Universal T cells and compositions and methods of their use

Abstract

The present invention provides a modified HLA-E single chain trimer construct and nucleic acid molecules encoding the same as well as cells and compositions comprising the same for increasing the persistence of at least one cell of interest or reducing the clearance of at least one cell of interest. In some embodiments, the invention also provides methods of preventing and / or eliminating allogeneic responses, allogeneic recognition, and / or allogeneic rejection. The invention also relates to a chimeric antigen receptor (CAR) cell or a T cell expressing an engineered TCR, said cell comprising a modified HLA-E single chain trimer construct and / or a nucleic acid molecule encoding the same.

Description

[0001] Citations of Related Applications

[0002] This application claims priority to U.S. Provisional Application Serial No. 63 / 371,144 filed on August 11, 2022 and U.S. Provisional Application Serial No. 63 / 382,766 filed on November 8, 2022, the disclosures of which are hereby incorporated by reference in their entireties.

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0004] This invention was made with government support under Grant No. CA244711 awarded by the National Institutes of Health. The government has certain rights in this invention. Background Art

[0005] Chimeric antigen receptor (CAR) T cell therapy is a form of T cell transfer therapy in which T cells are harvested from the patient and modified to express antigen receptors that are not normally expressed (Ahmad, A., 2020, International Journal of Molecular Sciences, 21 (12): 4303). However, the clinical development of CAR T cell products is often hindered by the low production and poor function of peripheral blood T cells in cancer patients (especially individuals who have received previous systemic therapy). Due to the tumor microenvironment and pre-treatment, the patient's T cell function is dysregulated (Thommen et al., Cancer Cell Vol. 33: 547-562, 2018) and the treatment time is delayed (Levine et al., Mol Ther Methods Clin Dev Vol. 4: 92-101, 2017). Other drawbacks of current CAR T cells are their sensitivity to host natural killer (NK) cell recognition due to the lack of major histocompatibility complex class I molecules (MHC I) and the risk of graft-versus-host disease (GvHD).

[0006] Therefore, there is a need in the art for a "universal" T cell product that can be engineered to optimize persistence and avoid rejection by the host immune system. The present invention meets this unmet need. Summary of the invention

[0007] In various aspects, the present invention provides a peptide comprising: a human leukocyte antigen (HLA) signal peptide or a fragment thereof, comprising at least one amino acid sequence having at least about 70% identity with an amino acid sequence selected from SEQ ID NOs: 1-10, 13, and 46; a modified beta-2-microglobulin (B2M) or a fragment thereof; and an HLA class I histocompatibility antigen alpha chain E (antigen, alpha chain E) (HLA-E) or a fragment thereof. In some embodiments, the peptide is a single-chain trimer.

[0008] In some embodiments, the HLA signal peptide includes at least one selected from the following: HLA-A*02:01 signal peptide or a fragment thereof, HLA-B*08:01 signal peptide or a fragment thereof, HLA-C*03:01 signal peptide or a fragment thereof, HLA-G signal peptide or a fragment thereof, HSP60 signal peptide or a fragment thereof, CMV Towne signal peptide or a fragment thereof, CMV AF1 signal peptide or a fragment thereof, CMV109b signal peptide or a fragment thereof, RL9HIV signal peptide or a fragment thereof, or Mtb44 signal peptide or a fragment thereof.

[0009] In some embodiments, the HLA-E is human HLA-E or a fragment thereof.

[0010] In some embodiments, the modified B2M includes at least one selected from the following: a modified B2M signal peptide or a fragment thereof or a leader-less modified B2M or a fragment thereof. In some embodiments, the modified B2M signal peptide or a fragment thereof is linked to an HLA signal peptide or a fragment thereof; the HLA signal peptide or a fragment thereof is linked to a leader-less modified B2M or a fragment thereof; and the leader-less modified B2M or a fragment thereof is linked to an HLA-E or a fragment thereof.

[0011] In some embodiments, the peptide further comprises at least one linker and at least one spacer.

[0012] In some embodiments, the peptide comprises an amino acid sequence that is at least about 70% identical to the amino acid sequence shown in SEQ ID NO:40.

[0013] In some embodiments, the peptide prevents, reduces or inhibits natural killer (NK) cell-mediated killing of at least one donor cell. In some embodiments, the peptide increases the persistence of at least one donor cell or reduces the clearance of at least one donor cell.

[0014] In various aspects, the present invention provides a composition comprising at least one peptide of the present invention. In some embodiments, the composition is a pharmaceutically acceptable composition.

[0015] In various aspects, the present invention also provides a nucleic acid molecule comprising: a nucleotide sequence encoding an HLA signal peptide or a fragment thereof, wherein the HLA signal peptide or a fragment thereof comprises at least one amino acid sequence having at least about 70% identity with an amino acid sequence selected from SEQ ID NO: 1-10, 13 and 46; a nucleotide sequence encoding a modified B2M or a fragment thereof; and a nucleotide sequence encoding HLA-E or a fragment thereof.

[0016] In some embodiments, the nucleotide sequence encoding the HLA signal peptide or a fragment thereof comprises at least one nucleotide sequence having at least about 70% identity to a nucleotide sequence selected from SEQ ID NOs: 14-24 and 47.

[0017] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that is at least about 70% identical to the nucleotide sequence shown in SEQ ID NO:41.

[0018] In other aspects, the present invention also provides a nucleic acid molecule comprising: a nucleotide sequence encoding an HLA signal peptide or a fragment thereof, the nucleotide sequence comprising at least one nucleotide sequence having at least about 70% identity with a nucleotide sequence selected from SEQ ID NO: 14-24 and 47; a nucleotide sequence encoding a modified B2M or a fragment thereof; and a nucleotide sequence encoding HLA-E or a fragment thereof.

[0019] In some embodiments, the nucleotide sequence encoding the modified B2M includes at least one selected from the group consisting of: a nucleotide sequence encoding a modified B2M signal peptide or a fragment thereof and a nucleotide sequence encoding a modified B2M or a fragment thereof without a leader sequence.

[0020] In some embodiments, a nucleotide sequence encoding a modified B2M signal peptide or a fragment thereof is linked to a nucleotide sequence encoding an HLA signal peptide or a fragment thereof; a nucleotide sequence encoding an HLA signal peptide or a fragment thereof is linked to a nucleotide sequence encoding a modified B2M or a fragment thereof without a leader sequence; and a nucleotide sequence encoding a modified B2M or a fragment thereof without a leader sequence is linked to a nucleotide sequence encoding HLA-E or a fragment thereof.

[0021] In some embodiments, the nucleic acid molecule further comprises at least one nucleotide sequence encoding a linker and at least one nucleotide sequence encoding a spacer.

[0022] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that is at least about 70% identical to the nucleotide sequence shown in SEQ ID NO:41.

[0023] In some embodiments, the nucleic acid molecule prevents, reduces or inhibits NK cell-mediated killing of at least one donor cell. In some embodiments, the nucleic acid molecule increases the persistence of at least one donor cell or reduces the clearance of at least one donor cell.

[0024] In various aspects, the present invention also provides a composition comprising at least one nucleic acid molecule of the present invention.

[0025] In other aspects, the present invention provides a genetically engineered cell comprising at least one nucleic acid molecule of the present invention.

[0026] In some embodiments, the genetically engineered cells are modified to not express at least one selected from the group consisting of: B2M, class II major histocompatibility complex transactivator (CIITA), or native T cell receptor (TCR).

[0027] In some embodiments, the genetically engineered cell is a triple knockout (TKO) cell that does not express at least one selected from the group consisting of: major histocompatibility complex (MHC) I, MHC II, or native TCR.

[0028] In some embodiments, the genetically engineered cells are selected from autologous cells, allogeneic cells, alloresponsive cells, T cells, induced pluripotent stem cells (IPSC), chimeric antigen receptor (CAR) cells, engineered TCR cells, or any combination thereof.

[0029] In some embodiments, the T cell is selected from an alloresponsive T cell, a T cell with an engineered TCR, an allospecific T cell, a T cell with an alloreactive TCR, a CAR T cell, an engineered TCR-expressing T cell, or any combination thereof.

[0030] In various aspects, the invention provides a method of preventing, reducing or eliminating rejection of an allogeneic or xenograft in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of at least one cell of the invention.

[0031] In various aspects, the invention provides a method of preventing, reducing or eliminating an allogeneic response in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of at least one cell of the invention.

[0032] In various aspects, the invention provides a method of depleting the level of alloresitive cells in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of at least one cell of the invention.

[0033] In various aspects, the invention provides a method of inducing allogeneic tolerance in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of at least one cell of the invention.

[0034] In various aspects, the present invention provides a method of improving the effectiveness of CAR cell therapy in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of at least one CAR cell of the present invention.

[0035] In various aspects, the present invention provides a method of improving the effectiveness of engineered TCR cell therapy in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of at least one engineered TCR cell of the present invention.

[0036] In some embodiments, the subject has at least one selected from the following: organ transplantation, tissue transplantation, cell transplantation, allogeneic transplantation, intestinal transplantation, reconstructive transplantation, autoimmune diseases or disorders, graft-versus-host disease (GvHD), diseases or disorders associated with at least one HLA receptor, diseases or disorders associated with at least one HLA-containing receptor, diseases or disorders associated with at least one MHC receptor, diseases or disorders associated with at least one MHC-containing receptor, diseases or disorders associated with the expression of alloresitive cells, diseases or disorders associated with organ transplantation, diseases or disorders associated with tissue transplantation, diseases or disorders associated with cell transplantation, diseases or disorders associated with allogeneic transplantation, diseases or disorders associated with intestinal transplantation, cancer, diseases or disorders associated with cancer, or diseases or disorders associated with reconstructive transplantation.

[0037] In various aspects, the invention provides a method of preventing or treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of at least one cell of the invention. In some embodiments, the method comprises depleting, reducing or eliminating the level of at least one selected from the following: allogeneic responsive cells, immune cells, T cells, B cells, NK cells, leukocytes, myeloid cells (myeloid cells, bone marrow cells) or plasma cells.

[0038] In some embodiments, the method is an engineered TCR cell therapy.

[0039] In some embodiments, the disease or condition is selected from a disease or condition associated with the expression of alloresitive cells, a disease or condition associated with at least one HLA receptor, a disease or condition associated with at least one HLA-containing receptor, a disease or condition associated with at least one MHC receptor, a disease or condition associated with at least one MHC-containing receptor, GvHD, an autoimmune disease or condition, a disease or condition associated with organ transplantation, a disease or condition associated with tissue transplantation, a disease or condition associated with cell transplantation, a disease or condition associated with allogeneic transplantation, a disease or condition associated with intestinal transplantation, a disease or condition associated with reconstructive transplantation, cancer, a disease or condition associated with cancer, or any combination thereof.

[0040] In some embodiments, the disease or condition is selected from the group consisting of cancer, a disease or condition associated with cancer, and any combination thereof.

[0041] In some embodiments, the disease or disorder associated with the expression of alloresitive cells is selected from allogeneic transplant rejection, immune rejection, chronic allogeneic rejection, engraftment rejection, transplant rejection, inflammation, inflammation caused by ischemia / reperfusion, infection, immune response to an allogeneic transplant, or any combination thereof.

[0042] In some embodiments, the subject has at least one selected from an organ transplant, a tissue transplant, a cell transplant, an allogeneic transplant, an intestinal transplant, or a reconstructive transplant.

[0043] In various aspects, the invention provides a method of preventing or treating cancer, a disease or condition associated with cancer, or a combination thereof in a subject in need thereof, the method comprising administering a therapeutically effective amount of at least one cell of the invention.

[0044] In some embodiments, the method is an engineered TCR cell therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The following detailed description of exemplary embodiments of the present invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the present invention, exemplary embodiments are shown in the accompanying drawings. However, it should be understood that the present invention is not limited to the precise arrangements and means of the embodiments shown in the accompanying drawings.

[0046] Figure 1Depicted is a schematic representation of universal CAR T (UCART) cells, which overcome the shortcomings of current CAR T cell manufacturing processes.

[0047] Figure 2 Depicted is a schematic representation of MHC I knockout (KO)-induced NK cell killing under normal conditions due to the "missing self" of NK cells (Karre et al., Immunol Today Vol. 11(7), 1990); and stress ligands recognized by NK cells, which are upregulated on the surface of T cells.

[0048] Figure 3 Depicted is a schematic representation of the prevention of UCART cell clearance by expression of the human leukocyte antigen (HLA) class I histocompatibility antigen alpha chain E (HLA-E) and its interaction with the cluster of differentiation 159 (CD159) NK receptor (NKG2A).

[0049] Figure 4 Depicted is a schematic representation of the design of the optimal HLA-E single-chain trimer (SCT).

[0050] Figure 5 Depicted is a schematic representation of mitigation of target cell lysis due to NKG2C / HLA-E interaction.

[0051] Figure 6 Depicted are representative results showing transduction of K562 with HLA-E single chain dimer (SCD) and SCT.

[0052] Figure 7 Depicted are representative production of single KOs of stress-ligands on K562 cells, such as the MHC class I polypeptide-related sequence A (MICA) KO.

[0053] Figure 8 Representative results are depicted demonstrating that single KO of NK cell ligands on HLA-Eα K562 cells does not reduce 51 Residual cleavage in Cr release assay.

[0054] Fig. 9 Representative results are depicted demonstrating that blocking DNAM-1 or 2B4 does not reduce NK cell activation.

[0055] Fig.10 Representative results are depicted demonstrating that the HLA-C*03:01 leader peptide is the optimal peptide for inhibiting NK cells from cytomegalovirus (CMV)- and CMV+ donors.

[0056] Fig.11Representative results are depicted demonstrating that HLA-E SCT presenting an HLA-C leader peptide resulted in the most significant NK cell suppression.

[0057] Fig.12 Representative results are depicted demonstrating that HLA-E+ SCT confers protection against NK cell-mediated killing.

[0058] Fig.13 Representative characterization of TKO HLA-E+ T cells is depicted. Phenotypes displayed on day 14 of T cell expansion. Prior to EP, bulk T cells were activated for 5 days with anti-CD3 / 28 beads at a 3:1 beads:cell ratio.

[0059] Fig.14 Representative results are depicted demonstrating that expression of HLA-E+ on TKO T cells inhibits "missing self" responses of NK cells and that the absence of MHC class I on TKO cells results in activation of NK cells due to "missing self" responses.

[0060] Fig.15 Representative results are depicted demonstrating that HLA-E+ SCT consistently reduces cell lysis mediated by NK cells from different donors.

[0061] Fig.16 Representative results are depicted demonstrating that activated NK cells are characterized by upregulation of CD69, CD25, and CD8.

[0062] Fig.17 Representative results are depicted, demonstrating the phenotype of activated NK cells versus resting NK cells.

[0063] Fig.18 Representative results are described demonstrating that NKG2A / C expression increases upon NK cell activation.

[0064] Fig.19 Depicted are representative results demonstrating inhibition of NK cells when co-cultured with HLA-E SCT+K562 target cells.

[0065] Fig. 20 Representative results are depicted demonstrating that blocking stress ligands on K562 did not significantly reduce NK cell activation.

[0066] Fig.21 Representative results are depicted demonstrating that HLA-E+ SCT suppressed both resting and activated NK cells.

[0067] Fig. 22 Depicted is a schematic representation of NK cell deficiency associated with active human CMV (HCMV) infection.

[0068] Fig.23 Schematic representation depicting human NK cell expression and separation of NKG2A+ subset from NKG2C+ subset.

[0069] Fig.24 Representative results are depicted demonstrating that NKG2A+ NK cells but not NKG2C+ NK cells were inhibited by HLA-E+ K562 cells.

[0070] Fig.25 A schematic representation of the generation of TKO HLA-E+ SCT cells is depicted.

[0071] Fig.26 Representative results are depicted demonstrating that TKO T cells cannot be recognized by unrelated donor T cells as there is no measurable alloreactivity.

[0072] Fig. 27 Representative HLA signal peptide alignments are depicted.

[0073] Fig.28 Representative nucleotide sequences encoding HLA signal peptide alignments are depicted.

[0074] Fig.29 Representative HLA-C signal peptide-HLA-E SCT sequences are depicted.

[0075] Fig.30 Draw for 4 hours 51 Representative results of a Cr release assay demonstrating that HLA-E presenting the HLA-C*03:01 leader peptide resulted in the most significant reduction in K562 lysis, suggesting that it is able to inhibit NK cell activation due to a "missing self" response. Shown at a 2:1 E:T ratio.

[0076] Fig.31 Representative results are depicted demonstrating that HLA-E+ K562 cells inhibit NK cells from different donors and that the effect depends on phenotypic differences in the donor NK cell populations.

[0077] Fig.32 Representative results are depicted, showing Fig.31 Surface expression of NKG2A and NKG2C of the donors shown in .

[0078] Fig.33 Representative results are depicted showing NKG2A and NKG2C surface expression of expanded NK cells from 16 different normal donors.

[0079] Fig.34 include Fig.34 A and Fig.34B, depicts representative results demonstrating that sorted NKG2A+ NK cells were inhibited by HLA-E+ K562 cells, and sorted NKG2C+ NK cells were not inhibited by HLA-E expression on K562. (**P<0.0025, ***P<0.000125; data are shown in 2:1 E:T ratio). Fig.34 A depicts representative results demonstrating that sorted NKG2A+ NK cells were inhibited by HLA-E+ K562 cells. Fig.34 B depicts representative results demonstrating that sorted NKG2C+ NK cells are not inhibited by HLA-E expression on K562.

[0080] Fig.35 Depicted are representative results demonstrating that degranulation of NKG2A+NK cells was reduced when co-cultured with HLA-E+K562 cells. Degranulation of NKG2C+NK cells was increased when co-cultured with HLA-E+K562, indicating NK cell activation.

[0081] Fig.36 Representative results are depicted demonstrating the editing strategy and efficiency for generating TKO HLA-E+ T cells.

[0082] Fig.37 Depicted are representative results demonstrating that TKO T cells were not recognized by allogeneic donor PBMCs, as no alloreactive responses were measured in mixed lymphocyte reactions (MLRs).

[0083] Fig.38 Representative results are depicted demonstrating that TKO CAR T cells are targeted and killed by NK cells due to a "missing self" response. HLA-E expression on TKO CAR T cells inhibits NK cell-mediated lysis.

[0084] Fig.39 Representative results are depicted demonstrating that HLA-E SCT expression exhibits a protective effect against NK activity in vivo. DETAILED DESCRIPTION

[0085] The present invention is based in part on the unexpected result that the expression of modified human leukocyte antigen (HLA) class I histocompatibility antigen alpha chain E (HLA-E) and the absence of beta-2-microglobulin (B2M), class II major histocompatibility complex transactivator (CIITA), and natural T cell receptor (TCR) alpha chain inhibits natural killer (NK) cells and increases the persistence of donor T cells in the host. Thus, in one aspect, the present invention provides a single chain trimer comprising modified B2M, HLA-E, and an HLA signal peptide or a fragment thereof, the HLA signal peptide or a fragment thereof comprising at least one amino acid sequence having at least about 70% identity with an amino acid sequence selected from SEQ ID NOs: 1-10, 13, and 46. In some embodiments, the single chain trimer comprises an amino acid sequence having at least about 70% identity with the amino acid sequence shown in SEQ ID NO:40.

[0086] In another aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a single-chain trimer comprising a modified B2M, HLA-E, and an HLA signal peptide or a fragment thereof, wherein the HLA signal peptide or a fragment thereof comprises at least one amino acid sequence having at least about 70% identity with an amino acid sequence selected from SEQ ID NOs: 1-10, 13, and 46. In another aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a modified B2M, a nucleotide sequence encoding HLA-E, and a nucleotide sequence encoding an HLA signal peptide or a fragment thereof, wherein the nucleotide sequence encoding the HLA signal peptide or a fragment thereof comprises at least one nucleotide sequence encoding sequence having at least about 70% identity with a nucleotide sequence selected from SEQ ID NOs: 14-24 and 47. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence having at least about 70% identity with the nucleotide sequence shown in SEQ ID NO: 41. In various embodiments, the present invention also provides a composition and a genetically engineered cell comprising at least a nucleic acid molecule of the present invention.

[0087] In some aspects, the present invention provides a method of preventing, reducing or eliminating alloresponses, allorecognition of donor cells, or rejection of allogeneic or xenogeneic transplants, a method of depleting alloresponsive cell levels, a method of inducing alloresponsive tolerance, a method of improving the effectiveness of CAR cell therapy and / or engineered TCR cell therapy, and / or a method of preventing or treating a disease or condition in a subject in need thereof using at least one genetically engineered cell of the present invention. In some embodiments, the subject has an organ transplant, a tissue transplant, a cell transplant, an allogeneic transplant, an intestinal transplant, a reconstructive transplant, cancer, and / or a cancer-related disease or condition.

[0088] definition

[0089] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described.

[0090] As used herein, each of the following terms has the meaning associated with it in this section.

[0091] The articles "a" and "an" are used herein to refer to one or more than one (ie, at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

[0092] As used herein, "about" when referring to a measurable value (such as an amount, a temporal duration, etc.) is meant to encompass variations of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% of the specified value, as such variations are suitable for performing the disclosed methods.

[0093] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any animal, in some embodiments a mammal, and in some embodiments a human, having a complement system, including humans who are in need of therapy for a condition or its sequelae, or who are susceptible to a condition or its sequelae. Individuals may include, for example, dogs, cats, pigs, cows, sheep, goats, horses, rats, monkeys, and mice, as well as humans.

[0094] The term "abnormal" when used in the context of organisms, tissues, cells, or components thereof, refers to those organisms, tissues, cells, or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) compared to those organisms, tissues, cells, or components thereof that display the "normal" (expected / steady-state) corresponding characteristic. A characteristic that is normal or expected for one cell, tissue type, or subject, may be abnormal for a different cell or tissue type.

[0095] A "disease" is a condition in a subject's health in which the subject is unable to maintain homeostasis and in which the subject's health will continue to deteriorate if the disease is not ameliorated.

[0096] In contrast, a "disorder" in a subject is a state of health in the subject in which the subject is able to maintain homeostasis, but in which the subject's state of health is not as good as it would be without the disorder. If left untreated, the disorder will not necessarily lead to a further decline in the subject's state of health.

[0097] "Cancer" used herein refers to the abnormal growth or division of cells. In general, the growth and / or life span of cancer cells exceed the growth and / or life span of normal cells and tissues around them, and are inconsistent with the growth and / or life span of normal cells and tissues around them. Cancer can be benign, pre-malignant or malignant. Cancer occurs in a variety of cells and tissues, including oral cavity (e.g., mouth, tongue, throat, etc.), digestive system (e.g., esophagus, stomach, small intestine, colon, rectum, liver, bile duct, gallbladder, pancreas, etc.), respiratory system (e.g., larynx, lung, bronchus, etc.), bones, joints, skin (e.g., basal cells, squamous cells, meningioma, etc.), breast, reproductive system (e.g., uterus, ovary, prostate, testicles, etc.), urinary system (e.g., bladder, kidney, ureter, etc.), eyes, nervous system (e.g., brain, etc.), endocrine system (e.g., thyroid, etc.), and hematopoietic system (e.g., lymphoma, myeloma, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, etc.).

[0098] A disease or condition is "reduced" if the severity of the signs or symptoms of the disease or condition, the frequency with which a patient experiences such signs or symptoms, or both, is reduced.

[0099] As used herein, "activated" refers to the state of a T cell that has been sufficiently stimulated to induce detectable cell proliferation. Activation can also be associated with induced cytokine production and detectable effector function. The term "activated T cell" refers in particular to a T cell that is undergoing cell division.

[0100] As used herein, the terms "inhibit" and "inhibition" refer to reducing, suppressing, weakening or blocking an activity or function by at least about 10% relative to a control value. In some embodiments, the activity is inhibited or blocked by at least about 50% compared to a control value. In some embodiments, the activity is inhibited or blocked by at least about 75%. In some embodiments, the activity is inhibited or blocked by at least about 95%.

[0101] As used herein, the term "autologous" refers to any material derived from the same individual that is to be later reintroduced into that individual.

[0102] "Allogeneic" refers to any material derived from a different animal of the same species as the individual into which the material is introduced. Two or more individuals are said to be allogeneic to each other when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be genetically different enough to produce antigenic interactions.

[0103] As used herein, the phrase "disease associated with the expression of alloresitive cells" includes, but is not limited to, diseases associated with the expression of alloresitive cells or conditions associated with cells expressing alloresitiveness, including, for example, allogeneic transplant rejection, immune rejection, chronic allogeneic rejection, implant rejection, transplant rejection, inflammation, inflammation caused by ischemia / reperfusion, infection, immune response to an allogeneic transplant, and any combination thereof.

[0104] "Xenogeneic" refers to a transplant that originates from an animal of a different species.

[0105] As used herein, "endogenous" refers to any material that originates from or is produced within an organism, cell, tissue, or system.

[0106] As used herein, the term "exogenous" refers to any material introduced from outside of an organism, cell, tissue or system or generated outside of an organism, cell, tissue or system.

[0107] As used herein, the term "immune cell" includes cells of hematopoietic origin and that play a role in immune response. Immune cells include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, dendritic cells, eosinophils, mast cells, basophils and granulocytes.

[0108] As used herein, the term "immune response" includes T cell-mediated and / or B cell-mediated immune responses that are affected by T cell costimulatory regulation. The term "immune response" further includes immune responses that are indirectly affected by T cell activation, such as antibody production (humoral response) and activation of cytokine responsive cells (such as macrophages).

[0109] As used herein, the term "T cell immune response" refers to the activation of antigen-specific T cells as measured by proliferation or expression of molecules on the cell surface or secretion of proteins such as cytokines.

[0110] As used herein, the term "T cell" refers to a lymphocyte (e.g., leukocyte) that plays a role in cell-mediated immunity. In some embodiments, the presence of a T cell receptor (TCR) on the cell surface distinguishes T cells from other lymphocytes. As known in the art, T cells do not usually present antigens, and rely on other lymphocytes (e.g., natural killer cells and B cells) to help antigen presentation. The types of T cells include: T helper cells (TH cells), memory T cells (Tcm, Tem or Temra), regulatory T cells (Treg), cytotoxic T cells (CTL), natural killer T cells (NK cells), γδT cells, and mucosal-associated invariant T cells (MAIT).

[0111] As used herein, the term "TCR" refers to "T cell receptor". A T cell receptor is a molecule on the surface of a T lymphocyte ("T cell"). In various embodiments, the receptor is an αβ-TCR receptor, which means that the T cell receptor comprises an alpha (α) and a beta (β) chain, which is usually expressed as part of a complex with a CD3 chain molecule.

[0112] As used herein, the term "B cell" refers to a cell produced in the bone marrow of an animal that expresses membrane-bound antibodies specific for an antigen. After interacting with an antigen, it differentiates into a plasma cell or memory B cell that produces antibodies specific for the antigen. "B cell" and "B lymphocyte" are used interchangeably. Both naive B cells and activated B cells are within the scope of the present invention.

[0113] As used herein, the term "chimeric antigen receptor" or alternatively "CAR" refers to a recombinant polypeptide construct, which at least includes an extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic signaling domain, and the cytoplasmic signaling domain includes a functional signaling domain derived from a stimulatory molecule as defined below. In one aspect, the stimulatory molecule is a ζ chain associated with a TCR complex. In one aspect, the cytoplasmic signaling domain further includes one or more functional signaling domains derived from at least one costimulatory molecule as defined below. In one aspect, the costimulatory molecule is selected from 41BB (i.e., CD137), CD3, and / or CD28. In one aspect, CAR includes a chimeric fusion protein, which includes an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain, and the intracellular signaling domain includes a functional signaling domain derived from a stimulatory molecule. In one aspect, CAR includes a chimeric fusion protein, which includes an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain, and the intracellular signaling domain includes a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In one aspect, CAR comprises a chimeric fusion protein, and the chimeric fusion protein comprises an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signal domain, and the intracellular signal domain comprises two functional signal domains derived from one or more costimulatory molecules and a functional signal domain derived from a stimulatory molecule. In one aspect, CAR comprises a chimeric fusion protein, and the chimeric fusion protein comprises an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signal domain, and the intracellular signal domain comprises at least two functional signal domains derived from one or more costimulatory molecules and a functional signal domain derived from a stimulatory molecule. In one aspect, CAR comprises an optional leader sequence at the amino terminus (N-ter) of the CAR fusion protein. In one aspect, CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally cut from the scFv domain during cell processing and CAR localization to the cell membrane.

[0114] The part of the CAR composition comprising an antibody or its antibody fragment can exist in various forms, wherein the antigen binding domain is expressed as part of a continuous polypeptide chain, the continuous polypeptide chain including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv) and a humanized antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85: 5879-5883; Bird et al., 1988, Science 242: 423-426). In one aspect, the antigen binding domain of the CAR composition of the present invention comprises an antibody fragment. In one embodiment, CAR comprises an antibody fragment containing scFv.

[0115] As used herein, a "signaling domain" is a functional portion of a protein that acts as an effector to transmit information within a cell through a defined signaling pathway to regulate cellular activity by producing second messengers or by responding to such messengers.

[0116] As used herein, the term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be polyclonal or monoclonal, multi-chain or single-chain, or complete immunoglobulins, and can be derived from natural or recombinant sources. Antibodies are typically tetramers of immunoglobulin molecules.

[0117] The term "antibody fragment" refers to at least a portion of an intact antibody or a recombinant variant thereof, and refers to an antigen binding domain, such as an antigen-determining variable region of an intact antibody, which is sufficient to confer recognition and specific binding to a target (such as an antigen) on the antibody fragment. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, scFv antibody fragments, linear antibodies, single domain antibodies such as sdAb (VL or VH), Camelidae VHH domains, and multispecific antibodies formed by antibody fragments. The term "scFv" refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are continuously connected by a short flexible polypeptide linker and can be expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise indicated, as used herein, a scFv may have the VL and VH variable regions in any order, e.g., with respect to the N-terminus and C-terminus of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

[0118] As used herein, "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and generally determines the class to which the antibody belongs.

[0119] As used herein, "antibody light chain" refers to the smaller of the two types of polypeptide chains present in antibody molecules in a naturally occurring conformation. Kappa and lambda light chains refer to the two major antibody light chain isotypes.

[0120] As used herein, the term "recombinant antibody" refers to an antibody produced using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or yeast expression system. The term should also be interpreted as referring to an antibody produced by synthesizing a DNA molecule encoding the antibody, and the DNA molecule expresses the antibody protein or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence is obtained using recombinant DNA or amino acid sequence technology available and well known in the art.

[0121] As used herein, the term "antigen" or "Ag" is defined as a molecule that triggers an immune response. This immune response may involve antibody production, or the activation of specific immune-competent cells, or both. It will be understood by those skilled in the art that any macromolecule (including almost all proteins or peptides) can be used as an antigen. In addition, antigens can be derived from recombinant or genomic DNA. It will be understood by those skilled in the art that any DNA containing a nucleotide sequence or a partial nucleotide sequence encoding a protein that triggers an immune response thus encodes the term "antigen" used herein. In addition, it will be understood by those skilled in the art that antigens do not have to be encoded only by the full-length nucleotide sequence of a gene. It is obvious that the present invention includes but is not limited to using partial nucleotide sequences of more than one gene, and these nucleotide sequences are arranged in various combinations to encode polypeptides that trigger a desired immune response. In addition, it will be understood by those skilled in the art that antigens do not need to be encoded by "genes" at all. It is obvious that antigens can be synthesized or can be derived from biological samples, or may be macromolecules other than polypeptides. Such biological samples may include but are not limited to tissue samples, cells, or fluids with other biological components.

[0122] As used herein, "antigen presenting cell" or "APC" refers to an immune system cell, such as a helper cell (e.g., B cell, dendritic cell, etc.), which displays foreign antigens complexed with HLA I, HLA II, MHC I, MHC II, or MHC III complexes on its surface. For example, T cells can recognize these complexes using their T-cell receptors (TCRs). APCs process antigens and present them to T cells.

[0123] The term "stimulation" refers to a primary response induced by the binding of a stimulatory molecule (e.g., a TCR complex, a BCR complex, etc.) to its cognate ligand, thereby mediating a signal transduction event (such as, but not limited to, signal transduction by a TCR complex and / or a BCR complex, etc.). Stimulation can mediate changes in the expression of certain molecules, such as downregulation of TGF-β and / or reorganization of cytoskeletal structure, etc.

[0124] The term "stimulatory molecule" used herein refers to a molecule expressed by a cell, which provides a primary cytoplasmic signal sequence that regulates the primary activation of TCR and / or BCR complexes in a stimulating manner for at least some aspects of cell signaling pathways. In one aspect, the primary signal is initiated by binding of, for example, TCR and / or BCR complexes to human leukocyte antigen (HLA) I, HLA II, major histocompatibility complex (MHC) I, MHC II or MHC III molecules loaded with peptides, and causes mediated cell responses, including but not limited to proliferation, activation, differentiation, etc. The primary cytoplasmic signal sequence that works in a stimulating manner can contain a signal motif referred to as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of primary cytoplasmic signal sequences containing ITAM useful in the present invention include those from TCR ζ, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CD5, CD22, CD79a, CD79b, CD278 (also referred to as "ICOS") and CD66d. In certain CARs of the invention, the cytoplasmic signal sequence derived from CD3-ζ is derived from a non-human species, such as a mouse, rodent, monkey, ape, etc.

[0125] The terms "effective amount" and "pharmaceutically effective amount" refer to an amount of a drug sufficient to provide a desired biological result. The result may be a reduction and / or alleviation of the signs, symptoms or causes of a disease or condition, or any other desired alteration of a biological system. One of ordinary skill in the art can determine the appropriate effective amount in any individual case using routine experimentation.

[0126] As used herein, the term "therapeutic" means to treat. A therapeutic effect is obtained by reducing, inhibiting, alleviating or eradicating the disease state.

[0127] "Therapeutic treatment" is treatment given to a subject who exhibits signs of a disease or condition with the goal of reducing or eliminating those signs.

[0128] As used herein, "treating a disease or condition" means reducing the frequency and / or severity of the signs and / or symptoms of the disease or condition experienced by a patient.

[0129] As used herein, the term "prevention" refers to the prophylactic or protective treatment of a disease or disease state.

[0130] As used herein, the phrases "biological sample," "sample" or "specimen" are intended to include any sample comprising cells, tissues, or body fluids in which expression of a nucleic acid or polypeptide can be detected. A biological sample can contain any biological material suitable for detecting a desired biomarker, and can include cells and / or non-cellular material obtained from an individual. Examples of such biological samples include, but are not limited to, blood, lymph, bone marrow, biopsies, and smears. Samples of a liquid nature are referred to herein as "body fluids." Biological samples can be obtained from a patient by a variety of techniques, including, for example, scraping or swabbing an area or using a needle to obtain body fluids. Methods for collecting various body samples are well known in the art.

[0131] "CDR" is defined as the complementarity determining region amino acid sequence of a TCR or TCR chain.

[0132] As used herein, "immunoassay" refers to any binding assay that uses antibodies capable of specifically binding to the target molecule to detect and quantify the target molecule.

[0133] The term "specific binding" used herein with respect to a polypeptide (e.g., TCR or TCR chain) refers to a polypeptide that recognizes and binds to a specific target molecule but does not substantially recognize or bind to other molecules in a sample. In some cases, the term "specific binding" or "specifically binds" is used to indicate that recognition and binding depend on the presence of a specific structure (e.g., antigenic determinant or epitope) on the target molecule.

[0134] The "coding region" of a gene is composed of nucleotide residues of the gene coding chain and nucleotides of the gene non-coding chain, which are homologous or complementary to the coding region of the mRNA molecule produced by gene transcription. The "coding region" of an mRNA molecule is also composed of nucleotide residues of the mRNA molecule, which match or encode a stop codon with the anticodon region of the transfer RNA molecule during translation of the mRNA molecule. Therefore, the coding region can include nucleotide residues that include codons for amino acid residues that are not present in the mature protein encoded by the mRNA molecule (e.g., amino acid residues in a protein export signal sequence).

[0135] The "complementarity" used in nucleic acid herein refers to the broad concept of sequence complementarity between the regions of two nucleic acid chains or between two regions of the same nucleic acid chain. It is known that the adenine residues of the first nucleic acid region can form specific hydrogen bonds ("base pairing") with the residues of the second nucleic acid region that are antiparallel to the first region (if the residues are thymine or uracil). Similarly, it is known that the cytosine residues of the first nucleic acid chain can base pair with the residues of the second nucleic acid chain that are antiparallel to the first chain (if the residues are guanine). If at least one nucleotide residue in the first region can base pair with the residues in the second region when the first region of the nucleic acid is arranged in an antiparallel manner with the second region of the same or different nucleic acids, the two regions are complementary. In some embodiments, the first region comprises the first part, and the second region comprises the second part, whereby, when the first and second parts are arranged in an antiparallel manner, at least about 50%, or at least about 75%, or at least about 90%, or at least about 95% of the nucleotide residues of the first part can base pair with the nucleotide residues in the second part. In some embodiments, all nucleotide residues in the first part can base pair with the nucleotide residues in the second part.

[0136] "Homologous" refers to the sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. When a position in the two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if the position in each of the two DNA molecules is occupied by adenine, the two molecules are homologous at that position. The percentage of homology between the two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in the two sequences are matched or homologous, the two sequences have 60% homology. Typically, the comparison is performed when the two sequences are aligned to provide maximum homology.

[0137] The term "variant" as used herein is a nucleic acid sequence or peptide sequence that is different in sequence from a reference nucleic acid sequence or peptide sequence, respectively, but retains the basic biological properties of the reference molecule. Changes in the sequence of nucleic acid variants may not change the amino acid sequence of the peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions and truncations. Changes in peptide variant sequences are generally limited or conservative, so that the sequences of the reference peptide and variant are generally very similar and identical in multiple regions. The amino acid sequences of variants and reference peptides may differ in any combination due to one or more substitutions, additions, deletions. The variants of nucleic acids or peptides may be naturally occurring, such as allelic variants, or may be variants that are not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides may be prepared by mutagenesis techniques or direct synthesis. In various embodiments, the variant sequence is at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 89%, at least 88%, at least 87%, at least 86%, or at least 85% identical to the reference sequence.

[0138] As used herein, the term "DNA" is defined as deoxyribonucleic acid.

[0139] "Encoding" refers to the following inherent properties of a specific nucleotide sequence in a polynucleotide (such as a gene, cDNA or mRNA): in biological processes, it is used as a template for the synthesis of other polymers and macromolecules with a determined nucleotide sequence (i.e., rRNA, tRNA and mRNA) or a determined amino acid sequence and the resulting biological properties. Therefore, if the transcription and translation of the mRNA corresponding to the gene produces a protein in a cell or other biological system, the gene encodes the protein. Both the coding strand (whose nucleotide sequence is the same as the mRNA sequence and is usually provided in the sequence table) and the non-coding strand (used as a template for transcription of a gene or cDNA) can be referred to as encoding a protein or other product of the gene or cDNA.

[0140] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. The phrase "nucleotide sequence encoding a protein or RNA" may also include introns, to the extent that a nucleotide sequence encoding a protein may contain introns in some versions.

[0141] "Isolated" means changed or removed from the natural state. For example, a nucleic acid or peptide naturally present in the normal environment of a living body is not "isolated", but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural environment is "isolated". An isolated nucleic acid or protein may exist in a substantially purified form, or may exist in a non-natural environment (such as, for example, a host cell).

[0142] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides that have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0143] In the context of the present invention, the following abbreviations for common nucleic acid bases are used: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0144] As used herein, the term "polynucleotide" is defined as a chain of nucleotides. In addition, nucleic acids are polymers of nucleotides. Therefore, nucleic acids and polynucleotides used herein are interchangeable. It is generally known to those skilled in the art that nucleic acids are polynucleotides, which can be hydrolyzed into monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed into nucleosides. Polynucleotides used herein include, but are not limited to, all nucleic acid sequences obtained by any method available in the art, including, but not limited to, recombinant methods (i.e., cloning nucleic acid sequences from a recombinant library or cell genome using common cloning techniques and PCR, etc.) and by synthetic methods.

[0145] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can make up a protein or peptide sequence. Polypeptides include any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains, for example, also commonly referred to as peptides, oligopeptides and oligomers in the art, and long chains, commonly referred to as proteins in the art, of which there are many types. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0146] As used herein, the term "RNA" is defined as ribonucleic acid.

[0147] As used herein, the term "recombinant DNA" is defined as DNA produced by joining DNA fragments from different sources.

[0148] As used herein, the term "recombinant polypeptide" is defined as a polypeptide produced using recombinant DNA methodologies.

[0149] As used herein, "conjugation" refers to the covalent attachment of one molecule to a second molecule.

[0150] As used herein, "operably linked" or "operatively linked" may refer to the expression of a gene being controlled by a promoter to which it is spatially linked. A promoter may be located 5' (upstream) or 3' (downstream) of a gene under its control. The distance between a promoter and a gene may be approximately the same as the distance between the promoter and the gene it controls in the gene from which it is derived. As is known in the art, variations in this distance may be accommodated without loss of promoter function.

[0151] As used herein, the term "promoter" is defined as a DNA sequence that is recognized by the cellular synthetic machinery or introduced synthetic machinery and is required to initiate specific transcription of a polynucleotide sequence.

[0152] As used herein, the term "regulation" may refer to any method that changes the level or activity of a substrate. Non-limiting examples of regulation of a protein include affecting expression (including transcription and / or translation), affecting folding, affecting degradation or protein turnover, and affecting the location of the protein. Non-limiting examples of regulation of an enzyme further include affecting enzyme activity. "Regulator" refers to a molecule whose activity includes affecting substrate levels or activity. A regulator may be direct or indirect. A regulator may play a role in activating or inhibiting or otherwise regulating its substrate.

[0153] As used herein, "vector" may refer to a nucleic acid sequence containing a replication origin. A vector may be a plasmid, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. A vector may be a DNA or RNA vector. A vector may be a self-replicating extrachromosomal vector or a vector that integrates into the host genome.

[0154] As used herein, "substantially purified" cells are cells that are substantially free of other cell types. Substantially purified cells also refer to cells that have been separated from other cell types with which they are normally associated in their naturally occurring state. In some cases, a substantially purified cell population refers to a homogenous cell population. In other cases, the term refers only to cells that have been separated from cells with which they are naturally associated in their naturally occurring state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0155] "Knockout" refers to the disruption of a specific single gene or allele of a gene from the genome by genetic manipulation. Thus, a "single allele knockout cell" refers to a cell in which a single allele of a gene is disrupted so that its gene product is not expressed.

[0156] The term "knockout construct" refers to a nucleic acid sequence intended to reduce or suppress the expression of a protein encoded by an endogenous DNA sequence in a cell. The nucleic acid sequence used as a knockout construct is generally composed of (1) DNA from some parts of a gene to be suppressed (exon sequence, intron sequence and / or promoter sequence) and (2) a marker sequence for detecting the presence of a knockout construct in a cell. The knockout construct is inserted into the cell and integrated with the genomic DNA of the cell, and the integration position prevents or interrupts the transcription of the natural DNA sequence. This insertion generally occurs by homologous recombination (that is, when the knockout construct is inserted into the cell, the region of the knockout construct homologous to the endogenous DNA sequence hybridizes with each other and recombines, so that the knockout construct is incorporated into the corresponding position of the endogenous DNA). The knockout construct nucleic acid sequence can include 1) all or part of the sequence of one or more exons and / or introns of the gene to be suppressed, 2) all or part of the promoter sequence of the gene to be suppressed, or 3) a combination thereof.

[0157] Range: Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that descriptions in range format are only for convenience and brevity, and should not be construed as rigid limitations on the scope of the invention. Therefore, the description of a range should be considered to have specifically disclosed all possible subranges within the range as well as single numerical values. For example, a range description (such as 1 to 6) should be considered to have specifically disclosed subranges within the range (such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc.) and single numbers (e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6). This applies regardless of the width of the range.

[0158] describe

[0159] The present invention provides a single-chain trimer comprising a modified B2M, HLA-E, and an HLA signal peptide or a fragment thereof, wherein the HLA signal peptide or a fragment thereof comprises at least one amino acid sequence having at least about 70% identity with an amino acid sequence selected from SEQ ID NOs: 1-10, 13, and 46. In some embodiments, the single-chain trimer comprises an amino acid sequence having at least about 70% identity with the amino acid sequence shown in SEQ ID NO: 40. In another aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a single-chain trimer comprising a modified B2M, HLA-E, and an HLA signal peptide or a fragment thereof, wherein the HLA signal peptide or a fragment thereof comprises at least one amino acid sequence having at least about 70% identity with an amino acid sequence selected from SEQ ID NOs: 1-10, 13, and 46. In another aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a modified B2M, a nucleotide sequence encoding HLA-E, and a nucleotide sequence encoding an HLA signal peptide or a fragment thereof, wherein the nucleotide sequence encoding the HLA signal peptide or a fragment thereof comprises at least one nucleotide sequence encoding sequence having at least about 70% identity with a nucleotide sequence selected from SEQ ID NOs: 14-24 and 47. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence having at least about 70% identity with the nucleotide sequence shown in SEQ ID NO: 41. In various embodiments, the present invention also provides a composition and a genetically engineered cell comprising at least the nucleic acid molecule of the present invention.

[0160] In certain aspects, the present invention provides a method of preventing, reducing or eliminating alloresponses, allorecognition of donor cells, or rejection of allogeneic or xenogeneic transplants, a method of depleting alloresponsive cell levels, a method of inducing alloresponsive tolerance, a method of improving the effectiveness of CAR therapy, TCR therapy, CAR cell therapy and / or engineered TCR cell therapy, a method of preventing or treating a disease or condition in a subject in need thereof using at least one genetically engineered cell of the present invention. In some embodiments, the subject has an organ transplant, a tissue transplant, a cell transplant, an allogeneic transplant, an intestinal transplant, a reconstructive transplant, cancer, and / or a disease or condition associated with cancer.

[0161] Peptides, nucleic acid molecules and compositions

[0162] In one aspect, the present invention relates in part to a peptide for reducing the removal of at least one cell of interest or increasing its persistence, the peptide comprising an HLA signal peptide or a fragment thereof, a modified B2M or a fragment thereof, and an HLA-E or a fragment thereof. In some embodiments, the peptide is a single-chain trimer. In another aspect, the present invention provides a nucleic acid molecule for reducing the removal of at least one cell of interest or increasing its persistence, the nucleic acid molecule comprising a nucleotide sequence encoding a peptide, the peptide comprising an HLA signal peptide or a fragment thereof, a modified B2M or a fragment thereof, and an HLA-E or a fragment thereof. In another aspect, a nucleic acid molecule for reducing the removal of at least one cell of interest or increasing its persistence, the nucleic acid molecule comprising a nucleotide sequence encoding an HLA signal peptide or a fragment thereof, a nucleotide sequence encoding a modified B2M or a fragment thereof, and a nucleotide sequence encoding an HLA-E or a fragment thereof. In various embodiments, at least one cell of interest is a donor cell, such as a T cell, a CAR T cell, a T cell expressing an engineered TCR, etc.

[0163] In some embodiments, HLA-E inhibits NK cell-mediated killing of donor cells. In some embodiments, HLA-E comprises a modified HLA-E or a fragment thereof. In some embodiments, HLA-E comprises human HLA-E or a fragment thereof. In some embodiments, HLA-E comprises HLA-E*0101 or a fragment thereof and / or HLA-E*0103 or a fragment thereof. For example, in some embodiments, HLA-E comprises a 64 to 1077 amino acid fragment of HLA-E*0101 shown in SEQ ID NO:37. In some embodiments, HLA-E comprises the amino acid sequence of HLA-E*0103 shown in SEQ ID NO:43.

[0164] In some embodiments, the modified B2M includes a B2M signal peptide or a fragment thereof and / or a B2M or a fragment thereof without a leader sequence. In one embodiment, the B2M signal peptide is a modified B2M signal peptide as shown in SEQ ID NO: 25. In some embodiments, the B2M without a leader sequence is a modified B2M without a leader sequence as shown in SEQ ID NO: 31.

[0165] In some embodiments, the modified B2M or fragment thereof is linked to HLA-E or a fragment thereof, an HLA signal peptide or a fragment thereof, or any combination thereof. In some embodiments, the modified B2M or fragment thereof is directly linked to HLA-E or a fragment thereof, an HLA signal peptide or a fragment thereof, or any combination thereof. In some embodiments, the modified B2M or fragment thereof is linked to HLA-E or a fragment thereof, an HLA signal peptide or a fragment thereof, or any combination thereof via a linker and / or a spacer.

[0166] In some embodiments, the nucleotide sequence encoding the modified B2M or a fragment thereof is linked to a nucleotide sequence encoding HLA-E or a fragment thereof, a nucleotide sequence encoding an HLA signal peptide or a fragment thereof, or any combination thereof. In some embodiments, the nucleotide sequence encoding the modified B2M or a fragment thereof is directly linked to a nucleotide sequence encoding HLA-E or a fragment thereof, a nucleotide sequence encoding an HLA signal peptide or a fragment thereof, or any combination thereof. In some embodiments, the nucleotide sequence encoding the modified B2M or a fragment thereof is linked to a nucleotide sequence encoding HLA-E or a fragment thereof, a nucleotide sequence encoding an HLA signal peptide or a fragment thereof, or any combination thereof via a linker and / or a spacer.

[0167] In various embodiments, the joint is any joint known in the art. In one embodiment, the joint is not a cleavable joint. In one embodiment, the joint is a cleavable joint. In some embodiments, the cleavable joint is a chemically cleavable joint, an enzyme-cleavable joint, a peptide-based joint, or any combination thereof. In some embodiments, the chemically cleavable joint is an acid-cleavable joint or a reducible joint. In one embodiment, the acid-cleavable joint is specifically designed to remain stable at the neutral pH of the blood circulation, but is hydrolyzed and releases cytotoxic drugs in the acidic environment of the cell compartment. In one embodiment, the reducible joint is designed to remain stable in the oxygen-rich environment in the blood stream, and is selectively cut in the reducing environment of the cell. In another embodiment, the peptide-based joint is designed to remain intact in the systemic circulation and is cut by a specific intracellular protease (such as cathepsin B). Examples of linkers include, but are not limited to, linkers containing lysosomal-specific protease cleavage sites, linkers containing mixed disulfides, aminoethoxyethoxyacetate (AEEA), β-glucuronide linkers, peptide linkers cleavable by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), dipeptide linkers (e.g., valine-citrulline (val-cit) or phenylalanine-lysine (phe-lys) linkers), aminocaproic acid, hydrazone, thiomaleimide, and dibenzocyclooctyne (DBCO). For example, in some embodiments, the linker comprises the amino acids shown in SEQ ID NO: 28. In some embodiments, the linker comprises the nucleotide sequence shown in SEQ ID No: 29-30. In some embodiments, the linker comprises the nucleotide sequence shown in SEQ ID No: 29.

[0168] In various embodiments, the spacer is any spacer known in the art. For example, in some embodiments, the spacer comprises the amino acids shown in SEQ ID NO:34. In some embodiments, the linker comprises the nucleotide sequence shown in SEQ ID No:35-36. In some embodiments, the linker comprises the nucleotide sequence shown in SEQ ID No:35.

[0169] In some embodiments, the peptide comprises a modB2M signal peptide (e.g., SEQ ID NO: 25), an HLA signal peptide described herein (e.g., SEQ ID NO: 1-10, 13, or 46), a modB2M without a leader sequence (e.g., SEQ ID NO: 31), and HLA-E (e.g., SEQ ID NO: 37 or 44). In one embodiment, the peptide comprises a linker or spacer between the HLA signal peptide and the modB2M without a leader sequence. In one embodiment, the peptide comprises a linker or spacer between modB2M without a leader sequence and HLA-E.

[0170] For example, in some embodiments, the modified B2M signal peptide or a fragment thereof shown in SEQ ID NO:25 is directly linked to the HLA signal peptide or a fragment thereof shown in SEQ ID NO:3; the HLA signal peptide or fragment is linked to the modified B2M or a fragment thereof without a leader sequence shown in SEQ ID NO:31 via the linker shown in SEQ ID NO:28; and the modified B2M or a fragment thereof without a leader sequence is linked to the HLA-E or a fragment thereof shown in SEQ ID NO:37 via the spacer shown in SEQ ID NO:34.

[0171] In other embodiments, the modified B2M signal peptide or a fragment thereof shown in SEQ ID NO:25 is directly linked to the HLA signal peptide or a fragment thereof shown in SEQ ID NO:3; the HLA signal peptide or fragment is linked to the modified B2M or a fragment thereof without a leader sequence shown in SEQ ID NO:31 via the linker shown in SEQ ID NO:28; and the modified B2M or a fragment thereof without a leader sequence is linked to the HLA-E or a fragment thereof shown in SEQ ID NO:44 via the spacer shown in SEQ ID NO:34.

[0172] In some embodiments, the nucleic acid molecule encoding the peptide of the present invention comprises a nucleotide sequence encoding a modB2M signal peptide (e.g., SEQ ID NO: 26 or 27), a nucleotide sequence encoding an HLA signal peptide described herein (e.g., SEQ ID NO: 14-24, 43 or 47), a nucleotide sequence encoding a modB2M without a leader sequence (e.g., SEQ ID NO: 32 or 33), and a nucleotide sequence encoding HLA-E (e.g., SEQ ID NO: 38, 39 or 45). In one embodiment, the nucleic acid molecule comprises a linker or spacer between the nucleotide sequence encoding the HLA signal peptide and the nucleotide sequence encoding the modB2M without a leader sequence. In one embodiment, the nucleic acid molecule comprises a linker or spacer between the nucleotide sequence encoding the modB2M without a leader sequence and the nucleotide sequence encoding HLA-E.

[0173] For example, in some embodiments, the nucleotide sequence encoding the modified B2M signal peptide shown in SEQ ID NO:26 or a fragment thereof is directly linked to the nucleotide sequence encoding the HLA signal peptide shown in SEQ ID NO:14 or a fragment thereof; the nucleotide sequence or fragment encoding the HLA signal peptide is linked to the nucleotide sequence encoding the modified B2M without a leader sequence or a fragment thereof shown in SEQ ID NO:32 via the linker shown in SEQ ID NO:29; and the nucleotide sequence encoding the modified B2M without a leader sequence or a fragment thereof is linked to the nucleotide sequence encoding HLA-E shown in SEQ ID NO:38 or a fragment thereof via the spacer shown in SEQ ID NO:35.

[0174] In some embodiments, the nucleotide sequence encoding the modified B2M signal peptide shown in SEQ ID NO:26 or a fragment thereof is directly linked to the nucleotide sequence encoding the HLA signal peptide shown in SEQ ID NO:14 or a fragment thereof; the nucleotide sequence or fragment encoding the HLA signal peptide is linked to the nucleotide sequence encoding the modified B2M without a leader sequence or a fragment thereof shown in SEQ ID NO:32 via the linker shown in SEQ ID NO:29; and the nucleotide sequence encoding the modified B2M without a leader sequence or a fragment thereof is linked to the nucleotide sequence encoding HLA-E shown in SEQ ID NO:45 or a fragment thereof via the spacer shown in SEQ ID NO:35.

[0175] In one embodiment, the length of the HLA signal peptide or fragment thereof is about 8 to about 24 amino acid residues, or about 9 to about 11 amino acid residues. In one embodiment of the present invention, the length of the modified HLA or fragment thereof is about 8 amino acid residues, about 9 amino acid residues, about 10 amino acid residues, about 11 amino acid residues, about 12 amino acid residues, about 13 amino acid residues, about 14 amino acid residues, about 15 amino acid residues, about 16 amino acid residues, about 17 amino acid residues, about 18 amino acid residues, about 19 amino acid residues, about 20 amino acid residues, about 21 amino acid residues, about 22 amino acid residues, about 23 amino acid residues, or about 24 amino acid residues.

[0176] In some embodiments, the HLA signal peptide or fragment thereof is an HLA-A signal peptide or fragment thereof, an HLA-A2 signal peptide or fragment thereof, an HLA-B signal peptide or fragment thereof, an HLA-B5 signal peptide or fragment thereof, an HLA-C signal peptide or fragment thereof, an HLA-DP signal peptide or fragment thereof, an HLA-DPA1 signal peptide or fragment thereof, an HLA-DPB1 signal peptide or fragment thereof, an HLA-DQ signal peptide or fragment thereof, an HLA-DQA1 signal peptide or fragment thereof, an HLA-DQA2 signal peptide or fragment thereof, an HLA-DQB1 signal peptide or fragment thereof, an HLA-DQB2 signal peptide or fragment thereof, an HLA-DR signal peptide or fragment thereof , HLA-DR3 signal peptide or a fragment thereof, HLA-DRA1 signal peptide or a fragment thereof, HLA-DRB1 signal peptide or a fragment thereof, HLA-DRB2 signal peptide or a fragment thereof, HLA-DRB3 signal peptide or a fragment thereof, HLA-DRB4 signal peptide or a fragment thereof, HLA-DRB5 signal peptide or a fragment thereof, HLA-DRB6 signal peptide or a fragment thereof, HLA-DRB7 signal peptide or a fragment thereof, HLA-DRB8 signal peptide or a fragment thereof, HLA-DRB9 signal peptide or a fragment thereof, HLA-E signal peptide or a fragment thereof, HLA-F signal peptide or a fragment thereof, HLA-G signal peptide or a fragment thereof, or any combination thereof.

[0177] In some embodiments, the HLA signal peptide or fragment thereof is a modified HLA signal peptide or fragment thereof. For example, in some embodiments, the HLA signal peptide comprises at least one selected from the following: HLA-A*02:01 signal peptide or fragment thereof, HLA-B*08:01 signal peptide or fragment thereof, HLA-C*03:01 signal peptide or fragment thereof, HLA-G signal peptide or fragment thereof, HSP60 signal peptide or fragment thereof, CMV Towne signal peptide or fragment thereof, CMV AF1 signal peptide or fragment thereof, CMV 109b signal peptide or fragment thereof, RL9 HIV signal peptide or fragment thereof, and Mtb44 signal peptide or fragment thereof.

[0178] In some embodiments, the HLA signal peptide comprises at least one amino acid sequence selected from SEQ ID NOs: 1-10, 13 and 46, or a fragment thereof. In some embodiments, the HLA signal peptide comprises at least one amino acid sequence substantially homologous to an amino acid sequence selected from SEQ ID NOs: 1-10, 13 and 46, or a fragment thereof. For example, in certain embodiments, the amino acid sequence has a degree of identity of at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% with respect to the original amino acid sequence.

[0179] In certain embodiments, the HLA signal peptide comprises an amino acid sequence having one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more mutations (such as point mutations) relative to an amino acid sequence selected from SEQ ID NOs: 1-10, 13 and 46.

[0180] In some embodiments, the peptides of the present invention (i.e., peptides comprising HLA signal peptides or fragments thereof, modified B2M or fragments thereof, and HLA-E or fragments thereof) comprise at least one amino acid sequence shown in SEQ ID NOs: 1-10, 13, 25, 28, 31, 34, 37, 40, 44 and 46, or fragments thereof. In some embodiments, the peptides of the present invention comprise at least one amino acid sequence substantially homologous to an amino acid sequence selected from SEQ ID NOs: 1-10, 13, 25, 28, 31, 34, 37, 40, 44 and / or 46, or fragments thereof. For example, in certain embodiments, the amino acid sequence has a degree of identity of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% to an amino acid sequence selected from SEQ ID NOs: 1-10, 13, 25, 28, 31, 34, 37, 40, 44 and / or 46, or a fragment thereof, with respect to the original amino acid sequence.

[0181] As known in the art, "similarity" between two polypeptides is determined by comparing the amino acid sequence of one polypeptide and its conservative amino acid surrogates with the sequence of a second polypeptide. Variants are defined as polypeptide sequences that are different from the original sequence, for example, less than 40% residue difference from each target segment of the original sequence, or less than 25% residue difference from each target segment of the original sequence, or less than 10% residue difference from each target segment of the original protein sequence, or only a few residue differences from each target segment of the original protein sequence, and at the same time having enough homology to the original sequence to retain the functionality of the original sequence and / or the ability to bind to ubiquitin or ubiquitinated proteins. The present invention includes amino acid sequences that are at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90% or 95% similar or identical to the original amino acid sequence. The degree of identity between two polypeptides is determined using computer algorithms and methods well known to those skilled in the art. For example, the identity between two amino acid sequences can be determined using the BLASTP algorithm (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990)).

[0182] Peptide of the present invention can be post-translationally modified. For example, post-translation modifications falling within the scope of the present invention include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding and proteolytic processing, etc. Some modifications or processing events need to introduce additional biological mechanisms. For example, processing events such as signal peptide cleavage and core glycosylation are checked by adding canine microsomal membranes or African clawed frog egg extract (U.S. Patent number 6,103,489) to standard translation reactions.

[0183] The peptides of the present invention may include non-natural amino acids formed by post-translational modification or by introducing non-natural amino acids during translation. There are many methods for introducing non-natural amino acids during protein translation. For example, special tRNAs, such as tRNAs with inhibitor properties, inhibitor tRNAs, have been used in site-directed non-natural amino acid replacement (SNAAR) processes. In SNAAR, unique codons are required on mRNA and inhibitor tRNAs for targeting non-natural amino acids to unique sites during protein synthesis (described in WO 90 / 05785). However, inhibitor tRNAs must not be recognized by aminoacyl tRNA synthetases present in the protein translation system. In some cases, after the tRNA molecule is aminoacylated, a chemical reaction that specifically modifies natural amino acids and does not significantly change the functional activity of the aminoacylated tRNA can be used to form non-natural amino acids. These reactions are referred to as post-aminoacylation modifications. For example, amine-specific photoaffinity tags can be used to modify the homologous tRNA (tRNA LYS ) is attached to the ε-amino group of the lysine.

[0184] Chemical methods can be used to prepare the peptides of the present invention. For example, peptides can be synthesized by solid phase techniques (Roberge JY et al (1995) Science 269: 202-204), cut from a resin, and purified by preparative high performance liquid chromatography. For example, automatic synthesis can be achieved using an ABI 431A peptide synthesizer (Perkin Elmer) according to the instructions provided by the manufacturer. Alternatively, peptides can be made by recombinant methods or by cutting from longer polypeptides. The composition of the peptide can be confirmed by amino acid analysis or sequencing.

[0185] Variants of peptides according to the present invention may be variants in which (i) one or more amino acid residues are replaced by conservative or non-conservative amino acid residues, and such replaced amino acid residues may or may not be amino acid residues encoded by the genetic code, (ii) one or more modified amino acid residues are present, for example, residues modified by attachment of substituents, (iii) the polypeptide is an alternative splice variant of the polypeptide of the present invention, (iv) a fragment of the polypeptide and / or (v) the polypeptide is fused to another polypeptide, such as the other polypeptide being a leader or secretory sequence or a sequence for purification (e.g., a His-tag) or for detection (e.g., an Sv5 epitope tag). Fragments include polypeptides produced by proteolytic cleavage (including multisite proteolysis) of the original sequence. Variants may be post-translationally modified or chemically modified. Such variants are considered to be within the scope of those skilled in the art according to the teachings herein.

[0186] The peptides of the present invention can be converted into pharmaceutically acceptable salts by reacting with inorganic acids (such as hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid, etc.) or organic acids (such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid, benzenesulfonic acid and toluenesulfonic acid).

[0187] In some embodiments, the nucleotide sequence encoding the HLA signal peptide comprises at least one nucleotide sequence encoding an amino acid sequence selected from SEQ ID NOs: 1-10, 13 and 46, or a fragment thereof. In some embodiments, the nucleotide sequence encoding the HLA signal peptide comprises at least one nucleotide sequence encoding an amino acid sequence substantially homologous to an amino acid sequence selected from SEQ ID NOs: 1-10, 13 and 46, or a fragment thereof. For example, in certain embodiments, the nucleotide sequence encoding the HLA signal peptide comprises at least one nucleotide sequence encoding an amino acid sequence having a degree of identity of at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% with respect to the original amino acid sequence.

[0188] In certain embodiments, the nucleotide sequence encoding the HLA signal peptide comprises at least one nucleotide sequence encoding an amino acid sequence having one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more mutations (such as point mutations) relative to an amino acid sequence selected from SEQ ID NOs: 1-10, 13 and 46.

[0189] In some embodiments, the nucleic acid molecule of the present invention (i.e., a nucleic acid molecule comprising a nucleotide sequence encoding a peptide comprising an HLA signal peptide or a fragment thereof, a modified B2M or a fragment thereof, and an HLA-E or a fragment thereof, or a nucleic acid molecule comprising a nucleotide sequence encoding an HLA signal peptide or a fragment thereof, a nucleotide sequence encoding a modified B2M or a fragment thereof, and a nucleotide sequence encoding an HLA-E or a fragment thereof) comprises at least one nucleotide sequence encoding at least one amino acid sequence as shown in SEQ ID NOs: 1-10, 13, 25, 28, 31, 34, 37 and 40, or a fragment thereof. In some embodiments, the nucleic acid molecule of the present invention comprises at least one nucleotide sequence encoding an amino acid sequence substantially homologous to an amino acid sequence selected from SEQ ID NOs: 1-10, 13, 25, 28, 31, 34, 37, 40, 44 and / or 46, or a fragment thereof. For example, in certain embodiments, the amino acid sequence encoded by the nucleotide sequence has a degree of identity of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% with respect to the original amino acid sequence and an amino acid sequence selected from SEQ ID NO: 1-10, 13, 25, 28, 31, 34, 37, 40, 44 and / or 46, or a fragment thereof.

[0190] In some embodiments, the nucleotide sequence encoding the HLA signal peptide comprises at least one nucleotide sequence selected from SEQ ID NOs: 14-24, 43 and 47, or a fragment thereof. In some embodiments, the nucleotide sequence encoding the HLA signal peptide comprises at least one nucleotide sequence comprising at least one nucleotide sequence substantially homologous to a nucleotide sequence selected from SEQ ID NOs: 14-24, 43 and 47. For example, in certain embodiments, the nucleotide sequence has a degree of identity of at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% with respect to the original nucleotide sequence.

[0191] In certain embodiments, the nucleotide sequence encoding the HLA signal peptide comprises a nucleotide sequence having one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more mutations (such as point mutations) relative to a nucleotide sequence selected from SEQ ID NOs: 14-24, 43 and 47.

[0192] In some embodiments, the nucleic acid molecule of the present invention (i.e., a nucleic acid molecule comprising a nucleotide sequence encoding a peptide comprising an HLA signal peptide or a fragment thereof, a modified B2M or a fragment thereof, and an HLA-E or a fragment thereof, or a nucleic acid molecule comprising a nucleotide sequence encoding an HLA signal peptide or a fragment thereof, a nucleotide sequence encoding a modified B2M or a fragment thereof, and a nucleotide sequence encoding an HLA-E or a fragment thereof) comprises at least one nucleotide sequence selected from SEQ ID NOs: 14-24, 26, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41-43, 45, and 47, or a fragment thereof. In some embodiments, the nucleic acid molecule of the present invention comprises at least one nucleotide sequence substantially homologous to a nucleotide sequence selected from SEQ ID NOs: 14-24, 26, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41-43, 45, and 47, or a fragment thereof. For example, in certain embodiments, the nucleotide sequence has a degree of identity of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% with respect to the original nucleotide sequence and a nucleotide sequence selected from SEQ ID NOs: 14-24, 26, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41-43, 45, and 47, or a fragment thereof.

[0193] Any of a variety of recombinant methods known in the art can be used to obtain an isolated nucleic acid sequence encoding a peptide of the invention, such as, for example, by screening a library from cells expressing the gene, by deriving the gene from a vector known to include the gene, or by isolating directly from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest can be produced synthetically, rather than by cloning.

[0194] The isolated nucleic acid can comprise any type of nucleic acid, including but not limited to DNA and RNA. For example, in one embodiment, the composition comprises an isolated DNA molecule, including, for example, an isolated cDNA molecule, which encodes a polypeptide of the present invention or its functional fragment. In one embodiment, the composition comprises an isolated RNA molecule encoding a polypeptide of the present invention or its functional fragment.

[0195] The nucleic acid molecules of the present invention may be modified to improve their stability in serum or cell culture growth medium. Modifications may be added to enhance the stability, functionality and / or specificity of the nucleic acid molecules of the present invention and to minimize their immunostimulatory properties. For example, to enhance stability, 3'-residues may be stabilized to prevent degradation, for example, they may be selected to consist of purine nucleotides (particularly adenosine or guanosine nucleotides). Alternatively, pyrimidine nucleotides may be substituted by modified analogs, for example, substitution of uridine by 2'-deoxythymidine is acceptable and will not affect the function of the molecule.

[0196] In one embodiment of the present invention, the nucleic acid molecule may contain at least one modified nucleotide analog. For example, the termini may be stabilized by incorporating modified nucleotide analogs.

[0197] The limiting examples of nucleotide analogs include sugar-and / or backbone-modified ribonucleotides (i.e., including modifications to the phosphate-sugar backbone). For example, the phosphodiester bond of natural RNA can be modified to include at least one nitrogen or sulfur heteroatom. In one embodiment of the backbone-modified ribonucleotide, the phosphate group connected to the adjacent ribonucleotide is replaced by a modified group (e.g., a thiophosphate group). In some embodiments of the sugar-modified ribonucleotide, the 2'OH group is replaced by a group selected from H, OR, R, halogen, SH, SR, NH2, NHR, NR2 or ON, wherein R is a C1-C6 alkyl, alkenyl or alkynyl, and the halogen is F, Cl, Br or I.

[0198] Other examples of modifications are nucleobase-modified ribonucleotides, i.e., ribonucleotides containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase. The bases may be modified to block the activity of adenosine deaminase. Exemplary modified nucleobases include, but are not limited to, uridine and / or cytidine modified at the 5-position, such as 5-(2-amino)propyluridine, 5-bromouridine; adenosine and / or guanosine modified at the 8-position, such as 8-bromoguanosine; deazanucleotides, such as 7-deazaadenosine; O- and N-alkylated nucleotides, such as N6-methyladenosine are suitable. It should be noted that the above modifications may be combined.

[0199] In some cases, the nucleic acid molecule comprises at least one of the following chemical modifications: 2'-H, 2'-O-methyl or 2'-OH modification of one or more nucleotides. In some embodiments, the nucleic acid molecule of the present invention may have enhanced resistance to nucleases. In order to improve nuclease resistance, the nucleic acid molecule may include, for example, 2'-modified ribose units and / or thiophosphate bonds. For example, 2'hydroxyl (OH) may be modified or replaced with a plurality of different "oxygen" or "deoxy" substituents. In order to improve nuclease resistance, the nucleic acid molecule of the present invention may include 2'-O-methyl, 2'-fluoro, 2'-O-methoxyethyl, 2'-O-aminopropyl, 2'-amino and / or thiophosphate bonds. Including locked nucleic acid (LNA), ethylene nucleic acid (ENA) (e.g., 2'-4'-ethylene bridged nucleic acid) and certain core base modifications (such as 2-amino-A, 2-thio (e.g., 2-thio-U), G-clamp modifications) may also increase the binding affinity to the target.

[0200] In one embodiment, the nucleic acid molecule includes 2'-modified nucleotides, such as 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) or 2'-ON-methylacetamido (2'-O-NMA). In one embodiment, the nucleic acid molecule includes at least one 2'-O-methyl modified nucleotide, and in some embodiments, all nucleotides of the nucleic acid molecule include a 2'-O-methyl modification.

[0201] The present invention also includes a vector having an isolated nucleic acid of the present invention inserted therein. The art is replete with suitable vectors for use in the present invention.

[0202] In short, the expression of natural or synthetic nucleic acids encoding peptides of the present invention is usually achieved by operably linking the nucleic acid encoding the peptide or part thereof to a promoter and incorporating the construct into an expression vector. The vector to be used is suitable for replication and optionally integration in eukaryotic cells. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters for regulating the expression of the desired nucleic acid sequence.

[0203] The vectors of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Gene delivery methods are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, 5,589,466, the entire contents of which are incorporated herein by reference. In another embodiment, the present invention provides a gene therapy vector.

[0204] The isolated nucleic acid of the present invention can be cloned into various types of vectors. For example, the nucleic acid can be cloned into vectors including but not limited to plasmids, phagemids, phage derivatives, animal viruses and cosmids. The target vectors include expression vectors, replication vectors, probe generation vectors and sequencing vectors.

[0205] In addition, the vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art, and is described, for example, in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. The virus that can be used as a vector includes but is not limited to retrovirus, adenovirus, adeno-associated virus, herpes virus and slow virus. In general, suitable vectors contain a replication origin, a promoter sequence, a convenient restriction endonuclease site and one or more selectable markers (such as WO 01 / 96584; WO 01 / 29058; and U.S. Patent number 6,326,193) that have a function in at least one organism.

[0206] A number of virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to a subject's cells in vivo or in vitro. Various retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Various adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0207] For example, vectors derived from retroviruses (such as lentiviruses) are suitable tools for realizing long-term gene transfer, because they allow long-term, stable integration of transgenics and their propagation in daughter cells. Lentiviral vectors have additional advantages than vectors derived from oncoretroviruses (such as murine leukemia viruses), because they can transduce non-proliferating cells, such as hepatocytes. They also have the additional advantage of low immunogenicity. In one embodiment, the composition includes a vector derived from adeno-associated virus (AAV). Adeno-associated virus (AAV) vectors have become powerful gene delivery tools for treating various diseases. AAV vectors have multiple features, making them very suitable for gene therapy, including lack of pathogenicity, minimal immunogenicity and the ability to transduce post-mitotic cells in a stable and effective manner. By selecting an appropriate combination of AAV serotype, promoter and delivery method, the expression of specific genes contained in the AAV vector can be specifically targeted to one or more types of cells.

[0208] In certain embodiments, the vector further comprises conventional control elements that are operably linked to the transgene in a manner that allows transcription, translation, and / or expression of the transgene in cells transfected with the plasmid vector or infected with the virus produced by the invention. As used herein, "operably linked" sequences include expression control sequences adjacent to the gene of interest and expression control sequences that act in trans or remotely to control the gene of interest. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; effective RNA processing signals, such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, when desired, sequences that enhance secretion of the encoded product. A large number of expression control sequences are known in the art, including natural, constitutive, inducible, and / or tissue-specific promoters, and can be utilized.

[0209] Additional promoter elements (e.g., enhancers) regulate the frequency of transcription initiation. Typically, they are located in the region 30-110 bp upstream of the start site, although recently multiple promoters have been shown to also contain functional elements downstream of the start site. The spacing between promoter elements is usually flexible, so when elements are inverted or moved relative to each other, promoter function is retained. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart, and then activity begins to decline. Depending on the promoter, each element seems to work collaboratively or independently to activate transcription.

[0210] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive high-level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr (Epstein-Barr) virus immediate early promoter, Rous (Rous) sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, hemoglobin promoter and creatine kinase promoter. Further, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also considered as part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on the expression of a polynucleotide sequence to which it is operably linked when such expression is desired, or can turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0211] The enhancer sequence found on the vector also regulates the expression of the gene contained therein. Usually, the enhancer is combined with a protein factor to enhance the transcription of the gene. The enhancer can be located upstream or downstream of the gene it regulates. The enhancer can also be tissue-specific, to enhance the transcription in a specific cell or tissue type. In one embodiment, the vector of the present invention comprises one or more enhancers to promote the transcription of the gene present in the vector.

[0212] In one embodiment, the isolated nucleic acid encoding the peptide of the present invention comprises in vitro transcribed (IVT) RNA. RNA is produced by in vitro transcription using a polymerase chain reaction (PCR)-generated template. Target DNA from any source can be directly converted into a template by PCR for in vitro mRNA synthesis using appropriate primers and RNA polymerase. The source of the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequences, or any other appropriate DNA source.

[0213] In one embodiment, the DNA to be used for PCR contains an open reading frame. The DNA can be from a naturally occurring DNA sequence in an organism's genome. In one embodiment, the DNA is a full-length target gene of a gene part. The gene can include some or all of a 5' and / or 3' untranslated region (UTR). The gene can include exons and introns. In one embodiment, the DNA to be used for PCR is a human gene. In another embodiment, the DNA to be used for PCR is a human gene including 5' and 3' UTR. DNA can alternatively be an artificial DNA sequence that is not usually expressed in a naturally occurring organism. An exemplary artificial DNA sequence is a DNA sequence containing a gene portion that is linked together to form an open reading frame encoding a fusion protein. The DNA portions that are linked together can be from a single organism or from more than one organism.

[0214] Genes that can be used as DNA sources for PCR include genes encoding polypeptides, which provide treatment or preventive effects for organisms, or can be used to diagnose diseases or illnesses in organisms. Examples of such genes are genes that can be used for short-term treatments, or genes in which there are safety issues regarding dosage or expression of genes. For example, for the treatment of cancer, autoimmune disorders, parasites, viruses, bacteria, fungi or other infections, the transgenic to be expressed can encode a polypeptide that acts as a ligand or receptor for immune system cells, or can stimulate or suppress the immune system of an organism. In some embodiments, it is not desirable to continuously stimulate the immune system for a long time, nor is it necessary to produce changes that continue after successful treatment, because this may then cause new problems. For the treatment of autoimmune disorders, it may be desirable to suppress or repress the immune system during a disease outbreak, but it cannot be suppressed for a long time, which may cause the patient to be overly sensitive to infection.

[0215] PCR is used to generate mRNA in vitro transcription templates for transfection. The method of performing PCR is well known in the art. Primers used for PCR are designed to have regions that are substantially complementary to the DNA region to be used as a PCR template. As used herein, "substantially complementary" refers to a nucleotide sequence in which most or all bases in the primer sequence are complementary, or one or more bases are non-complementary or mismatched. Substantially complementary sequences can anneal or hybridize with the expected DNA target under the annealing conditions used for PCR. Primers can be designed to be substantially complementary to any part of the DNA template. For example, primers can be designed to amplify gene portions (open reading frames) that are usually transcribed in cells, including 5' and 3' UTRs. Primers can also be designed to amplify gene portions encoding specific target domains. In one embodiment, primers are designed to amplify the coding region of human cDNA, including all or part of 5' and 3' UTRs. Primers useful for PCR are generated by synthetic methods well known in the art. "Forward primer" is a primer containing a nucleotide region that is substantially complementary to the nucleotides upstream of the DNA sequence to be amplified on the DNA template. As used herein, "upstream" refers to position 5 relative to the DNA sequence to be amplified of the coding strand. A "reverse primer" is a primer containing a nucleotide region that is substantially complementary to the double-stranded DNA template downstream of the DNA sequence to be amplified. "Downstream" as used herein refers to position 3' relative to the DNA sequence to be amplified of the coding strand.

[0216] Any DNA polymerase useful for PCR can be used in the methods disclosed herein. Reagents and polymerases are commercially available from a variety of sources.

[0217] Chemical structures with the ability to promote stability and / or translation efficiency can also be used. In one embodiment, RNA has 5' and 3' UTR. In one embodiment, the length of 5' UTR is between zero and 3000 nucleotides. The length of 5' and 3' UTR sequences to be added to the coding region can be changed by different methods, including but not limited to designing PCR primers that anneal to different regions of UTR. Using this method, those of ordinary skill in the art can modify the 5' and 3' UTR lengths required for achieving optimal translation efficiency after transfection of the transcribed RNA.

[0218] The 5' and 3' UTRs can be naturally occurring endogenous 5' and 3' UTRs of the target gene. Alternatively, a UTR sequence that is not endogenous to the target gene can be added by incorporating the UTR sequence into the forward and reverse primers or by any other modification of the template. The use of UTR sequences that are not endogenous to the target gene can be used to change the stability and / or translation efficiency of RNA. For example, it is known that AU-rich elements in 3' UTR sequences can reduce the stability of mRNA. Therefore, 3' UTR can be selected or designed to increase the stability of the transcribed RNA based on UTR characteristics well known in the art.

[0219] In one embodiment, the 5'UTR may contain a Kozak sequence of an endogenous gene. Alternatively, when a 5'UTR that is not endogenous to the target gene is added by PCR as described above, a consensus Kozak sequence can be redesigned by adding a 5'UTR sequence. The Kozak sequence can improve the translation efficiency of certain RNA transcripts, but it does not appear that all RNAs require a Kozak sequence to achieve efficient translation. It is known in the art that multiple mRNAs require a Kozak sequence. In other embodiments, the 5'UTR may be derived from an RNA virus whose RNA genome is stable in the cell. In other embodiments, various nucleotide analogs may be used in the 3' or 5'UTR to hinder exonuclease degradation of mRNA.

[0220] In order to be able to synthesize RNA from a DNA template without the need for gene cloning, a transcription promoter should be attached to the upstream of the sequence to be transcribed on the DNA template. When the sequence as an RNA polymerase promoter is added to the 5' end of the forward primer, the RNA polymerase promoter is integrated into the PCR product, upstream of the open reading frame to be transcribed. In one embodiment, the promoter is a T7 polymerase promoter, as described elsewhere herein. Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. The consensus nucleotide sequence for T7, T3 and SP6 promoters is known in the art.

[0221] In one embodiment, the mRNA has a cap on the 5' end and a 3' poly (A) tail, which determine ribosome binding, translation initiation, and stability of the mRNA in the cell. On a circular DNA template (e.g., plasmid DNA), RNA polymerase will produce long tandem products that are not suitable for expression in eukaryotic cells. Transcription of plasmid DNA linearized at the 3' UTR end will produce normal-sized mRNA, which is ineffective in eukaryotic transfection even after transcription.

[0222] On a linear DNA template, bacteriophage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003)).

[0223] The conventional method for integrating a polyA / T stretch into a DNA template is molecular cloning. However, polyA / T sequences integrated into plasmid DNA can lead to plasmid instability, which is why plasmid DNA templates obtained from bacterial cells are often heavily contaminated with deletions and other aberrations. This makes the cloning procedure not only laborious and time-consuming, but also generally unreliable. This is why a method that allows the construction of DNA templates with a polyA / T 3' stretch without the need for cloning is highly desirable.

[0224] The polyA / T segment of the transcription DNA template can be produced during PCR by using a reverse primer containing a polyT tail (such as a 100T tail) (size can be 50-5000T), or by any other method after PCR, including but not limited to DNA ligation or in vitro recombination. The poly (A) tail also provides stability to the RNA and reduces its degradation. Generally, the length of the poly (A) tail is positively correlated with the stability of the transcribed RNA. In one embodiment, the poly (A) tail is between 100 and 5000 adenosines.

[0225] The Poly (A) tail of the RNA can be further extended using a poly (A) polymerase (such as E. coli poly A polymerase (E-PAP)) after in vitro transcription. In one embodiment, the length of the poly (A) tail is increased from 100 nucleotides to between 300 and 400 nucleotides, resulting in an approximately two-fold increase in the translation efficiency of the RNA. In addition, attaching different chemical groups to the 3' end can increase mRNA stability. This attachment can contain modified / artificial nucleotides, aptamers, and other compounds. For example, poly (A) polymerase can be used to incorporate ATP analogs into the poly (A) tail. ATP analogs can further increase the stability of the RNA.

[0226] The 5' cap also provides stability to the RNA molecule. In one embodiment, the RNA produced by the methods disclosed herein includes a 5' cap. The 5' cap is provided using techniques known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29: 436-444 (2001); Stepinski, et al., RNA, 7: 1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330: 958-966 (2005)).

[0227] The RNA produced by the method disclosed herein can also contain an internal ribosome entry site (IRES) sequence. The IRES sequence can be any virus, chromosome or artificially designed sequence, which initiates the cap-independent binding of ribosomes to mRNA and promotes the initiation of translation. Any solute suitable for cell electroporation can be included, which can contain factors that promote cell permeability and vitality, such as sugars, peptides, lipids, proteins, antioxidants and surfactants.

[0228] In one aspect, the invention relates in part to a composition comprising at least one peptide of the invention and / or at least one nucleic acid molecule of the invention. For example, in some aspects, the composition comprises a DNA, RNA, mRNA or cDNA encoding one or more peptides described herein. In some embodiments, the composition reduces the clearance of at least one cell of interest.

[0229] In some embodiments, the composition increases the persistence of at least one cell of interest. Examples of such cells include, but are not limited to, donor cells, T cells, CAR cells, CAR T cells, T cells expressing engineered TCRs, and the like.

[0230] In some embodiments, the composition inhibits or reduces the level of at least one NK cell, alloresponsive cell, immune cell, T cell, B cell, NK cell, leukocyte, myeloid cell, or plasma cell associated with donor cells. In some embodiments, the composition inhibits NK cell-mediated killing of at least one donor cell.

[0231] In some embodiments, the composition prevents, reduces or suppresses an immune response against at least one donor cell.

[0232] It should be understood that the composition of the present invention can be prepared by methods well known in the art, including but not limited to chemical synthesis by solid phase synthesis and purification from other products of chemical reaction by HPLC, or produced by expressing the nucleic acid sequence (e.g. DNA sequence) encoding the peptide of the present invention in an in vitro translation system or living cells. In addition, the composition can include cellular components separated from biological samples. Separate and fully dialyze the composition to remove one or more unwanted small molecular weight molecules and / or freeze-drying so as to be more easily formulated into the required carrier. The peptide sequence can be synthesized by methods known to those of ordinary skill in the art, such as, for example, using an automatic peptide synthesizer to synthesize peptides, such as those automatic peptide synthesizers available from Applied Biosystems, Inc., Foster City, CA (Foster City, CA).

[0233] Longer peptides or polypeptides can also be prepared, for example, by recombinant methods. In some embodiments, nucleic acids encoding peptides described herein can be used, for example, to produce compositions for various compositions and methods of the present invention in vitro or in vivo. For example, in some embodiments, nucleic acids encoding peptides of the present invention are contained in vectors such as recombinant cells. The nucleic acids can be expressed to produce peptides or polypeptides comprising antigenic sequences. The peptides or polypeptides can be secreted from cells, or included as part of cells or contained within cells.

[0234] Modified cells

[0235] In various aspects, the present invention provides a genetically engineered cell comprising at least one peptide of the present invention and / or at least one nucleic acid molecule. In some embodiments, cells are genetically modified to contain or express peptides of the present invention. In some embodiments, cells are genetically modified to not express beta-2-microglobulin (B2M), class II major histocompatibility complex transactivator (CIITA) and / or natural T cell receptor (TCR). In certain embodiments, cells are genetically modified to reduce or eliminate MHC I, MHC II and / or natural TCR ab heterodimers. For example, in some embodiments, cells are triple knockout (TKO) cells that do not express MHC I, MHC II and / or natural TCR.

[0236] The TKO cells of the present invention can be prepared using any method known in the art. For example, in some embodiments, the efficient production of TKO cells can be promoted by genome editing technology, such as protein-based technology (e.g., zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), etc.) or RNA-based DNA recognition technology derived from the bacterial adaptive immune system (e.g., clustered regularly spaced short palindromic repeats (CRISPR) / Cas nuclease system). In some embodiments, TKO cells are achieved by using (i) Cas9 protein or expression vector or RNA encoding Cas9 protein and (ii) target-locus-specific guide RNA (gRNA) or expression vector encoding gRNA. In some embodiments, by using a method for specifically cutting a target sequence, TKO cells are achieved by introducing (i) guide RNA designed based on the target sequence and (ii) RNA encoding Cas protein into the cell (e.g., PCT International Publication No. WO / 2014 / 093661, which is incorporated herein by reference in its entirety).

[0237] In some embodiments, the method for knocking out a target gene in a cell is a method comprising the following steps: introducing a CRISPR-Cas system into a cell having one or more target genes, the CRISPR-Cas system being capable of producing (i) three or more guide RNAs for each of the one or more target genes and (ii) Cas proteins. In one embodiment of the invention, one or more target genes are knocked out by (i) targeting each of the three or more guide RNAs to the one or more target genes, and then (ii) causing the Cas protein to cut each of the one or more target genes.

[0238] CRISPR includes short repeats of dozens of base pairs, and is a locus that acts as a class of acquired immune system in prokaryotes. CRISPR-related (cas) gene clusters known to encode nucleases and helicases are present near CRISPR repeats. Foreign DNA is fragmented into a length of about 30 base pairs by the protein encoded by any cas gene cluster. In the case of inserting it into the CRISPR locus by any method, the fragment plays the role of immune memory. At the CRISPR locus, RNA is transcribed so that RNA is fragmented into smaller RNA (crRNA) with each foreign sequence by Cas protein. RNA guides another Cas protein to foreign DNA (or RNA derived from foreign DNA), so as to suppress the function of foreign DNA (or RNA derived from foreign DNA) with a mechanism similar to RNAi of eukaryotic cells. CRISPR-Cas system is applied to RNA-guided genome engineering at the cell level or individual level.

[0239] There are Type I, Type II, and Type III CRISPR / Cas. The type mainly used for genome editing is Type II CRISPR / Cas. In Type II, Cas9 is used as RGN. The Cas9 of Streptococcus pyogenes recognizes the three bases NGG as the Proto-spacer adjacent motif (PAM). Therefore, a sequence adjacent to two guanines can be cut upstream of it. This makes it possible to target almost any DNA sequence on the genome.

[0240] Among them, the CRISPR / Cas method allows the use of the Cas protein as a single protein to edit the genome simply by synthesizing a short gRNA homologous to the target DNA sequence as described above. Examples of Cas proteins include, but are not limited to, CAS1, CAS1B, CAS2, CAS3, CAS4, Cas5, Cas6, Cas7, Cas8, Cas9, 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, Csx15, Csf1, Csf2, Csf3, Csf4, homologues thereof, and mutants thereof. These enzymes are well known. For example, in one embodiment, the Cas protein is a Cas9 protein.

[0241] Examples of ways in which Cas proteins can be produced include (i) a form in which the Cas protein itself is introduced into a cell as a protein, (ii) a form in which RNA encoding the Cas protein is introduced into a cell, and (iii) a form in which a vector (such as a DNA vector) that can express the Cas protein in a cell is introduced into a cell. In some embodiments using a vector that can express a Cas protein in a cell, an expression vector containing (i) a DNA encoding the Cas protein and (ii) an expression regulatory sequence (such as a promoter) upstream of the DNA is used.

[0242] The components constituting the CRISPR-Cas system are not limited to any specific components, as long as guide RNA and Cas protein can be produced in cells. Examples of the manner in which guide RNA can be produced include (i) a form in which the guide RNA itself is introduced into the cell as RNA and (ii) a form in which a vector (such as a DNA vector) capable of expressing the guide RNA in the cell is introduced into the cell. In the case where the guide RNA itself is introduced into the cell, for example, the guide RNA can be obtained by chemical synthesis or in vitro transcription of the guide RNA. In some embodiments using a vector capable of expressing the guide RNA in a cell, an expression vector containing (i) DNA encoding the guide RNA and (ii) an expression control sequence (such as a promoter) upstream of the DNA is used.

[0243] The vector may be a nucleic acid molecule containing DNA, RNA, or both DNA and RNA. Specific examples of vectors include, but are not limited to, plasmid vectors and viral vectors (such as retroviral vectors, adenoviral vectors, and adeno-associated viral vectors). The vector may be a vector that replicates autonomously in a host cell into which the vector is introduced. Alternatively, the vector may be a vector that is integrated into the host cell genome when the vector is introduced into the host cell.

[0244] The type of expression control sequence to be used in the vector expressing guide RNA or Cas protein is not particularly limited. Any expression control sequence that works in the cell introduced into the expression vector can be used. Examples of expression control sequences include, but are not limited to, promoters, enhancers, internal ribosome entry sites (IRES) and any other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly (U) sequences). The expression control sequence can be a sequence that induces gene expression in a wide range of host cells, or can be a sequence that induces gene expression in certain host cells. Examples of tissue-specific promoters that induce gene expression only in specific host cells cover promoters that can be induced to express in desired tissues (such as muscle, nerve, bone, skin, blood), specific organs (e.g., liver and pancreas) and specific cell types (e.g., lymphocytes). Specific examples of promoters include, but are not limited to, pol III promoters, pol II promoters, pol I promoters and combinations thereof. Specific examples of pol III promoters include, but are not limited to, U6 promoters and HI promoters. Examples of poll promoters include, but are not limited to, the retroviral Rous sarcoma VIMS (RSV) LTR promoter, the cytomegalovirus (CMV) promoter, the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGR) promoter, and the EFL promoter.

[0245] In the case where an expression vector expressing a guide RNA or a Cas protein is to be used, at least four DNAs encoding three or more guide RNAs and Cas proteins may be contained in a single expression vector or in separate expression vectors. For example, the following is possible:

[0246] (1) A single expression vector is to be used, which contains all DNA encoding three or more guide RNAs and Cas proteins;

[0247] (2) The case where an expression vector containing all DNAs encoding three or more guide RNAs and an expression vector encoding a Cas protein are to be used;

[0248] (3) The case where three or more expression vectors each containing three or more guide RNAs and an expression vector containing a DNA encoding a Cas protein are to be used.

[0249] The type of cell to be introduced into the CRISPR-Cas system according to an embodiment of the present invention is not limited to any particular one. The cell can be a prokaryotic cell or a eukaryotic cell. In one embodiment, the cell is a eukaryotic cell. In another embodiment, the cell is an animal cell. In one embodiment, the animal cell is a mammalian cell (such as a mouse cell or a human cell).

[0250] When the CRISPR-Cas system is introduced into a cell, there is no particular limitation on which part of the cell the CRISPR-Cas system is introduced into. The CRISPR-Cas system can be introduced into the nucleus, or can be introduced into the cytoplasm. When the CRISPR-Cas system is introduced in the form of RNA, the CRISPR-Cas system can be introduced into the cytoplasm.

[0251] The CRISPR-Cas system can be introduced into cells through methods such as viral particles, liposomes, electroporation, microinjection, and conjugation.

[0252] The method for knocking out a target gene in a cell of the present invention can be used for producing knockout non-human organisms, gene therapy, drug screening, and diagnosis and prognosis of diseases. In some embodiments, the method is used to produce knockout non-human organisms.

[0253] In some embodiments, retroviral or lentiviral vectors are used to deliver nucleic acid sequences to cells. For example, retroviral and lentiviral vectors expressing the peptides of the invention can be delivered to different types of eukaryotic cells, tissues, and whole organisms using transduced cells as vectors or cell-free local or systemic delivery of encapsulated, bound, or naked vectors. The methods used can be used for any purpose where expression is desired or sufficiently stable.

[0254] In other embodiments, in vitro transcribed mRNA is used to deliver the nucleic acid sequence into the cell. Transfected cells can be used as carriers or cell-free local or systemic delivery encapsulation, binding or naked mRNA to deliver in vitro transcribed mRNA to different types of eukaryotic cells and tissues and whole organisms. The method used can be used for any purpose requiring or sufficient transient expression.

[0255] In some embodiments, the cell can be any suitable cell type capable of expressing the desired peptide. In some embodiments, the modified cell is used in a method of introducing the cell into a recipient. In some embodiments, the cell is autologous, allogeneic, isogenic, or xenogeneic to the recipient. In some embodiments, the cell is derived from a stem cell or a precursor cell. In some embodiments, the stem cell or precursor cell from which the modified cell is derived is autologous, allogeneic, isogenic, or xenogeneic to the recipient.

[0256] In one embodiment, the cell is an immune cell. Exemplary immune cells include, but are not limited to, T cells (including killer T cells, helper T cells, regulatory T cells, TCR-carrying T cells, allogeneic responsive T cells, allogeneic specific T cells, T cells carrying allogeneic reactive TCRs, and γδT cells), B cells, antigen presenting cells (APCs), NK cells, NK T cells, CAR T cells, and TCR-expressing T cells.

[0257] For example, in certain embodiments, the composition includes an immune cell that includes or expresses one or more peptides described herein (e.g., SCT).Exemplary immune cells that may include or express one or more CARs described herein or engineered TCRs include, but are not limited to, T cells (including killer T cells, helper T cells, regulatory T cells, and γδT cells), natural killer (NK) cells, NK T cells, CAR T cells, and T cells expressing engineered TCRs.Exemplary immune cells that may include or express one or more peptides described herein (e.g., SCT) include, but are not limited to, antigen presenting cells, dendritic cells, B cells, macrophages, Langerhans cells, T cells, NK cells, NK T cells, CAR T cells, and T cells expressing engineered TCRs.

[0258] In one embodiment, the cell is an antigen presenting cell (APC). For example, in certain embodiments, the composition comprises an APC modified to contain or express one or more peptides described herein (e.g., SCT). Exemplary APCs include, but are not limited to, dendritic cells (DCs), macrophages, Langerhans cells, B cells, and the like.

[0259] The disclosed compositions and methods can be applied to the modulation of T cell activity in basic research and treatment in the fields of cancer, stem cells, acute and chronic infections, and autoimmune diseases, including evaluating the ability of genetically modified T cells to kill target cancer cells.

[0260] Before amplification and genetic modification of the T cell of the present invention, a T cell source is obtained from a subject. T cells can be obtained from a plurality of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from infection site, ascites, pleural effusion, spleen tissue and tumor. In certain embodiments of the present invention, any number of T cell lines available in the art can be used. In certain embodiments of the present invention, any number of techniques known to persons skilled in the art (such as Ficoll) can be used. TM T cells are obtained from a blood unit collected from a subject by separation. In one embodiment, cells in individual circulating blood are obtained by apheresis. Apheresis products generally contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In one embodiment, cells collected by apheresis can be washed to remove plasma fractions and the cells are placed in an appropriate buffer or culture medium for subsequent processing steps. In one embodiment of the invention, cells are washed with phosphate buffered saline (PBS). In alternative embodiments, the washing solution lacks calcium, and may lack magnesium, or may lack a variety of (if not all) divalent cations. Again, surprisingly, the initial activation step in the absence of calcium can lead to amplified activation. As those of ordinary skill in the art can readily appreciate, the washing step can be completed by methods known to those skilled in the art, such as by using a semi-automatic "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics cell recycler 5) according to the manufacturer's instructions. After washing, the cells can be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free 2+ , Mg-free 2+ Alternatively, the apheresis sample can be depleted of unwanted components and the cells resuspended directly in culture medium.

[0261] In another embodiment, the erythrocytes are lysed and the monocytes are depleted, for example by PERCOLL TM T cells are separated from peripheral blood lymphocytes by gradient centrifugation or by counterflow centrifugal elutriation. Specific T cell subsets, such as CD3 + 、CD28+ 、CD4 + 、CD8 + 、CD45RA + and CD45RO + For example, in one embodiment, by binding to anti-CD3 / anti-CD28 (i.e., 3x28)-coupled beads (such as In one embodiment, the incubation time period is 24 hours. In another embodiment, the incubation time period is 24 hours. For separating T cells from patients with leukemia, longer incubation times (such as 24 hours) can be used to increase cell yield. In any case where T cells are less than other cell types, longer incubation times can be used to separate T cells, such as separating tumor infiltrating lymphocytes (TIL) from tumor tissue or immunocompromised individuals. Further, longer incubation times can be used to improve the efficiency of capturing CD8+T cells. Therefore, by simply shortening or extending the time that allows T cells to bind to CD3 / CD28 beads and / or by increasing or decreasing the ratio of beads to T cells (as further described herein), T cell subsets can be selected or not at the beginning of culture or at other time points during this process. In addition, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on beads or other surfaces, T cell subsets can be selected or not at the beginning of culture or at other desired time points. Those skilled in the art will recognize that multiple rounds of selection can also be used in the context of the present invention. In some embodiments, it may be necessary to perform a selection procedure and use "unselected" cells in the activation and amplification process. "Unselected" cells can also be subjected to a further round of selection.

[0262] Enrichment of T cell populations by negative selection can be achieved using a combination of antibodies directed against surface markers unique to the negatively selected cells. One approach is to perform cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies directed against cell surface markers present on the negatively selected cells. For example, to enrich for CD4 + For cells, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In certain embodiments, it may be desirable to enrich or positively select for cells that normally express CD4 + 、CD25 + 、CD62Lhi GITR + and FoxP3 + Alternatively, in certain embodiments, T regulatory cells are depleted by anti-C25 coupled beads or other similar selection methods.

[0263] In order to separate the desired cell population by positive or negative selection, the concentration of cells and surfaces (e.g., particles, such as beads, etc.) can be changed. In some embodiments, it may be necessary to significantly reduce the volume (i.e., increase the cell concentration) in which beads and cells are mixed together to ensure the maximum contact of cells and beads. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, a concentration greater than 100 million cells / ml is used. In a further embodiment, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45 or 50 million cells / ml is used. In another embodiment, a cell concentration of 75, 80, 85, 90, 95 or 100 million cells / ml is used. In a further embodiment, a concentration of 125 or 150 million cells / ml can be used. Using high concentrations can increase cell yield, cell activation and cell expansion. Further, the use of high cell concentrations allows for more efficient capture of cells that may weakly express the target antigen of interest, such as CD28-negative T cells, or from samples where multiple tumor cells are present (i.e., leukemic blood, tumor tissue, etc.). Such cell populations may have therapeutic value and may be worth obtaining. For example, the use of high concentrations of cells allows for more efficient selection of CD8 T cells that typically have weak CD28 expression. + T cells.

[0264] In a related embodiment, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surface (e.g., particles such as beads), interactions between the particles and cells are minimized. This selects for cells that express large amounts of the desired antigen to bind to the particles. For example, CD4 + T cells expressed higher levels of CD28 and were more sensitive to CD8 + T cells are captured more efficiently. In one embodiment, the concentration of cells used is 5×10 6 In other embodiments, the concentration used may be about 1×10 5 / ml to 1X 10 6 / ml, and any integer values ​​in between.

[0265] In other embodiments, cells can be incubated at 2-10°C or room temperature on a rotator at different speeds for different lengths of time.

[0266] The T cells for stimulation may also be frozen after the washing steps. Without wishing to be bound by theory, the freezing and subsequent thawing steps provide a more homogeneous product by removing granulocytes and to some extent monocytes from the cell population. After the washing steps to remove plasma and platelets, the cells may be suspended in a freezing solution. Although a variety of freezing solutions and parameters are known in the art and will be used herein, one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or medium containing 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or medium containing 31.25% Plasmalyte-A, 31.25% dextrose 5%, 0.45% NaCl, 10% dextran 40 and 5% dextrose, 20% human serum albumin, and 7.5% DMSO, or other suitable cell freezing medium containing, for example, Hespan and Plasmalyte A, and then freezing the cells to -80°C at a rate of 1°C per minute and storing in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing can be used as well as immediate uncontrolled freezing at -20°C or in liquid nitrogen.

[0267] In certain embodiments, cryopreserved cells are thawed and washed as described herein and allowed to stand at room temperature for one hour prior to activation using the methods of the invention.

[0268] It is also contemplated in the context of the present invention that a blood sample or apheresis product is collected from a subject in a time period before the amplified cells described herein may be needed. Therefore, the source of the cells to be amplified can be collected at any necessary time point, and the required cells, such as T cells, are separated and frozen for later use in T cell therapy, which is used for any number of diseases or conditions that can benefit from T cell therapy, such as those described herein. In one embodiment, a blood sample or apheresis is collected from a generally healthy subject. In certain embodiments, a blood sample or apheresis is collected from a generally healthy subject who is at risk of illness but not yet ill, and the cells of interest are separated and frozen for later use. In certain embodiments, T cells can be amplified, frozen and used later. In certain embodiments, samples are collected from patients shortly after a specific disease as described herein is diagnosed but before any treatment. In a further embodiment, cells are isolated from a blood sample or apheresis of a subject prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation therapy, immunosuppressants (such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506), antibodies or other immunoablative agents (such as CAMPATH, anti-CD3 antibodies, cyclophosphamide, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and radiation). These drugs either inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit p70S6 kinase (rapamycin), which is important for growth factor-induced signaling (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773, 1993). In a further embodiment, cells are isolated from the patient and frozen for later use in combination with bone marrow or stem cell transplantation, T cell ablative therapy (using chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH). In another embodiment, cells are isolated first and can be frozen for later use in treatment after B cell ablative therapy (such as an agent reactive with CD20, e.g., Rituxan).

[0269] In a further embodiment of the present invention, T cells are obtained directly from the patient after treatment. In this respect, it has been observed that after some cancer treatments, particularly after the use of drugs that damage the immune system, the quality of the T cells obtained may be optimal or improved for their in vitro expansion capacity during the period during which the patient can usually recover from treatment soon after treatment. Similarly, after using the methods described herein to perform ex vivo operations, these cells may be in a state of enhanced implantation and in vivo amplification. Therefore, in the context of the present invention, it is envisioned that blood cells are collected in this recovery phase, including other cells of T cells, dendritic cells or hematopoietic lineages. Further, in some embodiments, mobilization (e.g., mobilization with GM-CSF) and conditioning schemes can be used to create conditions conducive to the re-proliferation, recirculation, regeneration and / or amplification of specific cell types in subjects, especially during a specific time window after therapy. Illustrative cell types include T cells, B cells, dendritic cells and other cells of the immune system.

[0270] Whether before or after T cells are genetically modified to express the peptides of the invention, T cells can generally be activated and expanded using methods such as those described in U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005.

[0271] Typically, the T cells of the present invention are amplified by contacting a surface to which an agent and a ligand are attached, the agent stimulating CD3 / TCR complex-related signals, and the ligand stimulating co-stimulatory molecules on the surface of T cells. In some embodiments, T cell populations can be stimulated as described herein, such as by contacting an anti-CD3 antibody, or an antigen-binding fragment thereof, or an anti-CD2 antibody fixed on a surface, or by contacting a protein kinase C activator (e.g., bryostatin) together with a calcium ion carrier. In order to co-stimulate the auxiliary molecules on the surface of T cells, a ligand that binds to the auxiliary molecule is used. For example, a T cell population can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions suitable for stimulating T cell proliferation. In order to stimulate CD4 + T cells or CD8 + For the proliferation of T cells, anti-CD3 antibodies and anti-CD28 antibodies are used. Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, France), which can be used as other methods known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

[0272] In some embodiments, the primary stimulation signal and costimulatory signal for T cells can be provided by different protocols. For example, the agent providing each signal can be in solution or coupled to the surface. When coupled to the surface, the agent can be coupled to the same surface (i.e., formed in "cis") or to a separate surface (i.e., formed in "trans"). Alternatively, one agent can be coupled to the surface, and another agent is in solution. In one embodiment, the agent providing the costimulatory signal is combined with the cell surface, and the agent providing the primary activation signal is in solution or coupled to the surface. In some embodiments, both agents can be in solution. In another embodiment, the agent can be in soluble form, then cross-linked to the surface, such as cells expressing Fc receptors or antibodies or other binding agents to be combined with the agent. In this regard, for the artificial antigen presenting cells (aAPC) envisioned for activating and amplifying T cells in the present invention, see, for example, U.S. Patent Application Publication Nos. 2004 / 0101519 and 2006 / 0034810.

[0273] In one embodiment, two agents are immobilized on beads, either on the same bead, i.e., "cis," or on different beads, i.e., "trans." For example, the agent providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof, and the agent providing the co-stimulatory signal is an anti-CD28 antibody or an antigen-binding fragment thereof; and both agents are co-immobilized on the same bead in equal molecular numbers. In one embodiment, a 1:1 ratio of each antibody to the bead is used for CD4 +T cell expansion and T cell growth. In certain aspects of the invention, a ratio of anti-CD3:CD28 antibodies bound to beads is used such that an increase in T cell expansion is observed compared to the expansion observed using a 1:1 ratio. In one embodiment, an increase of about 1 to about 3 times is observed compared to the expansion observed using a 1:1 ratio. In one embodiment, the ratio of CD3:CD28 antibodies bound to beads ranges from 100:1 to 1:100 and all integer values ​​therebetween. In one aspect of the invention, there are more anti-CD28 antibodies bound to the particles than anti-CD3 antibodies, i.e., the ratio of CD3:CD28 is less than 1. In certain embodiments of the invention, the ratio of anti-CD28 antibodies bound to beads to anti-CD3 antibodies is greater than 2:1. In one embodiment, a 1:100 CD3:CD28 ratio of antibodies bound to beads is used. In another embodiment, a 1:75 CD3:CD28 ratio of antibodies bound to beads is used. In a further embodiment, a 1:50 CD3:CD28 ratio of antibodies bound to beads is used. In another embodiment, a 1:30 CD3:CD28 ratio of antibody bound to beads is used. In one embodiment, a 1:10 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, a 1:3 CD3:CD28 ratio of antibody bound to beads is used. In yet another embodiment, a 3:1 CD3:CD28 ratio of antibody bound to beads is used.

[0274] T cells or other target cells can be stimulated using a particle to cell ratio of 1:500 to 500:1 and any integer value therebetween. As one of ordinary skill in the art can readily appreciate, the particle to cell ratio can depend on the particle size relative to the target cell. For example, beads of small size can only bind to a few cells, while larger beads can bind to multiple cells. In certain embodiments, a cell to particle ratio ranges from 1:100 to 100:1 and any integer value therebetween, and in further embodiments, a ratio comprising 1:9 to 9:1 and any integer value therebetween can also be used to stimulate T cells. The ratio of anti-CD3 and anti-CD28 conjugated particles to T cells resulting in T cell stimulation can vary as described above, with exemplary values ​​including 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, and 15:1, with an exemplary ratio of at least 1:1 particles / T cell. In one embodiment, a ratio of particles to cells of 1:1 or less is used. In one embodiment, the ratio of particles to cells is 1:5. In further embodiments, the ratio of particles to cells can vary depending on the number of days of stimulation. For example, in one embodiment, the ratio of particles to cells on the first day is 1:1 to 10:1, and additional particles are added to the cells every day or every other day thereafter for up to 10 days, with a final ratio of 1:1 to 1:10 (based on the cell count on the day of addition). In one embodiment, the ratio of particles to cells on the first day of stimulation is 1:1, and is adjusted to 1:5 on the third and fifth days of stimulation. In another embodiment, particles are added every day or every other day, with a final ratio of 1:1 on the first day of stimulation, and a final ratio of 1:5 on the third and fifth days of stimulation. In another embodiment, the ratio of particles to cells on the first day of stimulation is 2:1, and is adjusted to 1:10 on the third and fifth days of stimulation. In another embodiment, particles are added every day or every other day, with a final ratio of 1:1 on the first day, and a final ratio of 1:10 on the third and fifth days of stimulation. It will be appreciated by those skilled in the art that a variety of other ratios may be suitable for the present invention. In some embodiments, the ratio will vary depending on the particle size and the cell size and type.

[0275] In a further embodiment of the invention, cells (such as T cells) are combined with agent-coated beads, beads and cells are subsequently separated, and cells are then cultured. In an alternative embodiment, the agent-coated beads and cells are not separated before culture, but cultured together. In a further embodiment, beads and cells are first concentrated by applying a force (such as a magnetic force) to increase the connection of cell surface markers, thereby inducing cell stimulation.

[0276] For example, cell surface proteins can be linked by contacting T cells with paramagnetic beads (3x28 beads) that allow anti-CD3 and anti-CD28 to attach. In one embodiment, cells (e.g., 10 4 Up to 10 9 T cells) and beads (e.g., M-450CD3 / CD28 T paramagnetic beads, ratio of 1: 1) combination. Again, it can be easily understood by those of ordinary skill in the art that any cell concentration can be used. For example, the target cells may be very rare in the sample, accounting for only 0.01% of the sample, or the entire sample (i.e. 100%) may contain the target cells of interest. Therefore, any number of cells is in the context of the present invention. In certain embodiments, it may be necessary to significantly reduce the volume in which the particles and cells are mixed together (i.e., increase the cell concentration) to ensure maximum contact between the cells and the particles. For example, in one embodiment, a concentration of about 2 billion cells / ml is used. In another embodiment, a concentration greater than 100 million cells / ml is used. In a further embodiment, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45 or 50 million cells / ml is used. In yet another embodiment, a cell concentration of 75, 80, 85, 90, 95 or 100 million cells / ml is used. In a further embodiment, a concentration of 125 or 150 million cells / ml can be used. Using high concentrations can increase cell yield, cell activation and cell expansion. In addition, using high cell concentrations allows more efficient capture of cells that may weakly express the target antigen of interest, such as CD28-negative T cells. Such cell populations may have therapeutic value and are desirable in some embodiments. For example, using high concentrations of cells allows more efficient selection of CD8+T cells that typically have weaker CD28 expression.

[0277] In one embodiment of the invention, the mixture can be cultured for several hours (about 3 hours) to about 14 days or any hour integer value therebetween. In another embodiment, the mixture can be cultured for 21 days. In one embodiment of the invention, beads and T cells are cultured together for about 8 days. In another embodiment, beads and T cells are cultured together for 2-3 days. Several stimulation cycles may also be required so that the culture time of T cells can be 60 days or longer. Conditions suitable for T cell culture include appropriate culture medium (such as minimum essential medium or RPMI culture medium 1640 or X-vivo 15, (Lonza)), and culture medium can contain factors necessary for proliferation and vigor, including serum (such as fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ and TNF-α or any other additives for cell growth known to those skilled in the art. Other additives for cell growth include, but are not limited to, surfactants, human plasma protein powder (plasmanate) and reducing agents, such as N-acetylcysteine ​​and 2-mercaptoethanol. Culture medium may include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15 and X-Vivo 20, Optimizer, wherein amino acids, sodium pyruvate and vitamins are added, serum-free or supplemented with an appropriate amount of serum (or plasma) or a group of determined hormones and / or cytokines sufficient to make T cells grow and amplify. Antibiotics (e.g., penicillin and streptomycin) are only included in experimental cultures, not in cell cultures to be injected into subjects. Target cells are maintained under conditions necessary to support growth, such as appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5% CO 2 ).

[0278] T cells exposed to different stimulation times may exhibit different characteristics. For example, a typical blood or apheresis peripheral blood mononuclear cell product has a greater than cytotoxic or suppressive T cell population (T C , CD8 + ) of the helper T cell population (T H , CD4 + ). In vitro expansion of T cells by stimulation of the CD3 and CD28 receptors results in a T cell population that consists primarily of T cells approximately 8-9 days prior to H After about 8-9 days, the T cell population contains more and more T C Therefore, depending on the purpose of treatment, a subject may be infused with a cell population that primarily contains T cells. H Similarly, if antigen-specific T cells have been isolated, CIf a subset of cells is expressed, then expanding that subset to a greater extent may be beneficial.

[0279] Furthermore, in addition to CD4 and CD8 markers, other phenotypic markers also vary significantly during cell expansion, but are largely reproducible. Therefore, this reproducibility enables the customization of activated T cell products for specific purposes.

[0280] In one embodiment, the cell comprises a chimeric antigen receptor (CAR). In certain embodiments disclosed herein, CAR generally comprises an antigen binding domain, a transmembrane domain, and an intracellular domain. In some embodiments, CAR comprises an antigen binding domain that is combined with a tumor-associated antigen or a tumor-specific antigen.

[0281] In various embodiments, the CAR can be any CAR molecule, including but not limited to a "first generation," "second generation," "third generation," "fourth generation," or "fifth generation" CAR (see, e.g., 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., Cancer Res. 65:9080-9088 (2005); Maher et al., Nat. Biotechnol. 20:70-75 (2002); Kershaw et al., Cancer Res. 14:113-114 (2003); al., J. Immunol. 173:2143-2150 (2004); Sadelain et al., Curr. Opin. Immunol. (2009); Hollyman et al., J. Immunother. 32: 169-180 (2009)).

[0282] "First generation" CAR for the present invention comprises an antigen binding domain fused to a transmembrane domain, such as a single chain variable fragment (scFv), which is fused to the cytoplasm / intracellular domain of the TCR chain. "First generation" CARs generally have an intracellular domain from a CD3 ζ-chain, which is the main transmitter of signals from endogenous TCRs. "First generation" CARs can provide de novo antigen recognition and cause activation of CD4+ and CD8+ T cells through their CD3 ζ chain signaling domains in a single fusion molecule, independent of HLA-mediated antigen presentation.

[0283] "Second generation" CAR used in the present invention includes an antigen binding domain, such as a single-chain variable fragment (scFv), which is fused to an intracellular signaling domain capable of activating T cells and a co-stimulatory domain intended to enhance T cell efficacy and persistence (Sadelain et al., Cancer Discov.3: 388-398 (2013)). Therefore, CAR design can combine antigen recognition with signal transduction, and these two functions are physiologically undertaken by two independent complex TCR heterodimers and CD3 complexes. "Second generation" CAR includes intracellular domains from various co-stimulatory molecules, such as CD28, 4-1BB, ICOS, OX40, etc., located at the cytoplasmic tail of CAR, to provide additional signals to cells.

[0284] "Second generation" CAR provides costimulation (e.g., through CD28 or 4-1BB domains) and activation (e.g., through CD3ζ signaling domains). Preclinical studies have shown that "second generation" CAR can improve the anti-tumor activity of T cells. For example, in clinical trials for CD19 molecules in patients with chronic lymphocytic leukemia (CLL) and acute lymphocytic leukemia (ALL), it was demonstrated that "second generation" CAR modified T cells have powerful effects (Davila et al., Oncoimmunol. 1 (9): 1577-1583 (2012)).

[0285] "Third generation" CARs provide multiple co-stimulatory (e.g., by including CD28 and 4-1BB domains) and activation (e.g., by including the CD3ζ activation domain).

[0286] "Fourth generation" CARs provide co-stimulation (e.g., through the CD28 or 4-1BB domains) and activation (e.g., through the CD3ζ signaling domain and a constitutive or inducible chemokine component).

[0287] “Fifth generation” CARs provide co-stimulation (e.g., through CD28 or 4-1BB domains) and activation (e.g., through the CD3ζ signaling domain, a constitutive or inducible chemokine component, and the intracellular domain of a cytokine receptor (e.g., IL-2Rβ)).

[0288] In various embodiments, the CAR can be included in a multivalent CAR system, such as a DualCAR or "TandemCAR" system. The multivalent CAR system includes a system or cell comprising multiple CARs and a system or cell comprising a bivalent / bispecific CAR targeting more than one antigen.

[0289] The present invention encompasses a variety of CAR cells that are engineered to inhibit or reduce the level of at least one target cell (e.g., allogeneic responsive cells, immune cells, T cells, B cells, natural killer cells, leukocytes, myeloid cells, plasma cells, etc.). In some embodiments, the CAR cell comprises at least one peptide and / or nucleic acid molecule of the present invention.

[0290] For example, in various embodiments, the CAR construct can include various CAR constructs known in the art, including but not limited to U.S. Pat. No. 8,906,682 B2, U.S. Pat. No. 8,911,993 B2, U.S. Pat. No. 8,916,381 B1, U.S. Pat. No. 8,975,071 B1, U.S. Pat. No. 9,101,584 B2, U.S. Pat. No. 9,102,760 B2, U.S. Pat. No. 9,102,761 B2, U.S. Pat. No. 9,328,156 B2, U.S. Pat. No. 9,464,140 B2, U.S. Pat. No. 9,481,728 B 2. CARs disclosed in U.S. Patent No. 9,499,629 B2, U.S. Patent No. 9,518,123 B2, U.S. Patent No. 9,540,445 B2, U.S. Patent No. 9,573,988 B2, U.S. Patent No. 10,040,846 B2, U.S. Patent No. 10,117,896 B2, U.S. Patent No. 10,174,095 B2, U.S. Patent No. 10,221,245 B2, U.S. Patent No. 10,308,717 B2, U.S. Patent Application No. 20170137783 A1, and / or U.S. Patent Application No. 20180258149 A1.

[0291] In one embodiment, the scFv portion of the CAR of the present invention is encoded by a transgenic, and the sequence of the transgenic has been codon optimized for expression in mammalian cells. In one embodiment, the entire CAR construct of the present invention is encoded by a transgenic, and the entire sequence of the transgenic has been codon optimized for expression in mammalian cells. Codon optimization refers to the discovery that the frequency of occurrence of synonymous codons (i.e., codons encoding the same amino acids) in coding DNA is biased in different species. This codon degeneracy allows the same polypeptide to be encoded by a variety of nucleotide sequences. Various codon optimization methods are known in the art, and include, for example, at least the methods disclosed in U.S. Patent Nos. 5,786,464 and 6,114,148.

[0292] In some embodiments, the CAR cell comprises an antibody or fragment thereof that is engineered to enhance binding to at least one target of interest. The target-specific binding domain may be any domain that is bound to a specific target, including but not limited to a target-specific binding domain derived from any one or more of the following: monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, HLA I molecules, HLA II molecules, MHC I molecules, MHC II molecules and their fragments, including but not limited to single-domain antibodies, such as heavy chain variable domains (VH), light chain variable domains (VL) and variable domains (VHH) of camelid-derived nanobodies, and alternative scaffolds used as antigen-binding domains as known in the art, such as recombinant fibronectin domains. In some cases, the target-specific binding domain is beneficially derived from the same species in which CAR will eventually be used. For example, for human use, it may be beneficial that the target-specific binding domain of CAR includes human or humanized residues of the target-specific binding domain of an antibody or its fragment. Therefore, in one embodiment, the target-specific binding domain portion includes a humanized antibody or its fragment.

[0293] In some embodiments, non-human antibodies are humanized, wherein specific sequences or regions of the antibody are modified to increase similarity to antibodies or fragments thereof produced naturally in humans. In one embodiment, the target-specific binding domain portion is humanized. In one embodiment, the antigen binding domain portion is humanized.

[0294] Humanized antibodies can be produced using a variety of techniques known in the art, including, but not limited to, CDR-grafting (see, e.g., European Patent No. EP 239,400; International Publication No. WO 91 / 09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089, each of which is incorporated herein by reference in its entirety), veneering or resurfacing (see, e.g., European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al., 1995, Protein Engineering, 8(7):915-92; and Roguska et al., 1996, Protein Engineering, 9(8):102-103; and Roguska et al., 1997, Protein Engineering, 11(6):113-114; and Roguska et al., 1998, Protein Engineering, 11(6):103-115; and Roguska et al., 1997, Protein Engineering, 11(6):102-116; and Roguska et al., 1997, Protein Engineering, 11(6):103-117; and Roguska et al., 1997, Protein Engineering, 11(6):103 ... al., 1994, PNAS, 91:969-973, each of which is incorporated herein by reference in its entirety); strand displacement (see, e.g., U.S. Patent No. 5,565,332, which is incorporated herein by reference in its entirety); and techniques disclosed, for example, in U.S. Patent Application Publication No. US2005 / 0042664; U.S. Patent Application Publication No. US2005 / 0048617; U.S. Patent No. 6,407,213; U.S. Patent No. 5,766,886; International Publication No. WO 9317105; Tan et al., J. Immunol., 169:1119-25 (2002); Caldas et al., Protein Eng., 13(5):353-60 (2000); Morea et al., Methods, 20(3):267-79 (2000); Baca et al., Protein Eng., 13(5):353-60 (2000); al., J. Biol. Chem., 272(16):10678-84 (1997); Roguska et al., Protein Eng., 9(10):895-904 (1996); Couto et al., Cancer Res., 55(23 Supp):5973s-5977s (1995); Couto et al., Cancer Res., 55(8):1717-22 (1995); Sandhu JS, Gene, 150(2):409-10 (1994); and Pedersen et al., J. Mol. Biol., 235(3):959-73 (1994), each of which is incorporated herein by reference in its entirety. Typically, framework residues in the framework region will be substituted with corresponding residues from the CDR donor antibody to alter (e.g., improve) antigen binding.These framework substitutions can be identified by methods well known in the art, such as by modeling the interactions of the CDR and framework residues to identify framework residues important for antigen binding, and by sequence comparison to identify unusual framework residues at specific positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323, which are incorporated herein by reference in their entirety.).

[0295] One or more amino acid residues from non-human sources remain in a humanized antibody or antibody fragment. These non-human amino acid residues are generally referred to as "import" residues, which are generally taken from "import" variable domains. As provided herein, a humanized antibody or antibody fragment comprises one or more CDRs from a non-human immunoglobulin molecule and a framework region, wherein the amino acid residues constituting the framework are completely or mostly from human germline. A variety of techniques for humanizing antibodies or antibody fragments are known in the art and can be essentially performed according to the method of Winter and coworkers (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)), by replacing rodent CDRs or CDR sequences with the corresponding sequences of human antibodies, i.e., CDR-grafting (EP 239,400; PCT Publication No. WO 91 / 09967; and U.S. Pat. Nos. 4,816,567; 6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640, the contents of which are incorporated herein by reference in their entireties). In such humanized chimeric antibodies and antibody fragments, substantially less than complete human variable domains are replaced by corresponding sequences from non-human species. In fact, humanized antibodies are usually human antibodies, in which some CDR residues and possibly some framework (FR) residues are replaced by residues from similar sites in rodent antibodies. The humanization of antibodies and antibody fragments can also be achieved by veneer or surface remodeling (EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology, 28 (4 / 5): 489-498; Studnicka et al., Protein Engineering, 7 (6): 805-814 (1994); and Roguska et al., PNAS, 91: 969-973 (1994)) or chain replacement (U.S. Patent No. 5,565,332), the contents of which are incorporated herein by reference in their entirety.

[0296] The human variable domains (light and heavy chains) selected for making humanized antibodies are to reduce antigenicity. According to the so-called "best fit" method, the variable domain sequences of rodent antibodies are screened for the entire library of known human variable domain sequences. The human sequences closest to rodents are then accepted as the human frameworks (FRs) of humanized antibodies (Sims et al., J.Immunol., 151: 2296 (1993); Chothia et al., J.Mol.Biol., 196: 901 (1987), the contents of which are incorporated herein by reference in their entirety). Another method uses a specific framework of the consensus sequence of all human antibodies derived from a specific subgroup of light or heavy chains. The same framework can be used for a variety of different humanized antibodies (see, e.g., Nicholson et al. Mol. Immun. 34(16-17): 1157-1165 (1997); Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); Presta et al., J. Immunol., 151: 2623 (1993), the contents of which are incorporated herein by reference in their entireties).

[0297] In various embodiments, the CAR composition comprising an antibody or fragment thereof is partially humanized, retaining high affinity for a target antigen, a target cell carrying an antigen, a target alloresponsive cell carrying an antigen, a target TCR, a target T cell carrying a TCR, a target alloresponsive T cell carrying a TCR, a target BCR, a target B cell carrying a BCR, a target alloresponsive B cell carrying a BCR, a target immune cell, a target T cell, a target B cell, a target natural killer cell, a target leukocyte, a target myeloid cell, a target plasma cell, or any combination thereof, as well as other favorable biological properties.

[0298] In one embodiment, humanized antibodies and antibody fragments are prepared by using the three-dimensional model analysis of parental sequences and various conceptual humanized products of parental and humanized sequences. The three-dimensional immunoglobulin model is generally available and is familiar to those skilled in the art. Computer programs can be used to illustrate and display the possible three-dimensional conformational structure of selected candidate immunoglobulin sequences. Checking these displays allows analysis of the possible effects of residues in the function of candidate immunoglobulin sequences, i.e., analyzing the residues that affect the ability of candidate immunoglobulin to bind target antigens. In this way, FR residues can be selected and combined from receptor and input sequence to realize required antibody or antibody fragment characteristics, such as an increase in affinity to the target antigen. Generally speaking, CDR residues directly and most substantially participate in affecting antigen binding.

[0299] Humanized antibodies or antibody fragments retain antigenic specificity similar to the original antibody. However, the methods disclosed herein can further improve the binding affinity and / or specificity of the antibody to human cells carrying the antigen. In one embodiment, the target-specific binding domain is characterized by a specific functional feature or property of the antibody or antibody fragment.

[0300] In one aspect, the invention relates to an antigen binding domain comprising an antibody or antibody fragment, wherein the antibody binding domain specifically binds to a target of interest or a fragment thereof. In one embodiment, the scFv is adjacent to the leader sequence and is in the same reading frame.

[0301] In one embodiment, the antibody fragments provided herein are single chain variable fragments (scFv). In various embodiments, the antibodies of the present invention can exist in a variety of other forms, including, for example, Fv, Fab and (Fab')2, and bifunctional (i.e., bispecific) hybrid antibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)).

[0302] ScFv can be prepared according to methods known in the art (see, for example, Bird et al., (1988) Science 242: 423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). ScFv molecules can be produced by connecting the VH and VL regions together using a flexible polypeptide linker. The scFv molecule comprises a flexible polypeptide linker (e.g., a Ser-Gly linker) having an optimized length and / or amino acid composition. The length of the flexible polypeptide linker can greatly affect how the variable regions of the scFv fold and interact with each other. In fact, if a short polypeptide linker (e.g., between 5-10 amino acids) is used, intrachain folding can be prevented. Interchain folding is also necessary to bind the two variable regions together to form a functional epitope binding site. For examples of linker orientations and sizes, see, e.g., Hollinger et al. 1993 Proc Natl Acad. Sci. USA 90:6444-6448, U.S. Patent Application Publication Nos. 2005 / 0100543, 2005 / 0175606, 2007 / 0014794, and PCT Publication Nos. WO 2006 / 020258 and WO 2007 / 024715, incorporated herein by reference.

[0303] scFv can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 or more amino acid residues between VL and VH regions. The flexible polypeptide linker sequence can comprise any naturally occurring amino acid. In some embodiments, the flexible polypeptide linker sequence comprises amino acids glycine and serine. In another embodiment, the flexible polypeptide linker sequence comprises a glycine and serine repeating group, such as (Gly4Ser)n, wherein n is a positive integer equal to or greater than 1. In one embodiment, the flexible polypeptide linker includes but is not limited to (Gly4Ser)4 or (Gly4Ser)3. Changes in the length of the flexible polypeptide linker can retain or enhance activity, resulting in better therapeutic effects in activity studies.

[0304] Stability and mutation

[0305] The stability of scFv molecules (e.g., soluble scFv) can be assessed with reference to the biophysical properties (e.g., thermal stability) of conventional control scFv molecules or full-length antibodies. In one embodiment, the thermal stability of humanized soluble scFv is about 0.1, about 0.25, about 0.5, about 0.75, about 1, about 1.25, about 1.5, about 1.75, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10 degrees Celsius, about 11 degrees Celsius, about 12 degrees Celsius, about 13 degrees Celsius, about 14 degrees Celsius, or about 15 degrees Celsius higher than the control binding molecule (e.g., conventional scFv molecule) in the assay.

[0306] The improved thermal stability of scFv is then given to the entire CAR construct, resulting in improved therapeutic properties of the CAR construct. Compared with conventional antibodies, the thermal stability of scFv can be increased by at least about 2 ° C or 3 ° C. In one embodiment, the thermal stability of scFv is increased by 1 ° C compared with conventional antibodies. In another embodiment, the thermal stability of scFv is increased by 2 ° C compared with conventional antibodies. In another embodiment, the thermal stability of scFv is increased by 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 ° C compared with conventional antibodies. For example, the scFv molecules disclosed herein can be compared with the scFv molecules or Fab fragments of antibodies derived from scFv VH and VL. Thermal stability can be measured using methods known in the art. For example, in one embodiment, Tm can be measured. Methods for measuring Tm and other methods for determining protein stability are described in more detail below.

[0307] Mutations in scFv (generated by humanization of soluble scFv or direct mutagenesis) alter the stability of scFv and improve the overall stability of scFv and CAR constructs. The stability of humanized scFv was compared with mouse scFv using measurements such as Tm, temperature denaturation, and temperature aggregation. Thus, the residues introduced by humanization increased the Tm of scFv by more than 10°C.

[0308] In one embodiment, scFv comprises at least one mutation produced by a humanization process, so that the mutated scFv gives the CAR construct improved stability. In various embodiments, scFv comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mutations produced by a humanization process, so that the mutated scFv gives the CAR construct improved stability. Any assay described herein can be used to determine the binding ability of the mutant scFv.

[0309] In some embodiments, the engineered TCR comprises one or more TCR chains (e.g., TCR alpha chains, TCR beta chains, TCR delta chains, and TCR gamma chains) that specifically bind to a target of interest, such as a target of interest on a cancer cell, alone or together. In one embodiment, the engineered TCR comprises a TCR alpha chain and a TCR beta chain, wherein the engineered TCR specifically binds to a target of interest.

[0310] Methods for assessing protein stability

[0311] In order to evaluate the stability of the scFv construct, the stability of the minimum domain (i.e., the scFv of the CAR construct) of the multidomain protein is predicted using the method and those described below. Such methods allow determination of multiple thermal unfolding transitions, wherein the most unstable domain is either first unfolded, or the overall stability threshold of the multidomain unit (i.e., a multidomain protein showing a single unfolding transition) that is cooperatively unfolded is limited. The most unstable domain can be identified by a variety of additional means. Mutagenesis can be performed to detect which domain limits overall stability. In addition, it may be possible to perform protease resistance of multidomain proteins under conditions of unfolding in the known most unstable domain by DSC or other spectral methods (Fontana, et al., 1997, Fold. Des., 2: R17-26; Dimasi et al., 2009, J. Mol. Biol. 393: 672-692). Once the most unstable domain is determined, the sequence encoding the domain (or part thereof) can be used as a test sequence in the method.

[0312] a) Thermal stability

[0313] The thermal stability of a composition can be analyzed using a variety of non-limiting biophysical or biochemical techniques known in the art. In certain embodiments, thermal stability is assessed by analyzing spectroscopy.

[0314] An exemplary analytical spectroscopic method is differential scanning calorimetry (DSC). DSC uses a calorimeter that is sensitive to the heat absorption that accompanies the unfolding of most proteins or protein domains (e.g., Sanchez-Ruiz et al., 1988, Biochemistry, 27: 1648-1652). To determine the thermal stability of a protein, a protein sample is inserted into a calorimeter and the temperature is raised until the Fab or scFv unfolds. The temperature at which the protein unfolds is indicative of overall protein stability.

[0315] Another exemplary analytical spectroscopy method is circular dichroism (CD) spectroscopy. CD spectroscopy measures the optical activity of a composition as temperature increases. Circular dichroism (CD) spectroscopy measures the difference in absorption of left-handed polarized light and right-handed polarized light due to structural asymmetry. Disordered or unfolded structures produce CD spectra that are very different from those of ordered or folded structures. CD spectra reflect the sensitivity of proteins to the denaturing effects of increased temperature and are therefore indicative of the thermal stability of proteins (van Mierlo and Steemsma, J. Biotechnol., 2000, 79: 281-298).

[0316] Another exemplary analytical spectroscopy method for measuring thermal stability is fluorescence emission spectroscopy (van Mierlo and Steemsma, supra). Another exemplary analytical spectroscopy method for measuring thermal stability is nuclear magnetic resonance (NMR) spectroscopy (eg, van Mierlo and Steemsma, supra).

[0317] The thermal stability of the composition can be measured by biochemical methods. An exemplary biochemical method for evaluating thermal stability is a thermal challenge assay. In a "thermal challenge assay", the composition is subjected to a series of elevated temperatures over a set time period. For example, in one embodiment, a test scFv molecule or a molecule comprising an scFv molecule is subjected to a series of elevated temperatures, such as for 1-1.5 hours. Then, the activity of the protein is determined by a related biochemical assay. For example, if the protein is a binding protein (e.g., a scFv or a polypeptide containing scFv), the binding activity of the binding protein can be determined by a functional or quantitative ELISA.

[0318] Such assays can be performed using E. coli and high throughput screening in high throughput formats and those disclosed in the Examples. scFv variant libraries can be created using methods known in the art. scFv expression can be induced, and scFvs can be subjected to thermal challenges. Binding assays can be performed on challenged test samples, and those scFvs that are stable can be scaled up and further characterized.

[0319] Thermal stability is assessed by measuring the melting temperature (Tm) of the composition using any of the above techniques (e.g., analytical spectroscopy techniques). The melting temperature is the temperature at the midpoint of the thermal transition curve, where 50% of the molecules of the composition are in a folded state (e.g., Dimasi et al., 2009, J. Mol Biol. 393:672-692). In one embodiment, the Tm value of the scFv is about 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C 8℃, 69℃, 70℃, 71℃, 72℃, 73℃, 74℃, 75℃, 76℃, 77℃, 78℃, 79℃, 80℃, 81℃, 82℃, 83℃, 84℃, 85℃, 86℃, 87℃, 88℃, 89℃, 90℃, 91℃, 92℃, 93℃, 94℃, 95℃, 96℃, 97℃, 98℃, 99℃, 100℃. In one embodiment, the Tm value of IgG is about 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C 8℃, 69℃, 70℃, 71℃, 72℃, 73℃, 74℃, 75℃, 76℃, 77℃, 78℃, 79℃, 80℃, 81℃, 82℃, 83℃, 84℃, 85℃, 86℃, 87℃, 88℃, 89℃, 90℃, 91℃, 92℃, 93℃, 94℃, 95℃, 96℃, 97℃, 98℃, 99℃, 100℃. In one embodiment, the Tm value of the multivalent antibody is about 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C 8℃, 69℃, 70℃, 71℃, 72℃, 73℃, 74℃, 75℃, 76℃, 77℃, 78℃, 79℃, 80℃, 81℃, 82℃, 83℃, 84℃, 85℃, 86℃, 87℃, 88℃, 89℃, 90℃, 91℃, 92℃, 93℃, 94℃, 95℃, 96℃, 97℃, 98℃, 99℃, 100℃.

[0320] Thermal stability is also assessed by measuring the specific heat or heat capacity (Cp) of the composition using analytical calorimetric techniques (e.g., DSC). The specific heat of a composition is the energy (e.g., in kcal / mol) required to raise the temperature of 1 mole of water by 1°C. A large Cp is a sign of a denatured or inactive protein composition. The change in heat capacity (ΔCp) of a composition is measured by determining the specific heat of the composition before and after its thermal transition. Thermal stability can also be assessed by measuring or determining other parameters of thermodynamic stability, including Gibbs free energy of expansion (ΔG), enthalpy of expansion (ΔH), or entropy of expansion (ΔS). The temperature (i.e., TC value) at which 50% of the composition retains its activity (e.g., binding activity) is determined using one or more of the above-mentioned biochemical assays (e.g., thermal challenge assays).

[0321] In addition, compared with unmutated scFv, mutations to scFv change the thermal stability of scFv. When humanized scFv is incorporated into a CAR construct, the humanized scFv imparts thermal stability to the entire anti-CAR construct. In one embodiment, the scFv comprises a single mutation that imparts thermal stability to the scFv. In another embodiment, the scFv comprises multiple mutations that impart thermal stability to the scFv. In one embodiment, multiple mutations in the scFv have an additive effect on the thermal stability of the scFv.

[0322] b) % Aggregation

[0323] The stability of a composition can be determined by measuring its aggregation tendency. Aggregation can be measured by a variety of non-limiting biochemical or biophysical techniques. For example, chromatography (e.g., size exclusion chromatography (SEC)) can be used to assess the aggregation of a composition. SEC separates molecules based on size. Semisolid beads of polymer gel are contained in the column, which allow ions and small molecules to enter its interior, but do not allow macromolecules to enter. When the protein composition is applied to the top of the column, tightly folded proteins (i.e., non-aggregated proteins) are distributed through a larger volume of solvent than is available to large protein aggregates. Therefore, large aggregates pass through the column faster, and in this way, the mixture can be separated or fractionated into its components. When each fraction is eluted from the gel, it can be quantified separately (e.g., by light scattering). Therefore, the % aggregation of the composition can be determined by comparing the concentration of a certain fraction with the total concentration of the protein applied to the gel. The stable composition eluted from the column is essentially a single fraction and is essentially a single peak in the elution curve or chromatogram.

[0324] c) Binding affinity

[0325] The stability of the composition can be assessed by determining the target binding affinity of the composition. A variety of methods for determining binding affinity are known in the art. An exemplary method for determining binding affinity employs surface plasmon resonance. Surface plasmon resonance is an optical phenomenon that allows real-time biospecific interactions to be analyzed by detecting changes in protein concentrations within a biosensor matrix, for example using a BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For further description, see Jonsson, U., et al. (1993) Ann. Biol. Clin. 51: 19-26; Jonsson, U., i (1991) Biotechniques 11: 620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8: 125-131; and Johnnson, B., et al. (1991) Anal. Biochem. 198: 268-277.

[0326] In various embodiments, the portion of the CAR composition of the present invention comprising an antibody or antibody fragment further comprises a heavy chain and a light chain variable region, wherein the heavy chain and the light chain variable region comprise an amino acid sequence homologous to the amino acid sequence of an antibody described herein, and wherein the antibody retains the desired functional properties of the antibody of the present invention. In one embodiment, the CAR composition comprises an antibody fragment. In one embodiment, the antibody fragment comprises scFv.

[0327] In various embodiments, the part of the antibody or antibody fragment comprising the CAR composition is engineered by modifying one or more amino acids in one or two variable regions (i.e., VH and / or VL).For example, the antibody or antibody fragment of the CAR composition is engineered by modifying one or more amino acids in one or more CDR regions and / or one or more framework regions.

[0328] In some embodiments, CAR cells include extracellular receptors, transmembrane domains and intracellular signaling domains. In one embodiment, the extracellular receptor is connected to the intracellular signaling domain. In one embodiment, the extracellular receptor is connected to the intracellular signaling domain by being connected to the transmembrane domain of the intracellular signaling domain.

[0329] In some embodiments, the CAR cell comprises a recombinant DNA construct comprising a sequence encoding a CAR disclosed therein.

[0330] Nucleic acid sequences encoding desired molecules can be obtained using recombinant methods known in the art, such as, for example, by screening a cell library expressing the gene, by deriving the gene from a vector known to include the gene, or by isolating directly from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest can be produced by synthesis rather than cloning.

[0331] Nucleic acid sequences encoding the desired molecules can be obtained using recombinant methods known in the art, such as, for example, by screening libraries of cells expressing the gene, by deriving the gene from a vector known to include the gene, or by isolating directly from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest can be produced by synthesis rather than cloning.

[0332] In some embodiments, in order to evaluate the expression of the peptide, the expression vector to be introduced into the cell may also contain a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from a cell population that is attempted to be transfected or infected by a viral vector. In other aspects, selectable markers may be carried on separate DNA fragments and used for co-transfection procedures. Both selectable markers and reporter genes may be flanked by appropriate regulatory sequences to enable them to be expressed in host cells. Useful selectable markers include, for example, antibiotic resistance genes, such as neo, etc.

[0333] Reporter gene is used to identify potential transfected cells and to evaluate the function of regulatory sequences. Generally speaking, reporter gene is a gene that does not exist or is not expressed in the recipient organism or tissue, and the expression of its encoded polypeptide is shown as some characteristics that are easy to detect, such as enzyme activity. After DNA is introduced into the recipient cell, the expression of reporter gene is detected at the appropriate time. Suitable reporter gene can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein gene (such as Ui-Tei et al., 2000FEBS Letters479:79-82). Suitable expression system is well-known and can be prepared or commercially obtained using known technology. Generally speaking, the construct with the minimum 5' flanking region showing the highest level reporter gene expression is identified as a promoter. Such promoter region can be connected to a reporter gene and used to evaluate the ability of medicament regulating promoter-driven transcription.

[0334] Methods for introducing genes into cells and expressing them are known in the art. In the context of expression vectors, vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast or insect cells, by any method in the art. For example, expression vectors can be transferred to host cells by physical, chemical or biological means.

[0335] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). An exemplary method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0336] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used methods for inserting genes into mammalian cells (e.g., human cells). Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, e.g., U.S. Patent Nos. 5,350,674 and 5,585,362.

[0337] Chemical methods for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads and lipid-based systems, including water-in-oil emulsions, micelles, mixed micelles and liposomes. An exemplary colloidal system for use as an in vitro and in vivo delivery vehicle is a liposome (e.g., an artificial membrane vesicle).

[0338] In the case of using a non-viral delivery system, an exemplary delivery vehicle is a liposome. Consider using a lipid formulation to introduce nucleic acid into a host cell (in vitro, in vitro or in vivo). In another aspect, nucleic acid can be associated with lipid. Nucleic acid associated with lipid can be encapsulated in the aqueous interior of liposome, dispersed in the lipid bilayer of liposome, attached to liposome by a connecting molecule associated with liposome and oligonucleotide, embedded in liposome, compounded with liposome, dispersed in a solution containing lipid, mixed with lipid, combined with lipid, included in lipid as a suspension, included in micelle or compounded with micelle or otherwise associated with lipid. Lipid, lipid / DNA or lipid / expression vector related compositions are not limited to any specific structure in solution. For example, they can be present in a double-layer structure, as micelle or have a "collapse" structure. They can also be simply dispersed in solution, and may form an aggregate of uneven size or shape. Lipid is a fatty substance, can be a naturally occurring or synthetic lipid. For example, lipids include fat droplets naturally present in the cytoplasm as well as the class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0339] Lipids suitable for use can be obtained from commercial sources. For example, dimyristoylphosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO; dihexadecyl phosphate ("DCP") can be obtained from K&K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristoylphosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the only solvent because it evaporates more easily than methanol. "Liposome" is a general term covering various monolayer and multilayer lipid vehicles formed by the generated closed lipid bilayer or aggregates. The characteristic of liposomes is that they have a vesicle structure having a phospholipid bilayer membrane and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. When phospholipids are suspended in an excess of aqueous solution, they form spontaneously. The lipid components undergo self-rearrangement before forming a closed structure and trap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions in which the structure in the solution is different from the normal vesicle structure are also contemplated. For example, lipids can present a micellar structure or exist only as a non-uniform aggregate of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0340] Regardless of the method used to introduce exogenous nucleic acid into the host cell, various assays can be performed to confirm the presence of the recombinant DNA sequence in the host cell. Such assays include, for example, "molecular biology" assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as by immunological methods (ELISA and Western blotting) or by assays described herein to detect the presence or absence of specific peptides to identify agents falling within the scope of the present invention.

[0341] RNA can be introduced into target cells using any of a variety of different methods, such as commercially available methods, including but not limited to electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg Germany), cationic liposome-mediated transfection using lipofection, polymer encapsulation, peptide-mediated transfection, or biolistic particle delivery systems, such as a "gene gun" (see, e.g., Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001)).

[0342] How to use

[0343] The present invention also relates in part to a method of reducing the removal of at least one target cell (e.g., donor cells, such as T cells, CAR cells, CAR T cells, T cells expressing engineered TCRs, etc.) or increasing its persistence. In various aspects, the present invention provides a method of preventing, reducing or eliminating allogeneic responses; a method of reducing, reducing, eliminating or depleting allogeneic responsive cell levels, and a method of inducing allogeneic tolerance or non-responsiveness of allogeneic cells. In various aspects, the present invention also provides a method of improving the effectiveness of CAR therapy or engineered TCR cell therapy.

[0344] In various aspects, the present invention also provides a method for preventing or treating various diseases or conditions. In some embodiments, the method includes administering any of the nucleic acid molecules, compositions and / or cells described herein that are effective for treatment. For example, in one embodiment, the method includes administering at least one genetically engineered TKO cell that is effective for expressing at least one peptide (e.g., SCT) of the present invention. In one embodiment, the method includes administering at least one CAR T cell that is effective for expressing at least one peptide (e.g., SCT) of the present invention. In one embodiment, the method includes administering a therapeutically effective amount of at least one T cell that expresses an engineered TCR that expresses at least one peptide (e.g., SCT) of the present invention.

[0345] In some embodiments, the disease or disorder comprises a disease or disorder associated with the expression of alloresitive cells, a disease or disorder associated with at least one HLA receptor, a disease or disorder associated with at least one HLA-containing receptor, a disease or disorder associated with at least one MHC receptor, a disease or disorder associated with at least one MHC-containing receptor, GvHD, an autoimmune disease or disorder, a disease or disorder associated with organ transplantation, a disease or disorder associated with tissue transplantation, a disease or disorder associated with cell transplantation, a disease or disorder associated with allogeneic transplantation, a disease or disorder associated with intestinal transplantation, a disease or disorder associated with reconstructive transplantation, cancer, a disease or disorder associated with cancer, or any combination thereof.

[0346] In various aspects, the present invention provides a method for preventing or treating cancer, a disease or condition associated with cancer, or a combination thereof in a subject in need thereof, the method comprising administering to the subject an effective amount of at least one peptide, nucleic acid molecule, cell, or composition of the present invention. For example, in one embodiment, the method is engineered TCR cell therapy.

[0347] In some aspects, the invention provides methods of administering to a subject an effective amount of at least one peptide, nucleic acid molecule, cell, or composition of the invention. In some embodiments, the subject suffers from cancer, a disease or condition associated with cancer, or a combination thereof.

[0348] The following are non-limiting examples of cancers that may be treated by the disclosed methods and compositions: acute lymphoblastic leukemia; acute myeloid leukemia; adrenocortical carcinoma; childhood adrenocortical carcinoma; appendix carcinoma; basal cell carcinoma; bile duct carcinoma, extrahepatic; bladder cancer; bone cancer; osteosarcoma and malignant fibrous histiocytoma; childhood brain stem glioma; adult brain tumors; brain tumors, brain stem glioma, children; brain tumors, atypical teratoid / rhabdoid tumors, CNS, children; CNS embryonal tumors; cerebellar astrocytoma; cerebral astrocytoma / glioblastoma; craniopharyngioma; ependymoblastoma; ependymoma; medulloblastoma; medullary epithelioma; moderately differentiated pineal parenchymal tumor; supratentorial primitive neuroectodermal tumor and pineoblastoma; visual Pathway and hypothalamic gliomas; Brain and spinal cord tumors; Breast cancer; Bronchial tumors; Burkitt's lymphoma; Carcinoid tumors; Gastrointestinal carcinoid tumors; CNS atypical teratoid / rhabdoid tumors; CNS embryonal tumors; CNS lymphomas; Cerebellar astrocytoma Brain astrocytoma / malignant glioma, children; Cervical cancer; Chordoma, children; Chronic lymphocytic leukemia; Chronic myeloid leukemia; Chronic myeloproliferative disorders; Colon cancer; Colorectal cancer; Craniopharyngioma; Cutaneous T-cell lymphoma; Esophageal cancer; Ewing's family of tumors; Extragonadal germ cell tumors; Extrahepatic bile duct cancer; Eye cancer, intraocular melanoma; Eye cancer, retinoblastoma; Gallbladder cancer; Stomach (gastric) cancer; Gastrointestinal carcinoid tumors; Gastrointestinal stromal tumors (gastric intestinal stromal tumor); germ cell tumor, extracranial; germ cell tumor, extragonadal; germ cell tumor, ovarian; gestational trophoblastic tumor; glioma; glioma, brainstem, pediatric; glioma, cerebral astrocytoma, pediatric; glioma, visual pathway and hypothalamus, pediatric; hairy cell leukemia; head and neck cancer; hepatocellular (liver) cancer; histiocytosis, Langerhans cell; Hodgkin lymphoma; hypopharyngeal cancer; glioma, hypothalamic and visual pathway; intraocular melanoma; islet cell tumor; kidney (renal cell) cancer; Langerhans cell histiocytosis; laryngeal cancer; leukemia, acute lymphocytic; leukemia, acute myeloid; leukemia, chronic lymphocytic; leukemia, chronic myeloid; leukemia, hairy cell; lip and oral cavity cancer; Liver cancer; Lung cancer, non-small cell; Lung cancer, small cell; Lymphoma, AIDS-related; Lymphoma, Burkitt; Lymphoma, cutaneous T-cell; Lymphoma, non-Hodgkin's lymphoma; Lymphoma, primary central nervous system; Macroglobulinemia, Waldenstrom; Malignant fibrous histiocytoma of bone and osteosarcoma; Medulloblastoma; Melanoma; Melanoma, intraocular (eye); Merkel cell carcinoma; Mesothelioma; Metastatic squamous neck carcinoma with occult primary; Oral cancer; Multiple endocrine neoplasms syndrome, (children); Multiple myeloma / plasma cell neoplasms; Fungal diseases; Mycosis fungoides; Myelodysplastic syndrome; Myelodysplastic / myeloproliferative disorders; Myeloid leukemia, chronic; Myeloid leukemia, acute, adult;Myeloid leukemia, acute, childhood; multiple myeloma; myeloproliferative disorders, chronic; cancer of the nasal cavity and paranasal sinuses; nasopharyngeal carcinoma; neuroblastoma; non-small cell lung cancer; oral cancer; oral cancer; oropharyngeal cancer; osteosarcoma and malignant fibrous histiocytoma of bone; ovarian cancer; ovarian epithelial cancer; ovarian germ cell tumors; ovarian tumors of low malignant potential; pancreatic cancer; pancreatic cancer, islet cell tumors; papillomatosis; parathyroid cancer; penile cancer; pharyngeal cancer; pheochromocytoma; moderately differentiated pineal parenchymal tumors; pineoblastoma and supratentorial primitive neuroectodermal tumors; pituitary tumors; plasma cell neoplasms / multiple myeloma; pleuropulmonary blastoma; primary central nervous system lymphoma; prostate cancer; rectal cancer; renal cell (kidney) cancer; renal pelvis and ureter, transitional cell carcinoma; chromosome 15 involved Respiratory tract cancer of the upper nut gene; retinoblastoma; rhabdomyosarcoma; salivary gland cancer; sarcoma, Ewing's family of tumors; sarcoma, Kaposi; sarcoma, soft tissue; sarcoma, uterine; Sezary syndrome; skin cancer (non-melanoma); skin cancer (melanoma); skin cancer, Merkel cell; small cell lung cancer; small intestine cancer; soft tissue sarcoma; squamous cell carcinoma with occult primary, metastatic squamous neck cancer; stomach (gastric) cancer; supratentorial primitive neuroectodermal tumor; T-cell lymphoma, skin; testicular cancer; pharyngeal cancer; thymoma and thymic carcinoma; thyroid cancer; transitional cell carcinoma of the renal pelvis and ureter; trophoblastic tumor, pregnancy; urethral cancer; uterine cancer, endometrial; uterine sarcoma; vaginal cancer; vulvar cancer; Waldenstrom's macroglobulinemia; and Wilms' tumor. ;

[0349] In some aspects, the present invention also provides methods including administering an effective amount of any peptide disclosed herein, nucleic acid molecules, cells, or compositions to a subject, wherein the subject has at least one donor cell. In some aspects, the present invention also provides methods including administering an effective amount of any peptide disclosed herein, nucleic acid molecules, cells, or compositions to a subject, wherein the subject needs to reduce the removal of at least one target cell (e.g., donor cells, such as T cells, CAR cells, CAR T cells, T cells expressing engineered TCRs, etc.) or increase its persistence.

[0350] In one embodiment, the present invention provides a method for preventing or treating a disease or condition associated with alloresponsive cells. In various embodiments, the disease or condition associated with the expression of alloresponsive cells is allograft rejection, immune rejection, chronic alloresponsive cells, implant rejection, transplant rejection, inflammation, inflammation caused by ischemia / reperfusion, infection, immune response to allograft, or any combination thereof. In various embodiments, the disease or condition associated with the expression of alloresponsive cells is caused by organ transplantation, cell transplantation, cell transplantation, allograft, intestinal transplantation, reconstructive transplantation, autoimmune disease or condition, disease or condition associated with at least one HLA receptor, disease or condition associated with at least one HLA-containing receptor, disease or condition associated with at least one MHC receptor, disease or condition associated with at least one MHC-containing receptor, or any combination thereof.

[0351] In various embodiments, the alloresponsive cells are cells carrying antigens, TCRs, BCRs, alloresponsive immune cells, alloresponsive specific immune cells, alloresponsive immune cells, autologous immune cells, immune cells carrying alloresponsive antigens, TCRs and / or BCRs, alloresponsive T cells, alloresponsive specific T cells, alloresponsive T cells, autologous T cells, T cells carrying alloresponsive TCRs, alloresponsive B cells, alloresponsive B cells, autologous B cells, B cells carrying alloresponsive BCRs, or any combination thereof. For example, in various embodiments, the alloresponsive T cells are T cells carrying TCRs, alloresponsive T cells, alloresponsive specific T cells, alloresponsive T cells, autologous T cells, T cells carrying alloresponsive TCRs, or any combination thereof. In various embodiments, the alloresponsive B cells are BCR-bearing B cells, alloresponsive B cells, allospecific B cells, allogeneic B cells, autologous B cells, B cells carrying alloreactive BCRs, or any combination thereof.

[0352] In one embodiment, the method comprises administering at least one cell genetically modified to express at least one peptide of the invention (eg, SCT), wherein the peptide specifically inhibits NK cells.

[0353] In addition, the present invention provides nucleic acid molecules and compositions and cells comprising the same and their use in agents or methods for preventing, reducing and / or eliminating alloresponses, methods for eliminating specific cell populations, methods for depleting the level of alloresponsive cells (e.g., T cells), or methods for inducing allotolerance and / or immune non-compliance to allogeneic transplants. For example, the nucleic acid molecules of the present invention can be used to treat a subject who has been treated for a disease or condition associated with the expression of alloresponsive T cells, wherein the subject who has been treated for the expression of alloresponsive T cells exhibits a disease associated with the expression of alloresponsive cells.

[0354] In one embodiment, the present invention relates to a vector comprising a nucleic acid molecule as described herein, which is operably linked to a promoter for expression in a mammalian cell (e.g., a mammalian T cell, a mammalian B cell, etc.). For example, in one embodiment, the present invention provides a recombinant cell expressing at least one peptide of the present invention (e.g., SCT) for preventing and / or treating a disease or condition associated with alloresponsive cells. In one embodiment, the cell of the present invention is capable of contacting an alloresponsive cell with at least one peptide of the present invention (e.g., SCT) expressed on its surface so as to inhibit at least one NK cell and inhibit alloresponse.

[0355] For example, in one embodiment, the present invention provides a recombinant T cell expressing at least one peptide of the present invention (eg, SCT) for use in preventing and / or treating a disease or condition associated with alloreactive T cells.

[0356] In one embodiment, the cells of the invention are capable of contacting allogeneic responsive cells with at least one peptide of the invention (e.g., SCT) expressed on their surface to inhibit at least one NK cell and inhibit allogeneic responses. In one embodiment, the cells of the invention are capable of contacting allogeneic responsive cells with at least one peptide of the invention (e.g., SCT) expressed on their surface to inhibit at least one NK cell and induce allogeneic tolerance.

[0357] In one embodiment, the invention encompasses cell therapy, wherein cells are modified to contain or express at least one peptide of the invention (eg, SCT), and the cells are infused into a recipient in need thereof.

[0358] In one embodiment, the present invention includes cell therapy, wherein the method includes administering to a subject a composition comprising cells (such as antigen presenting cells) comprising or expressing at least one peptide (e.g., SCT) of the present invention. For example, in one embodiment, the method includes administering a composition comprising antigen presenting cells loaded with at least one peptide (e.g., SCT) of the present invention and expressing the at least one peptide (e.g., SCT) on the surface.

[0359] In one embodiment, the present invention includes cell therapy, wherein the method includes administering to a subject a composition comprising cells activated or stimulated by antigen presenting cells, the antigen presenting cells comprising or expressing at least one peptide (e.g., SCT) of the present invention. For example, in one embodiment, the method includes contacting cells (such as naive T cells) with antigen presenting cells, the antigen presenting cells being loaded with at least one peptide (e.g., SCT) of the present invention and expressing the at least one peptide (e.g., SCT) on the surface; thereby activating the cells. The method includes administering to a subject a composition comprising activated cells. For example, in one embodiment, the method of the present invention includes the following steps:

[0360] (1) providing a naive T cell population; (2) providing a dendritic cell population; (3) loading or pulsing dendritic cells with one or more peptides of the present invention (e.g., SCT); (4) co-culturing naive T cells and loaded dendritic cells; and (5) isolating stimulated T cells. In one embodiment, the method further comprises the step of (6) administering the stimulated T cells to a subject in need thereof.

[0361] In certain embodiments, the modified cells (e.g., antigen presenting cells presenting at least one peptide of the invention (e.g., SCT)) are capable of preventing or reducing an immune response in vivo. For example, in certain embodiments, the modified cells are capable of preventing or reducing transplant rejection in a recipient.

[0362] In one embodiment, the modified T cells of the present invention can undergo strong in vivo T cell expansion and can last for a long time. For example, the modified T cells of the present invention can undergo strong in vivo T cell expansion and last for a long time at high levels in the blood and bone marrow and form specific memory T cells.

[0363] In some embodiments, the present invention includes a cell therapy, wherein the cell is genetically engineered to express engineered TCR or chimeric antigen receptor (CAR) and the engineered TCR cell or CAR cell (eg, CAR immune cell, CAR T cell, CAR B cell, etc.) is infused into a recipient in need. The infused cells can kill or dissolve allogeneic responsive cells in the recipient. Unlike antibody therapy, engineered TCR cells and CAR-modified cells can replicate in vivo, thereby producing long-term persistence, which can lead to sustained allogeneic response control. In various embodiments, after the cell is applied to the patient, the cell or its offspring applied to the patient continues in the patient for at least four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, thirteen months, fourteen months, fifteen months, sixteen months, seventeen months, eighteen months, nineteen months, twenty months, twenty-one months, twenty-two months, twenty-three months, two years, three years, four years or five years.

[0364] In one embodiment, the allogeneic response suppression caused by engineered TCR cells or CAR-modified cells can be an active or passive immune response, or alternatively can be due to a direct or indirect immune response. In one embodiment, engineered TCR cells or CAR-transduced cells respond to human allogeneic responsive cells expressing antigens, TCRs and / or BCRs, exhibiting specific proinflammatory cytokine secretion and potent cytolytic activity.

[0365] With respect to ex vivo immunization, prior to administration of the cells into a mammal, at least one of the following occurs in vitro: i) cell expansion, ii) introduction of a nucleic acid encoding a CAR into the cells, or iii) cell cryopreservation.

[0366] Ex vivo procedures are well known in the art and are discussed more fully below. In short, cells are isolated from mammals (e.g., humans) and genetically modified (i.e., in vitro transduction or transfection) with a vector expressing a CAR disclosed herein. CAR-modified cells can be administered to a mammalian recipient to provide therapeutic benefits. The mammalian recipient can be a human being, and the CAR-modified cells can be autologous relative to the recipient. Alternatively, the cell can be allogeneic, homologous or xenogeneic relative to the recipient.

[0367] In vitro expansion procedures for hematopoietic stem and progenitor cells are described in U.S. Pat. No. 5,199,942, which is incorporated herein by reference and can be applied to the cells of the present invention. Other suitable methods are known in the art, and therefore the present invention is not limited to any particular method for in vitro expansion of cells. In brief, the in vitro culture and expansion of cells comprises: (1) collecting CD34+ hematopoietic stem and progenitor cells from a peripheral blood harvest or bone marrow explant of a mammal; and (2) expanding such cells in vitro. In addition to the cell growth factors described in U.S. Pat. No. 5,199,942, other factors such as flt3 L, IL-1, IL-3, and c-kit ligand can also be used for the culture and expansion of cells.

[0368] In some embodiments, the CAR-modified cells and engineered TCR cells of the present invention are used to treat diseases, disorders and disorders associated with alloreactive cell expression. In various embodiments, the cells of the present invention are used to treat patients at risk of diseases, disorders and disorders associated with alloreactive cell expression. Therefore, the present invention provides a method for treating or preventing diseases, disorders and disorders associated with alloreactive cell expression, including administering a therapeutically effective amount of genetically engineered cells of the present invention to a subject in need.

[0369] The present invention also provides a method for inhibiting the proliferation of an allogeneic responsive cell population or reducing the population, the method comprising contacting the allogeneic responsive cell population with at least one genetically engineered cell of the present invention that is combined with the allogeneic responsive cell. In one embodiment, the present invention provides a method for inhibiting the proliferation of an allogeneic responsive cell population carrying an antigen, TCR and / or BCR or reducing the population, the method comprising contacting the allogeneic responsive cell population with at least one genetically engineered cell of the present invention that is combined with the antigen, TCR domain and / or BCR domain of the allogeneic responsive cell. In various embodiments, relative to a negative control, the cells of the present invention reduce the number, number, amount or percentage of cells and / or allogeneic responsive cells in a subject suffering from a disease or condition associated with allogeneic responsive cell expression or in an animal model of the disease or condition by at least 1%, at least 10%, at least 25%, at least 30%, at least 40%, at least 50%, at least 65%, at least 75%, at least 85%, at least 95%, or at least 99%. In one embodiment, the subject is human.

[0370] The present invention also provides a method for preventing, treating and / or managing a disease associated with alloresponsive cell expression, the method comprising administering to a subject in need thereof at least one genetically engineered cell of the present invention combined with an alloresponsive cell. The present invention also provides a method for preventing, treating and / or managing a disease associated with expression of a cell carrying an antigen, TCR, and / or BCR, the method comprising administering to a subject in need thereof at least one genetically engineered cell of the present invention combined with an antigen-, TCR-, and / or BCR-expressing cell. In one embodiment, the subject is human. Non-limiting examples of conditions associated with alloresponsive cell expression include autoimmune diseases or conditions (e.g., lupus), inflammatory diseases or conditions (e.g., allergies and asthma), diseases or conditions caused by transplantation, allograft rejection, immune rejection, chronic allograft rejection, implant rejection, transplant rejection, or any combination thereof, inflammation, inflammation caused by ischemia / reperfusion, infection, immune response to allografts, or any combination thereof.

[0371] The present invention provides a method for preventing recurrence of a disease or condition associated with the expression of allogeneic responsive cells, the method comprising administering to a subject in need thereof at least one genetically engineered cell of the present invention in combination with allogeneic responsive cells. In one embodiment, the method comprises administering to a subject in need thereof a plurality of effective amounts of at least one genetically engineered cell of the present invention in combination with allogeneic responsive cells. In one embodiment, the method comprises administering to a subject in need thereof an effective amount of at least one genetically engineered cell of the present invention in combination with allogeneic responsive cells and an effective amount of another therapy.

[0372] The compositions and genetically engineered cells of the invention can be administered alone or in combination with a diluent and / or other components (such as IL-2) or other cytokines or cell populations as a pharmaceutical composition.

[0373] In further embodiments, the compositions of the invention are used in patients together with (e.g., before, simultaneously with, or after) a transplant (e.g., a bone marrow transplant, an organ transplant, etc.). In certain embodiments, after the transplant, the subject receives an infusion of the expanded immune cells of the invention. In another embodiment, the expanded cells are administered before or after surgery.

[0374] Subjects contemplated for administration of the compositions and pharmaceutical compositions of the invention include, but are not limited to, humans and other primates, mammals (including commercially relevant mammals such as non-human primates, cattle, pigs, horses, sheep, cats, and dogs).

[0375] The dosage of the above treatment administered to the patient will vary depending on the exact nature of the condition being treated and the recipient of the treatment. The dosage for human administration can be adjusted according to practices generally recognized in the art. T cell administration dosage and scheduling strategies have been discussed (Ertl et al, 2011, Cancer Res, 71: 3175-81; Junghans, 2010, Journal of Translational Medicine, 8: 55).

[0376] The pharmaceutical composition of the present invention may comprise a composition as described herein, and one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise a buffer such as neutral buffered saline, phosphate buffered saline, etc.; a carbohydrate such as glucose, mannose, sucrose or dextran, mannitol; a protein; a polypeptide or amino acid such as glycine; an antioxidant; a chelating agent such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. In one embodiment, the composition of the present invention is formulated for intravenous administration.

[0377] The pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition and the type and severity of the patient's disease, although appropriate dosages can be determined through clinical trials.

[0378] When an "immunologically effective amount" or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by the physician taking into account individual differences in age, weight, degree of infection or metastasis, and condition of the patient (subject). In general, the dosage of the pharmaceutical composition comprising the cells described herein can be 10 4 Up to 10 9 cells / kg body weight, in some cases 10 5 Up to 10 6 Cells / kg body weight, including all integer values ​​within these ranges. The cell composition can also be administered multiple times at these doses. The cells can be administered using infusion techniques known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). A technician in the medical field can easily determine the optimal dose and treatment regimen for a particular patient by monitoring the patient's disease signs and adjusting the treatment accordingly.

[0379] In various embodiments, it may be necessary to administer the activated cells to the subject, then subsequently redraw blood (or perform apheresis), activate the cells therefrom according to the present invention, and reinfuse these activated and amplified cells into the patient. This process can be performed multiple times every few weeks. In various embodiments, cells can be activated from a blood draw of 10cc to 400cc. In various embodiments, cells can be activated from a blood draw of 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, or 100cc. Without being bound by theory, certain cell populations can be selected using this multiple blood draw / multiple re-infusion scheme.

[0380] The subject composition can be applied in any convenient manner, including by aerosol inhalation, injection, ingestion, blood transfusion, implantation or transplantation. Compositions as described herein can be administered to patients by arterial, subcutaneous, intradermal, intranodal, intramedullary, intramuscular, intravenous (iv) injection or intraperitoneal administration. In one embodiment, the T cell composition of the present invention is administered to patients by intradermal or subcutaneous injection. In one embodiment, the cell composition of the present invention is administered to patients by iv injection. The cell composition can be injected directly into a transplantation site, lymph node, or infection site.

[0381] In various embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art (wherein the cells are expanded to therapeutic levels) are administered to a patient in combination with any number of related treatment modalities (e.g., before, simultaneously, or after), including, but not limited to, treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab treatment for MS patients, or efalizumab treatment for psoriasis patients, or other treatments for PML patients. In some embodiments, the cells of the invention can be combined in a treatment regimen with chemotherapy, radiation therapy, immunosuppressants (such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506), antibodies or other immunoablative agents (such as CAMPATH, anti-CD3 antibodies or other antibody therapies), cytotoxins, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and radiation. Drugs that inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit p70S6 kinase (rapamycin), which is important for growth factor-induced signaling, can also be used (Liu et al., Cell 66: 807-815, 1991; Henderson et al., Immun. 73: 316-321, 1991; Bierer et al., Curr. Opin. Immun. 5: 763-773, 1993). In one embodiment, the cell composition of the invention is administered to the patient in combination with (e.g., before, at the same time, or after) a cell ablative therapy using a bone marrow transplant, a chemotherapeutic agent (such as fludarabine), external beam radiation therapy (XRT), cyclophosphamide, or an antibody (such as OKT3 or CAMPATH). In one embodiment, the cell composition of the invention is administered after B-cell ablative therapy (such as an agent reactive with CD20, e.g., Rituxan).

[0382] In one embodiment, the subject can receive blood cell removal, wherein ex vivo collection, enrichment or depletion of leukocytes to select and / or separate target cells, such as T cells. These cell isolates can be amplified and processed by methods known in the art so that one or more peptides of the present invention (e.g., SCT) can be introduced to produce HLA SCT cells of the present invention (e.g., HLA SCT immune cells, HLA SCT T cells, HLA SCT B cells, etc.). Subjects in need can then receive standard treatment with high-dose chemotherapy, followed by peripheral blood stem cell transplantation. In various embodiments, after or at the same time as the transplant, the subject receives an infusion of the amplified HLA SCT cells of the present invention (e.g., HLA SCT immune cells, HLA SCT T cells, HLA SCT B cells, etc.). In one embodiment, the amplified cells are administered before or after surgery.

[0383] Experimental Examples

[0384] The present invention will now be described with reference to the following examples. These examples are for illustrative purposes only, and the present invention should in no way be construed as being limited to only these examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0385] Without further description, it is believed that one of ordinary skill in the art can use the foregoing description and the following illustrative examples to make and utilize the compounds of the present invention and practice the claimed methods. Therefore, the following working examples should not be construed as limiting the remainder of the disclosure in any way.

[0386] Example 1: Decoy HLA-E SCT for allogeneic donor cells

[0387] To avoid host allogeneic rejection of donor CAR T cells, multiple gene edits were performed in this study to render donor T cells ineffective for beta-2 microglobulin (B2M), CIITA, and native TCRα chain, thereby eliminating cell surface MHC class I molecules, MHC class II molecules, and native TCR ab heterodimers. Elimination of cell surface MHC I and MHC II reduces the risk of allogeneic recognition by the recipient (host) immune system and avoids (or delays) rejection of transferred donor T cells. Elimination of cell surface native TCR ab heterodimers reduces the risk of graft-versus-host disease (GvHD), which is mediated by donor T cell recognition of host / recipient MHC molecules and is associated with moderate-to-severe morbidity and mortality. Gene editing of the B2M, CIITA, and TRAC loci generated triple knockout T cells (TKO), which can persist and avoid causing GvHD ( Figure 1). However, it is known that MHC I-deficient cells (of any lineage) are sensitized to NK cells from unrelated individuals and are rapidly eliminated after transfer into unrelated recipients ( Figure 2 ). Due to the "loss of self" hypothesis of NK cells, KO of MHC-I induces NK cell killing (Karre et al., Immunol Today Vol. 11(7), 1990), and NK cells recognize stress ligands that are upregulated on the surface of T cells (Lanier / Spies et al., Science Vol. 285(5428), 1999).

[0388] The studies described here have inserted HLA-E / β-2-microglobulin / peptide single-chain trimer (SCT) into TKO T cells to create "universal" T cells ( Figure 3 ). The data show that TKO T cells expressing HLA-E SCT (TKO / E-SCT) can avoid being recognized by the immune system (especially NK cells and T cells) and show improved persistence after transfer into patients. More specifically, the expression and interaction of HLA-E with NKG2A prevents the clearance of UCART cells because the interaction between NKG2A and NKG2C depends on peptides presented by HLA-E (Rolle et al., Cell Rep Vol. 24, 2018; Llano et al., Eur. J. Immunol. Vol. 28, 1998).

[0389] Furthermore, this study has utilized the SCT format as a facile platform to generate novel molecular entities that address a critical gap in the ability to engraft normal donor cells or tissues in unrelated allogeneic hosts. Figure 4 The identity of HLA-E*01:03 as well as the amino acid sequence and binding affinity (nM) are shown. This study designed and tested HLA-E single-chain trimer (ScT) as an inhibitory receptor for UCART cells and also identified key NK cell activation receptors ( Figure 5 ). Using LV gene transfer, a panel of K562 transfectants was established to develop a screening platform that allows functional assays using NK cells as effectors to determine the extent of protection against cytolysis ( Figures 6 to 9 ). Expression of the HLA-E / β-2-microglobulin single chain dimer (without peptide) resulted in low expression levels in cells lacking MHC class I, indicating that addition of the peptide sequence is required to achieve normal cell surface levels of expression. Next, seven HLA-E SCT constructs expressed in K562 were examined and used as standard 4h 51Targets in Chromium Release Assays. NK cells from multiple normal donors were used as effector cells to investigate the ability of HLA-E SCTs as baits to inhibit NK cell-mediated killing. It is well known that both NKG2A and NKG2C bind to HLA-E present on target cells. It is the combined signaling integration through the two NKG2 receptors that determines NK cell effector function.

[0390] like Fig.10 and Fig.11 As shown, NK cells from donor 517 were most significantly inhibited by the HLA-C*03:01 leader peptide construct. Inhibition of NK cell lysis was also observed with a variety of other SCT constructs tested; however, the C*0301 leader peptide provided the most reproducible inhibition ( Figure 8 and Figures 12 to 15 The experiment was repeated using a larger group of NK cell donors (n=5), and again, significant inhibition of K562 lysis mediated by HLA-E SCT with the C*03:01 leader peptide (aka K878) was seen ( Fig.15 ).

[0391] To better understand the differences in NK cell suppression mediated by K878 (HLA-E SCT), NKG2A and NKG2C expression were determined by flow cytometry ( Figures 16 to 20 NK517 NK cells only expressed NKG2A, and NK485 NK cells expressed significant NKG2C as either a single population or a NKG2A+ / NKG2C+ dual population. NK485 NK cells were also less susceptible to inhibition by HLA-E SCT. In contrast, NK517 NK cells were almost completely inhibited by HLA-E SCT ( Fig.15 and Fig.18 ). Further studies have shown that the NKG2A+ / NKG2C+ double positive subset is suppressed by HLA-E SCT, while NKG2C+ single positive NK cells are least suppressed by HLA-E SCT. This observation has been repeated in multiple settings using multiple donors ( Figure 21 to Figure 24 ).

[0392] Subsequent experiments used LV gene transfer to express HLA-E SCT encoding the C*03:01 leader peptide in TKO T cells ( Fig.25). Using standard molecular techniques for CRISPR-Cas9, normal donor purified T cells were first activated with anti-CD3 / CD28 beads, and 24 hours later, transduced with LV encoding HLA-E*01:03 (which encodes the C*03:01 leader peptide) and then returned to culture. Four days later, the T cells were de-beaded and then electroporated using a Maxcyte GTx instrument in the presence of Cas9 ribonucleoprotein (RNP) and sgRNAs for B2M, CIITA, and TRAC. Afterwards, the modified T cells were returned to culture for another 7 days and then purified using a cell sorter. The purified cells were >99% TKO as assessed by standard flow cytometry.

[0393] In a representative experiment, TKO cells (lacking MHC-I and MHC-II and CD3 / TCR) were readily recognized by NK cells as measured in a standard Cr51 lysis assay. In contrast, control (mock, WT) T cells expressing MHC-I were resistant to lysis as expected ( Fig.14 Next, the TKO cells were tested in a mixed lymphocyte reaction (MLR test) to determine whether the edited TKO cells stimulated normal donor T cells from an unrelated donor ( Fig.26 ). This is a commonly used alloreactivity test and is used to assess differences in tissue compatibility between two individuals. Fig.26 As shown, two normal donors were examined to assess possible alloreactivity. TKO cells and control autologous PBMCs from ND561 failed to stimulate responder T cells from ND 543, while unedited PBMC561 were highly stimulatory, as shown by incorporation of tritiated thymidine in a standard proliferation assay. Similarly, TKO cells and control autologous PBMCs from ND543 failed to stimulate responder T cells from ND561, while control allogeneic (ND543) PBMCs were highly stimulatory. This experiment provides conclusive evidence that TKO cells obtained by standard CRISPR-Cas9 gene editing are non-stimulatory in MLR assays.

[0394] In addition, follow-up studies further evaluated the ability of TKO cells expressing HLA-E SCT to avoid recognition by NK cells. This is a critical test to determine the efficacy of how HLA-E SCT protects TKO cells from NK cells. TKO cells lacking MHC-I were highly susceptible to NK cell lysis, while control mock / WT (unedited) T cells were less susceptible, which is consistent with previous experiments ( Fig.14). TKO cells expressing HLA-ESCT are resistant to lysis by NK cells. Therefore, TKO cells expressing HLA-ESCT are resistant to attack by allogeneic T cells and allogeneic NK cells in conventional immunological assays.

[0395] In summary, a long-standing problem in biomedical science is the inability to transplant cells, tissues, or organs from one person to another without immunosuppression. Allogeneic and xenotransplants are rapidly rejected by the host immune system, and the mechanisms involving the innate and adaptive immune systems have been well described. This study shows that HLA-E SCT presenting an HLA-C leader peptide resulted in significant NK cell suppression ( Figure 27 to Figure 29 ). In addition, due to the loss of self-response, TKO CAR T cells are targeted for NK cell-mediated killing, while HLA-E SCT expression on UCART cells inhibits NK cell-mediated killing. In addition, NK cell populations with higher NKG2C expression are less inhibited by HLA-E SCT than donors with higher NKG2A+ groups. Other studies have focused on evaluating various stress ligands (i.e., MICA / ULBP) on TKO cells using standard in vitro assays. In addition, studies have also focused on evaluating TKO / HLA-E SCT / CAR cells in humanized animal models with Nalm-6 leukemia and in vivo tests using hIL-15NSG mice to determine the in vivo persistence of UCART cells. Therefore, current studies have shown that HLA-E SCT protects target cells from NK-mediated killing, and therefore, the expression of this new type of SCT protects normal donor cells or tissues (genetically edited to eliminate MHC molecules) from being recognized by the immune system of unrelated allogeneic hosts. HLA-E SCT enables the engineering of any cell, tissue, or organ to allow transplantation into an unrelated host, such as another human.

[0396] The materials and methods used in these experiments are now described.

[0397] Standard 4h 51 Chromium release assay

[0398] NK cells from normal donor 517 were expanded in IL-2 / IL-15 for 10 days and then cultured in a standard 4 h 51 Cr release assays were used as effector cells at the indicated effector to target ratios (E:T ratios). Target cells studied (10,000 per well) included parental K562 and K562 transfected with various HLA-E single chain trimer constructs. 51Target cells were labeled with chromium for 1 hour, washed 3x, and then placed in a 96-well tray containing NK cells. All assays were performed in triplicate. After incubation for 4 h at 37°C, 5% CO2, the plates were removed from the incubator and centrifuged for 10 minutes. 50ul of supernatant was carefully removed and counted in a MicroBeta2LumiJET microplate counter (Perkin Elmer). Data are expressed as percentage specific lysis and reported as mean + / - SD. The percentage of lysis inhibition for each SCT was calculated based on the CPM of the maximum release (SDS) value obtained, and it was demonstrated that the leader peptides expressed by various SCTs all affect the inhibitory function of these molecules in NK activity ( Fig.11 ).

[0399] Real-time apoptotic cell death analysis

[0400] Real-time apoptotic cell death analysis (live cell imaging with cell impedance) was performed using the xCELLigence real-time cell analysis eSIGHT system (Agilent). mCherry-labeled K562 target cells (10,000 / well) were inoculated and allowed to adhere for 24 hours. Effector NK cells (normal donor 517) were added at an E:T ratio of 5:1 and the assay was continued for an additional 3 days. The total integrated intensity of red was measured every 15 minutes (real-time imaging) for 3 days. The decrease in intensity reflects the elimination of the target. Killing of K562 parental cells was evident (red line), while K562 cells expressing HLA-C*03:01 leader peptide / HLA-E SCT (also known as K878) were protected and survived in the presence of NK517 ( Fig.12 ). Parental K562 cells alone (no NK) also showed growth (control), while K562 cells died in the presence of SDS (control). Values ​​reflect the mean + / - SD of three replicate wells.

[0401] In a 3-day live cell imaging (xCELLigence) assay, HLA-E SCTs partially protected cells from killing by ND561 NK cells (21% NKG2C+). This data contrasts with that observed for ND517 (predominantly all NKs were NKG2A), where complete lytic protection by SCTs was observed ( Fig.12 The balance of NK populations in the donor (NKG2A vs. NKG2C) will affect the functional outcome of NK cells after joining with SCT ( Fig.24 ).

[0402] TKO-edited T cells

[0403] Normal donor T cells were activated with anti-CD3 / CD28 beads (Dynal) under standard conditions. Five days later, cells were gene-edited by electroporation (Maxcyte GTx) using sgRNAs for TRAC, B2M, and CIITA in the presence of Cas-9 RNP as described. In some experiments, triple knockout (TKO) edited T cells were transduced with lentivirus encoding HLA-E SCT (C*03:01 leader peptide) and maintained in medium containing IL-7 / IL-15. Unedited ND T cells were used as controls. Phenotypic analysis was performed on day 10 and analyzed by flow cytometry ( Fig.13 ).

[0404] Expression of HLA-E on TKO T cells

[0405] TKO T cells were sensitive to NK-mediated lysis and protected by HLA-E SCT expression. NK517 effector T cells were used for 4 h 51 Cr release assay to examine protection mediated by HLA-E SCT overexpression in normal donor TKO T cells. As a control, unedited (mock) allogeneic T cells were resistant to NK cell lysis, as were autologous mock (ND517) T cells. TKO CAR-T cells with HLA-E SCT were tested and shown to be resistant to NK cell-mediated killing. Control TKO CAR-T cells (red line) were sensitive to NK cell lysis ( Fig.14 ).

[0406] Using K562 HLA-E SCT target in 4h 51 NK cells from five normal donors were studied in a Cr release assay. Consistent protection was seen in all five donors ( Fig.15 ). As a control, NK cells from all donors killed the parental K562 cells.

[0407] NKG2A / NKG2C expression

[0408] Some donors express NKG2C, a known activating receptor that binds to HLA-E on target cells. CMV seronegative individuals are reported to have low levels of NKG2C+ NK cells (1-2%), while a subset (approximately 1 / 3) have significant amounts of NKG2C+ NK cells ( Fig.18 ).

[0409] Activation of NK cells by K562 cells + / - HLA-E SCT

[0410] Activation of NK cells by K562 cells + / - HLA-E SCT was examined using a flow cytometry-based assay. NK cells were mixed with K562 cells (1:1 ratio) for 6 hours and then stained with an antibody to CD107a to measure degranulation of the major subsets (NKG2A+ and NKG2C+). The bar graph summarizes representative results, demonstrating that HLA-E SCT provides protection for the NKG2A+ subset ( Fig.19 ). In contrast, HLA-E SCT did not confer protection against K562 in the NKG2C+ subset.

[0411] Stress ligand evaluation

[0412] Soluble NKG2D protein was used to block stress ligands (MICA, MICB, ULBP1-6) on K562 target cells, which interfered with NKG2D-mediated target cell recognition. The data showed that NKG2D protein had minimal efficacy in recognizing parental K562 and K878 (HLA-E SCT) target cells by NK cells from two donors ( Fig. 20 ).

[0413] Example 2: Determination of histocompatibility barriers between universal CAR T (UCART) cells and NK cells

[0414] CAR T cell adoptive therapy has been shown to be effective in treating hematological malignancies. However, the current CAR-T cell manufacturing process also faces challenges, one of which is manufacturing failure due to dysfunction of patient-derived T cells (Thommen et al., 2018, Cancer Cell, 33: 547-562). One proposed solution to overcome this limitation is to develop universal CAR-T (UCART) cells engineered from healthy, normal donor-derived T cells. Tissue compatibility barriers must be addressed when creating UCART cells. Ablation of α / βTCR surface expression can prevent graft-versus-host disease, while ablation of the surface expression of major histocompatibility complex (MHC) class I and class II molecules can reduce host-versus-graft responses (Abdelhakim et al., 2017, Biomedicines; Wang et al., 2015, Stem Cells Transl Med., 4: 1234-1245). These triple knockout (TKO) T cells serve as universal recipients for UCART cell development.

[0415] Importantly, one result of MHC class I ablation is NK cell activation due to "loss of self" response (Ljunggren et al., 1990, Immunol Today, 11: 237-244). HLA-E is a non-classical MHC class I molecule, known to interact with NK cell inhibitory receptor NKG2A, and its overexpression on UCART cells is confirmed in this article to alleviate NK cell activation. Since HLA-E / NKG2A interactions depend on the peptide presented by HLA-E, 10 HLA-E single-chain trimer (SCT) constructs expressing various peptide sequences are created to determine which HLA-E / peptide complexes can cause significant NK cell inhibition (Borrego et al., 1998, J Exp Med 1998, 187: 813-818). Ten HLA-E single-chain trimer (SCT) constructs for cell surface expression on K562 cells are created, and each construct encodes and presents different signal peptides. HLA-E+ K562 cells were used in preliminary experiments before moving lead candidate HLA-E constructs into TKO T cells. Functional assays were performed using primary human NK cells.

[0416] In preliminary experiments, K562 cells transduced to express these HLA-E / peptide complexes were used as a model for UCART cells. HLA-E SCT presenting the HLA-C leader peptide (HLA-E / C) resulted in significant inhibition of NK cells as determined by flow cytometry-based NK cell degranulation (CD107a). 51 The reduced lysis of K562 HLA-E / C-expressing target cells in the Cr release assay further validated the inhibitory effect of HLA-E / C complex on NK cell activation ( Fig.12 and Figure 30 to Figure 35 ). More specifically, Fig.30 Draw for 4 hours 51 Representative results of a Cr release assay demonstrating that HLA-E presenting the HLA-C*03:01 leader peptide resulted in the most significant reduction in K562 lysis, indicating its ability to inhibit NK cell activation due to a "loss of self" response. Shown at a 2:1 E:T ratio.

[0417] Fig.31 Representative results are further depicted demonstrating that HLA-E+ K562 cells inhibit NK cells from different donors and that the effect depends on phenotypic differences in the donor NK cell populations and Fig.32 Representative results are depicted, demonstrating that Fig.31 Surface expression of NKG2A and NKG2C of the donors shown in . Fig.33Representative results are depicted showing NKG2A and NKG2C surface expression of expanded NK cells from 16 different normal donors.

[0418] Fig.34 Include Fig.34 A and Fig.34 B, depicts representative results demonstrating that sorted NKG2A+ NK cells were inhibited by HLA-E+ K562 cells, and sorted NKG2C+ NK cells were not inhibited by HLA-E expression on K562. (**P<0.0025, ***P<0.000125; data are shown in 2:1 E:T ratio). Fig.34 A depicts representative results demonstrating that sorted NKG2A+ NK cells were inhibited by HLA-E+ K562 cells. Fig.34 B depicts representative results demonstrating that sorted NKG2C+ NK cells are not inhibited by HLA-E expression on K562. Fig.35 Representative results are depicted demonstrating decreased degranulation of NKG2A+NK cells when co-cultured with HLA-E+K562 cells. Increased degranulation of NKG2C+NK cells when co-cultured with HLA-E+K562 indicates NK cell activation.

[0419] Expression of HLA-E SCT on K562 cells consistently reduced NK cell-mediated lysis. The effect of HLA-E on NK cell activity depended on phenotypic differences in the donor NK cell population, with the NKG2A+ population showing the greatest inhibition. HLA-E+ K562 cells survived up to 48 hours after co-culture with NK cells. Finally, expression of the HLA-E / C complex on TKO T cells resulted in protection against NK lytic activity ( Figure 36 to Figure 38 ). More specifically, Fig.36 Representative results are depicted demonstrating the editing strategy and efficiency for generating TKO HLA-E+ T cells, and Fig.37 Depicted are representative results demonstrating that TKO T cells were not recognized by allogeneic donor PBMCs, as no alloreactive responses were measured in mixed lymphocyte reactions (MLRs). Fig.38 Representative results are depicted demonstrating that TKO CAR T cells are targeted and killed by NK cells due to a "loss of self" response. HLA-E expression on TKO CAR T cells inhibits NK cell-mediated lysis.

[0420] In summary, these data demonstrate the effectiveness of selected HLA-E / peptide complexes in inhibiting NK cell activation caused by "loss of self" responses. Modifications such as those described herein can prevent UCART cell clearance due to host recognition (NK cell activation), and can ultimately lead to more potent, safer and more durable UCART cell therapies.

[0421] Example 3: HLA-ESCT expression confers in vivo protection against NK activity

[0422] The in vivo protective effect of HLA-E SCT expression on lymphoma cell lines against human NK activity was evaluated in NOG hIL-15 mice. This immunodeficient mouse model expresses human IL-15, a cytokine that supports human NK cell transplantation. NK activity was evaluated for K562 (a lymphoma cell line lacking MHC class I) and an engineered K562 cell line expressing HLA-E SCT (designated as K878). Mice were injected IV with activated and amplified human NK cells, then K562 cells were injected in the left hind leg flank SQ, and K878 cells were injected in the right hind leg flank SQ to form a bilateral flank model.

[0423] Weekly bioluminescence imaging (BLI) revealed that HLA-E SCT expression suppressed NK cells, leading to growth of K878 relative to K562 at day 14, as determined by increased bioluminescent signal ( Fig.39 ). These results demonstrate that HLA-E SCT expression confers an in vivo protective advantage against human NK cell activity and further validate its expression in TKO T cells for use in manufacturing allogeneic T cell therapy.

[0424] In vivo function of HLA-E ScT NOG hIL-15 mice (Taconic Biosciences) were injected intraperitoneally (IP) with 20 mg / kg busulfan ( Fig.39 One week after NK injection, K562 and K562 HLA-E SCT+(K878) cells were suspended in a 1:1 mixture of PBS and Matrigel matrix (Corning) at 1e6 cells / μl ( Fig.39 ). Both tumor lines expressed the click beetle luciferase marker for bioluminescent imaging (BLI) tracking. One million K562 tumor cells were injected subcutaneously (SQ) in the left hind flank of each mouse, and 1e6 K878 tumor cells were injected SQ in the right hind flank of each mouse. BLI was measured 5 hours after tumor injection (day 0) and then once a week for 3 weeks ( Fig.39 ).

[0425] Example 4: Sequence

[0426] HLA-A*0201 signal peptide (SEQ ID NO: 1)

[0427] VMAPRTLVL

[0428] HLA-B*0801 signal peptide (SEQ ID NO: 2)

[0429] VMAPRTVLL

[0430] HLA-C signal peptide (SEQ ID NO: 3)

[0431] VMAPRTLIL

[0432] HLA-G signal peptide (SEQ ID NO:4)

[0433] VMAPRTLFL

[0434] Hsp60 signal peptide (SEQ ID NO: 5)

[0435] QMRPVSRVL

[0436] CMV Towne peptide (SEQ ID NO:6)

[0437] VMAPRTLLL

[0438] CMV AF1 peptide (SEQ ID NO:7)

[0439] VMAPRSLLL

[0440] CMV109b peptide (SEQ ID NO:8)

[0441] VMAPRILIL

[0442] RL9-HIV peptide (SEQ ID NO:9)

[0443] RMYSPTSIL

[0444] Mtb44 peptide (SEQ ID NO: 10)

[0445] RLPAKAPLL

[0446] K878_MA-2_005 (SEQ ID NO: 11)

[0447] TAATACGACTCACTATAGCAGCTCCCGGAGGTGCAAAA

[0448] K878_MA-1_004 (SEQ ID NO: 12)

[0449] TTCTAGCTCTAAAACTTTTGCACCTCCGGGAGCTG

[0450] Share (SEQ ID NO: 13)

[0451] VMAPRTLL

[0452] Nucleotide sequence encoding HLA-C signal peptide (SEQ ID NO: 14): GTGATGGCCCCAAGAACCCTGATCCTG

[0453] Nucleotide sequence encoding HLA-A*0201 signal peptide (SEQ ID NO: 15): GTGATGGCCCCCCGGACCCTGGTGCTG

[0454] Nucleotide sequence encoding HLA-B*0801 signal peptide (SEQ ID NO: 16): GTCATGGCGCCCCGAACCGTCCTCCTG

[0455] Nucleotide sequence encoding HLA-G signal peptide (SEQ ID NO: 17): GTGATGGCCCCAAGAACCCTGTTCCTG

[0456] Nucleotide sequence encoding Hsp60 peptide (SEQ ID NO: 18)

[0457] CAGATGAGACCGGTGTCCAGGGTACTG

[0458] Nucleotide sequence encoding CMV Towne peptide (SEQ ID NO: 19): GTGATGGCCCCCCGGACCCTGCTGCTG

[0459] Nucleotide sequence encoding CMV AF1 peptide (SEQ ID NO: 20) GTGATGGCCCCCCGGAGCCTGCTGCTG

[0460] Nucleotide sequence encoding CMV109b peptide (SEQ ID NO: 21)GTGATGGCCCCCCGGATCCTGATCCTG

[0461] Nucleotide sequence encoding RL9-HIV peptide (SEQ ID NO: 22)CGGATGTACAGCCCCACCAGCATCCTG

[0462] Nucleotide sequence encoding Mtb44 peptide (SEQ ID NO: 23)

[0463] CGGCTGCCCGCCAAGGCCCCCCTGCTG

[0464] Share (SEQ ID NO: 24)

[0465] GTGATGGCCCCCCGGACCCTGMT modB2M signal peptide (SEQ ID NO: 25)

[0466] MSRSVALAVLALLSLSGLEA

[0467] Nucleotide sequence encoding modB2M signal peptide (SEQ ID NO: 26)

[0468] ATGTCACGCTCTGTCGCTCTTGCAGTACTTGCCCTGTTGAGCCT CAGCGGACTCGAAGCC

[0469] The complementary nucleotide sequence of the nucleotide sequence encoding the modB2M signal peptide (SEQ ID NO: 27)

[0470] TACAGTGCGAGACAGCGAGAACGTCATGAACGGGACAACTCGG AGTCGCCTGAGCTTCGG

[0471] Linker 1 - amino acid sequence (SEQ ID NO: 28)

[0472] GGGASGGGGSGGGGS

[0473] Linker 1 - nucleotide sequence (SEQ ID NO: 29)

[0474] GGAGGAGGTGCGAGCGGTGGTGGAGGTAGCGGAGGTGGAGGAAGC

[0475] Linker 1 - complementary nucleotide sequence (SEQ ID NO: 30)

[0476] CCTCTCCACGCTCGCCACCACCTCCATCGCCTCCACCTCCTTCG

[0477] modB2M peptide without leader sequence (SEQ ID NO:31)

[0478] IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM

[0479] Nucleotide sequence encoding modB2M peptide without leader sequence (SEQ ID NO:32)

[0480] ATTCAAAGGACACCGAAAATCCAAGTATATAGCAGGCACCCCGCTGAAAACGGCAAAAGCAACTTTCTCAACTGTTACGTCTCCGGCTTCCACCCCAGTGATATCGAGGTCGATCTGCTCAAGAACGGCGAACGCATCGAGAAGGTTG AACACAGTGATCTGTCATTTAGTAAAGATTGGTCATTTTACCTTCTCTATTATACAGAGTTTACACCAACGGAGAAGGACGAATACGCATGTCGCGTTAATCACGTCACGCTTTCTCAACCTAAAATTGTGAAATGGGACCGTGATATG

[0481] The complementary nucleotide sequence of the nucleotide sequence encoding the modB2M peptide without the leader sequence (SEQ ID NO: 33)

[0482] TAAGTTTCCTGTGGCTTTTAGGTTCATATATCGTCCGTGGGGCGACTTTTGCCGTTTTCGTTGAAAGAGTTGACAATGCAGAGGCCGAAGGTGGGGTCACTATAGCTCCAGCTAGACGAGTTCTTGCCGCTTGCGTAGCTCTTCCAAC TTGTGTCACTAGACAGTAAATCATTTCTAACCAGTAAAATGGAAGAGATAATATGTCTCAAATGTGGTTGCCTCTTCCTGCTTATGCGTACAGCGCAATTAGTGCAGTGCGAAAGAGTTGGATTTTAACACTTTACCCTGGCACTATAC

[0483] Spacer-amino acid sequence (SEQ ID NO: 34)

[0484] GGGGSGGGGSGGGGSGGGGS

[0485] Spacer-nucleotide sequence (SEQ ID NO:35)

[0486] GGAGGCGGTGGGTCAGGTGGAGGTGGGTCTGGCGGAGGTGGA TCCGGTGGTGGAGGTAGT

[0487] Spacer-complementary nucleotide sequence (SEQ ID NO: 36)

[0488] CCTCCGCCACCCAGTCCACCTCCACCCAGACCGCCTCCACCTAG GCCACCACCTCCATCA

[0489] Heavy chain of HLA-E*0103 (SEQ ID NO:37)

[0490] GSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDGRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRWKPASQPTIPIVGIIAGLVLLGSVVSGAVVAAVIWRKKSSGGKGGSYSKAEWSDSAQGSESHSL

[0491] Nucleotide sequence encoding the heavy chain of HLA-E*0103 (SEQ ID NO:38)

[0492]

[0493] The complementary nucleotide sequence of the sequence encoding the heavy chain of HLA-E*0103 (SEQ ID NO: 39)

[0494]

[0495] Representative HLA-C signal peptide - HLA-E SCT (SEQ ID NO:40)

[0496] MSRSVALAVLALLSLSGLEAVMAPRTLILGGGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDGRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYLEDTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRWKPASQPTIPIVGIIAGLVLLGSVVSGAVVAAVIWRKKSSGGKGGSYSKAEWSDSAQGSESHSL

[0497] Nucleotide sequence encoding representative HLA-C signal peptide - HLA-E SCT (SEQ ID NO:41)

[0498]

[0499] The complementary nucleotide sequence of the nucleotide sequence encoding the representative HLA-C signal peptide-HLA-E SCT (SEQ ID NO: 42)

[0500]

[0501] The complementary nucleotide sequence of the nucleotide sequence encoding the HLA-C signal peptide (SEQ ID NO: 43)

[0502] CACTACCGGGGTTCTTGGGACTAGGAC

[0503] Heavy chain of HLA-E*0101 (SEQ ID NO:44)

[0504] GSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDRRFLRGYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYLEDTCVEWL HKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRWKPASQPTIPIVGIIAGLVLLGSVVSGAVVAAVIWRKKSSGGKGGSYSKAEWSDSAQGSESHSL

[0505] Nucleotide sequence encoding the heavy chain of HLA-E*0101 (SEQ ID NO: 45)

[0506]

[0507] Histone H2A peptide (SEQ ID NO:46)

[0508] RIIPRHLQL

[0509] Nucleotide sequence encoding histone H2A peptide (SEQ ID NO:47)

[0510] CGGATCATCCCCCGGCACCTGCAGCTG

[0511] The disclosure of each patent, patent application and publication cited herein is hereby incorporated by reference in its entirety. Although the present invention has been disclosed with reference to specific embodiments, it is apparent that other persons skilled in the art can design other embodiments and variations of the present invention without departing from the true spirit and scope of the present invention. The appended claims are intended to be interpreted as including all such embodiments and equivalent variations.

Claims

1. A peptide comprising: Human leukocyte antigen (HLA) signal peptide or fragment thereof, Modified beta-2-microglobulin (B2M) or a fragment thereof, and HLA class I histocompatibility antigen alpha chain E (HLA-E) or its fragment; in, The HLA signal peptide or fragment thereof comprises at least one amino acid sequence having at least about 70% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-10, 13 and 46.

2. The peptide according to claim 1, wherein The peptide is a single chain trimer.

3. The peptide according to claim 1, wherein The HLA signal peptide includes at least one selected from the group consisting of: HLA-A*02:01 signal peptide or a fragment thereof, HLA-B*08:01 signal peptide or a fragment thereof, HLA-C*03:01 signal peptide or a fragment thereof, HLA-G signal peptide or a fragment thereof, HSP60 signal peptide or a fragment thereof, CMV Towne signal peptide or a fragment thereof, CMVAF1 signal peptide or a fragment thereof, CMV 109b signal peptide or a fragment thereof, RL9HIV signal peptide or a fragment thereof, and Mtb44 signal peptide or a fragment thereof.

4. The peptide according to claim 1, wherein The HLA-E is human HLA-E or a fragment thereof.

5. The peptide according to claim 1, wherein The modified B2M includes at least one selected from the group consisting of a modified B2M signal peptide or a fragment thereof and a modified B2M or a fragment thereof without a leader sequence.

6. The peptide according to claim 5, wherein The modified B2M signal peptide or fragment thereof is linked to the HLA signal peptide or fragment thereof; The HLA signal peptide or fragment is linked to the modified B2M or fragment thereof without a leader sequence; and The modified B2M or fragment thereof without a leader sequence is linked to the HLA-E or fragment thereof.

7. The peptide according to claim 6, wherein The peptide further comprises at least one linker and at least one spacer.

8. The peptide according to claim 7, wherein The peptide comprises an amino acid sequence that is at least about 70% identical to the amino acid sequence shown in SEQ ID NO:

40.

9. The peptide according to claim 1, wherein The peptide prevents, reduces or inhibits Natural Killer (NK) cell-mediated killing of at least one donor cell.

10. The peptide according to claim 1, wherein The peptide increases the persistence of at least one donor cell or decreases the clearance of at least one donor cell.

11. A composition comprising at least one peptide according to any one of claims 1 to 10.

12. The composition according to claim 11, wherein The composition is a pharmaceutically acceptable composition.

13. A nucleic acid molecule comprising: A nucleotide sequence encoding a human leukocyte antigen (HLA) signal peptide or a fragment thereof, A nucleotide sequence encoding a modified beta-2-microglobulin (B2M) or a fragment thereof, and A nucleotide sequence encoding HLA class I histocompatibility antigen alpha chain E (HLA-E) or a fragment thereof; in, The HLA signal peptide or fragment thereof comprises at least one amino acid sequence having at least about 70% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-10, 13 and 46.

14. The nucleic acid molecule according to claim 14, wherein The nucleotide sequence encoding the HLA signal peptide or a fragment thereof comprises at least one nucleotide sequence having at least about 70% identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs: 14-24 and 47.

15. The nucleic acid molecule according to claim 14, wherein The nucleic acid molecule comprises a nucleotide sequence that is at least about 70% identical to the nucleotide sequence shown in SEQ ID NO:

41.

16. A nucleic acid molecule comprising: A nucleotide sequence encoding a human leukocyte antigen (HLA) signal peptide or a fragment thereof, A nucleotide sequence encoding a modified beta-2-microglobulin (B2M) or a fragment thereof, and A nucleotide sequence encoding HLA class I histocompatibility antigen alpha chain E (HLA-E) or a fragment thereof; in, The nucleotide sequence encoding the HLA signal peptide or a fragment thereof comprises at least one nucleotide sequence having at least about 70% identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs: 14-24 and 47.

17. The nucleic acid molecule according to claim 17, wherein The HLA signal peptide includes at least one selected from the group consisting of: HLA-A*02:01 signal peptide or a fragment thereof, HLA-B*08:01 signal peptide or a fragment thereof, HLA-C*03:01 signal peptide or a fragment thereof, HLA-G signal peptide or a fragment thereof, HSP60 signal peptide or a fragment thereof, CMV Towne signal peptide or a fragment thereof, CMV AF1 signal peptide or a fragment thereof, CMV 109b signal peptide or a fragment thereof, RL9HIV signal peptide or a fragment thereof, and Mtb44 signal peptide or a fragment thereof.

18. The nucleic acid molecule according to claim 17, wherein The HLA-E is human HLA-E or a fragment thereof.

19. The nucleic acid molecule according to claim 17, wherein The nucleotide sequence encoding the modified B2M includes at least one selected from the group consisting of a nucleotide sequence encoding a modified B2M signal peptide or a fragment thereof and a nucleotide sequence encoding a modified B2M or a fragment thereof without a leader sequence.

20. The nucleic acid molecule according to claim 20, wherein The nucleotide sequence encoding the modified B2M signal peptide or a fragment thereof is linked to the nucleotide sequence encoding the HLA signal peptide or a fragment thereof; A nucleotide sequence encoding an HLA signal peptide or a fragment thereof is linked to the nucleotide sequence encoding the modified B2M or a fragment thereof without a leader sequence; and The nucleotide sequence encoding the modified B2M or a fragment thereof without a leader sequence is linked to the nucleotide sequence encoding HLA-E or a fragment thereof.

21. The nucleic acid molecule according to claim 21, wherein The nucleic acid molecule further comprises at least one nucleotide sequence encoding a linker and at least one nucleotide sequence encoding a spacer.

22. The nucleic acid molecule according to claim 22, wherein The nucleic acid molecule comprises a nucleotide sequence that is at least about 70% identical to the nucleotide sequence shown in SEQ ID NO:

41.

23. The nucleic acid molecule according to claim 17, wherein The nucleic acid molecule prevents, reduces or inhibits Natural Killer (NK) cell-mediated killing of at least one donor cell.

24. The nucleic acid molecule according to claim 17, wherein The nucleic acid molecule increases the persistence of at least one donor cell or decreases the clearance of at least one donor cell.

25. A composition comprising at least one nucleic acid molecule according to any one of claims 14-25.

26. A genetically engineered cell comprising at least one nucleic acid molecule according to any one of claims 14-25.

27. The genetically engineered cell according to claim 27, wherein The genetically engineered cells are modified to not express at least one selected from the group consisting of: beta-2-microglobulin (B2M), class II major histocompatibility complex transactivator (CIITA), and natural T cell receptor (TCR).

28. The genetically engineered cell according to claim 28, wherein The genetically engineered cell is a triple knockout (TKO) cell that does not express at least one selected from the group consisting of major histocompatibility complex (MHC) I, MHC II, and natural TCR.

29. The genetically engineered cell according to claim 29, wherein The genetically engineered cells are selected from the group consisting of autologous cells, allogeneic cells, alloresponsive cells, T cells, induced pluripotent stem cells (IPSCs), chimeric antigen receptor (CAR) cells, and any combination thereof.

30. The genetically engineered cell according to claim 30, wherein The T cells are selected from the group consisting of alloresponsive T cells, T cells carrying engineered TCRs, allospecific T cells, T cells carrying alloreactive TCRs, CART cells, T cells expressing engineered TCRs, and any combination thereof.

31. A method of preventing, reducing or eliminating rejection of an allogeneic or xenogeneic transplant in a subject in need thereof, wherein: The method comprises administering to the subject a therapeutically effective amount of at least one cell of claim 27.

32. A method of preventing, reducing or eliminating an allogeneic response in a subject in need thereof, wherein: The method comprises administering to the subject a therapeutically effective amount of at least one cell of claim 27.

33. A method of depleting alloresponsive cell levels in a subject in need thereof, wherein: The method comprises administering to the subject a therapeutically effective amount of at least one cell of claim 27.

34. A method of inducing allogeneic tolerance in a subject in need thereof, wherein: The method comprises administering to the subject a therapeutically effective amount of at least one cell of claim 27.

35. A method for improving the effectiveness of CAR therapy in a subject in need thereof, wherein: The method comprises administering to the subject a therapeutically effective amount of at least one CAR cell of claim 30.

36. A method for improving the effectiveness of engineered TCR cell therapy in a subject in need thereof, wherein: The method comprises administering to the subject a therapeutically effective amount of at least one T cell of claim 31.

37. The method according to any one of claims 32 to 37, wherein: The subject has at least one selected from the group consisting of: organ transplantation, tissue transplantation, cell transplantation, allogeneic transplantation, intestinal transplantation, reconstructive transplantation, autoimmune diseases or disorders, graft-versus-host disease (GvHD), diseases or disorders associated with at least one HLA receptor, diseases or disorders associated with at least one HLA-containing receptor, diseases or disorders associated with at least one MHC receptor, diseases or disorders associated with at least one MHC-containing receptor, diseases or disorders associated with the expression of alloresitive cells, diseases or disorders associated with organ transplantation, diseases or disorders associated with tissue transplantation, diseases or disorders associated with cell transplantation, diseases or disorders associated with allogeneic transplantation, diseases or disorders associated with intestinal transplantation, diseases or disorders associated with reconstructive transplantation, cancer, and diseases or disorders associated with cancer.

38. A method of preventing or treating a disease or condition in a subject in need thereof, wherein: The method comprises administering to the subject a therapeutically effective amount of at least one cell of claim 27.

39. The method of claim 39, wherein: The method comprises depleting, reducing or eliminating the level of at least one selected from the group consisting of alloresponsive cells, immune cells, T cells, B cells, natural killer cells, leukocytes, myeloid cells and plasma cells.

40. The method of claim 39, wherein: The method is engineered TCR therapy.

41. The method of claim 39, wherein: The disease or condition is selected from the group consisting of cancer, a disease or condition associated with cancer, and any combination thereof.

42. The method of claim 39, wherein: The disease or disorder is selected from the group consisting of a disease or disorder associated with the expression of alloresitive cells, a disease or disorder associated with at least one HLA receptor, a disease or disorder associated with at least one HLA-containing receptor, a disease or disorder associated with at least one MHC receptor, a disease or disorder associated with at least one MHC-containing receptor, GvHD, an autoimmune disease or disorder, a disease or disorder associated with organ transplantation, a disease or disorder associated with tissue transplantation, a disease or disorder associated with cell transplantation, a disease or disorder associated with allogeneic transplantation, a disease or disorder associated with intestinal transplantation, a disease or disorder associated with reconstructive transplantation, cancer, a disease or disorder associated with cancer, and any combination thereof.

43. The method of claim 43, wherein: The disease or disorder associated with expression of alloresitive cells is selected from the group consisting of allogeneic transplant rejection, immune rejection, chronic allogeneic rejection, implant rejection, transplant rejection, inflammation, inflammation caused by ischemia / reperfusion, infection, immune response to allogeneic transplant, and any combination thereof.

44. The method of claim 39, wherein: The subject has at least one selected from the group consisting of an organ transplant, a tissue transplant, a cell transplant, an allogeneic transplant, an intestinal transplant, and a reconstructive transplant.

45. A method for preventing or treating cancer in a subject in need thereof, wherein: The method comprises administering to the subject a therapeutically effective amount of at least one cell of claim 27.

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