> 76

<211> 489

<212> PRT

<213> Artificial Sequence

<220>

<223> Anti-CD19 CAR

<400> 76

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu

20 25 30

Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr

50 55 60

Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro

65 70 75 80

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile

85 90 95

Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

130 135 140

Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser

145 150 155 160

Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Tyr Asn Ser Ala Leu Lys Ser

195 200 205

Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys

210 215 220

Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

245 250 255

Thr Ser Val Thr Val Ser Ser Thr Thr Thr Thr Pro Ala Pro Arg Pro Pro

260 265 270

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

275 280 285

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

290 295 300

Phe Ala Cys Asp Ile Tyr Phe Trp Val Leu Val Val Val Gly Gly Val

305 310 315 320

Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp

325 330 335

Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met

340 345 350

Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala

355 360 365

Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Val Lys Phe Ser Arg Ser

370 375 380

Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu

385 390 395 400

Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg

405 410 415

Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln

420 425 430

Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr

435 440 445

Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp

450 455 460

Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala

465 470 475 480

Leu His Met Gln Ala Leu Pro Pro Arg

485

<210> 77

<211> 369

<212> PRT

<213> Artificial Sequence

<220>

<223> Anti-BCMA CAR

<400> 77

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Leu

20 25 30

Ala Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg

35 40 45

Thr Phe Ser Thr Tyr Phe Met Ala Trp Phe Arg Gln Pro Pro Gly Lys

50 55 60

Gly Leu Glu Tyr Val Gly Gly Ile Arg Trp Ser Asp Gly Val Pro His

65 70 75 80

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala

85 90 95

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

100 105 110

Ala Val Tyr Phe Cys Ala Ser Arg Gly Ile Ala Asp Gly Ser Asp Phe

115 120 125

Gly Ser Tyr Gly Gln Gly Thr Gln Val Thr Val Ser Ser Pro Ala Lys

130 135 140

Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile

145 150 155 160

Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala

165 170 175

Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr

180 185 190

Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu

195 200 205

Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile

210 215 220

Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp

225 230 235 240

Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Gly Gly Cys Glu Leu

245 250 255

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

260 265 270

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

275 280 285

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

290 295 300

Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

305 310 315 320

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

325 330 335

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

340 345 350

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

355 360 365

Arg

<210> 78

<211> 511

<212> PRT

<213> Artificial Sequence

<220>

<223> Anti-BCMA CAR

<400> 78

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu

20 25 30

Val Gln Ala Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu His

35 40 45

Thr Phe Ser Ser His Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys

50 55 60

Glu Arg Glu Ser Val Ala Val Ile Gly Trp Arg Asp Ile Ser Thr Ser

65 70 75 80

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala

85 90 95

Lys Lys Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Ala Ala Arg Arg Ile Asp Ala Ala Asp Phe Asp

115 120 125

Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser

145 150 155 160

Gly Gly Gly Gly Ser Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu

165 170 175

Val Gln Ala Gly Asp Ser Leu Arg Leu Thr Cys Thr Ala Ser Gly Arg

180 185 190

Ala Phe Ser Thr Tyr Phe Met Ala Trp Phe Arg Gln Ala Pro Gly Lys

195 200 205

Glu Arg Glu Phe Val Ala Gly Ile Ala Trp Ser Gly Gly Ser Thr Ala

210 215 220

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala

225 230 235 240

Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr

245 250 255

Ala Val Tyr Tyr Cys Ala Ser Arg Gly Ile Glu Val Glu Glu Phe Gly

260 265 270

Ala Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Thr Ser Thr Arg

275 280 285

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

290 295 300

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

305 310 315 320

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile

325 330 335

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

340 345 350

Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

355 360 365

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

370 375 380

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg

385 390 395 400

Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln

405 410 415

Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp

420 425 430

Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro

435 440 445

Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp

450 455 460

Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg

465 470 475 480

Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr

485 490 495

Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505 510

<210> 79

<211> 496

<212> PRT

<213> Artificial Sequence

<220>

<223> Anti-BCMA CAR

<400> 79

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu

20 25 30

Val Gln Ala Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu His

35 40 45

Thr Phe Ser Ser His Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys

50 55 60

Glu Arg Glu Ser Val Ala Val Ile Gly Trp Arg Asp Ile Ser Thr Ser

65 70 75 80

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala

85 90 95

Lys Lys Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Ala Ala Arg Arg Ile Asp Ala Ala Asp Phe Asp

115 120 125

Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly

130 135 140

Ser Gly Gly Gly Gly Ser Ala Val Gln Leu Val Glu Ser Gly Gly Gly

145 150 155 160

Leu Val Gln Ala Gly Asp Ser Leu Arg Leu Thr Cys Thr Ala Ser Gly

165 170 175

Arg Ala Phe Ser Thr Tyr Phe Met Ala Trp Phe Arg Gln Ala Pro Gly

180 185 190

Lys Glu Arg Glu Phe Val Ala Gly Ile Ala Trp Ser Gly Gly Ser Thr

195 200 205

Ala Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

210 215 220

Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu Asp

225 230 235 240

Thr Ala Val Tyr Tyr Cys Ala Ser Arg Gly Ile Glu Val Glu Glu Phe

245 250 255

Gly Ala Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Thr Ser Thr

260 265 270

Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile

275 280 285

Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala

290 295 300

Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr

305 310 315 320

Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu

325 330 335

Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile

340 345 350

Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp

355 360 365

Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Gly Gly Cys Glu Leu

370 375 380

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

385 390 395 400

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

405 410 415

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

420 425 430

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

435 440 445

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

450 455 460

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

465 470 475 480

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490 495

<210> 80

<211> 493

<212> PRT

<213> Artificial Sequence

<220>

<223> Anti-BCMA CAR

<400> 80

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu

20 25 30

Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Glu

35 40 45

Ser Val Ser Val Ile Gly Ala His Leu Ile His Trp Tyr Gln Gln Lys

50 55 60

Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu

65 70 75 80

Thr Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Ala Ala Ile Tyr Tyr

100 105 110

Cys Leu Gln Ser Arg Ile Phe Pro Arg Thr Phe Gly Gln Gly Thr Lys

115 120 125

Leu Glu Ile Lys Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly

130 135 140

Glu Gly Ser Thr Lys Gly Gln Val Gln Leu Val Gln Ser Gly Ser Glu

145 150 155 160

Leu Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly

165 170 175

Tyr Thr Phe Thr Asp Tyr Ser Ile Asn Trp Val Arg Gln Ala Pro Gly

180 185 190

Gln Gly Leu Glu Trp Met Gly Trp Ile Asn Thr Glu Thr Arg Glu Pro

195 200 205

Ala Tyr Ala Tyr Asp Phe Arg Gly Arg Phe Val Phe Ser Leu Asp Thr

210 215 220

Ser Val Ser Thr Ala Tyr Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp

225 230 235 240

Thr Ala Val Tyr Tyr Cys Ala Arg Asp Tyr Ser Tyr Ala Met Asp Tyr

245 250 255

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ala Ala Thr Thr

260 265 270

Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln

275 280 285

Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala

290 295 300

Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala

305 310 315 320

Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr

325 330 335

Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln

340 345 350

Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser

355 360 365

Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys

370 375 380

Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln

385 390 395 400

Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu

405 410 415

Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg

420 425 430

Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met

435 440 445

Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly

450 455 460

Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp

465 470 475 480

Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 81

<211> 99

<212> PRT

<213> Artificial Sequence

<220>

<223> Mature Forms of B2M

<400> 81

Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg His Pro Ala Glu

1 5 10 15

Asn Gly Lys Ser Asn Phe Leu Asn Cys Tyr Val Ser Gly Phe His Pro

20 25 30

Ser Asp Ile Glu Val Asp Leu Leu Lys Asn Gly Glu Arg Ile Glu Lys

35 40 45

Val Glu His Ser Asp Leu Ser Phe Ser Lys Asp Trp Ser Phe Tyr Leu

50 55 60

Leu Tyr Tyr Thr Glu Phe Thr Pro Thr Glu Lys Asp Glu Tyr Ala Cys

65 70 75 80

Arg Val Asn His Val Thr Leu Ser Gln Pro Lys Ile Val Lys Trp Asp

85 90 95

Arg Asp Met

<210> 82

<211> 233

<212> PRT

<213> Artificial Sequence

<220>

<223> People NKG2A

<400> 82

Met Asp Asn Gln Gly Val Ile Tyr Ser Asp Leu Asn Leu Pro Pro Asn

1 5 10 15

Pro Lys Arg Gln Gln Arg Lys Pro Lys Gly Asn Lys Ser Ser Ile Leu

20 25 30

Ala Thr Glu Gln Glu Ile Thr Tyr Ala Glu Leu Asn Leu Gln Lys Ala

35 40 45

Ser Gln Asp Phe Gln Gly Asn Asp Lys Thr Tyr His Cys Lys Asp Leu

50 55 60

Pro Ser Ala Pro Glu Lys Leu Ile Val Gly Ile Leu Gly Ile Ile Cys

65 70 75 80

Leu Ile Leu Met Ala Ser Val Val Thr Ile Val Val Ile Pro Ser Thr

85 90 95

Leu Ile Gln Arg His Asn Asn Ser Ser Leu Asn Thr Arg Thr Gln Lys

100 105 110

Ala Arg His Cys Gly His Cys Pro Glu Glu Trp Ile Thr Tyr Ser Asn

115 120 125

Ser Cys Tyr Tyr Ile Gly Lys Glu Arg Arg Thr Trp Glu Glu Ser Leu

130 135 140

Leu Ala Cys Thr Ser Lys Asn Ser Ser Leu Leu Ser Ile Asp Asn Glu

145 150 155 160

Glu Glu Met Lys Phe Leu Ser Ile Ile Ser Pro Ser Ser Trp Ile Gly

165 170 175

Val Phe Arg Asn Ser Ser His His Pro Trp Val Thr Met Asn Gly Leu

180 185 190

Ala Phe Lys His Glu Ile Lys Asp Ser Asp Asn Ala Glu Leu Asn Cys

195 200 205

Ala Val Leu Gln Val Asn Arg Leu Lys Ser Ala Gln Cys Gly Ser Ser

210 215 220

Ile Ile Tyr His Cys Lys His Lys Leu

225 230

<210> 83

<211> 348

<212> PRT

<213> Artificial Sequence

<220>

<223> KIR2DL1

<400> 83

Met Ser Leu Leu Val Val Ser Met Ala Cys Val Gly Phe Phe Leu Leu

1 5 10 15

Gln Gly Ala Trp Pro His Glu Gly Val His Arg Lys Pro Ser Leu Leu

20 25 30

Ala His Pro Gly Arg Leu Val Lys Ser Glu Glu Thr Val Ile Leu Gln

35 40 45

Cys Trp Ser Asp Val Met Phe Glu His Phe Leu Leu His Arg Glu Gly

50 55 60

Met Phe Asn Asp Thr Leu Arg Leu Ile Gly Glu His His Asp Gly Val

65 70 75 80

Ser Lys Ala Asn Phe Ser Ile Ser Arg Met Thr Gln Asp Leu Ala Gly

85 90 95

Thr Tyr Arg Cys Tyr Gly Ser Val Thr His Ser Pro Tyr Gln Val Ser

100 105 110

Ala Pro Ser Asp Pro Leu Asp Ile Val Ile Ile Gly Leu Tyr Glu Lys

115 120 125

Pro Ser Leu Ser Ala Gln Leu Gly Pro Thr Val Leu Ala Gly Glu Asn

130 135 140

Val Thr Leu Ser Cys Ser Ser Arg Ser Ser Tyr Asp Met Tyr His Leu

145 150 155 160

Ser Arg Glu Gly Glu Ala His Glu Arg Arg Leu Pro Ala Gly Pro Lys

165 170 175

Val Asn Gly Thr Phe Gln Ala Asp Phe Pro Leu Gly Pro Ala Thr His

180 185 190

Gly Gly Thr Tyr Arg Cys Phe Gly Ser Phe His Asp Ser Pro Tyr Glu

195 200 205

Trp Ser Lys Ser Ser Ser Asp Pro Leu Leu Val Ser Val Thr Gly Asn Pro

210 215 220

Ser Asn Ser Trp Pro Ser Pro Thr Glu Pro Ser Ser Lys Thr Gly Asn

225 230 235 240

Pro Arg His Leu His Ile Leu Ile Gly Thr Ser Val Val Ile Ile Leu

245 250 255

Phe Ile Leu Leu Phe Phe Leu Leu His His Trp Cys Ser Asn Lys Lys

260 265 270

Asn Ala Ala Val Met Asp Gln Glu Ser Ala Gly Asn Arg Thr Ala Asn

275 280 285

Ser Glu Asp Ser Asp Glu Gln Asp Pro Gln Glu Val Thr Tyr Thr Gln

290 295 300

Leu Asn His Cys Val Phe Thr Gln Arg Lys Ile Thr Arg Pro Ser Gln

305 310 315 320

Arg Pro Lys Thr Pro Pro Thr Asp Ile Ile Val Tyr Thr Glu Leu Pro

325 330 335

Asn Ala Glu Ser Arg Ser Lys Val Val Ser Cys Pro

340 345

<210> 84

<211> 348

<212> PRT

<213> Artificial Sequence

<220>

<223> KIR2DL2

<400> 84

Met Ser Leu Met Val Val Ser Met Ala Cys Val Gly Phe Phe Leu Leu

1 5 10 15

Gln Gly Ala Trp Pro His Glu Gly Val His Arg Lys Pro Ser Leu Leu

20 25 30

Ala His Pro Gly Arg Leu Val Lys Ser Glu Glu Thr Val Ile Leu Gln

35 40 45

Cys Trp Ser Asp Val Arg Phe Glu His Phe Leu Leu His Arg Glu Gly

50 55 60

Lys Phe Lys Asp Thr Leu His Leu Ile Gly Glu His His Asp Gly Val

65 70 75 80

Ser Lys Ala Asn Phe Ser Ile Gly Pro Met Met Gln Asp Leu Ala Gly

85 90 95

Thr Tyr Arg Cys Tyr Gly Ser Val Thr His Ser Pro Tyr Gln Leu Ser

100 105 110

Ala Pro Ser Asp Pro Leu Asp Ile Val Ile Thr Gly Leu Tyr Glu Lys

115 120 125

Pro Ser Leu Ser Ala Gln Pro Gly Pro Thr Val Leu Ala Gly Glu Ser

130 135 140

Val Thr Leu Ser Cys Ser Ser Arg Ser Ser Tyr Asp Met Tyr His Leu

145 150 155 160

Ser Arg Glu Gly Glu Ala His Glu Cys Arg Phe Ser Ala Gly Pro Lys

165 170 175

Val Asn Gly Thr Phe Gln Ala Asp Phe Pro Leu Gly Pro Ala Thr His

180 185 190

Gly Gly Thr Tyr Arg Cys Phe Gly Ser Phe Arg Asp Ser Pro Tyr Glu

195 200 205

Trp Ser Asn Ser Ser Asp Pro Leu Leu Val Ser Val Ile Gly Asn Pro

210 215 220

Ser Asn Ser Trp Pro Ser Pro Thr Glu Pro Ser Ser Lys Thr Gly Asn

225 230 235 240

Pro Arg His Leu His Ile Leu Ile Gly Thr Ser Val Val Ile Ile Leu

245 250 255

Phe Ile Leu Leu Phe Phe Leu Leu His Arg Trp Cys Ser Asn Lys Lys

260 265 270

Asn Ala Ala Val Met Asp Gln Glu Ser Ala Gly Asn Arg Thr Ala Asn

275 280 285

Ser Glu Asp Ser Asp Glu Gln Asp Pro Gln Glu Val Thr Tyr Thr Gln

290 295 300

Leu Asn His Cys Val Phe Thr Gln Arg Lys Ile Thr Arg Pro Ser Gln

305 310 315 320

Arg Pro Lys Thr Pro Pro Thr Asp Ile Ile Val Tyr Thr Glu Leu Pro

325 330 335

Asn Ala Glu Ser Arg Ser Lys Val Val Ser Cys Pro

340 345

<210> 85

<211> 341

<212> PRT

<213> Artificial Sequence

<220>

<223> KIR2DL3

<400> 85

Met Ser Leu Met Val Val Ser Met Val Cys Val Gly Phe Phe Leu Leu

1 5 10 15

Gln Gly Ala Trp Pro His Glu Gly Val His Arg Lys Pro Ser Leu Leu

20 25 30

Ala His Pro Gly Pro Leu Val Lys Ser Glu Glu Thr Val Ile Leu Gln

35 40 45

Cys Trp Ser Asp Val Arg Phe Gln His Phe Leu Leu His Arg Glu Gly

50 55 60

Lys Phe Lys Asp Thr Leu His Leu Ile Gly Glu His His Asp Gly Val

65 70 75 80

Ser Lys Ala Asn Phe Ser Ile Gly Pro Met Met Gln Asp Leu Ala Gly

85 90 95

Thr Tyr Arg Cys Tyr Gly Ser Val Thr His Ser Pro Tyr Gln Leu Ser

100 105 110

Ala Pro Ser Asp Pro Leu Asp Ile Val Ile Thr Gly Leu Tyr Glu Lys

115 120 125

Pro Ser Leu Ser Ala Gln Pro Gly Pro Thr Val Leu Ala Gly Glu Ser

130 135 140

Val Thr Leu Ser Cys Ser Ser Arg Ser Ser Tyr Asp Met Tyr His Leu

145 150 155 160

Ser Arg Glu Gly Glu Ala His Glu Arg Arg Phe Ser Ala Gly Pro Lys

165 170 175

Val Asn Gly Thr Phe Gln Ala Asp Phe Pro Leu Gly Pro Ala Thr His

180 185 190

Gly Gly Thr Tyr Arg Cys Phe Gly Ser Phe Arg Asp Ser Pro Tyr Glu

195 200 205

Trp Ser Asn Ser Ser Asp Pro Leu Leu Val Ser Val Thr Gly Asn Pro

210 215 220

Ser Asn Ser Trp Pro Ser Pro Thr Glu Pro Ser Ser Glu Thr Gly Asn

225 230 235 240

Pro Arg His Leu His Val Leu Ile Gly Thr Ser Val Val Ile Ile Leu

245 250 255

Phe Ile Leu Leu Leu Phe Phe Leu Leu His Arg Trp Cys Cys Asn Lys

260 265 270

Lys Asn Ala Val Val Met Asp Gln Glu Pro Ala Gly Asn Arg Thr Val

275 280 285

Asn Arg Glu Asp Ser Asp Glu Gln Asp Pro Gln Glu Val Thr Tyr Ala

290 295 300

Gln Leu Asn His Cys Val Phe Thr Gln Arg Lys Ile Thr Arg Pro Ser

305 310 315 320

Gln Arg Pro Lys Thr Pro Pro Thr Asp Ile Ile Val Tyr Thr Glu Leu

325 330 335

Pro Asn Ala Glu Pro

340

<210> 86

<211> 377

<212> PRT

<213> Artificial Sequence

<220>

<223> KIR2DL4

<400> 86

Met Ser Met Ser Pro Thr Val Ile Ile Leu Ala Cys Leu Gly Phe Phe

1 5 10 15

Leu Asp Gln Ser Val Trp Ala His Val Gly Gly Gln Asp Lys Pro Phe

20 25 30

Cys Ser Ala Trp Pro Ser Ala Val Val Pro Gln Gly Gly His Ala Thr

35 40 45

Leu Arg Cys His Cys Arg Arg Gly Phe Asn Ile Phe Thr Leu Tyr Lys

50 55 60

Lys Asp Gly Val Pro Val Pro Glu Leu Tyr Asn Arg Ile Phe Trp Asn

65 70 75 80

Ser Phe Leu Ile Ser Pro Val Thr Pro Ala His Ala Gly Thr Tyr Arg

85 90 95

Cys Arg Gly Phe His Pro His Ser Pro Thr Glu Trp Ser Ala Pro Ser

100 105 110

Asn Pro Leu Val Ile Met Val Thr Gly Leu Tyr Glu Lys Pro Ser Leu

115 120 125

Thr Ala Arg Pro Gly Pro Thr Val Arg Ala Gly Glu Asn Val Thr Leu

130 135 140

Ser Cys Ser Ser Gln Ser Ser Phe Asp Ile Tyr His Leu Ser Arg Glu

145 150 155 160

Gly Glu Ala His Glu Leu Arg Leu Pro Ala Val Pro Ser Ile Asn Gly

165 170 175

Thr Phe Gln Ala Asp Phe Pro Leu Gly Pro Ala Thr His Gly Glu Thr

180 185 190

Tyr Arg Cys Phe Gly Ser Phe His Gly Ser Pro Tyr Glu Trp Ser Asp

195 200 205

Pro Ser Asp Pro Leu Pro Val Ser Val Thr Gly Asn Pro Ser Ser Ser

210 215 220

Trp Pro Ser Pro Thr Glu Pro Ser Phe Lys Thr Gly Ile Ala Arg His

225 230 235 240

Leu His Ala Val Ile Arg Tyr Ser Val Ala Ile Ile Leu Phe Thr Ile

245 250 255

Leu Pro Phe Phe Leu Leu His Arg Trp Cys Ser Lys Lys Lys Asn Ala

260 265 270

Ala Val Met Asn Gln Glu Pro Ala Gly His Arg Thr Val Asn Arg Glu

275 280 285

Asp Ser Asp Glu Gln Asp Pro Gln Glu Val Thr Tyr Ala Gln Leu Asp

290 295 300

His Cys Ile Phe Thr Gln Arg Lys Ile Thr Gly Pro Ser Gln Arg Ser

305 310 315 320

Lys Arg Pro Ser Thr Asp Thr Ser Val Cys Ile Glu Leu Pro Asn Ala

325 330 335

Glu Pro Arg Ala Leu Ser Pro Ala His Glu His His Ser Gln Ala Leu

340 345 350

Met Gly Ser Ser Arg Glu Thr Thr Ala Leu Ser Gln Thr Gln Leu Ala

355 360 365

Ser Ser Asn Val Pro Ala Ala Gly Ile

370 375

<210> 87

<211> 375

<212> PRT

<213> Artificial Sequence

<220>

<223> KIR2DL5

<400> 87

Met Ser Leu Met Val Val Ser Met Ala Cys Val Gly Phe Phe Leu Leu

1 5 10 15

Gln Gly Ala Trp Thr His Glu Gly Gly Gln Asp Lys Pro Leu Leu Ser

20 25 30

Ala Trp Pro Ser Ala Val Val Pro Arg Gly Gly His Val Thr Leu Leu

35 40 45

Cys Arg Ser Arg Leu Gly Phe Thr Ile Phe Ser Leu Tyr Lys Glu Asp

50 55 60

Gly Val Pro Val Pro Glu Leu Tyr Asn Lys Ile Phe Trp Lys Ser Ile

65 70 75 80

Leu Met Gly Pro Val Thr Pro Ala His Ala Gly Thr Tyr Arg Cys Arg

85 90 95

Gly Ser His Pro Arg Ser Pro Ile Glu Trp Ser Ala Pro Ser Asn Pro

100 105 110

Leu Val Ile Val Val Thr Gly Leu Phe Gly Lys Pro Ser Leu Ser Ala

115 120 125

Gln Pro Gly Pro Thr Val Arg Thr Gly Glu Asn Val Thr Leu Ser Cys

130 135 140

Ser Ser Arg Ser Ser Phe Asp Met Tyr His Leu Ser Arg Glu Gly Arg

145 150 155 160

Ala His Glu Pro Arg Leu Pro Ala Val Pro Ser Val Asn Gly Thr Phe

165 170 175

Gln Ala Asp Phe Pro Leu Gly Pro Ala Thr His Gly Gly Thr Tyr Tyr

180 185 190

Cys Phe Gly Ser Leu His Asp Ser Pro Tyr Glu Trp Ser Asp Pro Ser

195 200 205

Asp Pro Leu Leu Val Ser Val Thr Gly Asn Ser Ser Ser Ser Ser Ser

210 215 220

Ser Pro Thr Glu Pro Ser Ser Lys Thr Gly Ile Arg Arg His Leu His

225 230 235 240

Ile Leu Ile Gly Thr Ser Val Ala Ile Ile Leu Phe Ile Ile Leu Phe

245 250 255

Phe Phe Leu Leu His Cys Cys Cys Ser Asn Lys Lys Asn Ala Ala Val

260 265 270

Met Asp Gln Glu Pro Ala Gly Asp Arg Thr Val Asn Arg Glu Asp Ser

275 280 285

Asp Asp Gln Asp Pro Gln Glu Val Thr Tyr Ala Gln Leu Asp His Cys

290 295 300

Val Phe Thr Gln Thr Lys Ile Thr Ser Pro Ser Gln Arg Pro Lys Thr

305 310 315 320

Pro Pro Thr Asp Thr Thr Met Tyr Met Glu Leu Pro Asn Ala Lys Pro

325 330 335

Arg Ser Leu Ser Pro Ala His Lys His His Ser Gln Ala Leu Arg Gly

340 345 350

Ser Ser Arg Glu Thr Thr Ala Leu Ser Gln Asn Arg Val Ala Ser Ser

355 360 365

His Val Pro Ala Ala Gly Ile

370 375

<210> 88

<211> 444

<212> PRT

<213> Artificial Sequence

<220>

<223> KIR3DL1

<400> 88

Met Leu Leu Met Val Val Ser Met Ala Cys Val Gly Phe Phe Leu Val

1 5 10 15

Gln Arg Ala Gly Pro His Val Gly Gly Gln Asp Lys Pro Phe Leu Ser

20 25 30

Ala Trp Pro Ser Ala Val Val Pro Arg Gly Gly His Val Thr Leu Arg

35 40 45

Cys His Tyr Arg His Arg Phe Asn Asn Phe Met Leu Tyr Lys Glu Asp

50 55 60

Arg Ile His Val Pro Ile Phe His Gly Arg Leu Phe Gln Glu Ser Phe

65 70 75 80

Asn Met Ser Pro Val Thr Thr Ala His Ala Gly Asn Tyr Thr Cys Arg

85 90 95

Gly Ser His Pro His Ser Pro Thr Gly Trp Ser Ala Pro Ser Asn Pro

100 105 110

Val Val Ile Met Val Thr Gly Asn His Arg Lys Pro Ser Leu Leu Ala

115 120 125

His Pro Gly Pro Leu Val Lys Ser Gly Glu Arg Val Ile Leu Gln Cys

130 135 140

Trp Ser Asp Ile Met Phe Glu His Phe Phe Leu His Lys Glu Gly Ile

145 150 155 160

Ser Lys Asp Pro Ser Arg Leu Val Gly Gln Ile His Asp Gly Val Ser

165 170 175

Lys Ala Asn Phe Ser Ile Gly Pro Met Met Leu Ala Leu Ala Gly Thr

180 185 190

Tyr Arg Cys Tyr Gly Ser Val Thr His Thr Pro Tyr Gln Leu Ser Ala

195 200 205

Pro Ser Asp Pro Leu Asp Ile Val Val Thr Gly Pro Tyr Glu Lys Pro

210 215 220

Ser Leu Ser Ala Gln Pro Gly Pro Lys Val Gln Ala Gly Glu Ser Val

225 230 235 240

Thr Leu Ser Cys Ser Arg Ser Ser Tyr Asp Met Tyr His Leu Ser

245 250 255

Arg Glu Gly Gly Ala His Glu Arg Arg Leu Pro Ala Val Arg Lys Val

260 265 270

Asn Arg Thr Phe Gln Ala Asp Phe Pro Leu Gly Pro Ala Thr His Gly

275 280 285

Gly Thr Tyr Arg Cys Phe Gly Ser Phe Arg His Ser Pro Tyr Glu Trp

290 295 300

Ser Asp Pro Ser Asp Pro Leu Leu Val Ser Val Thr Gly Asn Pro Ser

305 310 315 320

Ser Ser Trp Pro Ser Pro Thr Glu Pro Ser Ser Lys Ser Gly Asn Pro

325 330 335

Arg His Leu His Val Leu Ile Gly Thr Ser Val Val Ile Ile Leu Phe

340 345 350

Ile Leu Leu Leu Phe Phe Leu Leu His Leu Trp Cys Ser Asn Lys Lys

355 360 365

Asn Ala Ala Val Met Asp Gln Glu Pro Ala Gly Asn Arg Thr Ala Asn

370 375 380

Ser Glu Asp Ser Asp Glu Gln Asp Pro Gln Glu Val Thr Tyr Thr Gln

385 390 395 400

Leu Asn His Cys Val Phe Thr Gln Arg Lys Ile Thr Arg Pro Ser Gln

405 410 415

Arg Pro Lys Thr Pro Pro Thr Asp Thr Ile Leu Tyr Thr Glu Leu Pro

420 425 430

Asn Ala Lys Pro Arg Ser Lys Val Val Ser Cys Pro

435 440

<210> 89

<211> 455

<212> PRT

<213> Artificial Sequence

<220>

<223> KIR3DL2

<400> 89

Met Ser Leu Thr Val Val Ser Met Ala Cys Val Gly Phe Phe Leu Leu

1 5 10 15

Gln Gly Ala Trp Pro Leu Met Gly Gly Gln Asp Lys Pro Phe Leu Ser

20 25 30

Ala Arg Pro Ser Thr Val Val Pro Arg Gly Gly His Val Ala Leu Gln

35 40 45

Cys His Tyr Arg Arg Gly Phe Asn Asn Phe Met Leu Tyr Lys Glu Asp

50 55 60

Arg Ser His Val Pro Ile Phe His Gly Arg Ile Phe Gln Glu Ser Phe

65 70 75 80

Ile Met Gly Pro Val Thr Pro Ala His Ala Gly Thr Tyr Arg Cys Arg

85 90 95

Gly Ser Arg Pro His Ser Leu Thr Gly Trp Ser Ala Pro Ser Asn Pro

100 105 110

Leu Val Ile Met Val Thr Gly Asn His Arg Lys Pro Ser Leu Leu Ala

115 120 125

His Pro Gly Pro Leu Leu Lys Ser Gly Glu Thr Val Ile Leu Gln Cys

130 135 140

Trp Ser Asp Val Met Phe Glu His Phe Phe Leu His Arg Glu Gly Ile

145 150 155 160

Ser Glu Asp Pro Ser Arg Leu Val Gly Gln Ile His Asp Gly Val Ser

165 170 175

Lys Ala Asn Phe Ser Ile Gly Pro Leu Met Pro Val Leu Ala Gly Thr

180 185 190

Tyr Arg Cys Tyr Gly Ser Val Pro His Ser Pro Tyr Gln Leu Ser Ala

195 200 205

Pro Ser Asp Pro Leu Asp Ile Val Ile Thr Gly Leu Tyr Glu Lys Pro

210 215 220

Ser Leu Ser Ala Gln Pro Gly Pro Thr Val Gln Ala Gly Glu Asn Val

225 230 235 240

Thr Leu Ser Cys Ser Ser Trp Ser Ser Tyr Asp Ile Tyr His Leu Ser

245 250 255

Arg Glu Gly Glu Ala His Glu Arg Arg Leu Arg Ala Val Pro Lys Val

260 265 270

Asn Arg Thr Phe Gln Ala Asp Phe Pro Leu Gly Pro Ala Thr His Gly

275 280 285

Gly Thr Tyr Arg Cys Phe Gly Ser Phe His Ala Leu Pro Cys Val Trp

290 295 300

Ser Asn Ser Ser Asp Pro Leu Leu Val Ser Val Thr Gly Asn Pro Ser

305 310 315 320

Ser Ser Trp Pro Ser Pro Thr Glu Pro Ser Ser Lys Ser Gly Ile Cys

325 330 335

Arg His Leu His Val Leu Ile Gly Thr Ser Val Val Ile Phe Leu Phe

340 345 350

Ile Leu Leu Leu Phe Phe Leu Leu Tyr Arg Trp Cys Ser Asn Lys Lys

355 360 365

Asn Ala Ala Val Met Asp Gln Glu Pro Ala Gly Asp Arg Thr Val Asn

370 375 380

Arg Gln Asp Ser Asp Glu Gln Asp Pro Gln Glu Val Thr Tyr Ala Gln

385 390 395 400

Leu Asp His Cys Val Phe Ile Gln Arg Lys Ile Ser Arg Pro Ser Gln

405 410 415

Arg Pro Lys Thr Pro Leu Thr Asp Thr Ser Val Tyr Thr Glu Leu Pro

420 425 430

Asn Ala Glu Pro Arg Ser Lys Val Val Ser Cys Pro Arg Ala Pro Gln

435 440 445

Ser Gly Leu Glu Gly Val Phe

450 455

<210> 90

<211> 410

<212> PRT

<213> Artificial Sequence

<220>

<223> KIR3DL3

<400> 90

Met Ser Leu Met Val Val Ser Met Ala Cys Val Gly Phe Phe Leu Leu

1 5 10 15

Glu Gly Pro Trp Pro His Val Gly Gly Gln Asp Lys Pro Phe Leu Ser

20 25 30

Ala Trp Pro Ser Thr Val Val Ser Glu Gly Gln His Val Thr Leu Gln

35 40 45

Cys Arg Ser Arg Leu Gly Phe Asn Glu Phe Ser Leu Ser Lys Glu Asp

50 55 60

Gly Met Pro Val Pro Glu Leu Tyr Asn Arg Ile Phe Arg Asn Ser Phe

65 70 75 80

Leu Met Gly Pro Val Thr Pro Ala His Ala Gly Thr Tyr Arg Cys Cys

85 90 95

Ser Ser His Pro His Ser Pro Thr Gly Trp Ser Ala Pro Ser Asn Pro

100 105 110

Val Val Ile Met Val Thr Gly Val His Arg Lys Pro Ser Leu Leu Ala

115 120 125

His Pro Gly Pro Leu Val Lys Ser Gly Glu Thr Val Ile Leu Gln Cys

130 135 140

Trp Ser Asp Val Arg Phe Glu Arg Phe Leu Leu His Arg Glu Gly Ile

145 150 155 160

Thr Glu Asp Pro Leu Arg Leu Val Gly Gln Leu His Asp Ala Gly Ser

165 170 175

Gln Val Asn Tyr Ser Met Gly Pro Met Thr Pro Ala Leu Ala Gly Thr

180 185 190

Tyr Arg Cys Phe Gly Ser Val Thr His Leu Pro Tyr Glu Leu Ser Ala

195 200 205

Pro Ser Asp Pro Leu Asp Ile Val Val Val Gly Leu Tyr Gly Lys Pro

210 215 220

Ser Leu Ser Ala Gln Pro Gly Pro Thr Val Gln Ala Gly Glu Asn Val

225 230 235 240

Thr Leu Ser Cys Ser Arg Ser Leu Phe Asp Ile Tyr His Leu Ser

245 250 255

Arg Glu Ala Glu Ala Gly Glu Leu Arg Leu Thr Ala Val Leu Arg Val

260 265 270

Asn Gly Thr Phe Gln Ala Asn Phe Pro Leu Gly Pro Val Thr His Gly

275 280 285

Gly Asn Tyr Arg Cys Phe Gly Ser Phe Arg Ala Leu Pro His Ala Trp

290 295 300

Ser Asp Pro Ser Asp Pro Leu Pro Val Ser Val Thr Gly Asn Ser Arg

305 310 315 320

Tyr Leu His Ala Leu Ile Gly Thr Ser Val Val Ile Ile Pro Phe Ala

325 330 335

Ile Leu Leu Phe Phe Leu Leu His Arg Trp Cys Ala Asn Lys Lys Asn

340 345 350

Ala Val Val Met Asp Gln Glu Pro Ala Gly Asn Arg Thr Val Asn Arg

355 360 365

Glu Asp Ser Asp Glu Gln Asp Pro Gln Glu Val Thr Tyr Ala Gln Leu

370 375 380

Asn His Cys Val Phe Thr Gln Arg Lys Ile Thr Arg Pro Ser Gln Arg

385 390 395 400

Pro Lys Thr Pro Pro Thr Asp Thr Ser Val

405 410

<210> 91

<211> 650

<212> PRT

<213> Artificial Sequence

<220>

<223> LIR1

<400> 91

Met Thr Pro Ile Leu Thr Val Leu Ile Cys Leu Gly Leu Ser Leu Gly

1 5 10 15

Pro Arg Thr His Val Gln Ala Gly His Leu Pro Lys Pro Thr Leu Trp

20 25 30

Ala Glu Pro Gly Ser Val Ile Thr Gln Gly Ser Pro Val Thr Leu Arg

35 40 45

Cys Gln Gly Gly Gln Glu Thr Gln Glu Tyr Arg Leu Tyr Arg Glu Lys

50 55 60

Lys Thr Ala Leu Trp Ile Thr Arg Ile Pro Gln Glu Leu Val Lys Lys

65 70 75 80

Gly Gln Phe Pro Ile Pro Ser Ile Thr Trp Glu His Ala Gly Arg Tyr

85 90 95

Arg Cys Tyr Tyr Gly Ser Asp Thr Ala Gly Arg Ser Glu Ser Ser Asp

100 105 110

Pro Leu Glu Leu Val Val Thr Gly Ala Tyr Ile Lys Pro Thr Leu Ser

115 120 125

Ala Gln Pro Ser Pro Val Val Asn Ser Gly Gly Asn Val Ile Leu Gln

130 135 140

Cys Asp Ser Gln Val Ala Phe Asp Gly Phe Ser Leu Cys Lys Glu Gly

145 150 155 160

Glu Asp Glu His Pro Gln Cys Leu Asn Ser Gln Pro His Ala Arg Gly

165 170 175

Ser Ser Arg Ala Ile Phe Ser Val Gly Pro Val Ser Pro Ser Arg Arg

180 185 190

Trp Trp Tyr Arg Cys Tyr Ala Tyr Asp Ser Asn Ser Pro Tyr Glu Trp

195 200 205

Ser Leu Pro Ser Asp Leu Leu Glu Leu Leu Val Leu Gly Val Ser Lys

210 215 220

Lys Pro Ser Leu Ser Val Gln Pro Gly Pro Ile Val Ala Pro Glu Glu

225 230 235 240

Thr Leu Thr Leu Gln Cys Gly Ser Asp Ala Gly Tyr Asn Arg Phe Val

245 250 255

Leu Tyr Lys Asp Gly Glu Arg Asp Phe Leu Gln Leu Ala Gly Ala Gln

260 265 270

Pro Gln Ala Gly Leu Ser Gln Ala Asn Phe Thr Leu Gly Pro Val Ser

275 280 285

Arg Ser Tyr Gly Gly Gln Tyr Arg Cys Tyr Gly Ala His Asn Leu Ser

290 295 300

Ser Glu Trp Ser Ala Pro Ser Asp Pro Leu Asp Ile Leu Ile Ala Gly

305 310 315 320

Gln Phe Tyr Asp Arg Val Ser Leu Ser Val Gln Pro Gly Pro Thr Val

325 330 335

Ala Ser Gly Glu Asn Val Thr Leu Leu Cys Gln Ser Gln Gly Trp Met

340 345 350

Gln Thr Phe Leu Leu Thr Lys Glu Gly Ala Ala Asp Asp Pro Trp Arg

355 360 365

Leu Arg Ser Thr Tyr Tyr Gln Ser Gln Lys Tyr Gln Ala Glu Phe Pro Met

370 375 380

Gly Pro Val Thr Ser Ala His Ala Gly Thr Tyr Tyr Arg Cys Tyr Gly Ser

385 390 395 400

Gln Ser Ser Lys Pro Tyr Leu Leu Thr His Pro Ser Asp Pro Leu Glu

405 410 415

Leu Val Val Ser Gly Pro Ser Gly Gly Pro Ser Ser Pro Thr Thr Gly

420 425 430

Pro Thr Ser Thr Ser Gly Pro Glu Asp Gln Pro Leu Thr Pro Thr Gly

435 440 445

Ser Asp Pro Gln Ser Gly Leu Gly Arg His Leu Gly Val Val Ile Gly

450 455 460

Ile Leu Val Ala Val Ile Leu Leu Leu Leu Leu Leu Leu Leu Leu Phe

465 470 475 480

Leu Ile Leu Arg His Arg Arg Gln Gly Lys His Trp Thr Ser Thr Gln

485 490 495

Arg Lys Ala Asp Phe Gln His Pro Ala Gly Ala Val Gly Pro Glu Pro

500 505 510

Thr Asp Arg Gly Leu Gln Trp Arg Ser Ser Pro Ala Ala Asp Ala Gln

515 520 525

Glu Glu Asn Leu Tyr Ala Ala Val Lys His Thr Gln Pro Glu Asp Gly

530 535 540

Val Glu Met Asp Thr Arg Ser Pro His Asp Glu Asp Pro Gln Ala Val

545 550 555 560

Thr Tyr Ala Glu Val Lys His Ser Arg Pro Arg Arg Glu Met Ala Ser

565 570 575

Pro Pro Ser Pro Leu Ser Gly Glu Phe Leu Asp Thr Lys Asp Arg Gln

580 585 590

Ala Glu Glu Asp Arg Gln Met Asp Thr Glu Ala Ala Ala Ser Glu Ala

595 600 605

Pro Gln Asp Val Thr Tyr Ala Gln Leu His Ser Leu Thr Leu Arg Arg

610 615 620

Glu Ala Thr Glu Pro Pro Pro Ser Gln Glu Gly Pro Ser Pro Ala Val

625 630 635 640

Pro Ser Ile Tyr Ala Thr Leu Ala Ile His

645 650

<210> 92

<211> 164

<212> PRT

<213> Artificial Sequence

<220>

<223> CD3-zeta

<400> 92

Met Lys Trp Lys Ala Leu Phe Thr Ala Ala Ile Leu Gln Ala Gln Leu

1 5 10 15

Pro Ile Thr Glu Ala Gln Ser Phe Gly Leu Leu Asp Pro Lys Leu Cys

20 25 30

Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu Thr Ala

35 40 45

Leu Phe Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr

50 55 60

Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg

65 70 75 80

Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met

85 90 95

Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn

100 105 110

Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met

115 120 125

Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly

130 135 140

Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala

145 150 155 160

Leu Pro Pro Arg

<210> 93

<211> 339

<212> DNA

<213> Artificial Sequence

<220>

<223> CD3-zeta

<400> 93

agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttggggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgctaa 339

<210> 94

<211> 220

<212> PRT

<213> Artificial Sequence

<220>

<223> CD28

<400> 94

Met Leu Arg Leu Leu Leu Ala Leu Asn Leu Phe Pro Ser Ile Gln Val

1 5 10 15

Thr Gly Asn Lys Ile Leu Val Lys Gln Ser Pro Met Leu Val Ala Tyr

20 25 30

Asp Asn Ala Val Asn Leu Ser Cys Lys Tyr Ser Tyr Asn Leu Phe Ser

35 40 45

Arg Glu Phe Arg Ala Ser Leu His Lys Gly Leu Asp Ser Ala Val Glu

50 55 60

Val Cys Val Val Tyr Gly Asn Tyr Ser Gln Gln Leu Gln Val Tyr Ser

65 70 75 80

Lys Thr Gly Phe Asn Cys Asp Gly Lys Leu Gly Asn Glu Ser Val Thr

85 90 95

Phe Tyr Leu Gln Asn Leu Tyr Val Asn Gln Thr Asp Ile Tyr Phe Cys

100 105 110

Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser

115 120 125

Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro

130 135 140

Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly

145 150 155 160

Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile

165 170 175

Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met

180 185 190

Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro

195 200 205

Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser

210 215 220

<210> 95

<211> 321

<212> DNA

<213> Artificial Sequence

<220>

<223> CD28

<400> 95

attgaagtta tgtatcctcc tccttaccta gacaatgaga agagcaatgg aaccattatc 60

catgtgaaag ggaaacacct ttgtccaagt cccctatttc ccggaccttc taagcccttt 120

tgggtgctgg tggtggttgg tggagtcctg gcttgctata gcttgctagt aacagtggcc 180

tttattattt tctgggtgag gagtaagagg agcaggctcc tgcacagtga ctacatgaac 240

atgactcccc gccgccccgg gcccacccgc aagcattacc agccctatgc cccaccacgc 300

gacttcgcag cctatcgctc c 321

<210> 96

<211> 255

<212> PRT

<213> Artificial Sequence

<220>

<223> 4-1BB

<400> 96

Met Gly Asn Ser Cys Tyr Asn Ile Val Ala Thr Leu Leu Leu Val Leu

1 5 10 15

Asn Phe Glu Arg Thr Arg Ser Leu Gln Asp Pro Cys Ser Asn Cys Pro

20 25 30

Ala Gly Thr Phe Cys Asp Asn Asn Arg Asn Gln Ile Cys Ser Pro Cys

35 40 45

Pro Pro Asn Ser Phe Ser Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile

50 55 60

Cys Arg Gln Cys Lys Gly Val Phe Arg Thr Arg Lys Glu Cys Ser Ser

65 70 75 80

Thr Ser Asn Ala Glu Cys Asp Cys Thr Pro Gly Phe His Cys Leu Gly

85 90 95

Ala Gly Cys Ser Met Cys Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu

100 105 110

Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln

115 120 125

Lys Arg Gly Ile Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys

130 135 140

Ser Val Leu Val Asn Gly Thr Lys Glu Arg Asp Val Val Cys Gly Pro

145 150 155 160

Ser Pro Ala Asp Leu Ser Pro Gly Ala Ser Ser Val Thr Pro Pro Ala

165 170 175

Pro Ala Arg Glu Pro Gly His Ser Pro Gln Ile Ile Ser Phe Phe Leu

180 185 190

Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu

195 200 205

Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

210 215 220

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

225 230 235 240

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

245 250 255

<210> 97

<211> 277

<212> PRT

<213> Artificial Sequence

<220>

<223> OX40

<400> 97

Met Cys Val Gly Ala Arg Arg Leu Gly Arg Gly Pro Cys Ala Ala Leu

1 5 10 15

Leu Leu Leu Gly Leu Gly Leu Ser Thr Val Thr Gly Leu His Cys Val

20 25 30

Gly Asp Thr Tyr Pro Ser Asn Asp Arg Cys Cys His Glu Cys Arg Pro

35 40 45

Gly Asn Gly Met Val Ser Arg Cys Ser Arg Ser Gln Asn Thr Val Cys

50 55 60

Arg Pro Cys Gly Pro Gly Phe Tyr Asn Asp Val Val Ser Ser Lys Pro

65 70 75 80

Cys Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly Ser Glu Arg Lys

85 90 95

Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Arg Cys Arg Ala Gly

100 105 110

Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp Cys Ala Pro Cys

115 120 125

Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala Cys Lys Pro Trp

130 135 140

Thr Asn Cys Thr Leu Ala Gly Lys His Thr Leu Gln Pro Ala Ser Asn

145 150 155 160

Ser Ser Asp Ala Ile Cys Glu Asp Arg Asp Pro Pro Ala Thr Gln Pro

165 170 175

Gln Glu Thr Gln Gly Pro Pro Ala Arg Pro Ile Thr Val Gln Pro Thr

180 185 190

Glu Ala Trp Pro Arg Thr Ser Gln Gly Pro Ser Thr Arg Pro Val Glu

195 200 205

Val Pro Gly Gly Arg Ala Val Ala Ala Ile Leu Gly Leu Gly Leu Val

210 215 220

Leu Gly Leu Leu Gly Pro Leu Ala Ile Leu Leu Ala Leu Tyr Leu Leu

225 230 235 240

Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly

245 250 255

Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser

260 265 270

Thr Leu Ala Lys Ile

275

<210> 98

<211> 199

<212> PRT

<213> Artificial Sequence

<220>

<223> ICOS

<400> 98

Met Lys Ser Gly Leu Trp Tyr Phe Phe Leu Phe Cys Leu Arg Ile Lys

1 5 10 15

Val Leu Thr Gly Glu Ile Asn Gly Ser Ala Asn Tyr Glu Met Phe Ile

20 25 30

Phe His Asn Gly Gly Val Gln Ile Leu Cys Lys Tyr Pro Asp Ile Val

35 40 45

Gln Gln Phe Lys Met Gln Leu Leu Lys Gly Gly Gln Ile Leu Cys Asp

50 55 60

Leu Thr Lys Thr Lys Gly Ser Gly Asn Thr Val Ser Ile Lys Ser Leu

65 70 75 80

Lys Phe Cys His Ser Gln Leu Ser Asn Asn Ser Val Ser Phe Phe Leu

85 90 95

Tyr Asn Leu Asp His Ser His Ala Asn Tyr Tyr Phe Cys Asn Leu Ser

100 105 110

Ile Phe Asp Pro Pro Pro Phe Lys Val Thr Leu Thr Gly Gly Tyr Leu

115 120 125

His Ile Tyr Glu Ser Gln Leu Cys Cys Gln Leu Lys Phe Trp Leu Pro

130 135 140

Ile Gly Cys Ala Ala Phe Val Val Val Cys Ile Leu Gly Cys Ile Leu

145 150 155 160

Ile Cys Trp Leu Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro

165 170 175

Asn Gly Glu Tyr Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser

180 185 190

Arg Leu Thr Asp Val Thr Leu

195

<210> 99

<211> 93

<212> PRT

<213> Artificial Sequence

<220>

<223> DAP10

<400> 99

Met Ile His Leu Gly His Ile Leu Phe Leu Leu Leu Leu Pro Val Ala

1 5 10 15

Ala Ala Gln Thr Thr Pro Gly Glu Arg Ser Ser Leu Pro Ala Phe Tyr

20 25 30

Pro Gly Thr Ser Gly Ser Cys Ser Gly Cys Gly Ser Leu Ser Leu Pro

35 40 45

Leu Leu Ala Gly Leu Val Ala Ala Asp Ala Val Ala Ser Leu Leu Ile

50 55 60

Val Gly Ala Val Phe Leu Cys Ala Arg Pro Arg Arg Ser Pro Ala Gln

65 70 75 80

Glu Asp Gly Lys Val Tyr Ile Asn Met Pro Gly Arg Gly

85 90

<210> 100

<211> 260

<212> PRT

<213> Artificial Sequence

<220>

<223> CD27

<400> 100

Met Ala Arg Pro His Pro Trp Trp Leu Cys Val Leu Gly Thr Leu Val

1 5 10 15

Gly Leu Ser Ala Thr Pro Ala Pro Lys Ser Cys Pro Glu Arg His Tyr

20 25 30

Trp Ala Gln Gly Lys Leu Cys Cys Gln Met Cys Glu Pro Gly Thr Phe

35 40 45

Leu Val Lys Asp Cys Asp Gln His Arg Lys Ala Ala Gln Cys Asp Pro

50 55 60

Cys Ile Pro Gly Val Ser Phe Ser Pro Asp His His Thr Arg Pro His

65 70 75 80

Cys Glu Ser Cys Arg His Cys Asn Ser Gly Leu Leu Val Arg Asn Cys

85 90 95

Thr Ile Thr Ala Asn Ala Glu Cys Ala Cys Arg Asn Gly Trp Gln Cys

100 105 110

Arg Asp Lys Glu Cys Thr Glu Cys Asp Pro Leu Pro Asn Pro Ser Leu

115 120 125

Thr Ala Arg Ser Ser Gln Ala Leu Ser Pro His Pro Gln Pro Thr His

130 135 140

Leu Pro Tyr Val Ser Glu Met Leu Glu Ala Arg Thr Ala Gly His Met

145 150 155 160

Gln Thr Leu Ala Asp Phe Arg Gln Leu Pro Ala Arg Thr Leu Ser Thr

165 170 175

His Trp Pro Pro Gln Arg Ser Leu Cys Ser Ser Asp Phe Ile Arg Ile

180 185 190

Leu Val Ile Phe Ser Gly Met Phe Leu Val Phe Thr Leu Ala Gly Ala

195 200 205

Leu Phe Leu His Gln Arg Arg Lys Tyr Arg Ser Asn Lys Gly Glu Ser

210 215 220

Pro Val Glu Pro Ala Glu Pro Cys His Tyr Ser Cys Pro Arg Glu Glu

225 230 235 240

Glu Gly Ser Thr Ile Pro Ile Gln Glu Asp Tyr Arg Lys Pro Glu Pro

245 250 255

Ala Cys Ser Pro

260

<210> 101

<211> 595

<212> PRT

<213> Artificial Sequence

<220>

<223> CD30

<400> 101

Met Arg Val Leu Leu Ala Ala Leu Gly Leu Leu Phe Leu Gly Ala Leu

1 5 10 15

Arg Ala Phe Pro Gln Asp Arg Pro Phe Glu Asp Thr Cys His Gly Asn

20 25 30

Pro Ser His Tyr Tyr Asp Lys Ala Val Arg Arg Cys Cys Tyr Arg Cys

35 40 45

Pro Met Gly Leu Phe Pro Thr Gln Gln Cys Pro Gln Arg Pro Thr Asp

50 55 60

Cys Arg Lys Gln Cys Glu Pro Asp Tyr Tyr Leu Asp Glu Ala Asp Arg

65 70 75 80

Cys Thr Ala Cys Val Thr Cys Ser Arg Asp Asp Leu Val Glu Lys Thr

85 90 95

Pro Cys Ala Trp Asn Ser Ser Arg Val Cys Glu Cys Arg Pro Gly Met

100 105 110

Phe Cys Ser Thr Ser Ala Val Asn Ser Cys Ala Arg Cys Phe Phe His

115 120 125

Ser Val Cys Pro Ala Gly Met Ile Val Lys Phe Pro Gly Thr Ala Gln

130 135 140

Lys Asn Thr Val Cys Glu Pro Ala Ser Pro Gly Val Ser Pro Ala Cys

145 150 155 160

Ala Ser Pro Glu Asn Cys Lys Glu Pro Ser Ser Gly Thr Ile Pro Gln

165 170 175

Ala Lys Pro Thr Pro Val Ser Pro Ala Thr Ser Ser Ala Ser Thr Met

180 185 190

Pro Val Arg Gly Gly Thr Arg Leu Ala Gln Glu Ala Ala Ser Lys Leu

195 200 205

Thr Arg Ala Pro Asp Ser Pro Ser Ser Val Gly Arg Pro Ser Ser Asp

210 215 220

Pro Gly Leu Ser Pro Thr Gln Pro Cys Pro Glu Gly Ser Gly Asp Cys

225 230 235 240

Arg Lys Gln Cys Glu Pro Asp Tyr Tyr Leu Asp Glu Ala Gly Arg Cys

245 250 255

Thr Ala Cys Val Ser Cys Ser Arg Asp Asp Leu Val Glu Lys Thr Pro

260 265 270

Cys Ala Trp Asn Ser Ser Arg Thr Cys Glu Cys Arg Pro Gly Met Ile

275 280 285

Cys Ala Thr Ser Ala Thr Asn Ser Cys Ala Arg Cys Val Pro Tyr Pro

290 295 300

Ile Cys Ala Ala Glu Thr Val Thr Lys Pro Gln Asp Met Ala Glu Lys

305 310 315 320

Asp Thr Thr Phe Glu Ala Pro Pro Leu Gly Thr Gln Pro Asp Cys Asn

325 330 335

Pro Thr Pro Glu Asn Gly Glu Ala Pro Ala Ser Thr Ser Pro Thr Gln

340 345 350

Ser Leu Leu Val Asp Ser Gln Ala Ser Lys Thr Leu Pro Ile Pro Thr

355 360 365

Ser Ala Pro Val Ala Leu Ser Ser Thr Gly Lys Pro Val Leu Asp Ala

370 375 380

Gly Pro Val Leu Phe Trp Val Ile Leu Val Leu Val Val Val Val Gly

385 390 395 400

Ser Ser Ala Phe Leu Leu Cys His Arg Arg Ala Cys Arg Lys Arg Ile

405 410 415

Arg Gln Lys Leu His Leu Cys Tyr Pro Val Gln Thr Ser Gln Pro Lys

420 425 430

Leu Glu Leu Val Asp Ser Arg Pro Arg Arg Ser Ser Thr Gln Leu Arg

435 440 445

Ser Gly Ala Ser Val Thr Glu Pro Val Ala Glu Glu Arg Gly Leu Met

450 455 460

Ser Gln Pro Leu Met Glu Thr Cys His Ser Val Gly Ala Ala Tyr Leu

465 470 475 480

Glu Ser Leu Pro Leu Gln Asp Ala Ser Pro Ala Gly Gly Pro Ser Ser

485 490 495

Pro Arg Asp Leu Pro Glu Pro Arg Val Ser Thr Glu His Thr Asn Asn

500 505 510

Lys Ile Glu Lys Ile Tyr Ile Met Lys Ala Asp Thr Val Ile Val Gly

515 520 525

Thr Val Lys Ala Glu Leu Pro Glu Gly Arg Gly Leu Ala Gly Pro Ala

530 535 540

Glu Pro Glu Leu Glu Glu Glu Leu Glu Ala Asp His Thr Pro His Tyr

545 550 555 560

Pro Glu Gln Glu Thr Glu Pro Pro Leu Gly Ser Cys Ser Asp Val Met

565 570 575

Leu Ser Val Glu Glu Glu Gly Lys Glu Asp Pro Leu Pro Thr Ala Ala

580 585 590

Ser Gly Lys

595

<210> 102

<211> 277

<212> PRT

<213> Artificial Sequence

<220>

<223> CD40

<400> 102

Met Val Arg Leu Pro Leu Gln Cys Val Leu Trp Gly Cys Leu Leu Thr

1 5 10 15

Ala Val His Pro Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu

20 25 30

Ile Asn Ser Gln Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val

35 40 45

Ser Asp Cys Thr Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu

50 55 60

Ser Glu Phe Leu Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His

65 70 75 80

Lys Tyr Cys Asp Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr

85 90 95

Ser Glu Thr Asp Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr

100 105 110

Ser Glu Ala Cys Glu Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly

115 120 125

Phe Gly Val Lys Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu

130 135 140

Pro Cys Pro Val Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys

145 150 155 160

Cys His Pro Trp Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln

165 170 175

Ala Gly Thr Asn Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu

180 185 190

Arg Ala Leu Val Val Ile Pro Ile Ile Phe Gly Ile Leu Phe Ala Ile

195 200 205

Leu Leu Val Leu Val Phe Ile Lys Lys Val Ala Lys Lys Pro Thr Asn

210 215 220

Lys Ala Pro His Pro Lys Gln Glu Pro Gln Glu Ile Asn Phe Pro Asp

225 230 235 240

Asp Leu Pro Gly Ser Asn Thr Ala Ala Pro Val Gln Glu Thr Leu His

245 250 255

Gly Cys Gln Pro Val Thr Gln Glu Asp Gly Lys Glu Ser Arg Ile Ser

260 265 270

Val Gln Glu Arg Gln

275

<210> 103

<211> 235

<212> PRT

<213> Artificial Sequence

<220>

<223> CD8

<400> 103

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Ser Gln Phe Arg Val Ser Pro Leu Asp Arg Thr

20 25 30

Trp Asn Leu Gly Glu Thr Val Glu Leu Lys Cys Gln Val Leu Leu Ser

35 40 45

Asn Pro Thr Ser Gly Cys Ser Trp Leu Phe Gln Pro Arg Gly Ala Ala

50 55 60

Ala Ser Pro Thr Phe Leu Leu Tyr Leu Ser Gln Asn Lys Pro Lys Ala

65 70 75 80

Ala Glu Gly Leu Asp Thr Gln Arg Phe Ser Gly Lys Arg Leu Gly Asp

85 90 95

Thr Phe Val Leu Thr Leu Ser Asp Phe Arg Arg Glu Asn Glu Gly Tyr

100 105 110

Tyr Phe Cys Ser Ala Leu Ser Asn Ser Ile Met Tyr Phe Ser His Phe

115 120 125

Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg

130 135 140

Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg

145 150 155 160

Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly

165 170 175

Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr

180 185 190

Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His

195 200 205

Arg Asn Arg Arg Arg Val Cys Lys Cys Pro Arg Pro Val Val Lys Ser

210 215 220

Gly Asp Lys Pro Ser Leu Ser Ala Arg Tyr Val

225                 230                 235

<210> 104

<211> 458

<212> PRT

<213> Artificial Sequence

<220>

<223> CD4

<400> 104

Met Asn Arg Gly Val Pro Phe Arg His Leu Leu Leu Val Leu Gln Leu

1 5 10 15

Ala Leu Leu Pro Ala Ala Thr Gln Gly Lys Lys Val Val Leu Gly Lys

20 25 30

Lys Gly Asp Thr Val Glu Leu Thr Cys Thr Ala Ser Gln Lys Lys Ser

35 40 45

Ile Gln Phe His Trp Lys Asn Ser Asn Gln Ile Lys Ile Leu Gly Asn

50 55 60

Gln Gly Ser Phe Leu Thr Lys Gly Pro Ser Lys Leu Asn Asp Arg Ala

65 70 75 80

Asp Ser Arg Arg Ser Leu Trp Asp Gln Gly Asn Phe Pro Leu Ile Ile

85 90 95

Lys Asn Leu Lys Ile Glu Asp Ser Asp Thr Tyr Ile Cys Glu Val Glu

100 105 110

Asp Gln Lys Glu Glu Val Gln Leu Leu Val Phe Gly Leu Thr Ala Asn

115 120 125

Ser Asp Thr His Leu Leu Gln Gly Gln Ser Leu Thr Leu Thr Leu Glu

130 135 140

Ser Pro Pro Gly Ser Ser Pro Ser Val Gln Cys Arg Ser Pro Arg Gly

145 150 155 160

Lys Asn Ile Gln Gly Gly Lys Thr Leu Ser Val Ser Gln Leu Glu Leu

165 170 175

Gln Asp Ser Gly Thr Trp Thr Cys Thr Val Leu Gln Asn Gln Lys Lys

180 185 190

Val Glu Phe Lys Ile Asp Ile Val Val Leu Ala Phe Gln Lys Ala Ser

195 200 205

Ser Ile Val Tyr Lys Lys Glu Gly Glu Gln Val Glu Phe Ser Phe Pro

210 215 220

Leu Ala Phe Thr Val Glu Lys Leu Thr Gly Ser Gly Glu Leu Trp Trp

225 230 235 240

Gln Ala Glu Arg Ala Ser Ser Ser Lys Ser Trp Ile Thr Phe Asp Leu

245 250 255

Lys Asn Lys Glu Val Ser Val Lys Arg Val Thr Gln Asp Pro Lys Leu

260 265 270

Gln Met Gly Lys Lys Leu Pro Leu His Leu Thr Leu Pro Gln Ala Leu

275 280 285

Pro Gln Tyr Ala Gly Ser Gly Asn Leu Thr Leu Ala Leu Glu Ala Lys

290 295 300

Thr Gly Lys Leu His Gln Glu Val Asn Leu Val Val Met Arg Ala Thr

305 310 315 320

Gln Leu Gln Lys Asn Leu Thr Cys Glu Val Trp Gly Pro Thr Ser Pro

325 330 335

Lys Leu Met Leu Ser Leu Lys Leu Glu Asn Lys Glu Ala Lys Val Ser

340 345 350

Lys Arg Glu Lys Ala Val Trp Val Leu Asn Pro Glu Ala Gly Met Trp

355 360 365

Gln Cys Leu Leu Ser Asp Ser Gly Gln Val Leu Leu Glu Ser Asn Ile

370 375 380

Lys Val Leu Pro Thr Trp Ser Thr Pro Val Gln Pro Met Ala Leu Ile

385 390 395 400

Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly Leu Gly Ile

405 410 415

Phe Phe Cys Val Arg Cys Arg His Arg Arg Arg Gln Ala Glu Arg Met

420 425 430

Ser Gln Ile Lys Arg Leu Leu Ser Glu Lys Lys Thr Cys Gln Cys Pro

435 440 445

His Arg Phe Gln Lys Thr Cys Ser Pro Ile

450 455

<210> 105

<211> 22

<212> PRT

<213> Artificial Sequence

<220>

<223> 2A Self-cleaving Peptide - P2A

<220>

<221> MISC_Features

<222> (1)..(3)

<223> The GSG at the N-terminus of the 2A peptide is optional.

<400> 105

Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val

1 5 10 15

Glu Glu Asn Pro Gly Pro

20

<210> 106

<211> 21

<212> PRT

<213> Artificial Sequence

<220>

<223> 2A Self-cleaving Peptide - T2A

<220>

<221> MISC_Features

<222> (1)..(3)

<223> The GSG at the N-terminus of peptide 2A is optional.

<400> 106

Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu

1 5 10 15

Glu Asn Pro Gly Pro

20

<210> 107

<211> 23

<212> PRT

<213> Artificial Sequence

<220>

<223> 2A Self-cleaving Peptide - E2A

<220>

<221> MISC_Features

<222> (1)..(3)

<223> The GSG at the N-terminus of peptide 2A is optional.

<400> 107

Gly Ser Gly Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp

1 5 10 15

Val Glu Ser Asn Pro Gly Pro

20

<210> 108

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> 2A Self-cleaving Peptide - F2A

<220>

<221> MISC_Features

<222> (1)..(3)

<223> The GSG at the N-terminus of peptide 2A is optional.

<400> 108

Gly Ser Gly Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala

1 5 10 15

Gly Asp Val Glu Ser Asn Pro Gly Pro

20 25

<210> 109

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Connect subsequences

<220>

<221> Repeat

<222> (1)..(5)

<223> A 5-amino acid peptide can be repeated 1, 2, 3, 4, or 5 times.

<400> 109

Glu Ala Ala Ala Lys

1 5

<210> 110

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Connect subsequences

<220>

<221> Repeat

<222> (1)..(5)

<223> A 5-amino acid peptide can be repeated 3, 4, or 5 times.

<400> 110

Glu Ala Ala Ala Lys

1 5

<210> 111

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Connect subsequences

<220>

<221> Repeat

<222> (1)..(5)

<223> A 5-amino acid peptide can be repeated 1, 2, 3, 4, or 5 times.

<400> 111

Gly Gly Gly Gly Ser

1 5

<210> 112

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Connect subsequences

<220>

<221> Repeat

<222> (1)..(5)

<223> A 5-amino acid peptide can be repeated 3, 4, or 5 times.

<400> 112

Gly Gly Gly Gly Ser

1 5

<210> 113

<211> 2

<212> PRT

<213> Artificial Sequence

<220>

<223> Connect subsequences

<220>

<221> Repeat

<222> (1)..(1)

<223> G can be repeated 1, 2, 3, 4, or 5 times, but not less than 1.

<400> 113

Gly Ser

1

<210> 114

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> Connect subsequences

<400> 114

Gly Gly Gly Gly Ser Gly Gly Ser Gly Ser Gly Gly Gly Ser

1 5 10

<210> 115

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Common parts of the signal peptides of fusion proteins

<220>

<221> MISC_Features

<222> (1)..(1)

<223> X = Val or Ile

<220>

<221> MISC_Features

<222> (2)..(2)

<223> X = Thr, Ala, Met, or Leu

<220>

<221> MISC_Features

<222> (3)..(3)

<223> X = Ala, Pro, Lys, or Asn

<220>

<221> MISC_Features

<222> (4)..(4)

<223> X = Pro, Leu, or Thr

<220>

<221> MISC_Features

<222> (5)..(5)

<223> X = Arg, Gln, or Lys

<220>

<221> MISC_Features

<222> (6)..(6)

<223> X = Thr or Ala

<220>

<221> MISC_Features

<222> (7)..(7)

<223> X = Leu, Ile, Val, or Pro

<220>

<221> MISC_Features

<222> (8)..(8)

<223> X = Val, Leu, Ile, Phe, or Thr

<400> 115

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu

1 5

<210> 116

<211> 21

<212> PRT

<213> Artificial Sequence

<220>

<223> CD8

<400> 116

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 117

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> HLA-C restricted presentation peptide

<400> 117

Ile Ile Asp Lys Ser Gly Ser Thr Val

1 5

<210> 118

<211> 113

<212> PRT

<213> Artificial Sequence

<220>

<223> DAP12

<400> 118

Met Gly Gly Leu Glu Pro Cys Ser Arg Leu Leu Leu Leu Pro Leu Leu

1 5 10 15

Leu Ala Val Ser Gly Leu Arg Pro Val Gln Ala Gln Ala Gln Ser Asp

20 25 30

Cys Ser Cys Ser Thr Val Ser Pro Gly Val Leu Ala Gly Ile Val Met

35 40 45

Gly Asp Leu Val Leu Thr Val Leu Ile Ala Leu Ala Val Tyr Phe Leu

50 55 60

Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Ala Glu Ala Ala Thr Arg

65 70 75 80

Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly

85 90 95

Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr

100 105 110

Lys

<210> 119

<211> 216

<212> PRT

<213> Artificial Sequence

<220>

<223> NKG2D

<400> 119

Met Gly Trp Ile Arg Gly Arg Arg Ser Arg His Ser Trp Glu Met Ser

1 5 10 15

Glu Phe His Asn Tyr Asn Leu Asp Leu Lys Lys Ser Asp Phe Ser Thr

20 25 30

Arg Trp Gln Lys Gln Arg Cys Pro Val Val Lys Ser Lys Cys Arg Glu

35 40 45

Asn Ala Ser Pro Phe Phe Phe Cys Cys Phe Ile Ala Val Ala Met Gly

50 55 60

Ile Arg Phe Ile Ile Met Val Ala Ile Trp Ser Ala Val Phe Leu Asn

65 70 75 80

Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys

85 90 95

Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln

100 105 110

Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met

115 120 125

Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp

130 135 140

Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile

145 150 155 160

Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro

165 170 175

Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr

180 185 190

Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr

195 200 205

Tyr Ile Cys Met Gln Arg Thr Val

210 215

<210> 120

<211> 567

<212> PRT

<213> Artificial Sequence

<220>

<223> Megf10

<400> 120

Met Val Ile Ser Leu Asn Ser Cys Leu Ser Phe Ile Cys Leu Leu Leu

1 5 10 15

Cys His Trp Ile Gly Thr Ala Ser Pro Leu Asn Leu Glu Asp Pro Asn

20 25 30

Val Cys Ser His Trp Glu Ser Tyr Ser Val Thr Val Gln Glu Ser Tyr

35 40 45

Pro His Pro Phe Asp Gln Ile Tyr Tyr Thr Ser Cys Thr Asp Ile Leu

50 55 60

Asn Trp Phe Lys Cys Thr Arg His Arg Val Ser Tyr Arg Thr Ala Tyr

65 70 75 80

Arg His Gly Glu Lys Thr Met Tyr Arg Arg Lys Ser Gln Cys Cys Pro

85 90 95

Gly Phe Tyr Glu Ser Gly Glu Met Cys Val Pro His Cys Ala Asp Lys

100 105 110

Cys Val His Gly Arg Cys Ile Ala Pro Asn Thr Cys Gln Cys Glu Pro

115 120 125

Gly Trp Gly Gly Thr Asn Cys Ser Ser Ala Cys Asp Gly Asp His Trp

130 135 140

Gly Pro His Cys Thr Ser Arg Cys Gln Cys Lys Asn Gly Ala Leu Cys

145 150 155 160

Asn Pro Ile Thr Gly Ala Cys His Cys Ala Ala Gly Phe Arg Gly Trp

165 170 175

Arg Cys Glu Asp Arg Cys Glu Gln Gly Thr Tyr Gly Asn Asp Cys His

180 185 190

Gln Arg Cys Gln Cys Gln Asn Gly Ala Thr Cys Asp His Val Thr Gly

195 200 205

Glu Cys Arg Cys Pro Pro Gly Tyr Thr Gly Ala Phe Cys Glu Asp Leu

210 215 220

Cys Pro Pro Gly Lys His Gly Pro Gln Cys Glu Gln Arg Cys Pro Cys

225 230 235 240

Gln Asn Gly Gly Val Cys His His Val Thr Gly Glu Cys Ser Cys Pro

245 250 255

Ser Gly Trp Met Gly Thr Val Cys Gly Gln Pro Cys Pro Glu Gly Arg

260 265 270

Phe Gly Lys Asn Cys Ser Gln Glu Cys Gln Cys His Asn Gly Gly Thr

275 280 285

Cys Asp Ala Ala Thr Gly Gln Cys His Cys Ser Pro Gly Tyr Thr Gly

290 295 300

Glu Arg Cys Gln Asp Glu Cys Pro Val Gly Thr Tyr Gly Val Leu Cys

305 310 315 320

Ala Glu Thr Cys Gln Cys Val Asn Gly Gly Lys Cys Tyr His Val Ser

325 330 335

Gly Ala Cys Leu Cys Glu Ala Gly Phe Ala Gly Glu Arg Cys Glu Ala

340 345 350

Arg Leu Cys Pro Glu Gly Leu Tyr Gly Ile Lys Cys Asp Lys Arg Cys

355 360 365

Pro Cys His Leu Glu Asn Thr His Ser Cys His Pro Met Ser Gly Glu

370 375 380

Cys Ala Cys Lys Pro Gly Trp Ser Gly Leu Tyr Cys Asn Glu Thr Cys

385 390 395 400

Ser Pro Gly Phe Tyr Gly Glu Ala Cys Gln Gln Ile Cys Ser Cys Gln

405 410 415

Asn Gly Ala Asp Cys Asp Ser Val Thr Gly Lys Cys Thr Cys Ala Pro

420 425 430

Gly Phe Lys Gly Ile Asp Cys Ser Thr Pro Cys Pro Leu Gly Thr Tyr

435 440 445

Gly Ile Asn Cys Ser Ser Arg Cys Gly Cys Lys Asn Asp Ala Val Cys

450 455 460

Ser Pro Val Asp Gly Ser Cys Thr Cys Lys Ala Gly Trp His Gly Val

465 470 475 480

Asp Cys Ser Ile Arg Cys Pro Ser Gly Thr Trp Gly Phe Gly Cys Asn

485 490 495

Leu Thr Cys Gln Cys Leu Asn Gly Gly Ala Cys Asn Thr Leu Asp Gly

500 505 510

Thr Cys Thr Cys Ala Pro Gly Trp Arg Gly Glu Lys Cys Glu Leu Pro

515 520 525

Cys Gln Asp Gly Thr Tyr Gly Leu Asn Cys Ala Glu Arg Cys Asp Cys

530 535 540

Ser His Ala Asp Gly Cys His Pro Thr Thr Gly His Cys Arg Cys Leu

545 550 555 560

Pro Gly Trp Ser Gly Leu Phe

565

<210> 121

<211> 86

<212> PRT

<213> Artificial Sequence

<220>

<223> FcR-gamma

<400> 121

Met Ile Pro Ala Val Val Leu Leu Leu Leu Leu Leu Val Glu Gln Ala

1 5 10 15

Ala Ala Leu Gly Glu Pro Gln Leu Cys Tyr Ile Leu Asp Ala Ile Leu

20 25 30

Phe Leu Tyr Gly Ile Val Leu Thr Leu Leu Tyr Cys Arg Leu Lys Ile

35 40 45

Gln Val Arg Lys Ala Ala Ile Thr Ser Tyr Glu Lys Ser Asp Gly Val

50 55 60

Tyr Thr Gly Leu Ser Thr Arg Asn Gln Glu Thr Tyr Glu Thr Leu Lys

65 70 75 80

His Glu Lys Pro Pro Gln

85

<210> 122

<211> 2

<212> PRT

<213> Artificial Sequence

<220>

<223> Connect subsequences

<220>

<221> Repeat

<222> (1)..(2)

<223> (GnS)m; n=1, 2, 3, 4, and 5; m=1, 2, 3, 4, or 5;

In (GnS), G can be repeated 1, 2, 3, 4, or 5 times, but not less than 1.

(GnS) can be repeated 1, 2, 3, 4, or 5 times, but not less than 1.

<400> 122

Gly Ser

1

<210> 123

<211> 1461

<212> DNA

<213> Artificial Sequence

<220>

<223> CJP CAR19 DNA Sequence

<400> 123

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggacatcc agatgacaca gactacatcc tccctgtctg cctctctggg agacagagtc 120

accatcagtt gcagggcaag tcaggacatt agtaaatatt taaattggta tcagcagaaa 180

ccagatggaa ctgttaaact cctgatctac catacatcaa gattacactc aggagtccca 240

tcaaggttca gtggcagtgg gtctggaaca gattattctc tcaccattag caacctggag 300

caagaagata ttgccactta cttttgccaa cagggtaata cgcttccgta cacgttcgga 360

ggggggacca agctggagat cacaggtggc ggtggctcgg gcggtggtgg gtcgggtggc 420

ggcggatctg aggtgaaact gcaggagtca ggacctggcc tggtggcgcc ctcacagagc 480

ctgtccgtca catgcactgt ctcaggggtc tcattacccg actatggtgt aagctggatt 540

cgccagcctc cacgaaaggg tctggagtgg ctgggagtaa tatggggtag tgaaaccaca 600

tactataatt cagctctcaa atccagactg accatcatca aggacaactc caagagccaa 660

gttttcttaa aaatgaacag tctgcaaact gatgacacag ccatttacta ctgtgccaaa 720

cattattact acggtggtag ctatgctatg gactactggg gccaaggaac ctcagtcacc 780

gtctcctcaa ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 840

cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 900

agggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 960

gtccttctcc tgtcactggt tatcaccctt tactgcaaac ggggcagaaa gaaactcctg 1020

tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt 1080

agctgccgat ttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 1140

agcgcagacg cccccgcgta caagcagggc cagaaccagc tctataacga gctcaatcta 1200

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1260

ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1320

atggcggagg cctacagtga gattggggatg aaaggcgagc gccggagggg caaggggcac 1380

gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1440

caggccctgccccctcgcta a 1461

<210> 124

<211> 656

<212> PRT

<213> Artificial Sequence

<220>

<223> CJP-P2A-CM amino acid sequence

<400> 124

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu

20 25 30

Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr

50 55 60

Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro

65 70 75 80

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile

85 90 95

Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

130 135 140

Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser

145 150 155 160

Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Tyr Asn Ser Ala Leu Lys Ser

195 200 205

Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys

210 215 220

Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

245 250 255

Thr Ser Val Thr Val Ser Ser Thr Thr Thr Thr Pro Ala Pro Arg Pro Pro

260 265 270

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

275 280 285

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

290 295 300

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg

325 330 335

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln

340 345 350

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu

355 360 365

Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

370 375 380

Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

385 390 395 400

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

405 410 415

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

420 425 430

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

435 440 445

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

450 455 460

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

465 470 475 480

Gln Ala Leu Pro Pro Arg Gly Arg Arg Lys Arg Gly Ser Gly Ala Thr

485 490 495

Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly

500 505 510

Pro Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu

515 520 525

Ser Gly Leu Glu Ala Val Met Ala Pro Arg Thr Val Leu Leu Gly Gly

530 535 540

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Gln Arg

545 550 555 560

Thr Pro Lys Ile Gln Val Tyr Ser Arg His Pro Ala Glu Asn Gly Lys

565 570 575

Ser Asn Phe Leu Asn Cys Tyr Val Ser Gly Phe His Pro Ser Asp Ile

580 585 590

Glu Val Asp Leu Leu Lys Asn Gly Glu Arg Ile Glu Lys Val Glu His

595 600 605

Ser Asp Leu Ser Phe Ser Lys Asp Trp Ser Phe Tyr Leu Leu Tyr Tyr

610 615 620

Thr Glu Phe Thr Pro Thr Glu Lys Asp Glu Tyr Ala Cys Arg Val Asn

625 630 635 640

His Val Thr Leu Ser Gln Pro Lys Ile Val Lys Trp Asp Arg Asp Met

645 650 655

<210> 125

<211> 1971

<212> DNA

<213> Artificial Sequence

<220>

<223> CJP-P2A-CM DNA sequence

<400> 125

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggacatcc agatgacaca gactacatcc tccctgtctg cctctctggg agacagagtc 120

accatcagtt gcagggcaag tcaggacatt agtaaatatt taaattggta tcagcagaaa 180

ccagatggaa ctgttaaact cctgatctac catacatcaa gattacactc aggagtccca 240

tcaaggttca gtggcagtgg gtctggaaca gattattctc tcaccattag caacctggag 300

caagaagata ttgccactta cttttgccaa cagggtaata cgcttccgta cacgttcgga 360

ggggggacca agctggagat cacaggtggc ggtggctcgg gcggtggtgg gtcgggtggc 420

ggcggatctg aggtgaaact gcaggagtca ggacctggcc tggtggcgcc ctcacagagc 480

ctgtccgtca catgcactgt ctcaggggtc tcattacccg actatggtgt aagctggatt 540

cgccagcctc cacgaaaggg tctggagtgg ctgggagtaa tatggggtag tgaaaccaca 600

tactataatt cagctctcaa atccagactg accatcatca aggacaactc caagagccaa 660

gttttcttaa aaatgaacag tctgcaaact gatgacacag ccatttacta ctgtgccaaa 720

cattattact acggtggtag ctatgctatg gactactggg gccaaggaac ctcagtcacc 780

gtctcctcaa ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 840

cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 900

agggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 960

gtccttctcc tgtcactggt tatcaccctt tactgcaaac ggggcagaaa gaaactcctg 1020

tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt 1080

agctgccgat ttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 1140

agcgcagacg cccccgcgta caagcagggc cagaaccagc tctataacga gctcaatcta 1200

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1260

ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1320

atggcggagg cctacagtga gattggggatg aaaggcgagc gccggagggg caaggggcac 1380

gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1440

caggccctgc cccctcgcgg ccgccgcaag cgcggctccg gtgccacgaa cttctctctg 1500

ttaaagcaag caggagacgt ggaagaaaac cccggtccta tgtctcgctc cgtggcattg 1560

gctgtgctcg cgctactctc tctttctggt ctcgaagctg ttatggctcc gcggactgtg 1620

ctgttaggtg gtggcggttc cggtggtggc ggttctggtg gtggcggctc catccagcgt 1680

acgccaaaga ttcaggttta ctcacgtcat ccagcagaga atggaaagtc aaatttcctg 1740

aattgctatg tgtctgggtt tcatccatcc gacattgaag ttgacttact gaagaatgga 1800

gagagaattg aaaaagtgga gcattcagac ttgtctttca gcaaggactg gtctttctat 1860

ctcttgtact acactgaatt caccccccact gaaaaagatg agtatgcctg ccgtgtgaac 1920

catgtgactt tgtcacagcc caagatagtt aagtggggatc gcgacatgta a 1971

<210> 126

<211> 656

<212> PRT

<213> Artificial Sequence

<220>

<223> CJP-P2A-CM12 amino acid sequence

<400> 126

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu

20 25 30

Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr

50 55 60

Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro

65 70 75 80

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile

85 90 95

Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

130 135 140

Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser

145 150 155 160

Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Tyr Asn Ser Ala Leu Lys Ser

195 200 205

Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys

210 215 220

Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

245 250 255

Thr Ser Val Thr Val Ser Ser Thr Thr Thr Thr Pro Ala Pro Arg Pro Pro

260 265 270

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

275 280 285

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

290 295 300

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg

325 330 335

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln

340 345 350

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu

355 360 365

Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

370 375 380

Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

385 390 395 400

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

405 410 415

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

420 425 430

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

435 440 445

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

450 455 460

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

465 470 475 480

Gln Ala Leu Pro Pro Arg Gly Arg Arg Lys Arg Gly Ser Gly Ala Thr

485 490 495

Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly

500 505 510

Pro Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu

515 520 525

Ser Gly Leu Glu Ala Val Met Ala Pro Arg Thr Leu Phe Leu Gly Gly

530 535 540

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Gln Arg

545 550 555 560

Thr Pro Lys Ile Gln Val Tyr Ser Arg His Pro Ala Glu Asn Gly Lys

565 570 575

Ser Asn Phe Leu Asn Cys Tyr Val Ser Gly Phe His Pro Ser Asp Ile

580 585 590

Glu Val Asp Leu Leu Lys Asn Gly Glu Arg Ile Glu Lys Val Glu His

595 600 605

Ser Asp Leu Ser Phe Ser Lys Asp Trp Ser Phe Tyr Leu Leu Tyr Tyr

610 615 620

Thr Glu Phe Thr Pro Thr Glu Lys Asp Glu Tyr Ala Cys Arg Val Asn

625 630 635 640

His Val Thr Leu Ser Gln Pro Lys Ile Val Lys Trp Asp Arg Asp Met

645 650 655

<210> 127

<211> 1971

<212> DNA

<213> Artificial Sequence

<220>

<223> CJP-P2A-CM12 DNA sequence

<400> 127

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggacatcc agatgacaca gactacatcc tccctgtctg cctctctggg agacagagtc 120

accatcagtt gcagggcaag tcaggacatt agtaaatatt taaattggta tcagcagaaa 180

ccagatggaa ctgttaaact cctgatctac catacatcaa gattacactc aggagtccca 240

tcaaggttca gtggcagtgg gtctggaaca gattattctc tcaccattag caacctggag 300

caagaagata ttgccactta cttttgccaa cagggtaata cgcttccgta cacgttcgga 360

ggggggacca agctggagat cacaggtggc ggtggctcgg gcggtggtgg gtcgggtggc 420

ggcggatctg aggtgaaact gcaggagtca ggacctggcc tggtggcgcc ctcacagagc 480

ctgtccgtca catgcactgt ctcaggggtc tcattacccg actatggtgt aagctggatt 540

cgccagcctc cacgaaaggg tctggagtgg ctgggagtaa tatggggtag tgaaaccaca 600

tactataatt cagctctcaa atccagactg accatcatca aggacaactc caagagccaa 660

gttttcttaa aaatgaacag tctgcaaact gatgacacag ccatttacta ctgtgccaaa 720

cattattact acggtggtag ctatgctatg gactactggg gccaaggaac ctcagtcacc 780

gtctcctcaa ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 840

cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 900

agggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 960

gtccttctcc tgtcactggt tatcaccctt tactgcaaac ggggcagaaa gaaactcctg 1020

tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt 1080

agctgccgat ttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 1140

agcgcagacg cccccgcgta caagcagggc cagaaccagc tctataacga gctcaatcta 1200

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1260

ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1320

atggcggagg cctacagtga gattggggatg aaaggcgagc gccggagggg caaggggcac 1380

gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1440

caggccctgc cccctcgcgg ccgccgcaag cgcggctccg gtgccacgaa cttctctctg 1500

ttaaagcaag caggagacgt ggaagaaaac cccggtccta tgtctcgctc cgtggcattg 1560

gctgtgctcg cgctactctc tctttctggt ctcgaagctg ttatggctcc gcggactctg 1620

ttcttaggtg gtggcggttc cggtggtggc ggttctggtg gtggcggctc catccagcgt 1680

acgccaaaga ttcaggttta ctcacgtcat ccagcagaga atggaaagtc aaatttcctg 1740

aattgctatg tgtctgggtt tcatccatcc gacattgaag ttgacttact gaagaatgga 1800

gagagaattg aaaaagtgga gcattcagac ttgtctttca gcaaggactg gtctttctat 1860

ctcttgtact acactgaatt caccccccact gaaaaagatg agtatgcctg ccgtgtgaac 1920

catgtgactt tgtcacagcc caagatagtt aagtggggatc gcgacatgta a 1971

<210> 128

<211> 656

<212> PRT

<213> Artificial Sequence

<220>

<223> CJP-P2A-CM13 amino acid sequence

<400> 128

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu

20 25 30

Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr

50 55 60

Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro

65 70 75 80

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile

85 90 95

Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

130 135 140

Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser

145 150 155 160

Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Tyr Asn Ser Ala Leu Lys Ser

195 200 205

Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys

210 215 220

Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

245 250 255

Thr Ser Val Thr Val Ser Ser Thr Thr Thr Thr Pro Ala Pro Arg Pro Pro

260 265 270

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

275 280 285

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

290 295 300

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg

325 330 335

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln

340 345 350

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu

355 360 365

Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

370 375 380

Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

385 390 395 400

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

405 410 415

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

420 425 430

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

435 440 445

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

450 455 460

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

465 470 475 480

Gln Ala Leu Pro Pro Arg Gly Arg Arg Lys Arg Gly Ser Gly Ala Thr

485 490 495

Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly

500 505 510

Pro Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu

515 520 525

Ser Gly Leu Glu Ala Val Met Ala Pro Arg Thr Leu Val Leu Gly Gly

530 535 540

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Gln Arg

545 550 555 560

Thr Pro Lys Ile Gln Val Tyr Ser Arg His Pro Ala Glu Asn Gly Lys

565 570 575

Ser Asn Phe Leu Asn Cys Tyr Val Ser Gly Phe His Pro Ser Asp Ile

580 585 590

Glu Val Asp Leu Leu Lys Asn Gly Glu Arg Ile Glu Lys Val Glu His

595 600 605

Ser Asp Leu Ser Phe Ser Lys Asp Trp Ser Phe Tyr Leu Leu Tyr Tyr

610 615 620

Thr Glu Phe Thr Pro Thr Glu Lys Asp Glu Tyr Ala Cys Arg Val Asn

625 630 635 640

His Val Thr Leu Ser Gln Pro Lys Ile Val Lys Trp Asp Arg Asp Met

645 650 655

<210> 129

<211> 1971

<212> DNA

<213> Artificial Sequence

<220>

<223> CJP-P2A-CM13 DNA sequence

<400> 129

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggacatcc agatgacaca gactacatcc tccctgtctg cctctctggg agacagagtc 120

accatcagtt gcagggcaag tcaggacatt agtaaatatt taaattggta tcagcagaaa 180

ccagatggaa ctgttaaact cctgatctac catacatcaa gattacactc aggagtccca 240

tcaaggttca gtggcagtgg gtctggaaca gattattctc tcaccattag caacctggag 300

caagaagata ttgccactta cttttgccaa cagggtaata cgcttccgta cacgttcgga 360

ggggggacca agctggagat cacaggtggc ggtggctcgg gcggtggtgg gtcgggtggc 420

ggcggatctg aggtgaaact gcaggagtca ggacctggcc tggtggcgcc ctcacagagc 480

ctgtccgtca catgcactgt ctcaggggtc tcattacccg actatggtgt aagctggatt 540

cgccagcctc cacgaaaggg tctggagtgg ctgggagtaa tatggggtag tgaaaccaca 600

tactataatt cagctctcaa atccagactg accatcatca aggacaactc caagagccaa 660

gttttcttaa aaatgaacag tctgcaaact gatgacacag ccatttacta ctgtgccaaa 720

cattattact acggtggtag ctatgctatg gactactggg gccaaggaac ctcagtcacc 780

gtctcctcaa ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 840

cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 900

agggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 960

gtccttctcc tgtcactggt tatcaccctt tactgcaaac ggggcagaaa gaaactcctg 1020

tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt 1080

agctgccgat ttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 1140

agcgcagacg cccccgcgta caagcagggc cagaaccagc tctataacga gctcaatcta 1200

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1260

ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1320

atggcggagg cctacagtga gattggggatg aaaggcgagc gccggagggg caaggggcac 1380

gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1440

caggccctgc cccctcgcgg ccgccgcaag cgcggctccg gtgccacgaa cttctctctg 1500

ttaaagcaag caggagacgt ggaagaaaac cccggtccta tgtctcgctc cgtggcattg 1560

gctgtgctcg cgctactctc tctttctggt ctcgaagctg ttatggctcc gcggactctg 1620

gtgttaggtg gtggcggttc cggtggtggc ggttctggtg gtggcggctc catccagcgt 1680

acgccaaaga ttcaggttta ctcacgtcat ccagcagaga atggaaagtc aaatttcctg 1740

aattgctatg tgtctgggtt tcatccatcc gacattgaag ttgacttact gaagaatgga 1800

gagagaattg aaaaagtgga gcattcagac ttgtctttca gcaaggactg gtctttctat 1860

ctcttgtact acactgaatt caccccccact gaaaaagatg agtatgcctg ccgtgtgaac 1920

catgtgactt tgtcacagcc caagatagtt aagtggggatc gcgacatgta a 1971

<210> 130

<211> 656

<212> PRT

<213> Artificial Sequence

<220>

<223> CJP-P2A-CM14 amino acid sequence

<400> 130

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu

20 25 30

Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr

50 55 60

Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro

65 70 75 80

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile

85 90 95

Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

130 135 140

Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser

145 150 155 160

Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Tyr Asn Ser Ala Leu Lys Ser

195 200 205

Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys

210 215 220

Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

245 250 255

Thr Ser Val Thr Val Ser Ser Thr Thr Thr Thr Pro Ala Pro Arg Pro Pro

260 265 270

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

275 280 285

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

290 295 300

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg

325 330 335

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln

340 345 350

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu

355 360 365

Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

370 375 380

Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

385 390 395 400

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

405 410 415

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

420 425 430

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

435 440 445

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

450 455 460

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

465 470 475 480

Gln Ala Leu Pro Pro Arg Gly Arg Arg Lys Arg Gly Ser Gly Ala Thr

485 490 495

Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly

500 505 510

Pro Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu

515 520 525

Ser Gly Leu Glu Ala Val Met Ala Pro Arg Thr Leu Ile Leu Gly Gly

530 535 540

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Gln Arg

545 550 555 560

Thr Pro Lys Ile Gln Val Tyr Ser Arg His Pro Ala Glu Asn Gly Lys

565 570 575

Ser Asn Phe Leu Asn Cys Tyr Val Ser Gly Phe His Pro Ser Asp Ile

580 585 590

Glu Val Asp Leu Leu Lys Asn Gly Glu Arg Ile Glu Lys Val Glu His

595 600 605

Ser Asp Leu Ser Phe Ser Lys Asp Trp Ser Phe Tyr Leu Leu Tyr Tyr

610 615 620

Thr Glu Phe Thr Pro Thr Glu Lys Asp Glu Tyr Ala Cys Arg Val Asn

625 630 635 640

His Val Thr Leu Ser Gln Pro Lys Ile Val Lys Trp Asp Arg Asp Met

645 650 655

<210> 131

<211> 1971

<212> DNA

<213> Artificial Sequence

<220>

<223> CJP-P2A-CM14 DNA sequence

<400> 131

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggacatcc agatgacaca gactacatcc tccctgtctg cctctctggg agacagagtc 120

accatcagtt gcagggcaag tcaggacatt agtaaatatt taaattggta tcagcagaaa 180

ccagatggaa ctgttaaact cctgatctac catacatcaa gattacactc aggagtccca 240

tcaaggttca gtggcagtgg gtctggaaca gattattctc tcaccattag caacctggag 300

caagaagata ttgccactta cttttgccaa cagggtaata cgcttccgta cacgttcgga 360

ggggggacca agctggagat cacaggtggc ggtggctcgg gcggtggtgg gtcgggtggc 420

ggcggatctg aggtgaaact gcaggagtca ggacctggcc tggtggcgcc ctcacagagc 480

ctgtccgtca catgcactgt ctcaggggtc tcattacccg actatggtgt aagctggatt 540

cgccagcctc cacgaaaggg tctggagtgg ctgggagtaa tatggggtag tgaaaccaca 600

tactataatt cagctctcaa atccagactg accatcatca aggacaactc caagagccaa 660

gttttcttaa aaatgaacag tctgcaaact gatgacacag ccatttacta ctgtgccaaa 720

cattattact acggtggtag ctatgctatg gactactggg gccaaggaac ctcagtcacc 780

gtctcctcaa ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 840

cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 900

agggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 960

gtccttctcc tgtcactggt tatcaccctt tactgcaaac ggggcagaaa gaaactcctg 1020

tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt 1080

agctgccgat ttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 1140

agcgcagacg cccccgcgta caagcagggc cagaaccagc tctataacga gctcaatcta 1200

ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1260

ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1320

atggcggagg cctacagtga gattggggatg aaaggcgagc gccggagggg caaggggcac 1380

gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1440

caggccctgc cccctcgcgg ccgccgcaag cgcggctccg gtgccacgaa cttctctctg 1500

ttaaagcaag caggagacgt ggaagaaaac cccggtccta tgtctcgctc cgtggcattg 1560

gctgtgctcg cgctactctc tctttctggt ctcgaagctg ttatggctcc gcggactctg 1620

atcttaggtg gtggcggttc cggtggtggc ggttctggtg gtggcggctc catccagcgt 1680

acgccaaaga ttcaggttta ctcacgtcat ccagcagaga atggaaagtc aaatttcctg 1740

aattgctatg tgtctgggtt tcatccatcc gacattgaag ttgacttact gaagaatgga 1800

gagagaattg aaaaagtgga gcattcagac ttgtctttca gcaaggactg gtctttctat 1860

ctcttgtact acactgaatt caccccccact gaaaaagatg agtatgcctg ccgtgtgaac 1920

catgtgactt tgtcacagcc caagatagtt aagtggggatc gcgacatgta a 1971

<210> 132

<211> 611

<212> PRT

<213> Artificial Sequence

<220>

<223> 1G4-TCR amino acid sequence

<400> 132

Met Glu Thr Leu Leu Gly Leu Leu Ile Leu Trp Leu Gln Leu Gln Trp

1 5 10 15

Val Ser Ser Lys Gln Glu Val Thr Gln Ile Pro Ala Ala Leu Ser Val

20 25 30

Pro Glu Gly Glu Asn Leu Val Leu Asn Cys Ser Phe Thr Asp Ser Ala

35 40 45

Ile Tyr Asn Leu Gln Trp Phe Arg Gln Asp Pro Gly Lys Gly Leu Thr

50 55 60

Ser Leu Leu Leu Ile Gln Ser Ser Gln Arg Glu Gln Thr Ser Gly Arg

65 70 75 80

Leu Asn Ala Ser Leu Asp Lys Ser Ser Gly Arg Ser Thr Leu Tyr Ile

85 90 95

Ala Ala Ser Gln Pro Gly Asp Ser Ala Thr Tyr Leu Cys Ala Val Arg

100 105 110

Pro Leu Tyr Gly Gly Ser Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser

115 120 125

Leu Ile Val His Pro Tyr Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln

130 135 140

Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp

145 150 155 160

Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr

165 170 175

Ile Thr Asp Lys Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser

180 185 190

Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn

195 200 205

Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro

210 215 220

Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp

225 230 235 240

Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu

245 250 255

Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp

260 265 270

Ser Ser Arg Ala Lys Arg Ser Gly Ser Gly Glu Gly Arg Gly Ser Leu

275 280 285

Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Ser Ile Gly

290 295 300

Leu Leu Cys Cys Ala Ala Leu Ser Leu Leu Trp Ala Gly Pro Val Asn

305 310 315 320

Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys Thr Gly Gln

325 330 335

Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His Glu Tyr Met Ser

340 345 350

Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu Ile His Tyr Ser

355 360 365

Val Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro Asn Gly Tyr Asn

370 375 380

Val Ser Arg Ser Thr Thr Thr Glu Asp Phe Pro Leu Arg Leu Leu Ser Ala

385 390 395 400

Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Ser Tyr Val Gly

405 410 415

Asn Thr Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg Leu Thr Val Leu

420 425 430

Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro

435 440 445

Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu

450 455 460

Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn

465 470 475 480

Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Pro Leu Lys

485 490 495

Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu

500 505 510

Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys

515 520 525

Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp

530 535 540

Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg

545 550 555 560

Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu Ser

565 570 575

Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala

580 585 590

Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp

595 600 605

Ser Arg Gly

610

<210> 133

<211> 1836

<212> DNA

<213> Artificial Sequence

<220>

<223> 1G4-TCR DNA sequence

<400> 133

atggagaccc tcttgggcct gcttatcctt tggctgcagc tgcaatgggt gagcagcaaa 60

caggaggtga cgcagattcc tgcagctctg agtgtcccag aaggagaaaa cttggttctc 120

aactgcagtt tcactgatag cgctatttac aacctccagt ggtttaggca ggaccctggg 180

aaaggtctca catctctgtt gcttattcag tcaagtcaga gagagcaaac aagtggaaga 240

cttaatgcct cgctggataa atcatcagga cgtagtactt tatacattgc agcttctcag 300

cctggtgact cagccaccta cctctgtgct gtgaggcccc tgtacggagg aagctacata 360

cctacatttg gaagaggaac cagccttatt gttcatccgt atatccagaa ccctgaccct 420

gccgtgtacc agctgagaga ctctaaatcc agtgacaagt ctgtctgcct attcaccgat 480

tttgattctc aaacaaatgt gtcacaaagt aaggattctg atgtgtatat cacagacaaa 540

tgcgtgctag acatgaggtc tatggacttc aagagcaaca gtgctgtggc ctggagcaac 600

aaatctgact ttgcatgtgc aaacgccttc aacaacagca ttatccaga agacaccttc 660

ttccccagcc cagaaagttc ctgtgatgtc aagctggtcg agaaaagctt tgaaacagat 720

acgaacctaa actttcaaaa cctgtcagtg attgggttcc gaatcctcct cctgaaagtg 780

gccgggttta atctgctcat gacgctgcgg ctgtggtcca gccgggccaa gcggtccggc 840

tccggagagg gcaggggatc tctccttatact tgtggcgacg tggaggagaa ccccggcccc 900

atgagcatcg gcctcctgtg ctgtgcagcc ttgtctctcc tgtgggcagg tccagtgaat 960

gctggtgtca ctcagacccc aaaattccag gtcctgaaga caggacagag catgacactg 1020

cagtgtgccc aggatatgaa ccatgaatac atgtcctggt atcgacaaga cccaggcatg 1080

gggctgaggc tgattcatta ctcagttggt gctggtatca ctgaccaagg agaagtcccc 1140

aatggctaca atgtctccag atcaaccaca gaggatttcc cgctcaggct gctgtcggct 1200

gctccctccc agacatctgt gtacttctgt gccagcagtt acgtcgggaa caccggggag 1260

ctgttttttg gagaaggctc taggctgacc gtactggagg acctgaagaa cgtgttcccc 1320

cccgaggtgg ccgtgttcga gcccagcgag gccgagatca gccacacccca gaaagccacc 1380

ctggtgtgcc tggccaccgg cttctacccc gatcacgtgg agctgtcttg gtgggtgaac 1440

ggcaaagagg tgcactccgg cgtctgcacc gaccctcagc ccctgaaaga gcagcccgcc 1500

ctgaacgaca gccggtactg cctgtcctcc cggctgagag tgtctgctac attctggcag 1560

aatccccgga accacttccg gtgccaggtg cagttctacg gcctgagcga gaacgacgag 1620

tggacccagg acaggccaa gcccgtgacc cagatcgtgt ccgccgaggc ctggggcaga 1680

gccgactgcg gcttcaccag cgagagctac cagcagggcg tgctgtctgc caccatcctg 1740

tacgagatcc tgctgggcaa ggccaccctg tacgccgtgc tggtgtccgc cctggtgctg 1800

atggccatgg tgaagcggaa ggacagcaga ggctga 1836

<210> 134

<211> 780

<212> PRT

<213> Artificial Sequence

<220>

<223> 1G4-P2A-CM amino acid sequence

<400> 134

Met Glu Thr Leu Leu Gly Leu Leu Ile Leu Trp Leu Gln Leu Gln Trp

1 5 10 15

Val Ser Ser Lys Gln Glu Val Thr Gln Ile Pro Ala Ala Leu Ser Val

20 25 30

Pro Glu Gly Glu Asn Leu Val Leu Asn Cys Ser Phe Thr Asp Ser Ala

35 40 45

Ile Tyr Asn Leu Gln Trp Phe Arg Gln Asp Pro Gly Lys Gly Leu Thr

50 55 60

Ser Leu Leu Leu Ile Gln Ser Ser Gln Arg Glu Gln Thr Ser Gly Arg

65 70 75 80

Leu Asn Ala Ser Leu Asp Lys Ser Ser Gly Arg Ser Thr Leu Tyr Ile

85 90 95

Ala Ala Ser Gln Pro Gly Asp Ser Ala Thr Tyr Leu Cys Ala Val Arg

100 105 110

Pro Leu Tyr Gly Gly Ser Tyr Ile Pro Thr Phe Gly Arg Gly Thr Ser

115 120 125

Leu Ile Val His Pro Tyr Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln

130 135 140

Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp

145 150 155 160

Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr

165 170 175

Ile Thr Asp Lys Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser

180 185 190

Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn

195 200 205

Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro

210 215 220

Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp

225 230 235 240

Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu

245 250 255

Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp

260 265 270

Ser Ser Arg Ala Lys Arg Ser Gly Ser Gly Glu Gly Arg Gly Ser Leu

275 280 285

Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Ser Ile Gly

290 295 300

Leu Leu Cys Cys Ala Ala Leu Ser Leu Leu Trp Ala Gly Pro Val Asn

305 310 315 320

Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys Thr Gly Gln

325 330 335

Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His Glu Tyr Met Ser

340 345 350

Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu Ile His Tyr Ser

355 360 365

Val Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro Asn Gly Tyr Asn

370 375 380

Val Ser Arg Ser Thr Thr Thr Glu Asp Phe Pro Leu Arg Leu Leu Ser Ala

385 390 395 400

Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Ser Tyr Val Gly

405 410 415

Asn Thr Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg Leu Thr Val Leu

420 425 430

Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro

435 440 445

Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu

450 455 460

Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn

465 470 475 480

Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Pro Leu Lys

485 490 495

Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu

500 505 510

Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys

515 520 525

Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp

530 535 540

Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg

545 550 555 560

Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu Ser

565 570 575

Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala

580 585 590

Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp

595 600 605

Ser Arg Gly Arg Arg Lys Arg Gly Ser Gly Ala Thr Asn Phe Ser Leu

610 615 620

Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Ser Arg

625 630 635 640

Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu Ser Gly Leu Glu

645 650 655

Ala Val Met Ala Pro Arg Thr Val Leu Leu Gly Gly Gly Gly Ser Gly

660 665 670

Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Gln Arg Thr Pro Lys Ile

675 680 685

Gln Val Tyr Ser Arg His Pro Ala Glu Asn Gly Lys Ser Asn Phe Leu

690 695 700

Asn Cys Tyr Val Ser Gly Phe His Pro Ser Asp Ile Glu Val Asp Leu

705 710 715 720

Leu Lys Asn Gly Glu Arg Ile Glu Lys Val Glu His Ser Asp Leu Ser

725 730 735

Phe Ser Lys Asp Trp Ser Phe Tyr Leu Leu Tyr Tyr Thr Glu Phe Thr

740 745 750

Pro Thr Glu Lys Asp Glu Tyr Ala Cys Arg Val Asn His Val Thr Leu

755 760 765

Ser Gln Pro Lys Ile Val Lys Trp Asp Arg Asp Met

770 775 780

<210> 135

<211> 2343

<212> DNA

<213> Artificial Sequence

<220>

<223> 1G4-P2A-CM DNA sequence

<400> 135

atggagaccc tcttgggcct gcttatcctt tggctgcagc tgcaatgggt gagcagcaaa 60

caggaggtga cgcagattcc tgcagctctg agtgtcccag aaggagaaaa cttggttctc 120

aactgcagtt tcactgatag cgctatttac aacctccagt ggtttaggca ggaccctggg 180

aaaggtctca catctctgtt gcttattcag tcaagtcaga gagagcaaac aagtggaaga 240

cttaatgcct cgctggataa atcatcagga cgtagtactt tatacattgc agcttctcag 300

cctggtgact cagccaccta cctctgtgct gtgaggcccc tgtacggagg aagctacata 360

cctacatttg gaagaggaac cagccttatt gttcatccgt atatccagaa ccctgaccct 420

gccgtgtacc agctgagaga ctctaaatcc agtgacaagt ctgtctgcct attcaccgat 480

tttgattctc aaacaaatgt gtcacaaagt aaggattctg atgtgtatat cacagacaaa 540

tgcgtgctag acatgaggtc tatggacttc aagagcaaca gtgctgtggc ctggagcaac 600

aaatctgact ttgcatgtgc aaacgccttc aacaacagca ttatccaga agacaccttc 660

ttccccagcc cagaaagttc ctgtgatgtc aagctggtcg agaaaagctt tgaaacagat 720

acgaacctaa actttcaaaa cctgtcagtg attgggttcc gaatcctcct cctgaaagtg 780

gccgggttta atctgctcat gacgctgcgg ctgtggtcca gccgggccaa gcggtccggc 840

tccggagagg gcaggggatc tctccttatact tgtggcgacg tggaggagaa ccccggcccc 900

atgagcatcg gcctcctgtg ctgtgcagcc ttgtctctcc tgtgggcagg tccagtgaat 960

gctggtgtca ctcagacccc aaaattccag gtcctgaaga caggacagag catgacactg 1020

cagtgtgccc aggatatgaa ccatgaatac atgtcctggt atcgacaaga cccaggcatg 1080

gggctgaggc tgattcatta ctcagttggt gctggtatca ctgaccaagg agaagtcccc 1140

aatggctaca atgtctccag atcaaccaca gaggatttcc cgctcaggct gctgtcggct 1200

gctccctccc agacatctgt gtacttctgt gccagcagtt acgtcgggaa caccggggag 1260

ctgttttttg gagaaggctc taggctgacc gtactggagg acctgaagaa cgtgttcccc 1320

cccgaggtgg ccgtgttcga gcccagcgag gccgagatca gccacacccca gaaagccacc 1380

ctggtgtgcc tggccaccgg cttctacccc gatcacgtgg agctgtcttg gtgggtgaac 1440

ggcaaagagg tgcactccgg cgtctgcacc gaccctcagc ccctgaaaga gcagcccgcc 1500

ctgaacgaca gccggtactg cctgtcctcc cggctgagag tgtctgctac attctggcag 1560

aatccccgga accacttccg gtgccaggtg cagttctacg gcctgagcga gaacgacgag 1620

tggacccagg acaggccaa gcccgtgacc cagatcgtgt ccgccgaggc ctggggcaga 1680

gccgactgcg gcttcaccag cgagagctac cagcagggcg tgctgtctgc caccatcctg 1740

tacgagatcc tgctgggcaa ggccaccctg tacgccgtgc tggtgtccgc cctggtgctg 1800

atggccatgg tgaagcggaa ggacagcaga ggccgccgca agcgcggctc cggtgccacg 1860

aacttctctc tgttaaagca agcaggagac gtggaagaaa accccggtcc tatgtctcgc 1920

tccgtggcat tggctgtgct cgcgctactc tctctttctg gtctcgaagc tgttatggct 1980

ccgcggactg tgctgttagg tggtggcggt tccggtggtg gcggttctgg tggtggcggc 2040

tccatccagc gtacgccaaa gattcaggtt tactcacgtc atccagcaga gaatggaaag 2100

tcaaatttcc tgaattgcta tgtgtctggg tttcatccat ccgacattga agttgactta 2160

ctgaagaatg gagagaat tgaaaaagtg gagcattcag acttgtcttt cagcaaggac 2220

tggtctttct atctcttgta ctacactgaa ttcaccccca ctgaaaaaga tgagtatgcc 2280

tgccgtgtga accatgtgac tttgtcacag cccaagatag ttaagtggga tcgcgacatg 2340

taa 2343

<210> 136

<211> 373

<212> PRT

<213> Artificial Sequence

<220>

<223> BCMA CAR amino acid sequence

<400> 136

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu

20 25 30

Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu

35 40 45

Thr Phe Ser Glu Tyr Thr Met Gly Trp Phe Arg Gln Pro Pro Gly Lys

50 55 60

Glu Arg Glu Phe Val Ala Ala Ile Ser Gly Gly Gly Ile Pro Tyr Tyr

65 70 75 80

Arg Thr Thr Val Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ala Gln

85 90 95

Asn Thr Val Ala Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala

100 105 110

Val Tyr Cys Ala Ala Asp Ala Arg Ala Val Met Thr Val Thr Pro

115 120 125

Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys

130 135 140

Thr Pro Lys Pro Gln Asp Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr

145 150 155 160

Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala

165 170 175

Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe

180 185 190

Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val

195 200 205

Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys

210 215 220

Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr

225 230 235 240

Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu

245 250 255

Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro

260 265 270

Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly

275 280 285

Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro

290 295 300

Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr

305 310 315 320

Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly

325 330 335

Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln

340 345 350

Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln

355 360 365

Ala Leu Pro Pro Arg

370

<210> 137

<211> 1119

<212> DNA

<213> Artificial Sequence

<220>

<223> BCMA CAR DNA sequence

<400> 137

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccgcaggtgc agctcgtgga gtctggggga ggattggtgc aggctggggg ctcgctcaga 120

ctctcctgcg cagcctctgg acttaccttc agtgaatata ccatgggctg gttccgccag 180

cctccaggga aggagcgtga atttgtagcg gctattagtg gaggtggtat cccatactat 240

agaaccaccg tgaagggtcg attctccatc tccagagaca acgcccagaa cacggtcgct 300

ctggagatga acagcctgaa acctgaggac acggccgttt attactgtgc agcagatgcg 360

cgtgcggtga tgactgtgac tcccaactac tggggccagg ggacccaggt caccgtctcc 420

tcagaaccca agacaccaaa accacaagac accacgacgc cagcgccgcg accaccaaca 480

ccggcgccca ccatcgcgtc gcagcccctg tccctgcgcc cagaggcgtg ccggccagcg 540

gcggggggcg cagtgcacac gaggggggctg gacttcgcct gtgatatcta catctgggcg 600

cccttggccg ggacttgtgg ggtccttctc ctgtcactgg ttatcaccct ttatgcaaa 660

cggggcagaa agaaactcct gtatatattc aaacaaccat ttatgagacc agtacaaact 720

actcaagagg aagatggctg tagctgccga tttccagaag aagaagaagg aggatgtgaa 780

ctgagagtga agttcagcag gagcgcagac gcccccgcgt accagcaggg ccagaaccag 840

ctctataacg agctcaatct aggacgaaga gaggagtacg atgttttgga caagagacgt 900

ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 960

aatgaactgc agaaagataa gatggcggag gcctacagtg agatggggat gaaaggcgag 1020

cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 1080

acctacgacg cccttcacat gcaggccctg ccccctcgc 1119

<210> 138

<211> 543

<212> PRT

<213> Artificial Sequence

<220>

<223> BCMA CAR-CM amino acid sequence

<400> 138

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu

20 25 30

Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu

35 40 45

Thr Phe Ser Glu Tyr Thr Met Gly Trp Phe Arg Gln Pro Pro Gly Lys

50 55 60

Glu Arg Glu Phe Val Ala Ala Ile Ser Gly Gly Gly Ile Pro Tyr Tyr

65 70 75 80

Arg Thr Thr Val Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ala Gln

85 90 95

Asn Thr Val Ala Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala

100 105 110

Val Tyr Cys Ala Ala Asp Ala Arg Ala Val Met Thr Val Thr Pro

115 120 125

Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys

130 135 140

Thr Pro Lys Pro Gln Asp Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr

145 150 155 160

Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala

165 170 175

Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe

180 185 190

Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val

195 200 205

Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys

210 215 220

Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr

225 230 235 240

Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu

245 250 255

Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro

260 265 270

Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly

275 280 285

Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro

290 295 300

Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr

305 310 315 320

Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly

325 330 335

Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln

340 345 350

Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln

355 360 365

Ala Leu Pro Pro Arg Gly Arg Arg Lys Arg Gly Ser Gly Ala Thr Asn

370 375 380

Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro

385 390 395 400

Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu Ser

405 410 415

Gly Leu Glu Ala Val Met Ala Pro Arg Thr Val Leu Leu Gly Gly Gly

420 425 430

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Gln Arg Thr

435 440 445

Pro Lys Ile Gln Val Tyr Ser Arg His Pro Ala Glu Asn Gly Lys Ser

450 455 460

Asn Phe Leu Asn Cys Tyr Val Ser Gly Phe His Pro Ser Asp Ile Glu

465 470 475 480

Val Asp Leu Leu Lys Asn Gly Glu Arg Ile Glu Lys Val Glu His Ser

485 490 495

Asp Leu Ser Phe Ser Lys Asp Trp Ser Phe Tyr Leu Leu Tyr Tyr Thr

500 505 510

Glu Phe Thr Pro Thr Glu Lys Asp Glu Tyr Ala Cys Arg Val Asn His

515 520 525

Val Thr Leu Ser Gln Pro Lys Ile Val Lys Trp Asp Arg Asp Met

530 535 540

<210> 139

<211> 1629

<212> DNA

<213> Artificial Sequence

<220>

<223> BCMA CAR-CM DNA sequence

<400> 139

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccgcaggtgc agctcgtgga gtctggggga ggattggtgc aggctggggg ctcgctcaga 120

ctctcctgcg cagcctctgg acttaccttc agtgaatata ccatgggctg gttccgccag 180

cctccaggga aggagcgtga atttgtagcg gctattagtg gaggtggtat cccatactat 240

agaaccaccg tgaagggtcg attctccatc tccagagaca acgcccagaa cacggtcgct 300

ctggagatga acagcctgaa acctgaggac acggccgttt attactgtgc agcagatgcg 360

cgtgcggtga tgactgtgac tcccaactac tggggccagg ggacccaggt caccgtctcc 420

tcagaaccca agacaccaaa accacaagac accacgacgc cagcgccgcg accaccaaca 480

ccggcgccca ccatcgcgtc gcagcccctg tccctgcgcc cagaggcgtg ccggccagcg 540

gcggggggcg cagtgcacac gaggggggctg gacttcgcct gtgatatcta catctgggcg 600

cccttggccg ggacttgtgg ggtccttctc ctgtcactgg ttatcaccct ttatgcaaa 660

cggggcagaa agaaactcct gtatatattc aaacaaccat ttatgagacc agtacaaact 720

actcaagagg aagatggctg tagctgccga tttccagaag aagaagaagg aggatgtgaa 780

ctgagagtga agttcagcag gagcgcagac gcccccgcgt accagcaggg ccagaaccag 840

ctctataacg agctcaatct aggacgaaga gaggagtacg atgttttgga caagagacgt 900

ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 960

aatgaactgc agaaagataa gatggcggag gcctacagtg agatggggat gaaaggcgag 1020

cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 1080

acctacgacg cccttcacat gcaggccctg ccccctcgcg gccgccgcaa gcgcggctcc 1140

ggtgccacga acttctctct gttaaagcaa gcaggagacg tggaagaaaa ccccggtcct 1200

atgtctcgct ccgtggcatt ggctgtgctc gcgctactct ctctttctgg tctcgaagct 1260

gttatggctc cgcggactgt gctgttaggt ggtggcggtt ccggtggtgg cggttctggt 1320

ggtggcggct ccatccagcg tacgccaaag attcaggttt actcacgtca tccagcagag 1380

aatggaaagt caaatttcct gaattgctat gtgtctgggt ttcatccatc cgacattgaa 1440

gttgacttac tgaagaatgg agagaatt gaaaaagtgg agcattcaga cttgtctttc 1500

agcaaggact ggtctttcta tctcttgtac tacactgaat tcaccccac tgaaaaagat 1560

gagtatgcct gccgtgtgaa ccatgtgact ttgtcacagc ccaagatagt taagtggggat 1620

cgcgacatg 1629

<210> 140

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> NY-ESO-1 derived peptides

<400> 140

Ser Leu Leu Met Trp Ile Thr Gln Cys

1 5

Claims (74)

1. A fusion protein comprising, from the N-terminus to the C-terminus, a presenting peptide, a linker, and a human β-2-microglobulin (β2M) peptide, wherein the presenting peptide is an HLA-E-restricted presenting peptide selected from the group consisting of SEQ ID NOs:29-38. 2. The fusion protein according to claim 1, wherein the fusion protein has 120-180 amino acids. 3. The fusion protein according to claim 1, wherein the amino acid sequence of the linker is (EAAAK)n, where n = 3, 4 or 5 (SEQ ID NO: 110), and wherein the linker has 5 to 30 amino acids. 4. The fusion protein according to claim 1, wherein the amino acid sequence of the linker is (GGGGS)n, where n = 3, 4 or 5 (SEQ ID NO: 112), and wherein the linker has 5 to 30 amino acids. 5. The fusion protein according to claim 4, wherein the amino acid sequence of the linker is SEQ ID NO: 1 or 2. 6. The fusion protein according to claim 1, wherein the amino acid sequence of the β2M peptide is SEQ ID NO:81. 7. The fusion protein according to claim 1, wherein the amino acid sequence of the presenting peptide is VMAPRTVLL (SEQ ID NO: 38). 8. A fusion protein, wherein the amino acid sequence of the fusion protein is selected from the group consisting of SEQ ID NOs:5 and 13-18. 9. A nucleic acid that encodes a fusion protein according to any one of claims 1 to 8. 10. A nucleic acid comprising (i) a first segment encoding the fusion protein of claim 1; and (ii) a second segment encoding a synthetic receptor. 11. The nucleic acid according to claim 10, wherein the synthetic receptor is selected from the group consisting of chimeric antigen receptor (CAR), T-cell receptor (TCR), TCR receptor fusion construct (TRuC), T-cell antigen conjugate (TAC), antibody TCR receptor (AbTCR), and chimeric CD3 receptor. 12. The nucleic acid according to claim 10, wherein the synthetic receptor is a CAR. 13. The nucleic acid of claim 10, wherein the synthetic receptor comprises an antigen-binding domain that specifically binds to tumor antigens. 14. The nucleic acid according to claim 13, wherein the tumor antigen is selected from the group consisting of CD19, CD20, CD22, CD30, CD123, CD138, CD33, CD70, BCMA, CS1, C-Met, IL13Ra2, EGFRvIII, CEA, Her2, GD2, MAGE, GPC3, mesothelin, PSMA, ROR1, EGFR, MUC1 and NY-ESO-1. 15. The nucleic acid according to claim 13, wherein the tumor antigen is CD19 or BCMA. 16. The nucleic acid of claim 10, wherein the synthetic receptor comprises an antigen-binding domain that specifically binds to viral antigens. 17. The nucleic acid according to claim 16, wherein the viral antigen is EBV or HPV. 18. The nucleic acid of claim 10, wherein the first fragment and the second fragment are linked by a polynucleotide encoding a 2A peptide. 19. The nucleic acid according to claim 18, wherein the 2A peptide is a P2A peptide, a T2A peptide, an F2A peptide, or an E2A peptide. 20. The nucleic acid according to claim 18, wherein the synthetic receptor, the 2A peptide, and the fusion protein are encoded from the N-terminus to the C-terminus. 21. The nucleic acid according to claim 10, wherein the first fragment and the second fragment are linked by an IRES sequence. 22. A vector comprising the nucleic acid according to claim 9. 23. The vector according to claim 22, wherein it is a viral vector. 24. The vector according to claim 23, wherein the viral vector is a lentiviral vector, an adenovirus vector, or an adeno-associated virus vector. 25. A vector comprising the nucleic acid according to claim 10. 26. The vector according to claim 25, wherein it is a viral vector. 27. The vector according to claim 26, wherein the viral vector is a lentiviral vector, an adenovirus vector, or an adeno-associated virus vector. 28. A genetically engineered cell that expresses the fusion protein according to any one of claims 1 to 8. 29. A genetically engineered cell comprising the nucleic acid according to claim 9. 30. A genetically engineered cell comprising the vector according to claim 22. 31. A genetically engineered cell comprising the nucleic acid according to claim 10. 32. A genetically engineered cell comprising the vector according to claim 25. 33. The cell according to claim 28, further expressing a synthetic receptor. 34. The cell of claim 29, further comprising a second nucleic acid encoding a synthetic receptor. 35. The cell of claim 34, wherein the synthetic receptor is selected from the group consisting of CAR, TCR, TRUC, TAC, AbTCR and chimeric CD3 receptor. 36. The cell of claim 35, wherein the synthetic receptor is a CAR. 37. The cell of claim 34, wherein the synthetic receptor comprises an antigen-binding domain that specifically binds to tumor antigens. 38. The cell of claim 37, wherein the tumor antigen is selected from the group consisting of CD19, CD20, CD22, CD30, CD123, CD138, CD33, CD70, BCMA, CS1, C-Met, IL13Ra2, EGFRvIII, CEA, Her2, GD2, MAGE, GPC3, mesothelin, PSMA, ROR1, EGFR, MUC1, and NY-ESO-1. 39. The cell of claim 38, wherein the tumor antigen is CD19 or BCMA. 40. The cell of claim 39, wherein the synthetic receptor is a CAR having an antigen-binding domain that specifically binds to CD19 or BCMA, wherein the amino acid sequence of the CAR is selected from the group consisting of SEQ ID NOs:74-80 and 136. 41. The cell of claim 38, wherein the synthetic receptor is a TCR having an antigen-binding domain that specifically binds to NY-ESO-1, wherein the amino acid sequence of the TCR is SEQ ID NO:132. 42. The cell of claim 34, wherein the synthetic receptor comprises an antigen-binding domain that specifically binds to viral antigens. 43. The cell of claim 42, wherein the viral antigen is EBV or HPV. 44. The cell of claim 29, wherein the fusion protein forms a complex with endogenous MHC heavy chains on the cell surface. 45. The cell of claim 29, wherein the cell lacks endogenous expression of at least one gene encoding a class I MHC molecule or MHC-like molecule on the cell surface. 46. The cell of claim 45, wherein the cell lacks endogenous expression of class I MHC molecules on the cell surface, the class I MHC molecules being selected from the group consisting of HLA-A heavy chain, HLA-B heavy chain, HLA-C heavy chain, HLA-E heavy chain, HLA-F heavy chain and HLA-G heavy chain. 47. The cell of claim 45, wherein the cell lacks endogenous expression of β2M on the cell surface. 48. The cell of claim 45, wherein the cell lacks endogenous expression of MHC-like molecules on the cell surface, the MHC-like molecules being selected from the group consisting of CD1 heavy chain, MR1 heavy chain, FcRn heavy chain and UL18. 49. The cell of claim 29, wherein the cell is an immune cell. 50. The immune cell of claim 49, wherein the immune cell is a leukocyte. 51. The immune cells of claim 50, wherein the leukocytes are selected from the group consisting of T cells, NK cells, NKT cells, B cells, plasma cells, dendritic cells, neutrophils, monocytes, macrophages and granulocytes. 52. The immune cell according to claim 51, wherein the leukocyte is an NK cell. 53. The immune cell according to claim 51, wherein the leukocyte is a T cell. 54. The immune cell of claim 53, wherein at least one gene encoding a component of the TCR complex is inactivated in the T cell. 55. A pharmaceutical composition having a cell- and pharmaceutically acceptable carrier as described in claim 29. 56. Use of the cell according to claim 29 in the preparation of a medicament for allogeneic transplantation. 57. A pharmaceutical composition having a cell and pharmaceutically acceptable carrier as described in claim 34. 58. Use of the cell according to claim 34 in the preparation of a medicament for allogeneic transplantation. 59. Use of the cell according to claim 37 in the preparation of a medicament for treating cancer. 60. Use of a cell in the preparation of a medicament for allogeneic transplantation, wherein the cell comprises a nucleic acid encoding the fusion protein of claim 1. 61. Use of a cell in the preparation of a medicament for allogeneic transplantation, wherein the cell expresses the fusion protein of claim 1. 62. A method for genetically engineering cells to prepare a medicament for allogeneic transplantation, the method comprising transducing the cells with a nucleic acid encoding the fusion protein of claim 1. 63. A method for genetically engineering cells to prepare a drug for allogeneic transplantation, the method comprising transducing the cells with the nucleic acid according to claim 9. 64. A method for genetically engineering cells to prepare a drug for allogeneic transplantation, the method comprising transducing the cells using the vector according to claim 22. 65. The method of claim 63, further comprising: inactivating at least one gene in the cell that encodes a class I MHC molecule or an MHC-like molecule. 66. The method according to claim 65, wherein (1) the inactivating gene encodes a class I MHC molecule, the class I MHC molecule being selected from the group consisting of HLA-A heavy chain, HLA-B heavy chain, HLA-C heavy chain, HLA-E heavy chain, HLA-F heavy chain and HLA-G heavy chain; (2) wherein the inactivating gene encodes β2M; or (3) wherein the inactivating gene encodes an MHC-like molecule, the MHC-like molecule being selected from the group consisting of CD1 heavy chain, MR1 heavy chain, FcRn heavy chain and UL18. 67. The method of claim 66, wherein the gene is inactivated by DNA cutting, DNA cutting and repair, base editing, guided editing, RNA interference, or RNA editing. 68. The method of claim 67, wherein the gene is inactivated by DNA cleavage, the DNA cleavage using a rare endonuclease selected from the group consisting of RNA-directed endonucleases, TAL nucleases, homing nucleases, zinc finger nucleases, and Mega-TAL nucleases. 69. The method of claim 67, wherein the gene is inactivated by DNA splicing and repair. 70. The method of claim 67, wherein the gene is inactivated using a CRISPR-Cas system. 71. The method of claim 70, wherein the CRISPR-Cas system is a CRISPR-Cas9 system. 72. The method of claim 63, wherein the cell is an immune cell. 73. The method according to claim 72, wherein the immune cells are selected from the group consisting of T cells, NK cells, NKT cells, B cells, plasma cells, monocytes, macrophages, dendritic cells and granulocytes. 74. Use of a genetically engineered cell in the preparation of a medicament for allogeneic transplantation, said cell being prepared according to the method of claim 63.