Method for protecting transplanted tissue from rejection - Patents.com

Abstract

The present invention includes compositions and methods for HLA-A2-specific chimeric antigen receptors (CARs). In certain embodiments, HLA-A2-specific CARs are expressed on regulatory T cells. In certain embodiments, HLA-AR-specific CARs protect transplanted tissue from rejection.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 477,815, filed March 28, 2017, which is incorporated by reference in its entirety. [Background technology]

[0002] 2. Background of the Invention Transplant rejection occurs when the recipient's immune system attacks the transplanted tissue or organ. Rejection is generally mediated by alloreactive T cells present in the recipient that recognize donor allo- or xenoantigens. Host T cells can recognize allograft human leukocyte antigens (HLA) or associated binding peptides. Alloreactive T cells are stimulated by donor antigen-presenting cells (APCs), which express both allogeneic MHC and costimulatory activity. Alloreactive CD4 + T cells produce cytokines that exacerbate the cytolytic CD8 response to alloantigens. Unwanted alloreactive T cell responses in patients (allograft rejection, graft-versus-host disease) are typically addressed with immunosuppressive drugs such as prednisone, azathioprine, and cyclosporine A. Unfortunately, these drugs generally must be maintained for the patient's lifetime, and they have numerous dangerous side effects, including systemic immunosuppression.

[0003] Peripheral blood contains a small population of T cell lymphocytes that express a T regulatory phenotype ("Treg"), i.e., are positive for both CD4 and CD25 antigens. There are several subsets of Treg cells. A subset of regulatory cells arises in the thymus. Thymus-derived Treg cells function by a cytokine-independent mechanism involving cell-cell contact. They are essential for the induction and maintenance of self-tolerance and the prevention of autoimmunity. These regulatory cells prevent the activation and proliferation of autoreactive T cells that have escaped the thymic defect or recognize extrathymic antigens, and therefore are important for homeostasis and immune regulation, as well as for the protection of the host against the development of autoimmunity. Thus, immunoregulatory CD4 + CD25 + T cells are often called "professional suppressor cells."

[0004] There is a need for novel compositions and methods that suppress alloreactions in vivo and protect transplanted tissue from rejection. The present invention fulfills this need. Summary of the Invention

[0005] overview As described herein, the present invention relates to compositions and methods for protecting transplanted tissue from rejection using an HLA-A2 specific CAR.

[0006] In one aspect, the present invention includes an engineered immune cell or a precursor thereof comprising a chimeric antigen receptor (CAR) having affinity for HLA-A2. In some embodiments, the CAR comprises an HLA-A2 binding domain and a CD8 hinge domain.

[0007] Another aspect of the present invention includes an engineered immune cell or a precursor thereof comprising a chimeric antigen receptor (CAR) having affinity for HLA-A2. In some embodiments, the CAR comprises an HLA-A2 binding domain, a CD8 hinge domain, a CD8 signal peptide, a CD28 transmembrane domain, a CD28 costimulatory domain, and a CD3 zeta intracellular domain.

[0008] The present invention is based on the discovery that genetically modified immune cells containing the HLA-A2-specific chimeric antigen receptor (CAR) of the present invention exhibit specificity for HLA-A2, HLA-A28, and / or HLA-A68. The present invention is also based on the discovery that genetically modified regulatory T cells containing the subject HLA-A2-specific CAR exhibit potent immunosuppressive activity.

[0009] Thus, in certain aspects, the present invention provides modified immune cells, or precursors thereof, that comprise a chimeric antigen receptor (CAR) with affinity for HLA-A2.

[0010] In one aspect, an engineered immune cell, or a precursor thereof, is provided that comprises a chimeric antigen receptor (CAR) having affinity for HLA-A2, where the CAR comprises an HLA-A2 binding domain.

[0011] In certain exemplary embodiments, the CAR further comprises a signal peptide.

[0012] In certain exemplary embodiments, the signal peptide is the CD8 signal peptide.

[0013] In certain exemplary embodiments, the CAR further comprises a hinge domain.

[0014] In certain exemplary embodiments, the hinge domain is a CD8 hinge domain.

[0015] In certain exemplary embodiments, the HLA-A2 binding domain is selected from the group consisting of an antibody, a Fab, or an scFv.

[0016] In certain exemplary embodiments, the HLA-A2 binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:3.

[0017] In certain exemplary embodiments, the HLA-A2 binding domain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:8.

[0018] In certain exemplary embodiments, the HLA-A2 binding domain comprises a spacer sequence.

[0019] In certain exemplary embodiments, the HLA-A2 binding domain comprises the amino acid sequence shown in SEQ ID NO:1.

[0020] In certain exemplary embodiments, the CAR comprises a transmembrane domain and an intracellular domain.

[0021] In certain exemplary embodiments, the transmembrane domain comprises a CD28 transmembrane domain.

[0022] In certain exemplary embodiments, the transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:17.

[0023] In certain exemplary embodiments, the intracellular domain comprises a CD28 intracellular domain and a CD3 zeta intracellular domain.

[0024] In certain exemplary embodiments, the CD28 intracellular domain comprises the amino acid sequence set forth in SEQ ID NO:19.

[0025] In certain exemplary embodiments, the CD3 zeta intracellular domain comprises the amino acid sequence set forth in SEQ ID NO:21.

[0026] In certain exemplary embodiments, the CD8 hinge comprises the amino acid sequence set forth in SEQ ID NO:15.

[0027] In certain exemplary embodiments, the CD8 signal peptide comprises the amino acid sequence set forth in SEQ ID NO:13.

[0028] In certain exemplary embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:23.

[0029] In certain exemplary embodiments, the HLA-A2 binding domain cross-reacts with HLA-A28.

[0030] In certain exemplary embodiments, the HLA-A2 binding domain cross-reacts with HLA-A68.

[0031] In certain exemplary embodiments, the modified cells are modified regulatory T cells.

[0032] In certain exemplary embodiments, the modified cells are autologous cells.

[0033] In certain exemplary embodiments, the modified cells are derived from a human.

[0034] In another aspect, an engineered immune cell or a precursor thereof is provided that comprises a chimeric antigen receptor (CAR) having affinity for HLA-A2, wherein the CAR comprises a CD8 signal peptide, an HLA-A2 binding domain, a CD8 hinge domain, a CD28 transmembrane domain, a CD28 costimulatory domain, and a CD3 zeta intracellular domain.

[0035] In certain exemplary embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:23.

[0036] In certain exemplary embodiments, the HLA-A2 binding domain cross-reacts with HLA-A28.

[0037] In certain exemplary embodiments, the HLA-A2 binding domain cross-reacts with HLA-A68.

[0038] In certain exemplary embodiments, the modified cells are modified regulatory T cells.

[0039] In certain exemplary embodiments, the modified cells are autologous cells.

[0040] In certain exemplary embodiments, the modified cells are derived from a human.

[0041] In another aspect, an isolated nucleic acid is provided comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) having affinity for HLA-A2, wherein the CAR comprises an HLA-A2 binding domain.

[0042] In certain exemplary embodiments, the CAR further comprises a signal peptide.

[0043] In certain exemplary embodiments, the signal peptide is the CD8 signal peptide.

[0044] In certain exemplary embodiments, the CAR further comprises a hinge domain.

[0045] In certain exemplary embodiments, the hinge domain is a CD8 hinge domain.

[0046] In certain exemplary embodiments, an isolated nucleic acid is provided comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) having affinity for HLA-A2, wherein the CAR comprises a CD8 signal peptide, an HLA-A2 binding domain, and a CD8 hinge domain.

[0047] In certain exemplary embodiments, the CAR comprises a CD28 transmembrane domain.

[0048] In certain exemplary embodiments, the CAR comprises a CD28 costimulatory domain.

[0049] In certain exemplary embodiments, the CAR comprises a CD3 zeta intracellular domain.

[0050] In certain exemplary embodiments, the isolated nucleic acid comprises the nucleic acid sequence set forth in SEQ ID NO: 24. In another aspect, there is provided an expression construct comprising an isolated nucleic acid of the aspects described herein.

[0051] In certain exemplary embodiments, the expression construct comprises the EF-1α promoter.

[0052] In certain exemplary embodiments, the expression construct comprises a rev response element (RRE).

[0053] In certain exemplary embodiments, the expression construct comprises a Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE).

[0054] In certain exemplary embodiments, the expression construct comprises a cPPT sequence.

[0055] In certain exemplary embodiments, the expression construct comprises an EF-1α promoter, a rev response element (RRE), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and a cPPT sequence.

[0056] In certain exemplary embodiments, the expression construct is a viral vector selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector.

[0057] In certain exemplary embodiments, the expression construct is a lentiviral vector.

[0058] In certain exemplary embodiments, the lentiviral vector is a self-inactivating lentiviral vector.

[0059] In another aspect, a method is provided for generating an engineered immune cell or a progenitor thereof of the aspects described herein, comprising introducing a nucleic acid of the aspects described herein or an expression construct of the aspects described herein into an immune cell.

[0060] In another aspect, a method is provided for achieving an immunosuppressive effect in a subject in need thereof, comprising administering to the subject an effective amount of an engineered immune cell or a precursor cell thereof of an aspect described herein.

[0061] In certain exemplary embodiments, the subject is suffering from an alloreaction and / or an autoimmune response.

[0062] In certain exemplary embodiments, an allogeneic or autoimmune response occurs following tissue transplantation, and the method suppresses, blocks, or inhibits graft-versus-host disease in the subject.

[0063] In certain exemplary embodiments, the modified cells are modified regulatory T cells.

[0064] In certain exemplary embodiments, the modified cells are autologous cells.

[0065] In certain exemplary embodiments, the modified cells are derived from a human.

[0066] In another aspect, a method is provided for achieving a prophylactic therapeutic effect in a subject in need thereof, comprising administering to the subject an effective amount of an engineered immune cell or a precursor thereof of an aspect described herein prior to the onset of an allogeneic or autoimmune response.

[0067] In certain exemplary embodiments, an allogeneic or autoimmune response occurs following tissue transplantation, and the method suppresses, blocks, or inhibits graft-versus-host disease in the subject.

[0068] In certain exemplary embodiments, the modified cells are modified regulatory T cells.

[0069] In certain exemplary embodiments, the modified cells are autologous cells.

[0070] In certain exemplary embodiments, the modified cells are derived from a human.

[0071] In another aspect, there is provided a method for achieving an immunosuppressive effect in a subject in need thereof, the subject having an alloreaction or an autoimmune response, the method comprising administering to the subject an engineered regulatory T cell comprising a chimeric antigen receptor (CAR) having affinity for HLA-A2, the CAR comprising a CD8 signal peptide, an HLA-A2 binding domain, a CD8 hinge domain, a CD28 transmembrane domain, a CD28 costimulatory domain, and a CD3 zeta intracellular domain.

[0072] In certain exemplary embodiments, an allogeneic or autoimmune response occurs following tissue transplantation, and the method suppresses, blocks, or inhibits graft-versus-host disease in the subject.

[0073] In certain exemplary embodiments, the modified cells are modified regulatory T cells.

[0074] In certain exemplary embodiments, the modified cells are autologous cells.

[0075] In certain exemplary embodiments, the modified cells are derived from a human.

[0076] In another aspect, a method of treating diabetes in a subject in need thereof is provided, comprising administering to the subject an effective amount of an engineered immune cell or a precursor cell thereof of any aspect described herein.

[0077] In certain exemplary embodiments, the diabetes is type 1 diabetes.

[0078] In certain exemplary embodiments, the modified cells are modified regulatory T cells.

[0079] In certain exemplary embodiments, the modified cells are autologous cells.

[0080] In certain exemplary embodiments, the modified cells are derived from a human.

[0081] In another aspect, a method of treating diabetes in a subject in need thereof is provided, comprising administering to the subject an engineered regulatory T cell comprising a chimeric antigen receptor (CAR) having affinity for HLA-A2, wherein the CAR comprises a CD8 signal peptide, an HLA-A2 binding domain, a CD8 hinge domain, a CD28 transmembrane domain, a CD28 costimulatory domain, and a CD3 zeta intracellular domain.

[0082] In certain exemplary embodiments, the diabetes is type 1 diabetes.

[0083] In certain exemplary embodiments, the modified cells are modified regulatory T cells.

[0084] In certain exemplary embodiments, the modified cells are autologous cells.

[0085] In certain exemplary embodiments, the modified cells are derived from a human. [The present invention 1001] A modified immune cell or a precursor thereof comprising a chimeric antigen receptor (CAR) having affinity for HLA-A2, wherein the CAR comprises a CD8 signal peptide, an HLA-A2 binding domain, and a CD8 hinge domain. [The present invention 1002] 1002. The modified cell of claim 1001, wherein said HLA-A2 binding domain is selected from the group consisting of an antibody, a Fab, or an scFv. [The present invention 1003] Any of the modified cells of the invention, wherein the HLA-A2 binding domain comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:3. [The present invention 1004] Any of the modified cells of the invention, wherein the HLA-A2 binding domain comprises a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:8. [The present invention 1005] The modified cell of any of claims 1003 or 1004, wherein said HLA-A2 binding domain comprises a spacer sequence. [The present invention 1006] Any of the modified cells of the invention, wherein the HLA-A2 binding domain comprises the amino acid sequence shown in SEQ ID NO:1. [The present invention 1007] Any of the modified cells of the present invention, wherein the CAR comprises a transmembrane domain and an intracellular domain. [The present invention 1008] The modified cell of the present invention, wherein the transmembrane domain comprises a CD28 transmembrane domain. [The present invention 1009] The modified cell of any of claims 1007 or 1008, wherein the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:17. [The present invention 1010] The modified cell of any of claims 1007 to 1009, wherein the intracellular domain comprises a CD28 intracellular domain and a CD3ζ intracellular domain. [The present invention 1011] The modified cell of the present invention, wherein the CD28 intracellular domain comprises the amino acid sequence shown in SEQ ID NO:19. [The present invention 1012] The modified cell of any of claims 1010 or 1011, wherein the CD3 zeta intracellular domain comprises the amino acid sequence shown in SEQ ID NO:21. [The present invention 1013] Any of the modified cells of the invention, wherein the CD8 hinge comprises the amino acid sequence shown in SEQ ID NO:15. [The present invention 1014] Any of the modified cells of the invention, wherein the CD8 signal peptide comprises the amino acid sequence shown in SEQ ID NO:13. [The present invention 1015] 1. An engineered immune cell, or a precursor thereof, comprising a chimeric antigen receptor (CAR) having affinity for HLA-A2, wherein the CAR comprises a CD8 signal peptide, an HLA-A2 binding domain, a CD8 hinge domain, a CD28 transmembrane domain, a CD28 costimulatory domain, and a CD3 zeta intracellular domain. [The present invention 1016] Any of the modified cells of the present invention, wherein the CAR comprises the amino acid sequence shown in SEQ ID NO:23. [The present invention 1017] Any of the modified cells of the present invention, wherein the HLA-A2 binding domain cross-reacts with HLA-A28. [The present invention 1018] Any of the modified cells of the present invention, wherein the HLA-A2 binding domain cross-reacts with HLA-A68. [The present invention 1019] The modified cell of any of claims 1001 to 1018, which is a modified regulatory T cell. [The present invention 1020] Any of the modified cells of the present invention which are autologous cells. [The present invention 1021] Any of the modified cells of the present invention derived from a human. [The present invention 1022] 1. An isolated nucleic acid comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) having affinity for HLA-A2, wherein the CAR comprises a CD8 signal peptide, an HLA-A2 binding domain, and a CD8 hinge domain. [The present invention 1023] The isolated nucleic acid of claim 1022, wherein the CAR comprises a CD28 transmembrane domain. [The present invention 1024] The isolated nucleic acid of any of claims 1022 or 1023, wherein the CAR comprises a CD28 costimulatory domain. [The present invention 1025] The isolated nucleic acid of any of claims 1022 to 1024, wherein the CAR comprises a CD3ζ intracellular domain. [The present invention 1026] An isolated nucleic acid according to any one of claims 1022 to 1025, comprising the nucleic acid sequence shown in SEQ ID NO:24. [The present invention 1027] An expression construct comprising any one of the isolated nucleic acids of the present invention 1022 to 1026. [The present invention 1028] An expression construct of the present invention 1027, comprising the EF-1α promoter. [The present invention 1029] An expression construct of any of 1027 or 1028 of the present invention comprising a rev response element (RRE). [The present invention 1030] An expression construct according to any one of claims 1027 to 1029, comprising a Woodchuck Hepatitis Virus post-transcriptional regulatory element (WPRE). [The present invention 1031] An expression construct according to any one of claims 1027 to 1030, comprising a cPPT sequence. [The present invention 1032] An expression construct of any of claims 1027 to 1031, comprising an EF-1α promoter, a rev response element (RRE), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and a cPPT sequence. [The present invention 1033] The expression construct of any of claims 1027 to 1032, which is a viral vector selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector. [The present invention 1034] An expression construct of any one of claims 1027 to 1033, which is a lentiviral vector. [The present invention 1035] The expression construct of the present invention, wherein said lentiviral vector is a self-inactivating lentiviral vector. [The present invention 1036] A method for generating a modified immune cell or a precursor cell thereof according to any one of claims 1001 to 1021, comprising the step of introducing into said immune cell a nucleic acid according to any one of claims 1022 to 1026, or an expression construct according to any one of claims 1027 to 1035. [The present invention 1037] A method for achieving an immunosuppressive effect in a subject in need thereof, comprising the step of administering to the subject an effective amount of a modified immune cell or a precursor cell thereof according to any one of the present inventions 1001 to 1021. [The present invention 1038] The method of claim 1037, wherein the subject is suffering from an alloreaction and / or an autoimmune reaction. [The present invention 1039] A method for achieving a prophylactic therapeutic effect in a subject in need thereof, comprising the step of administering to the subject an effective amount of a modified immune cell or a precursor cell thereof according to any one of the present inventions 1001 to 1021 before an alloreaction or an autoimmune reaction develops. [The present invention 1040] 1. A method for achieving an immunosuppressive effect in a subject in need thereof, the subject having an alloreaction or an autoimmune response, the method comprising administering to the subject an engineered regulatory T cell comprising a chimeric antigen receptor (CAR) having affinity for HLA-A2, the CAR comprising a CD8 signal peptide, an HLA-A2 binding domain, a CD8 hinge domain, a CD28 transmembrane domain, a CD28 costimulatory domain, and a CD3 zeta intracellular domain. [The present invention 1041] The method of any of claims 1037 to 1040, wherein said alloreaction or autoimmune response occurs following tissue transplantation, and said method suppresses, blocks, or inhibits graft-versus-host disease in said subject. [The present invention 1042] The method according to any one of claims 1037 to 1041, wherein the modified cells are modified regulatory T cells. [The present invention 1043] The method according to any one of claims 1037 to 1042, wherein the modified cells are autologous cells. [The present invention 1044] The method according to any one of claims 1037 to 1043, wherein the modified cell is derived from a human. [The present invention 1045] A method for treating diabetes in a subject in need thereof, comprising the step of administering to the subject an effective amount of a modified immune cell or a precursor cell thereof according to any one of claims 1001 to 1021 of the present inventions. [The present invention 1046] 1. A method of treating diabetes in a subject in need thereof, the method comprising administering to the subject an engineered regulatory T cell comprising a chimeric antigen receptor (CAR) having affinity for HLA-A2, the CAR comprising a CD8 signal peptide, an HLA-A2 binding domain, a CD8 hinge domain, a CD28 transmembrane domain, a CD28 costimulatory domain, and a CD3 zeta intracellular domain. [The present invention 1047] The method of any one of claims 1045 or 1046, wherein said diabetes is type 1 diabetes. [The present invention 1048] The method according to any one of claims 1045 to 1047, wherein the modified cells are modified regulatory T cells. [The present invention 1049] The method according to any one of claims 1045 to 1048, wherein the modified cells are autologous cells. [The present invention 1050] The method according to any one of claims 1045 to 1049, wherein the modified cell is derived from a human. [Brief description of the drawings]

[0086] The following detailed description of specific embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, there are shown in the drawings exemplary embodiments. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0087] [Figure 1] FIG. 1 is a schematic diagram of an HLA-A2-specific CAR that mediates antigen-specific suppression when expressed on human regulatory T cells. [Diagram 2] A series of plots illustrating primary human CD8+ T cells washed 3 times with OPTI-MEM reduced serum medium, resuspended at 100×106 cells / mL, mixed with 10 μg of in vitro transcribed RNA encoding an HLA-A2 specific CAR, and electroporated. After 16 hours, cells were stained with F(ab')2 fragment specific anti-biotin-SP (long spacer) AffiniPure goat anti-human IgG, followed by streptavidin-PE and anti-human CD8. Cells were fixed in 2% paraformaldehyde and analyzed on an LSRII flow cytometer. [Diagram 3]FIG. 3 is a series of plots illustrating cells electroporated as in FIG. 2 and mixed with human donor PBMCs of various HLA haplotypes at a 3:1 ratio for 6 hours in the presence of GolgiPlug Protein Transport Inhibitor. Cells were fixed, permeabilized, and stained with α-IL-2 and α-TNF-α antibodies. [Figure 4] A pair of plots illustrating regulatory T cells isolated from human cord blood donors via CD4+ RosetteSep followed by CD25 magnetic positive selection. Tregs were stimulated with α-CD3 / α-CD28 beads and expanded in XVIVO15 with 5% human AB serum containing 1×GlutaMAX and 300 IU / mL IL-2. 48 hours after the first stimulation, Tregs were transduced with lentiviral vectors to express 3PF12-28z CAR or irrelevant CAR. Cells were left to rest until day 14, when antigen-specific suppression was assessed by mixing Tregs with allogeneic T cells expressing CFSE-labeled A2-SL9 WT TCR (HIV-specific TCR) and K562 cells transgenically expressing HLA-A2 at a ratio of 8:1:0.5 (Teff:Treg:K562). To examine nonspecific suppressive function, Tregs were mixed with CFSE-labeled allogeneic PBMCs and α-CD3 stimulatory beads at a ratio of 8:1:3 (Teff:Treg:Beads). After 5 days of cell culture, the dilution of CFSE in CD8+ T cells was measured. [Diagram 5] Illustrates human C-peptide levels in response to 3PF12-28z or CD19-28z CAR-transduced T cells, or non-transduced negative control T cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0088] Detailed Description definition Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. Although any method and material similar or equivalent to those described herein can be used to practice the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology is used.

[0089] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting.

[0090] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0091] As used herein, "about" when referring to a measurable value, such as an amount, duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, as such variations are reasonable in practicing the disclosed methods.

[0092] "Activation" as used herein refers to the state of T cells that are sufficiently stimulated to induce detectable cell proliferation. Activation can also be associated with induced cytokine production and detectable effector function. The term "activated T cells" refers, inter alia, to T cells that are undergoing cell division.

[0093] As used herein, "alleviating" a disease means reducing the severity of one or more symptoms of the disease.

[0094] "Allogeneic" refers to a graft derived from a different animal of the same species.

[0095] "Alloantigen" refers to an antigen that is present only in some individuals of a species and is capable of inducing the production of alloantibodies by individuals who lack it.

[0096] The term "antibody" as used herein refers to an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be intact immunoglobulins derived from natural or recombinant sources, or immune-reactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies in the present invention can exist in a wide variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies (scFv) and humanized antibodies (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).

[0097] The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigen-determining variable region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0098] "Antibody heavy chain," as used herein, refers to the larger of two polypeptide chains that are present in all antibody molecules in their naturally occurring conformations.

[0099] "Antibody light chain," as used herein, refers to the smaller of the two polypeptide chains present in all antibody molecules in their naturally occurring conformations. The alpha light chain and the beta light chain refer to the two major antibody light chain isotypes.

[0100] The term "synthetic antibody," as used herein, means an antibody produced using recombinant DNA techniques, such as, for example, an antibody expressed by a bacteriophage as described herein. The term should also be taken to mean a DNA molecule encoding an antibody, produced by synthesis of a DNA molecule expressing the antibody protein or by synthesis of an amino acid sequence specifying the antibody, which DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence techniques available and well known in the art.

[0101] The term "antigen" or "Ag" as used herein is defined as a molecule that elicits an immune response. This immune response may involve either antibody production or activation of specific immunocompetent cells, or both. Those skilled in the art will appreciate that virtually any macromolecule, including any protein or peptide, can serve as an antigen. Moreover, antigens can be derived from recombinant or genomic DNA. Those skilled in the art will appreciate that any DNA that includes a nucleotide sequence or partial nucleotide sequence that encodes a protein that elicits an immune response therefore encodes an "antigen" as that term is used herein. Moreover, those skilled in the art will appreciate that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It will be readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of multiple genes, and that these nucleotide sequences are arranged in various combinations that elicit the desired immune response. Moreover, those skilled in the art will appreciate that an antigen need not be encoded by a "gene" at all. It will be readily apparent that an antigen can be synthetically produced or derived from a biological sample. Such biological samples may include, but are not limited to, a tissue sample, a tumor sample, a cell, or a biological fluid.

[0102] As used herein, the term "autologous" is meant to refer to any material derived from the same individual that is later reintroduced into that individual.

[0103] "Homogeneous" refers to any material derived from different animals of the same species.

[0104] The term "chimeric antigen receptor" or "CAR" as used herein refers to an artificial T cell receptor that is engineered to be expressed on immune effector cells and specifically bind to an antigen. CARs can be used as a therapy by adoptive cell transfer. T cells are removed from a patient and modified to express a receptor specific to a particular form of antigen. In some embodiments, CARs have specificity for a selected target, for example, human leukocyte antigen (HLA). CARs can also include an extracellular domain that includes an intracellular activation domain, a transmembrane domain, and an antigen-binding region. In some aspects, CARs include an extracellular domain that includes an anti-HLA binding domain fused to a CD8 hinge domain, a CD28 transmembrane domain and an intracellular domain, and a CD3 zeta domain.

[0105] The term "cleavage" refers to, for example, the breakage of covalent bonds or the hydrolysis of peptide bonds in the backbone of a nucleic acid molecule. Cleavage can be initiated by a variety of methods, including but not limited to the enzymatic or chemical hydrolysis of phosphodiester bonds. Both single-strand and double-strand breaks are possible. Double-strand breaks can occur as a result of two separate single-strand break events. DNA cleavage can result in the production of either blunt ends or sticky ends. In certain embodiments, fusion polypeptides can be used to target cleaved double-stranded DNA.

[0106] As used herein, the term "conservative sequence modification" is intended to refer to amino acid modifications that do not significantly affect or change the binding properties of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibody of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue that has a similar side chain. Families of amino acid residues that have similar side chains are defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the CDR regions of an antibody can be replaced with other amino acid residues from the same side chain family, and the altered antibodies can be tested for their ability to bind antigen using the functional assays described herein.

[0107] "Costimulatory ligand," as that term is used herein, includes molecules on antigen-presenting cells (e.g., aAPCs, dendritic cells, B cells, etc.) that specifically bind to a cognate costimulatory molecule on a T cell, thereby providing signals that mediate T cell responses, including, but not limited to, proliferation, activation, differentiation, etc., in addition to the primary signal provided by engagement of the TCR / CD3 complex with, for example, a peptide-loaded MHC molecule. Costimulatory ligands can include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, agonists or antibodies that bind to the Toll ligand receptor, and ligands that specifically bind to B7-H3. Costimulatory ligands also include antibodies that specifically bind to costimulatory molecules present on T cells, such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, among others, and ligands that specifically bind to CD83.

[0108] "Costimulatory molecule" refers to the cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors.

[0109] A "costimulatory signal," as used herein, refers to a signal that, in combination with a primary signal, such as TCR / CD3 ligation, leads to T cell proliferation and / or up- or down-regulation of key molecules.

[0110] A "disease" is an animal's health condition in which the animal is unable to maintain homeostasis and the animal's health will continue to deteriorate if the disease is not improved. On the other hand, an "injury" in an animal is a health condition in which the animal is able to maintain homeostasis, but the animal's health condition is less favorable than it would be in the absence of the disorder. If left untreated, the disorder does not necessarily cause the animal's health condition to further deteriorate.

[0111] "Donor antigen" refers to an antigen expressed by the donor tissue to be transplanted into the recipient.

[0112] "Recipient antigen" refers to the target for an immune response against a donor antigen.

[0113] The term "downregulation," as used herein, refers to the reduction or elimination of gene expression of one or more genes.

[0114] "Effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, material or composition as described herein that is effective to achieve a particular biological result or provides a therapeutic or prophylactic benefit. Such results may include, but are not limited to, an amount that, when administered to a mammal, causes a detectable level of immune suppression or tolerance compared to the immune response detected in the absence of the composition of the present invention. Immune response can be readily assessed by many art-recognized methods. Those skilled in the art will understand that the amount of the composition administered herein will vary and can be readily determined based on a number of factors, such as the disease or condition being treated, the age and health and physical condition of the mammal being treated, the severity of the disease, the particular compound being administered, and the like.

[0115] "Encoding" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, either with a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence and biological properties resulting therefrom. Thus, a gene codes for a protein if transcription and translation of the mRNA corresponding to the gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to code for the protein or other product of the gene or cDNA.

[0116] As used herein, "endogenous" refers to any substance that is derived from or produced within an organism, cell, tissue, or system.

[0117] The term "epitope" as used herein is defined as a small chemical molecule on an antigen that can elicit an immune response, including a B-cell response and / or a T-cell response. An antigen can have one or more epitopes. Most antigens have many epitopes; that is, they are multivalent. In general, an epitope is approximately about 10 amino acids and / or sugars in size. Preferably, an epitope is about 4-18 amino acids, more preferably about 5-16 amino acids, even more preferably about 6-14 amino acids, more preferably about 7-12, and most preferably about 8-10 amino acids. In general, one skilled in the art will understand that the overall three-dimensional structure of the molecule, rather than the specific linear sequence, is the primary criterion of antigen specificity, and thus, this distinguishes one epitope from another. Based on the present disclosure, a peptide used in the present invention can be an epitope.

[0118] As used herein, the term "exogenous" refers to any substance that is introduced from or produced outside an organism, cell, tissue, or system.

[0119] The term "expand" as used herein refers to an increase in number, such as an increase in the number of T cells. In one embodiment, T cells expanded ex vivo are increased in number relative to the number originally present in the culture. In another embodiment, T cells expanded ex vivo are increased in number relative to other cell types in the culture. The term "ex vivo" as used herein refers to cells removed from a living organism (e.g., a human) and grown outside of the organism (e.g., in a culture dish, test tube, or bioreactor).

[0120] The term "expression", as used herein, is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0121] "Expression vector" refers to a vector that contains a recombinant polynucleotide that includes an expression control sequence operably linked to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., Sendai virus, lentivirus, retrovirus, adenovirus and adeno-associated virus) that incorporate a recombinant polynucleotide.

[0122] "HLA-A2" refers to a human leukocyte antigen within the HLA-A serogroup. HLA-A is one of the three major types of MHC class I cell surface receptors. MHC class I molecules are one of two major classes of major histocompatibility complex (MHC) molecules found on the cell surface of cells. The function of MHC class I molecules is to present peptide fragments of non-self proteins from inside the cell to immune cells (e.g., cytotoxic T cells), thereby triggering a rapid response from the immune system against the specific non-self antigens presented.

[0123] "HLA-A28" refers to a human leukocyte antigen within the HLA-A serogroup.

[0124] "HLA-A68" refers to a human leukocyte antigen within the HLA-A serogroup. The alpha "A" chain is encoded by the HLA-A*68 allele group, and the beta chain is encoded by the beta-2 microglobulin (B2M) locus.

[0125] "Homologous" as used herein refers to the subunit sequence identity between two polymer molecules, for example, between two nucleic acid molecules, for example, between two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomer subunit; for example, if a position in each of the two DNA molecules is occupied by adenine, they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; for example, if half of the positions in the two sequences (for example, 5 positions in a polymer of 10 subunits length) are homologous, the two sequences are 50% homologous; if 90% of the positions (for example, 9 out of 10) are matching or homologous, the two sequences are 90% homologous.

[0126] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from a non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Moreover, humanized antibodies can contain residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are from a human immunoglobulin sequence. The humanized antibody will also optimally comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332: 323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

[0127] "Fully human" refers to an immunoglobulin, eg, an antibody, where the entire molecule is of human origin or consists of an amino acid sequence identical to a human form of the antibody.

[0128] "Identity" as used herein refers to the subunit sequence identity between two polymer molecules, particularly between two amino acid molecules, for example, between two polypeptide molecules. When two amino acid sequences have the same residue at the same position; for example, if a position in each of the two polypeptide molecules is occupied by arginine, they are identical at that position. The identity or degree to which two amino acid sequences have the same residue at the same position in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions; for example, if half of the positions in the two sequences (e.g., 5 positions in a 10 amino acid long polymer) are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 out of 10) are identical or matched, the two amino acid sequences are 90% identical.

[0129] The term "immunoglobulin" or "Ig" as used herein is defined as a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes called BCR (B cell receptor) or antigen receptor. The five members of this class of proteins are IgA, IgG, IgM, IgD and IgE. IgA is the major antibody present in bodily secretions such as saliva, tears, breast milk, gastrointestinal secretions, and mucous secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the main immunoglobulin produced in the primary immune response in most subjects. It is the most efficient immunoglobulin in agglutination, complement fixation and other antibody responses, and is important in defense against bacteria and viruses. IgD is an immunoglobulin with no known antibody function, but may serve as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity by triggering the release of mediators from mast cells and basophils upon exposure to allergens.

[0130] The term "immune response," as used herein, is defined as a cellular response to an antigen that occurs when lymphocytes identify an antigen molecule as foreign and induce the formation of antibodies and / or activate lymphocytes to eliminate the antigen.

[0131] The term "immunostimulatory" as used herein refers to increasing the overall immune response.

[0132] The term "immunosuppressive" as used herein refers to reducing the overall immune response.

[0133] As used herein, "instruction material" includes publications, records, diagrams, or any other medium of expression that can be used to communicate the usefulness of the compositions and methods of the invention. The instruction material of the kits of the invention may, for example, be attached to a container containing the nucleic acid, peptide, and / or composition of the invention, or may be shipped together with a container containing the nucleic acid, peptide, and / or composition. Alternatively, the instruction material may be shipped separately from the container, with the intention that the instruction material and the compound are used together by the recipient.

[0134] "Isolated" means altered or removed from a natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form or can exist in a non-native environment, such as a host cell.

[0135] The term "knockdown," as used herein, refers to a reduction in gene expression of one or more genes.

[0136] The term "knockout," as used herein, refers to the elimination of gene expression of one or more genes.

[0137] "Lentivirus" as used herein refers to a genus of the Retroviridae family.Lentiviruses are unique among retroviruses in that they can infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of host cells, making them one of the most efficient methods of gene delivery vectors.HIV, SIV and FIV are all examples of lentiviruses.Vectors derived from lentiviruses provide a means to achieve significant levels of gene transfer in vivo.

[0138] The term "limited toxicity" as used herein refers to peptides, polynucleotides, cells and / or antibodies of the invention that exhibit a substantial lack of negative biological effects, anti-tumor effects, or substantial negative physiological symptoms on healthy cells, non-tumor cells, non-disease cells, non-target cells, or populations of such cells, either in vitro or in vivo.

[0139] The term "modified" as used herein refers to an altered state or structure of a molecule or cell of the invention. Molecules can be modified in many ways, including chemically, structurally, and functionally. Cells can be modified through the introduction of nucleic acids.

[0140] The term "modulate" as used herein means to mediate a detectable increase or decrease in the level of response in a subject, compared to the level of response in a subject in the absence of treatment or compound and / or compared to the level of response in an otherwise identical but untreated subject. This term encompasses mediating a beneficial therapeutic response in a subject, preferably a human, by disrupting and / or affecting a natural signal or response.

[0141] In the context of the present invention, the following abbreviations are used for the commonly occurring nucleobases: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0142] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA can also include introns, to the extent that nucleotide sequences that encode proteins may contain introns in some versions.

[0143] "Parenteral" administration of the immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im) or intrasternal injection or infusion techniques.

[0144] The term "polynucleotide" as used herein is defined as a chain of nucleotides. Moreover, nucleic acid is a polymer of nucleotides. Thus, nucleic acid and polynucleotide are interchangeable as used herein. Those skilled in the art have the general knowledge that nucleic acid is a polynucleotide and can be hydrolyzed into monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotide includes, but is not limited to, all nucleic acid sequences obtained by any means available in the art, including but not limited to recombinant means, i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques and PCR™, etc., and synthetic means.

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

[0146] The term "self-antigen" as used herein is defined as the antigen expressed by host cell or tissue.Self-antigen can be tumor antigen, but in certain embodiments, it is expressed in both normal and tumor cells.Those skilled in the art will easily understand that self-antigen can be overexpressed in cells.

[0147] The term "specifically binds" as used herein with respect to an antibody means an antibody that recognizes a specific antigen in a sample but does not substantially recognize or bind to other molecules. For example, an antibody that specifically binds to an antigen from one species may also bind to antigens from one or more species. However, such cross-species reactivity does not in itself change the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of that antigen. However, such cross-reactivity does not in itself change the classification of the antibody as specific. In some cases, the term "specifically binds" or "specifically binds" can be used with respect to the interaction of an antibody, protein or peptide with a second chemical species to mean that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a particular protein structure rather than the entire protein. If an antibody is specific for epitope "A", the presence of a molecule containing epitope A (or free unlabeled A) will reduce the amount of labeled A bound to the antibody in a reaction containing labeled "A" and the antibody.

[0148] The term "stimulation" refers to a primary response induced by the binding of a stimulatory molecule (e.g., the TCR / CD3 complex) to its cognate ligand, thereby mediating a signaling event, such as, but not limited to, signaling through the TCR / CD3 complex. Stimulation can mediate altered expression of certain molecules, such as downregulation of TGF-β and / or remodeling of cytoskeletal structure.

[0149] "Stimulatory molecule," as that term is used herein, means a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell.

[0150] "Stimulatory ligand," as used herein, refers to a ligand that, when present on an antigen-presenting cell (e.g., aAPC, dendritic cell, B cell, etc.), can specifically bind to a cognate binding partner (herein referred to as a "stimulatory molecule") on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, etc. Stimulatory ligands are well known in the art and include, among others, peptide-loaded MHC class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0151] The term "subject" is intended to include a living organism (e.g., a mammal) in which an immune response can be elicited. A "subject" or "patient" as used herein may be a human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline, and murine mammals. Preferably, the subject is a human.

[0152] As used herein, a "substantially purified" cell is a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell that is separated from other cell types that it normally associates with in its naturally occurring state. In some cases, a population of substantially purified cells refers to a homogenous cell population. In other cases, the term simply refers to cells that are separated from the cells that they naturally associate with in their natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0153] "Target site" or "target sequence" refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule can specifically bind under conditions sufficient for binding to occur.

[0154] As used herein, the term "T cell receptor" or "TCR" refers to a complex of membrane proteins involved in the activation of T cells in response to the presentation of antigen. TCRs are responsible for recognizing antigens bound to major histocompatibility complex molecules. TCRs are composed of a heterodimer of alpha (a) and beta (β) chains, but in some cells, TCRs consist of gamma and delta (γ / δ) chains. TCRs can exist in α / β and γ / δ forms, which have structurally similar but distinct anatomical locations and functions. Each chain is composed of two extracellular domains, a variable domain and a constant domain. In some embodiments, TCRs can be engineered on any cell that contains a TCR, including, for example, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and γδ T cells.

[0155] The term "therapeutic" as used herein means treatment and / or prophylaxis. A therapeutic effect is obtained by suppression, amelioration, or eradication of a disease state.

[0156] "Graft" refers to a biocompatible lattice or a donor tissue, organ, or cells to be transplanted. Examples of transplants may include, but are not limited to, skin cells or tissue, bone marrow, and solid organs such as the heart, pancreas, kidney, lung, and liver. Grafts can also refer to any material to be administered to a host. For example, a graft can refer to a nucleic acid or a protein.

[0157] The terms "transfected" or "transformed" or "transduced" as used herein refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed or transduced with an exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0158] "Treating" a disease, as that term is used herein, means reducing the frequency or severity of at least one sign or symptom of the disease or disorder that affects a subject.

[0159] A "vector" is a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the inside of a cell. Numerous vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids and viruses. Thus, the term "vector" includes self-replicating plasmids or viruses. This term should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, etc.

[0160] "Xenogeneic" refers to any material derived from an animal of a different species.

[0161] Ranges: Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numbers within that range. For example, the description of a range such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, and individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0162] explanation The present invention includes compositions and methods for protecting transplanted tissue from rejection using HLA-A2-specific CARs. HLA-A2-specific CARs contain antigen-binding domains that bind to HLA-A2, HLA-A28, and / or HLA-A68. When expressed on human regulatory T cells (Tregs), HLA-A2-specific CARs mediate antigen-specific suppression. HLA-A2-specific CARs can redirect regulatory T cells to tissues expressing HLA-A2, HLA-A28, and / or HLA-A68, mediating tolerance.

[0163] The present invention includes HLA-A2 specific CAR and its use in suppressing alloreaction.Alloreaction is caused by donor MHC class I molecules, which are ubiquitously expressed in allografts, for example during organ transplantation.The present invention is based on the finding that regulatory T cells comprising HLA-A2 specific CAR can suppress alloreaction in an antigen-specific manner.

[0164] Chimeric antigen receptors (CARs) The present invention provides compositions and methods for engineered immune cells or their precursors, such as engineered regulatory T cells, comprising a chimeric antigen receptor (CAR) with affinity for HLA-A2. The subject CAR of the present invention comprises an antigen binding domain (e.g., HLA-A2 binding domain), a transmembrane domain, and an intracellular domain. The subject CAR of the present invention may optionally comprise a hinge domain and / or a signal peptide. In some embodiments, the signal peptide is a CD8 signal peptide. Thus, the subject CAR of the present invention comprises an antigen binding domain (e.g., HLA-A2 binding domain), a hinge domain, a transmembrane domain, and an intracellular domain. In some embodiments, the subject CAR of the present invention comprises a signal peptide, an antigen binding domain (e.g., HLA-A2 binding domain), a hinge domain, a transmembrane domain, and an intracellular domain. In some embodiments, each of the domains of the subject CAR is separated by a linker.

[0165] The antigen binding domain can be operably linked to another domain of the CAR, for example, a transmembrane domain or an intracellular domain, both of which are described elsewhere herein, for expression in a cell. In one embodiment, a first nucleic acid sequence encoding the antigen binding domain is operably linked to a second nucleic acid encoding the transmembrane domain, which is further operably linked to a third nucleic acid sequence encoding the intracellular domain.

[0166] The antigen binding domains described herein can be combined with any of the transmembrane domains described herein, any of the intracellular or cytoplasmic domains described herein, or any of the other domains described herein that can be included in a CAR of the invention.

[0167] In one aspect, the present invention includes an isolated HLA-A2-specific chimeric antigen receptor (CAR), comprising a CD8 signal peptide, an HLA-A2 VH domain, a spacer sequence, an HLA-A2 VL domain, a CD8 hinge region, a CD28 transmembrane domain, a CD28 costimulatory domain, and a CD3 zeta intracellular domain. In another aspect, the present invention includes an isolated nucleic acid encoding an HLA-A2-specific CAR, wherein the CAR comprises a CD8 signal peptide, an HLA-A2 VH domain, a spacer sequence, an HLA-A2 VL domain, a CD8 hinge region, a CD28 transmembrane domain, a CD28 costimulatory domain, and a CD3 zeta intracellular domain. Another aspect of the present invention includes an isolated polypeptide comprising a CD8 signal peptide, an HLA-A2 VH domain, a spacer sequence, an HLA-A2 VL domain, a CD8 hinge region, a CD28 transmembrane domain, and a CD3 zeta intracellular domain.

[0168] Another aspect of the present invention includes a genetically modified T cell comprising an isolated nucleic acid encoding an HLA-A2 specific CAR, wherein the CAR comprises a CD8 signal peptide, an HLA-A2 VH domain, a spacer sequence, an HLA-A2 VL domain, a CD8 hinge region, a CD28 transmembrane domain, a CD28 costimulatory domain, and a CD3 zeta intracellular domain. In some embodiments, the genetically modified immune cell (e.g., T cell) of the present invention comprises an HLA-A2 CAR, wherein the CAR comprises a CD8 signal peptide, an HLA-A2 VH domain, a spacer sequence, an HLA-A2 VL domain, a CD8 hinge, a CD28 transmembrane domain, a CD28 costimulatory domain, and a CD3 zeta intracellular domain. In some embodiments, the genetically modified immune cell (e.g., T cell) of the present invention or a precursor thereof comprises a chimeric antigen receptor (CAR) having affinity for HLA-A2. The CAR contains an HLA-A2 binding domain, a CD8 hinge domain, a CD8 signal peptide, a CD28 transmembrane domain, a CD28 costimulatory domain, and a CD3ζ intracellular domain.

[0169] In certain embodiments of the invention, the CAR is encoded by the nucleic acid sequence of SEQ ID NO: 24. In other embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 23.

[0170] In certain embodiments, the genetically modified T cells are T regulatory (Treg) cells.

[0171] The sequences of the individual domains are found in Table 1.

[0172] [Table 1] TIFF0007618192000002.tif222153TIFF0007618192000003.tif228153

[0173] Thus, the subject CAR may be a CAR with affinity for HLA-A2, comprising an HLA-A2 binding domain comprising the amino acid sequence set forth in SEQ ID NO:1. The subject HLA-A2 CAR may further comprise a hinge domain comprising the amino acid sequence set forth in SEQ ID NO:15. The subject HLA-A2 CAR may further comprise a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:17. The subject HLA-A2 CAR may further comprise an intracellular domain comprising the amino acid sequence set forth in SEQ ID NO:17. The subject HLA-A2 CAR may comprise the amino acid sequence set forth in SEQ ID NO:23.

[0174] In certain embodiments, the subject HLA-A2 CARs of the present invention comprise affinity for HLA-A2, HLA-A28, and / or HLA-A68, independent of peptide presentation by these MHC class I molecules. For example, in certain embodiments, the specificity of the subject HLA-A2 CARs of the present invention is not affected by presentation of any peptides by HLA-A2 molecules.

[0175] Antigen-binding domain The antigen-binding domain of CAR is the extracellular region of CAR for binding to specific target antigens, including proteins, carbohydrates and glycolipids.In some embodiments, CAR comprises affinity to target antigens on target cells.Target antigens can comprise any type of protein or epitope thereof associated with target cells.For example, CAR can comprise affinity to target antigens on target cells, which indicates a certain state of target cells.

[0176] In one embodiment, the CAR of the present invention comprises an antigen binding domain that binds to HLA-A2, HLA-A28, and / or HLA-A68. In another embodiment, the antigen binding domain of the present invention comprises an antibody or fragment thereof that binds to HLA-A2, HLA-A28, and / or HLA-A68 molecules. Preferably, the antigen binding domain is an scFv antibody that binds to HLA-A2, HLA-A28, and / or HLA-A68 molecules. The selection of the antigen binding domain depends on the type and number of antigens present on the surface of the target cell. For example, an antigen binding domain can be selected that recognizes an antigen that acts as a cell surface marker on the target cell that is associated with a particular state of the target cell.

[0177] As described herein, a CAR of the present disclosure having affinity for a specific target antigen on a target cell may comprise a target-specific binding domain. In some embodiments, the target-specific binding domain is a mouse target-specific binding domain, e.g., the target-specific binding domain is of mouse origin. In some embodiments, the target-specific binding domain is a human target-specific binding domain, e.g., the target-specific binding domain is of human origin. In exemplary embodiments, a CAR of the present disclosure having affinity for HLA-A2 on a target cell may comprise an HLA-A2 binding domain. In some embodiments, the HLA-A2 binding domain is a mouse HLA-A2 binding domain, e.g., the HLA-A2 binding domain is of mouse origin. In some embodiments, the HLA-A2 binding domain is a humanized HLA-A2 binding domain. In some embodiments, the HLA-A2 binding domain is a human HLA-A2 binding domain, e.g., the HLA-A2 binding domain is of human origin.

[0178] In some embodiments, the CAR of the present disclosure may have affinity for one or more target antigens on one or more target cells. In some embodiments, the CAR may have affinity for one or more target antigens on a target cell. In such embodiments, the CAR is a bispecific CAR or a multispecific CAR. In some embodiments, the CAR comprises one or more target-specific binding domains that confer affinity for one or more target antigens. In some embodiments, the CAR comprises one or more target-specific binding domains that confer affinity for the same target antigen. For example, a CAR that comprises one or more target-specific binding domains that have affinity for the same target antigen can also bind to separate epitopes of the target antigen. When multiple target-specific binding domains are present in a CAR, the binding domains can be arranged tandemly and separated by a linker peptide. For example, in a CAR that comprises two target-specific binding domains, the binding domains are covalently connected to each other on a single polypeptide chain through an oligo- or polypeptide linker, an Fc hinge region, or a membrane hinge region.

[0179] The antigen binding domain can include any domain that binds to an antigen, including, but not limited to, a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody, and any fragment thereof. Thus, in one embodiment, the antigen binding domain portion comprises a mammalian antibody or a fragment thereof. In another embodiment, the antigen binding domain of the CAR is selected from the group consisting of an anti-HLA-A2 antibody or a fragment thereof. In some embodiments, the antigen binding domain is selected from the group consisting of an antibody, an antigen binding fragment (Fab) and a single chain variable fragment (scFv). In some embodiments, the HLA-A2 binding domain of the present invention is selected from the group consisting of an HLA-A2 specific antibody, an HLA-A2 specific Fab and an HLA-A2 specific scFv. In one embodiment, the HLA-A2 binding domain is an HLA-A2 specific antibody. In one embodiment, the HLA-A2 binding domain is an HLA-A2 specific Fab. In one embodiment, the HLA-A2 binding domain is an HLA-A2 specific scFv.

[0180] As used herein, the term "single-chain variable fragment" or "scFv" refers to a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulins (e.g., mouse or human) covalently linked to form a VH::VL heterodimer. The heavy (VH) and light (VL) chains are either directly connected or connected by a peptide-encoding linker connecting the N-terminus of the VH and the C-terminus of the VL, or the C-terminus of the VH and the N-terminus of the VL. In some embodiments, the antigen-binding domain (e.g., HLA-A2 binding domain) comprises an scFv having the following configuration from N-terminus to C-terminus: VH-linker-VL. In some embodiments, the antigen-binding domain (e.g., HLA-A2 binding domain) comprises an scFv having the following configuration from N-terminus to C-terminus: VL-linker-VH. Those skilled in the art will be able to select the appropriate configuration for use in the present invention.

[0181] The linker is usually glycine-rich for flexibility and serine or threonine-rich for solubility. The linker can link the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. Non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem. 80(6):1910-1917 (2008) and WO 2014 / 087010, the contents of which are incorporated herein by reference in their entirety. A variety of linker sequences are known in the art, including, but not limited to, glycine serine (GS) linkers such as TIFF0007618192000004.tif4164, where n represents an integer of at least 1. An exemplary linker sequence is: TIFF0007618192000005.tif18140 and the like. A person skilled in the art will be able to select an appropriate linker sequence for use in the present invention. In one embodiment, the antigen-binding domain of the present invention (e.g., HLA-A2 binding domain) comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL are represented by the amino acid sequence TIFF0007618192000006.tif4128, which is separated by a linker sequence having the nucleic acid sequence It can be coded by TIFF0007618192000007.tif4128.

[0182] Despite the removal of the constant region and the introduction of the linker, the scFv protein retains the specificity of the original immunoglobulin. As described by Huston, et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988), a single chain Fv polypeptide antibody can be expressed from a nucleic acid comprising a sequence encoding a VH and a sequence encoding a VL. See also U.S. Patent Nos. 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Publication Nos. 20050196754 and 20050196754. Antagonist scFvs with inhibitory activity have been described (see, e.g., Zhao et al., Hyrbidoma (Larchmt) 2008 27(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle 2012 August 12; Shieh et al., J Imunol 2009 183(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63; Fife et al., J Clin Invst 2006 116(8):2252-61; Brocks et al., Immunotechnology 1997 3(3):173-84; Moosmayer et al., Ther Immunol 1995 2(10:31-40)). Agonistic scFvs with stimulatory activity have been described (see, e.g., Peter et al., J Bioi Chem 2003 25278(38):36740-7; Xie et al., Nat Biotech 1997 15(8):768-71; Ledbetter et al., Crit Rev Immunol 1997 17(5-6):427-55; Ho et al., BioChim Biophys Acta 2003 1638(3):257-66).

[0183] As used herein, "Fab" refers to the fragment of an antibody structure that binds to an antigen but is monovalent and does not have an Fc portion; for example, antibodies digested with the enzyme papain produce two Fab fragments and an Fc fragment (e.g., heavy chain (H) constant region; the Fc region that does not bind to antigen).

[0184] As used herein, "F(ab')2" refers to an antibody fragment produced by pepsin digestion of a whole IgG antibody, which has two antigen-binding (ab') (bivalent) regions, each (ab') region containing two separate amino acid chains, a portion of a H chain and a light (L) chain, linked by an S-S bond to bind to an antigen, and the remaining portions of the H chain linked together. The "F(ab')2" fragment can be split into two individual Fab' fragments.

[0185] In some cases, the antigen binding domain can be derived from the same species in which the CAR will ultimately be used. For example, for use in humans, the antigen binding domain of the CAR can comprise a human antibody or fragment thereof as described elsewhere herein.

[0186] In an exemplary embodiment, the HLA-A2 CAR of the present invention comprises an HLA-A2 binding domain, e.g., an HLA-A2-specific scFv. In one embodiment, the HLA-A2 binding domain comprises the amino acid sequence shown in SEQ ID NO:1.

[0187] In one embodiment, the HLA-A2 binding domain comprises a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:8. The light chain variable region of the HLA-A2 binding domain comprises three light chain complementarity determining regions (CDRs). As used herein, "complementarity determining region" or "CDR" refers to the region of the variable chain of an antigen binding molecule that binds to a specific antigen. Thus, the HLA-A2 binding domain may comprise a light chain variable region comprising CDR1 comprising the amino acid sequence shown in SEQ ID NO:10; CDR2 comprising the amino acid sequence shown in SEQ ID NO:11; and CDR3 comprising the amino acid sequence shown in SEQ ID NO:12.

[0188] In one embodiment, the HLA-A2 binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 3. The HLA-A2 binding domain may comprise a heavy chain variable region comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 6; and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7.

[0189] Acceptable variations of the HLA-A2 binding domain while maintaining specific binding to HLA-A2 will be known to those skilled in the art. For example, in some embodiments, the HLA-A2 binding domain comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% sequence identity to any of the amino acid sequences shown in SEQ ID NOs: 3, 5-8 and 10-12. For example, in some embodiments, the HLA-A2 binding domain is encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% sequence identity to any of the nucleic acid sequences set forth in SEQ ID NOs:2, 4 and 9.

[0190] The antigen binding domain can be operably linked to another domain of the CAR, such as a transmembrane domain or an intracellular domain, both of which are described elsewhere herein. In one embodiment, the nucleic acid encoding the antigen binding domain is operably linked to a nucleic acid encoding the transmembrane domain and a nucleic acid encoding the intracellular domain.

[0191] An antigen binding domain described herein, e.g., an antibody or fragment thereof that binds to HLA-A2, HLA-A28, and / or HLA-A68, can be combined with any of the transmembrane domains described herein, any of the intracellular or cytoplasmic domains described herein, or any of the other domains described herein that may be included in a CAR.

[0192] Transmembrane domain With regard to the transmembrane domain, the CAR of the present invention (e.g., HLA-A2 CAR) can be designed to include a transmembrane domain that connects the antigen-binding domain of the CAR to the intracellular domain. The transmembrane domain of the subject CAR is a region that can penetrate the plasma membrane of a cell (e.g., an immune cell or its precursor). The transmembrane domain is for insertion into a cell membrane, e.g., a eukaryotic cell membrane. In some embodiments, the transmembrane domain is interposed between the antigen-binding domain and the intracellular domain of the CAR.

[0193] In one embodiment, the transmembrane domain is naturally associated with one or more of the domains in the CAR. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domain to the transmembrane domain of the same or different surface membrane protein in order to minimize interaction with other members of the receptor complex.

[0194] The transmembrane domain can be derived from either natural or synthetic sources.When the source is natural, the domain can be derived from any membrane-binding or transmembrane protein, for example, type I transmembrane protein.When the source is synthetic, the transmembrane domain can be any artificial sequence, for example, an artificial hydrophobic sequence, that facilitates the insertion of CAR into cell membrane. Examples of transmembrane regions of particular use in the present invention include, but are not limited to, transmembrane domains derived from (i.e., comprising at least the transmembrane regions of) the α, β or ζ chains of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8 and TLR9. In some embodiments, the transmembrane domain may be synthetic, in which case it will primarily comprise hydrophobic residues such as leucine and valine. Preferably, at each end of the synthetic transmembrane domain, a triad of phenylalanine, tryptophan and valine is found.

[0195] The transmembrane domains described herein can be combined with any of the antigen binding domains described herein, any of the intracellular domains described herein, or any of the other domains described herein that can be included in the subject CAR.

[0196] In some embodiments, the transmembrane domain further comprises a hinge region. The CAR of the subject of the present invention may also comprise a hinge region. The hinge region of the CAR is a hydrophilic region located between the antigen binding domain and the transmembrane domain. In some embodiments, this domain facilitates proper protein folding for the CAR. The hinge region is an optional component for the CAR. The hinge region may comprise a domain selected from an antibody Fc fragment, an antibody hinge region, an antibody CH2 region, an antibody CH3 region, an artificial hinge sequence, or a combination thereof. Examples of hinge regions include, but are not limited to, the CD8a hinge, an artificial hinge composed of a polypeptide that may be as small as three glycines (Gly), and the CH1 and CH3 domains of IgG (such as human IgG4).

[0197] In some embodiments, the CAR of the present disclosure includes a hinge region that connects the antigen binding domain to the transmembrane domain, which in turn connects it to the intracellular domain. The hinge region can preferably assist the antigen binding domain to recognize and bind to the target antigen on the target cell (see, e.g., Hudecek et al., Cancer Immunol. Res. (2015) 3(2): 125-135). In some embodiments, the hinge region is a flexible domain, thereby allowing the antigen binding domain to have a structure that optimally recognizes the specific structure and density of the target antigen on a cell, such as a tumor cell (Hudecek et al., supra). The flexibility of the hinge region allows it to adopt many different conformations.

[0198] In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. In some embodiments, the hinge region is a receptor-derived hinge region polypeptide (e.g., a CD8-derived hinge region).

[0199] The hinge region can have a length of about 4 amino acids to about 50 amino acids, for example, about 4 aa to about 10 aa, about 10 aa to about 15 aa, about 15 aa to about 20 aa, about 20 aa to about 25 aa, about 25 aa to about 30 aa, about 30 aa to about 40 aa, or about 40 aa to about 50 aa.

[0200] A suitable hinge region can be readily selected and can be of any of a number of suitable lengths, for example, comprising from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 to 15 amino acids, from 3 to 12 amino acids, for example, from 4 to 10 amino acids, from 5 to 9 amino acids, from 6 to 8 amino acids, or from 7 to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0201] For example, the hinge region may be a glycine polymer (G) n , glycine-serine polymers (e.g. TIFF0007618192000008.tif4128, where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are less structured and therefore can serve as neutral tethers between components. Glycine polymers can be used; glycine has significantly more access to the phi-psi space than the equivalent alanine and is much less restricted than residues with longer side chains (see, e.g., Scheraga, Rev. Computational. Chem. (1992) 2: 73-142). Exemplary hinge regions include: The amino acid sequence may include, but is not limited to, TIFF0007618192000009.tif18133, etc.

[0202] In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. Immunoglobulin hinge region amino acid sequences are known in the art; see, for example, Tan et al., Proc. Natl. Acad. Sci. USA (1990) 87(1):162-166; and Huck et al., Nucleic Acids Res. (1986) 14(4): 1779-1789. As a non-limiting example, an immunoglobulin hinge region can include one of the following amino acid sequences: TIFF0007618192000010.tif4153 (see, e.g., Glaser et al., J. Biol. Chem. (2005) 280:41494-41503); TIFF0007618192000011.tif39143; etc.

[0203] The hinge region can comprise the amino acid sequence of a human IgG1, IgG2, IgG3 or IgG4 hinge region. In one embodiment, the hinge region can comprise one or more amino acid substitutions and / or insertions and / or deletions compared to a wild-type (naturally occurring) hinge region. For example, His229 of the human IgG1 hinge can be replaced with Tyr, thereby resulting in a hinge region having the sequence see, e.g., Yan et al., J. Biol. Chem. (2012) 287: 5891-5897. In one embodiment, the hinge region can comprise an amino acid sequence derived from human CD8, or a variant thereof.

[0204] In one embodiment, the transmembrane domain comprises a CD28 transmembrane domain. In another embodiment, the transmembrane domain comprises a CD8 hinge domain and a CD28 transmembrane domain. In some embodiments, the subject CAR comprises a CD8 hinge region having the amino acid sequence shown in SEQ ID NO:15, which can be encoded by the nucleic acid sequence shown in SEQ ID NO:16. In some embodiments, the subject CAR comprises a CD28 transmembrane domain having the amino acid sequence shown in SEQ ID NO:17, which can be encoded by the nucleic acid sequence shown in SEQ ID NO:18. In some embodiments, the transmembrane domain comprises a CD8 hinge region and a CD28 transmembrane domain.

[0205] Acceptable variations of the transmembrane and / or hinge domains while maintaining the intended function will be known to those of skill in the art. For example, in some embodiments, the hinge and / or transmembrane domains comprise an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% sequence identity to any of the amino acid sequences set forth in SEQ ID NOs:15 and / or 17. For example, in some embodiments, the hinge domain and / or transmembrane domain are encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% sequence identity to any of the nucleic acid sequences set forth in SEQ ID NOs:16 and / or 18.

[0206] The transmembrane domain may be combined with any hinge domain and / or may comprise one or more of the transmembrane domains described herein.

[0207] The transmembrane domains described herein, e.g., the alpha, beta or zeta chains of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8 and TLR9, can be combined with any of the antigen binding domains described herein, any of the intracellular or cytoplasmic domains described herein, or any of the other domains described herein that may be included in a CAR.

[0208] In one embodiment, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. Preferably, a triad of phenylalanine, tryptophan and valine is found at each end of the synthetic transmembrane domain.

[0209] A spacer domain may be incorporated between the extracellular and transmembrane domains of the CAR, or between the intracellular and transmembrane domains of the CAR. As used herein, the term "spacer domain" generally refers to any oligo- or polypeptide that functions to link the transmembrane domain to either the extracellular or intracellular domain within a polypeptide chain. A spacer domain may comprise up to 300 amino acids, e.g., 10-100 amino acids, or 25-50 amino acids. In some embodiments, the spacer domain may be a short, e.g., 2-10 amino acid long, oligo- or polypeptide linker. For example, a glycine-serine duo provides a particularly suitable linker between the transmembrane and intracellular signaling domains of the subject CAR.

[0210] Intracellular domain The CAR of the present invention also comprises an intracellular signaling domain. The terms "intracellular signaling domain" and "intracellular domain" are used interchangeably herein. The intracellular signaling domain of CAR is responsible for activating at least one effector function of the cell (e.g., immune cell) in which the CAR is expressed. The intracellular signaling domain transmits effector function signals and directs the cell (e.g., immune cell) to carry out its special function, such as damaging and / or destroying target cells.

[0211] The intracellular domain or otherwise cytoplasmic domain of the CAR comprises an intracellular domain similar or identical to the chimeric intracellular signaling molecule described elsewhere herein, and is responsible for the activation of the cell in which the CAR is expressed. In one embodiment, the intracellular domain comprises CD3zeta. In another embodiment, the intracellular domain comprises CD28 and CD3zeta.

[0212] Examples of intracellular domains for use in the present invention include, but are not limited to, the cytoplasmic portion of a surface receptor, a costimulatory molecule, and any molecules that act in concert to initiate signaling in a T cell, as well as any derivatives or variants of these elements and any synthetic sequences having the same functional capabilities.

[0213] Examples of intracellular signaling domains include, but are not limited to, the zeta chain of the T cell receptor complex or any of its homologs, such as the eta chain, FcsRI gamma and beta chains, MB1 (Iga) chain, B29 (Ig) chain, human CD3 zeta chain, CD3 polypeptides (Δ, δ and ε), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.), and other molecules involved in T cell transduction such as CD2, CD5 and CD28. In one embodiment, the intracellular signaling domain can be human CD3 zeta chain, FcyRIII, FcsRI, the cytoplasmic tail of an Fc receptor, an immunoreceptor tyrosine-based activation motif (ITAM)-bearing cytoplasmic receptor, and combinations thereof.

[0214] In one embodiment, the intracellular domain of the CAR comprises at least one signaling domain from any portion of one or more costimulatory molecules, e.g., CD3, CD8, CD27, CD28, ICOS, 4-IBB, PD-1, any derivative or variant thereof, any synthetic sequence thereof having the same functional capability, and any combination thereof.

[0215] Other examples of intracellular domains are TCR, CD3ζ, CD3γ, CD3δ, CD3ε, CD86, common FcRγ, FcRβ (FcεRib), CD79a, CD79b, FcγRlla, DAP10, DAP 12, T cell receptor (TCR), CD8, CD27, CD28, 4-1BB (CD137), OX9, OX40, CD30, CD40, PD-1, ICOS, KIR family proteins, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, CD83-specific binding ligand, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD lib, ITGAX, CD 11c, ITGBl, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT The co-stimulatory molecules include fragments or domains derived from one or more molecules or receptors, including, but not limited to, AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, other co-stimulatory molecules described herein, any derivative, variant, or fragment thereof, any synthetic sequence of a co-stimulatory molecule having the same functional capability, and any combination thereof.

[0216] Additional examples of intracellular domains include the intracellular signaling domains of several different other immune signaling receptors, including, but not limited to, first, second and third generation T cell signaling proteins, including, but not limited to, CD3, B7 family costimulatory receptors and tumor necrosis factor receptor (TNFR) superfamily receptors (see, e.g., Park and Brentjens, J. Clin. Oncol. (2015) 33(6): 651-653). Additionally, intracellular signaling domains can include signaling domains used by NK cells and NKT cells (see, e.g., Hermanson and Kaufman, Front. Immunol. (2015) 6: 195), such as the signaling domains of NKp30(B7-H6) (see, e.g., Zhang et al., J. Immunol. (2012) 189(5): 2290-2299), and DAP 12 (see, e.g., Topfer et al., J. Immunol. (2015) 194(7): 3201-3212), NKG2D, NKp44, NKp46, DAP10, and CD3z.

[0217] Intracellular signaling domains suitable for use in the subject CARs of the present invention include any desired signaling domain that provides a distinct and detectable signal (e.g., increased production of one or more cytokines by the cell; altered transcription of a target gene; altered activity of a protein; altered cell behavior, e.g., cell death; cell proliferation; cell differentiation; cell survival; modulation of cell signaling responses; etc.) in response to activation of the CAR (i.e., activated by an antigen and a dimerization agent). In some embodiments, the intracellular signaling domain comprises at least one (e.g., one, two, three, four, five, six, etc.) ITAM motif as described below. In some embodiments, the intracellular signaling domain comprises a DAP10 / CD28-type signaling chain. In some embodiments, the intracellular signaling domain is not covalently attached to the membrane-bound CAR, but is instead diffused in the cytoplasm.

[0218] The intracellular signaling domain suitable for use in the subject CAR of the present invention comprises an immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptide. In some embodiments, the ITAM motif is repeated twice within the intracellular signaling domain, where the first and second instances of the ITAM motif are separated from each other by 6-8 amino acids. In one embodiment, the intracellular signaling domain of the subject CAR comprises three ITAM motifs.

[0219] In some embodiments, the intracellular signaling domain comprises a signaling domain of a human immunoglobulin receptor containing an immunoreceptor tyrosine-based activation motif (ITAM), such as, but not limited to, FcγRI, FcγRIIA, FcγRIIC, FcγRIIIA, FcRL5 (see, e.g., Gillis et al., Front. Immunol. (2014) 5:254).

[0220] A suitable intracellular signaling domain can be an ITAM motif-containing portion derived from a polypeptide containing an ITAM motif. For example, a suitable intracellular signaling domain can be an ITAM motif-containing domain derived from any ITAM motif-containing protein. Thus, a suitable intracellular signaling domain does not need to contain the entire sequence of the entire protein from which it is derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to, DAP12, FCER1G (Fcε receptor Iγ chain), CD3D (CD3δ), CD3E (CD3ε), CD3G (CD3γ), CD3Z (CD3ζ), and CD79A (antigen receptor complex-associated protein α chain).

[0221] In one embodiment, the intracellular signaling domain is derived from DAP12 (also known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP; PLOSL; DNAX-activating protein 12; KAR associated protein; TYRO protein tyrosine kinase binding protein; killer activating receptor associated protein; killer activating receptor associated protein; etc.). In one embodiment, the intracellular signaling domain is derived from FCER1G (also known as FCRG; Fcε receptor Iγ chain; Fc receptor γ chain; fc-εRI-γ; fcRγ; fceRlγ; high affinity immunoglobulin ε receptor subunit γ; immunoglobulin E receptor, high affinity γ chain; etc.). In one embodiment, the intracellular signaling domain is derived from T cell surface glycoprotein CD3δ chain (also known as CD3D; CD3-DELTA; T3D; CD3 antigen, δ subunit; CD3δ; CD3d antigen, δ polypeptide (TiT3 complex); OKT3, δ chain; T cell receptor T3 δ chain; T cell surface glycoprotein CD3 δ chain; etc.). In one embodiment, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 epsilon chain (also known as CD3e, T cell surface antigen T3 / Leu-4 epsilon chain, T cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3 epsilon, T3e, etc.). In one embodiment, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 gamma chain (also known as CD3G, T cell receptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.). In one embodiment, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 zeta chain (also known as CD3Z, T cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.). In one embodiment, the intracellular signaling domain is derived from CD79A (also known as B cell antigen receptor complex-associated protein alpha chain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membrane glycoprotein; ig-α; membrane-associated immunoglobulin-associated protein; surface IgM-associated protein; etc.).In one embodiment, the intracellular signaling domain suitable for use in the FN3 CAR of the present disclosure comprises a DAP10 / CD28 type signaling chain. In one embodiment, the intracellular signaling domain suitable for use in the FN3 CAR of the present disclosure comprises a ZAP70 polypeptide. In some embodiments, the intracellular signaling domain comprises the cytoplasmic signaling domain of TCRzeta, FcRgamma, FcRbeta, CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b or CD66d. In one embodiment, the intracellular signaling domain in the CAR comprises the cytoplasmic signaling domain of human CD3zeta.

[0222] Typically, the entire intracellular signaling domain can be used, but in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain so long as it transmits an effector function signal. The intracellular signaling domain includes any truncated portion of the intracellular signaling domain that is sufficient to transmit an effector function signal.

[0223] The intracellular signaling domains described herein can be combined with any of the antigen binding domains described herein, any of the transmembrane domains described herein, or any of the other domains described herein that can be included in a CAR.

[0224] In one embodiment, the intracellular domain of the subject CAR comprises a CD28 intracellular domain comprising the amino acid sequence depicted in SEQ ID NO:19, which can be encoded by the nucleic acid sequence depicted in SEQ ID NO:20. In one embodiment, the intracellular domain of the subject CAR comprises a CD3 zeta domain comprising the amino acid sequence depicted in SEQ ID NO:21, which can be encoded by the nucleic acid sequence depicted in SEQ ID NO:22. In an exemplary embodiment, the intracellular domain of the subject CAR comprises a CD28 domain and a CD3 zeta domain.

[0225] Acceptable variations of the intracellular domain while maintaining specific activity will be known to those of skill in the art. For example, in some embodiments, the intracellular domain comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% sequence identity to any of the amino acid sequences set forth in SEQ ID NOs:19 and / or 21. For example, in some embodiments, the intracellular domain is encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% sequence identity to any of the nucleic acid sequences set forth in SEQ ID NOs:20 and / or 22.

[0226] In another embodiment, a spacer domain can be incorporated between the antigen binding domain and the transmembrane domain of the CAR, or between the intracellular domain and the transmembrane domain of the CAR. As used herein, the term "spacer domain" generally refers to any oligo- or polypeptide that functions to link the transmembrane domain to either the antigen binding domain or the intracellular domain within a polypeptide chain. In one embodiment, the spacer domain can comprise up to 300 amino acids, preferably 10-100 amino acids, most preferably 25-50 amino acids. In another embodiment, a short, preferably 2-10 amino acid long, oligo- or polypeptide linker can form a link between the transmembrane domain and the intracellular domain of the CAR. One example of a linker comprises a glycine-serine dyad.

[0227] CAR sequence The subject CAR of the present invention may be a CAR having affinity for HLA-A2. In one embodiment, the HLA-A2 CAR of the present invention comprises the amino acid sequence shown in SEQ ID NO:23, which may be encoded by the nucleic acid sequence shown in SEQ ID NO:24.

[0228] Acceptable variations of CARs while maintaining specific activity will be known to those of skill in the art. For example, in some embodiments, the CAR comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% sequence identity to the amino acid sequence shown in SEQ ID NO:23. For example, in some embodiments, the CAR is encoded by a nucleic acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:24.

[0229] Thus, the CAR of the present invention comprises an HLA-A2 binding domain and a transmembrane domain.In one embodiment, the CAR comprises an HLA-A2 binding domain and a transmembrane domain, and the transmembrane domain comprises a CD8 hinge region.In one embodiment, the CAR comprises an HLA-A2 binding domain and a transmembrane domain, and the transmembrane domain comprises a CD28 transmembrane domain.In one embodiment, the CAR comprises an HLA-A2 binding domain and a transmembrane domain, and the transmembrane domain comprises a CD8 hinge region and a CD28 transmembrane domain.

[0230] Thus, the CAR of the present invention comprises an HLA-A2 binding domain, a transmembrane domain and an intracellular domain.In one embodiment, the CAR comprises an HLA-A2 binding domain, a transmembrane domain and an intracellular domain, and the intracellular domain comprises a CD28 domain.In one embodiment, the CAR comprises an HLA-A2 binding domain, a transmembrane domain and an intracellular domain, and the intracellular domain comprises a CD3ζ domain.In one embodiment, the CAR comprises an HLA-A2 binding domain, a transmembrane domain and an intracellular domain, and the intracellular domain comprises a CD28 domain and a CD3ζ domain.

[0231] Thus, the CAR of interest of the present invention comprises an HLA-A2 binding domain, a CD8 hinge region, a CD28 transmembrane domain, a CD28 intracellular domain and a CD3 zeta intracellular domain.

[0232] Thus, the present invention provides modified immune cells or precursors thereof, e.g., modified regulatory T cells, comprising a chimeric antigen receptor (CAR) having affinity for HLA-A2 as described herein.

[0233] Human antibodies It may be preferred that the antigen-binding domain of CAR comprises a human antibody or a fragment thereof. Fully human antibodies are particularly desirable for therapeutic treatment of human subjects. Human antibodies can be produced by a variety of methods known in the art, including phage display methods (including improvements to these techniques) using antibody libraries derived from human immunoglobulin sequences. See also U.S. Patent Nos. 4,444,887 and 4,716,111; and PCT publications WO 98 / 46645, WO 98 / 50433, WO 98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33735 and WO 91 / 10741; each of which is incorporated herein by reference in its entirety.

[0234] Human antibodies can also be produced using transgenic mice that are incapable of expressing functional endogenous immunoglobulins but can express human immunoglobulin genes. For example, human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, human variable, constant and diversity regions may be introduced into mouse embryonic stem cells in addition to human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. For example, it has been described that homozygous deletion of antibody heavy chain joining region (JH) genes in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring that express human antibodies. The transgenic mice are immunized in the usual manner with a selected antigen, such as all or a portion of a polypeptide of the invention. Antibodies against selected targets can be obtained from immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes carried by the transgenic mice rearrange during B cell differentiation and subsequently undergo class switching and somatic mutation. Thus, using such techniques, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies, including but not limited to IgG1 (γ1) and IgG3. For a review of this technology for producing human antibodies, see Lonberg and Huszar (Int. Rev. Immunol., 13:65-93 (1995)).For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies, as well as protocols for producing such antibodies, see, e.g., PCT Publication Nos. WO 98 / 24893, WO 96 / 34096, and WO 96 / 33735; and U.S. Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; and 5,939,598, each of which is incorporated herein by reference in its entirety. In addition, companies such as Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.) may be engaged in providing human antibodies against a selected antigen using technology similar to that described above. For specific discussion of the transfer of human germ-line immunoglobulin gene arrays into germ-line mutant mice, which will result in the production of human antibodies upon antigen challenge, see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993); and Duchosal et al., Nature, 355:258 (1992).

[0235] Human antibodies can also be derived from phage display libraries (Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581-597 (1991); Vaughan et al., Nature Biotech., 14:309 (1996)). Using phage display technology (McCafferty et al., Nature, 348:552-553 (1990)), human antibodies and antibody fragments can be produced in vitro from immunoglobulin variable (V) domain gene repertoires from unimmunized donors. According to this technique, antibody V domain genes are cloned in frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as Ml 3 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Since the filamentous particle contains a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also results in the selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of B cells. Phage display can be performed in a wide variety of formats; for their reviews, see, for example, Johnson, Kevin S, and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). Several sources of V gene segments can be used for phage display. Clackson et al., Nature, 352:624-628 (1991) isolated a wide variety of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of unimmunized mice.Repertoires of V genes from unimmunized human donors can be constructed and antibodies to a wide variety of antigens (including self-antigens) can be isolated essentially following the techniques described in Marks et al., J. Mol. Biol., 222:581-597 (1991), or Griffith et al., EMBO J., 12:725-734 (1993). See also U.S. Patent Nos. 5,565,332 and 5,573,905, each of which is incorporated herein by reference in its entirety.

[0236] Human antibodies may also be generated by in vitro activated B cells (see U.S. Patent Nos. 5,567,610 and 5,229,275, each of which is incorporated by reference in its entirety.) Human antibodies may also be generated in vitro using hybridoma techniques, such as, but not limited to, those described in Roder et al. (Methods Enzymol., 121:140-167 (1986)).

[0237] Humanized antibodies Alternatively, in some embodiments, non-human antibodies can be humanized, in which certain sequences or regions of the antibody are modified to increase similarity with the antibodies naturally produced in humans. For example, in the present invention, the antibody or its fragment can include the scFv of a mammal other than human. In one embodiment, the antigen-binding domain portion is humanized.

[0238] Humanized antibodies can be prepared by a number of techniques, including 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 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., 1994, PNAS, 91:969-973, each of which is incorporated by reference in its entirety), chain shuffling (see, e.g., U.S. Pat. No. 5,565,332).No. 6,407,213, U.S. Pat. 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., 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. Often, framework residues in the framework regions are substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, for example, 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 particular 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 by reference in their entireties).

[0239] A humanized antibody has one or more amino acid residues introduced into it that are derived from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically obtained from an "import" variable domain. Thus, a humanized antibody comprises one or more CDRs from a non-human immunoglobulin molecule and a framework region from a human. Antibody humanization is well known in the art and can be performed by essentially substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody, i.e., CDR grafting, 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)) (EP 239,400; PCT Publication No. WO 2003 / 0231112). 91 / 09967; and U.S. Patent 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 entirety). In such humanized chimeric antibodies, substantially non-intact human variable domains are replaced by corresponding sequences from non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some framework (FR) residues are replaced by residues from analogous sites in rodent antibodies.Humanization of antibodies can also be achieved by veneering or resurfacing (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 shuffling (U.S. Patent No. 5,565,332), the contents of which are incorporated herein by reference in their entireties.

[0240] The selection of both light and heavy human variable domains to be used in the production of humanized antibodies is to reduce antigenicity. According to the so-called "best-fit" method, the sequence of the variable domain of rodent antibodies is screened against the entire library of known human variable domain sequences. The human sequence that is closest to that of rodent is then accepted as the human framework (FR) 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 derived from the consensus sequence of all human antibodies of a specific subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (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).

[0241] Antibodies can be humanized with retention of high affinity for the target antigen and other favorable biological properties. According to one aspect of the invention, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences.

[0242] Three-dimensional immunoglobulin models are generally available and are familiar to those skilled in the art. Computer programs are available that illustrate and display the possible three-dimensional conformations of selected candidate immunoglobulin sequences. Examination of these displays allows analysis of the possible role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the residues that affect the ability of the candidate immunoglobulin to bind to a target antigen. In this way, FR residues can be selected and combined from the recipient and import sequences to achieve the desired antibody characteristic, such as increased affinity for the target antigen. In general, CDR residues are directly and most substantially involved in influencing antigen binding.

[0243] Humanized antibodies retain similar antigen specificity as the original antibody. However, certain humanization methods can be used to improve the binding affinity and / or specificity of the antibody to the target antigen using the method of "directed evolution" as described by Wu et al., J. Mol. Biol., 294:151 (1999), the contents of which are incorporated herein by reference in their entirety.

[0244] Nucleic acids and expression vectors The present invention provides a nucleic acid encoding a CAR having affinity for HLA-A2.As described herein, the subject CAR comprises an antigen binding domain (e.g., an HLA-A2 binding domain), a transmembrane domain and an intracellular domain.Thus, the present invention provides a nucleic acid encoding the antigen binding domain (e.g., an HLA-A2 binding domain), a transmembrane domain and an intracellular domain of the subject CAR.

[0245] In an exemplary embodiment, the nucleic acid encoding the HLA-A2 CAR of the present invention is encoded by the nucleic acid sequence shown in SEQ ID NO:24.

[0246] In some embodiments, the nucleic acids of the present disclosure may be operably linked to transcriptional control elements, such as promoters and enhancers. Suitable promoter and enhancer elements are known to those of skill in the art.

[0247] For expression in bacterial cells, suitable promoters include, but are not limited to, lacI, lacZ, T3, T7, gpt, lambda P and trc. For expression in eukaryotic cells, suitable promoters include, but are not limited to, light and / or heavy chain immunoglobulin gene promoter and enhancer elements; cytomegalovirus immediate early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoter; promoters present in long terminal repeats from retroviruses; mouse metallothionein-I promoter; and various tissue-specific promoters known in the art. Suitable reversible promoters, including reversible inducible promoters, are known in the art. Such reversible promoters may be isolated and can be derived from many organisms, e.g., eukaryotes and prokaryotes. Modification of reversible promoters from a first organism for use in a second organism (e.g., first prokaryote and second eukaryote, first eukaryote and second prokaryote, etc.) is well known in the art.Such reversible promoters, and systems based on such reversible promoters but also including additional regulatory proteins, include promoters regulated by alcohol (e.g., the alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator protein (A1cR), etc.), promoters regulated by tetracycline (e.g., promoter systems including TetActivators, TetON, TetOFF, etc.), promoters regulated by steroids (e.g., the rat glucocorticoid receptor promoter system, the human estrogen receptor promoter system, the retinoid promoter system, thyroid promoter system, ecdysone promoter system, mifepristone promoter system, etc.), metal-regulated promoters (e.g., metallothionein promoter system, etc.), pathogenicity-regulated promoters (e.g., salicylic acid-regulated promoters, ethylene-regulated promoters, benzothiadiazole-regulated promoters, etc.), temperature-regulated promoters (e.g., heat shock-inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), light-regulated promoters, synthetic inducible promoters, etc., but are not limited to these.

[0248] In some embodiments, the promoter is a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, or a NK cell-specific promoter.For example, the CD4 gene promoter can be used; see, for example, Salmon et al. Proc. Natl. Acad. Sci. USA (1993) 90:7739; and Marodon et al. (2003) Blood 101:3416.As another example, the CD8 gene promoter can be used.NK cell-specific expression can be achieved by using the NcrI (p46) promoter; see, for example, Eckelhart et al. Blood (2011) 117:1565.

[0249] For expression in yeast cells, suitable promoters are constitutive promoters such as the ADH1 promoter, the PGK1 promoter, the ENO promoter, the PYK1 promoter, etc.; or regulatable promoters such as the GAL1 promoter, the GAL10 promoter, the ADH2 promoter, the PHOS promoter, the CUP1 promoter, the GALT promoter, the MET25 promoter, the MET3 promoter, the CYC1 promoter, the HIS3 promoter, the ADH1 promoter, the PGK promoter, the GAPDH promoter, the ADC1 promoter, the TRP1 promoter, the URA3 promoter, the LEU2 promoter, the ENO promoter, the TP1 promoter, and the AOX1 (e.g., for use in Pichia). The selection of the appropriate vector and promoter is well within the level of one skilled in the art. Suitable promoters for use in prokaryotic host cells include the bacteriophage T7 RNA polymerase promoter; the trp promoter; the lac operon promoter; hybrid promoters, such as the lac / tac hybrid promoter, the tac / trc hybrid promoter, the trp / lac promoter, the T7 / lac promoter; the trc promoter; the tac promoter, and the like; the araBAD promoter; in vivo regulated promoters, such as the ssaG promoter or related promoters (see, e.g., U.S. Patent Publication No. 20040131637), the pagC promoter (Pulkkinen and Miller, J. Bacteriol. (1991) 173(1): 86-93; Alpuche-Aranda et al., Proc. Natl. Acad. Sci. USA (1992) 89(21): 10079-83), the nirB promoter (Harborne et al. Mol. Micro. (1992) 6:2805-2813) (e.g., Dunstan et al., Infect. Immun. (1999) 67:5133-5141; McKelvie et al., Vaccine (2004) 22:3243-3255; and Chatfield et al., Biotechnol.(1992) 10:888-892); sigma70 promoters, e.g., the consensus sigma70 promoter (see, e.g., GenBank Accession Nos. AX798980, AX798961 and AX798183); stationary phase promoters, e.g., the dps promoter, the spv promoter, and the like; promoters from the pathogenicity island SPI-2 (see, e.g., WO96 / 17951); the actA promoter (see, e.g., Shetron-Rama et al., Infect. Immun. (2002) 70:1087-1096); the rpsM promoter (see, e.g., Valdivia and Falkow Mol. Microbiol. (1996). 22:367); the tet promoter (see, e.g., Hillen, W. and Wissmann, A. (1989) In Saenger, W. and Heinemann, U. (eds), Topics in Molecular and Structural Biology, Protein--Nucleic Acid Interaction. Macmillan, London, UK, Vol. 10, pp. 143-162); SP6 promoter (see, e.g., Melton et al., Nucl. Acids Res.(1984) 12:7035); and the like. Suitable strong promoters for use in prokaryotes, such as Escherichia coli, include, but are not limited to, Trc, Tac, T5, T7, and PLambda. Non-limiting examples of operators for use in bacterial host cells include the lactose promoter operator (the LacI repressor protein changes conformation upon contact with lactose, thereby preventing the Lad repressor protein from binding to the operator), the tryptophan promoter operator (when complexed with tryptophan, the TrpR repressor protein has a conformation that binds to the operator; in the absence of tryptophan, the TrpR repressor protein has a conformation that does not bind to the operator), and the tac promoter operator (see, e.g., deBoer et al., Proc. Natl. Acad. Sci. USA (1983) 80:21-25).

[0250] Other examples of suitable promoters include the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked to it. However, other constitutive promoter sequences may be used, including but not limited to the 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 virus immediate early promoter, Rous sarcoma virus promoter, EF-1α promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also envisioned as part of the present invention. The use of an inducible promoter provides a molecular switch that can enable expression of an operably linked polynucleotide sequence when such expression is desired and disable expression when expression is not desired. Examples of inducible promoters include, but are not limited to, a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0251] In some embodiments, the locus or construct or transgene containing a suitable promoter is irreversibly switched through the induction of an inducible system. Suitable systems for the induction of irreversible switches are well known in the art, for example, induction of irreversible switches can utilize Cre-lox mediated recombination (see, for example, Fuhrmann-Benzakein, et al., Proc. Natl. Acad. Sci. USA (2000) 28:e99, the disclosure of which is incorporated herein by reference). Any suitable combination of recombinases, endonucleases, ligases, recombination sites, etc. known in the art may be used in the generation of irreversibly switchable promoters. The methods, mechanisms and requirements for performing site-specific recombination described elsewhere herein are useful in the generation of irreversibly switched promoters and are well known in the art. See, e.g., Grindley et al. Annual Review of Biochemistry (2006) 567-605; and Tropp, Molecular Biology (2012) (Jones & Bartlett Publishers, Sudbury, Mass.), the disclosures of which are incorporated herein by reference.

[0252] In some embodiments, the nucleic acid of the present disclosure further comprises a nucleic acid sequence encoding a CAR-inducible expression cassette. In one embodiment, the CAR-inducible expression cassette is for the production of a transgenic polypeptide product that is released upon CAR signaling. See, for example, Chmielewski and Abken, Expert Opin. Biol. Ther. (2015) 15(8): 1145-1154; and Abken, Immunotherapy (2015) 7(5): 535-544.

[0253] The nucleic acids of the present disclosure may be present in an expression vector and / or a cloning vector. Expression vectors may include a selection marker, an origin of replication, and other features that provide for replication and / or maintenance of the vector. Suitable expression vectors include, for example, plasmids, viral vectors, and the like. Numerous suitable vectors and promoters are known to those of skill in the art; many are commercially available for generating recombinant constructs of interest. The following vectors are provided by way of example, but should in no way be construed as limiting: Bacteria: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).

[0254] Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker operable in the expression host may be present. Suitable expression vectors include viral vectors (e.g., vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest. Opthalmol. Vis. Sci. (1994) 35: 2543-2549; Borras et al., Gene Ther. (1999) 6: 515-524; Li and Davidson, Proc. Natl. Acad. Sci. USA (1995) 92: 7700-7704; Sakamoto et al., H. Gene Ther. (1999) 5: 1088-1097; WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655); adeno-associated virus (see, e.g., Ali et al., Hum. Gene Ther. (1998) 9: 81-86, Flannery et al., Proc. Natl. Acad. Sci. USA (1997) 94: 6916-6921; Bennett et al., Invest. Opthalmol. Vis. Sci. (1997) 38: 2857-2863; Jomary et al., Gene Ther. (1997) 4:683 690, Rolling et al., Hum. Gene Ther. (1999) 10: 641-648; Ali et al., Hum. Mol. Genet. (1996) 5: 591-594; Srivastava in WO 93 / 09239, Samulski et al. al., J. Vir. (1989) 63: 3822-3828; Mendelson et al., Virol. (1988) 166: 154-165; and Flotte et al., Proc. Natl. Acad. Sci.USA (1993) 90: 10613-10617); SV40; herpes simplex virus; viral vectors based on human immunodeficiency virus (see, e.g., Miyoshi et al., Proc. Natl. Acad. Sci. USA (1997) 94: 10319-23; Takahashi et al., J. Virol. (1999) 73: 7812-7816); retroviral vectors (e.g., murine leukemia virus, spleen necrosis virus), and vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like.

[0255] Additional expression vectors suitable for use include, but are not limited to, lentivirus vectors, gamma retrovirus vectors, foamy virus vectors, adeno-associated virus vectors, adenovirus vectors, poxvirus vectors, herpes virus vectors, engineered hybrid virus vectors, transposon-mediated vectors, etc. Viral vector technology is well known in the art and described, for example, in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY), and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.

[0256] Generally, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0257] In some embodiments, an expression vector (e.g., lentiviral vector) may be used to introduce the CAR into immune cells or their precursors (e.g., T cells). Thus, the expression vector (e.g., lentiviral vector) of the present invention may comprise a nucleic acid encoding a CAR. In some embodiments, the expression vector (e.g., lentiviral vector) will comprise additional elements that aid in the functional expression of the CAR encoded therein. In some embodiments, the expression vector comprising the nucleic acid encoding the CAR further comprises a mammalian promoter. In one embodiment, the vector further comprises an elongation factor-1-alpha promoter (EF-1α promoter). The use of the EF-1α promoter may increase the efficiency of expression of a downstream transgene (e.g., a nucleic acid sequence encoding a CAR). A physiological promoter (e.g., EF-1α promoter) may be less likely to induce integration-mediated genotoxicity and may negate the ability of a retroviral vector to transform stem cells. Other physiological promoters suitable for use in vectors (e.g., lentiviral vectors) are known to those of skill in the art and may be incorporated into the vectors of the present invention. In some embodiments, the vector (e.g., lentiviral vector) further comprises non-essential cis-acting sequences that may improve titer and gene expression. One non-limiting example of a non-essential cis-acting sequence is the central polypurine tract and central termination sequence (cPPT / CTS), which is important for efficient reverse transcription and nuclear import. Other non-essential cis-acting sequences are known to those skilled in the art and may be incorporated into the vector of the present invention (e.g., lentiviral vector). In some embodiments, the vector further comprises a post-transcriptional regulatory element. The post-transcriptional regulatory element may improve RNA translation, improve transgene expression, and stabilize the RNA transcript. One example of a post-transcriptional regulatory element is the Woodchuck Hepatitis Virus post-transcriptional regulatory element (WPRE). Thus, in some embodiments, the vector for the present invention further comprises a WPRE sequence. A variety of post-transcriptional regulatory elements are known to those skilled in the art and may be incorporated into the vector of the present invention (e.g., lentiviral vector).The vector of the present invention may further comprise additional elements such as a rev response element (RRE) for RNA transport, a packaging sequence and 5' and 3' long terminal repeats (LTR). The term "long terminal repeat" or "LTR" refers to a domain of base pairs located at the end of retroviral DNA, including the U3, R and U5 regions. LTRs generally provide functions necessary for retroviral gene expression (e.g., promoting gene transcription, initiation and polyadenylation of its product) and viral replication. In one embodiment, the vector of the present invention (e.g., lentiviral vector) comprises a 3'U3 deleted LTR. Thus, the vector of the present invention (e.g., lentiviral vector) may comprise any combination of elements described herein to enhance the efficiency of functional expression of the transgene. For example, the vector of the present invention (e.g., lentiviral vector) may comprise a WPRE sequence, a cPPT sequence, an RRE sequence, a 5'LTR, a 3'U3 deleted LTR' in addition to the nucleic acid encoding for CAR.

[0258] The vector of the present invention can be a self-inactivating vector. As used herein, the term "self-inactivating vector" refers to a vector whose 3'LTR enhancer promoter region (U3 region) is modified (e.g., by deletion or substitution). Self-inactivating vectors can prevent viral transcription beyond the first round of viral replication. As a result, self-inactivating vectors can infect and then integrate into host genome (e.g., mammalian genome) only once, but cannot be further passaged. Therefore, self-inactivating vectors can greatly reduce the risk of generating replicative viruses.

[0259] In some embodiments, the nucleic acid of the present invention can be RNA, for example, in vitro synthesized RNA. Methods for in vitro synthesis of RNA are known to those skilled in the art; any known method can be used to synthesize RNA comprising a sequence encoding a CAR of the present disclosure. Methods for introducing RNA into a host cell are known in the art. See, for example, Zhao et al. Cancer Res. (2010) 15: 9053. Introducing RNA comprising a nucleotide sequence encoding a CAR of the present disclosure into a host cell can be performed in vitro or ex vivo or in vivo. For example, host cells (e.g., NK cells, cytotoxic T lymphocytes, etc.) can be electroporated in vitro or ex vivo with RNA comprising a nucleotide sequence encoding a CAR of the present disclosure.

[0260] To evaluate the expression of a polypeptide or its part, the expression vector to be introduced into a cell may also contain either a selection marker gene or a reporter gene, or both, which facilitates identifying and selecting expressing cells from a group of cells to be transfected or infected through a viral vector.In some embodiments, the selection marker may be carried on a separate piece of DNA and may be used in a co-transfection procedure.Both the selection marker and the reporter gene may be flanked by appropriate regulatory sequences to allow expression in the host cell.Useful selection markers include, but are not limited to, antibiotic resistance genes.

[0261] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences.Generally, reporter genes are genes that are not present in or expressed by recipient organisms or tissues and code for polypeptides whose expression is revealed by some easily detectable properties, such as enzymatic activity.The expression of reporter genes is evaluated at a suitable time after DNA is introduced into recipient cells.Suitable reporter genes may include, but are not limited to, genes that code for luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82).

[0262] Methods for generating modified immune cells The present invention provides methods for producing / generating modified immune cells or their precursors (e.g., regulatory T cells). The cells are generally engineered by introducing a nucleic acid encoding a CAR of interest (e.g., an HLA-A2 CAR).

[0263] Methods for introducing nucleic acids into cells include physical, biological and chemical methods. Physical methods for introducing polynucleotides such as RNA into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. RNA can be introduced into target cells using commercially available methods including 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). RNA can also be introduced into cells using cationic liposome-mediated transfection using lipofection, using polymer encapsulation, using peptide-mediated transfection, or using biolistic particle delivery systems such as "gene guns" (see, e.g., Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001)).

[0264] Biological methods for introducing polynucleotide of interest into host cells include the use of DNA vectors and RNA vectors.Viral vectors, especially retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells.Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, etc.See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0265] In some embodiments, the nucleic acid encoding the subject CAR of the present invention is introduced into cells by an expression vector. Provided herein is an expression vector comprising a nucleic acid encoding the subject CAR (e.g., HLA-A2 CAR). Suitable expression vectors include lentivirus vectors, gamma retrovirus vectors, foamy virus vectors, adeno-associated virus (AAV) vectors, adenovirus vectors, engineered hybrid viruses, naked DNA, including but not limited to transposon-mediated vectors, such as Integrases, including Sleeping Beauty, Piggybak, and Phi31. Some other suitable expression vectors include herpes simplex virus (HSV) and retrovirus expression vectors.

[0266] Adenoviral expression vectors are based on adenoviruses, which have low capacity for integration into genomic DNA but high efficiency for transfection of host cells. Adenoviral expression vectors contain sufficient adenoviral sequences to (a) support the packaging of expression vectors and (b) ultimately express the CAR of interest in host cells. In some embodiments, the adenoviral genome is a 36 kb linear double-stranded DNA, into which a foreign DNA sequence (e.g., the nucleic acid encoding the CAR of interest) can be inserted to replace the large piece of adenoviral DNA to produce the expression vector of the present invention (see, for example, Danthinne and Imperiale, Gene Therapy (2000) 7(20): 1707-1714).

[0267] Another expression vector is based on adeno-associated virus, which utilizes adenovirus binding system.This AAV expression vector has high integration frequency into host genome.AAV expression vector can infect non-dividing cells, which makes it useful for gene delivery to mammalian cells, for example, in tissue culture or in vivo.AAV vector has a wide host range for infectivity.The details of the generation and use of AAV vector are described in U.S. Patent No. 5,139,941 and U.S. Patent No. 4,797,368.

[0268] Retroviral expression vectors can integrate into host genomes, deliver large amounts of foreign genetic material, infect a wide range of species and cell types, and be packaged into specialized cell lines.Retroviral vectors are constructed by inserting nucleic acid (e.g., nucleic acid encoding the target CAR) into the viral genome at a specific location to produce a virus that is replication-defective.Retroviral vectors can infect a wide range of cell types, but require host cell division for integration and stable expression of the target CAR.

[0269] Lentiviral vectors are derived from lentiviruses, which are complex retroviruses that contain common retroviral genes gag, pol and env, as well as other genes with regulatory or structural functions (see, for example, U.S. Patent Nos. 6,013,516 and 5,994,136). Some examples of lentiviruses include human immunodeficiency virus (HIV-1, HIV-2) and simian immunodeficiency virus (SIV). Lentiviral vectors have been generated by multiple attenuation of HIV pathogenicity genes, for example, genes env, vif, vpr, vpu and nef are deleted, making the vector biologically safe. Lentiviral vectors can infect non-dividing cells and can be used for both in vivo and ex vivo gene transfer and expression, for example, of nucleic acid encoding a CAR of interest (see, for example, U.S. Patent No. 5,994,136).

[0270] The expression vector comprising the nucleic acid of the present disclosure can be introduced into the host cell by any means known to those skilled in the art. The expression vector can optionally include a viral sequence for transfection. Alternatively, the expression vector can be introduced by fusion, electroporation, biolistics, transfection, lipofection, etc. Before the introduction of the expression vector, the host cell can be grown and expanded in culture, followed by appropriate treatment for the introduction and integration of the vector. The host cell can then be expanded and screened by the marker present in the vector. Various markers that can be used are known in the art and can include hprt, neomycin resistance, thymidine kinase, hygromycin resistance, etc. As used herein, the terms "cell", "cell line" and "cell culture" can be used interchangeably. In some embodiments, the host cell is an immune cell or its precursor, such as a T cell, a NK cell, or a NKT cell.

[0271] The present invention also provides genetically engineered cells that contain and stably express the CAR of the present disclosure.In some embodiments, the genetically engineered cells are genetically engineered T-lymphocytes (T cells), naive T cells (TN), memory T cells (e.g., central memory T cells (TCM), effector memory cells (TEM)), natural killer cells (NK cells), and macrophages that can generate therapeutically relevant progeny.In one embodiment, the genetically engineered cells are autologous cells.

[0272] The modified cells (e.g., containing the subject CAR) may be produced by stably transfecting an expression vector containing the nucleic acid of the present disclosure into a host cell. Additional methods for generating the modified cells of the present disclosure include, but are not limited to, chemical transformation methods (e.g., using calcium phosphate, dendrimers, liposomes, and / or cationic polymers), non-chemical transformation methods (e.g., electroporation, phototransformation, gene electrotransfer and / or hydrodynamic delivery), and / or particle-based methods (e.g., impalefection using a gene gun and / or magnetofection). Transfected cells expressing the subject CAR of the present disclosure may be expanded ex vivo.

[0273] Physical methods for introducing an expression vector into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.

[0274] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0275] Lipids suitable for use can be obtained from commercial suppliers. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol ("DMPG") and other lipids may 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 generic term that encompasses a wide variety of unilamellar and multilamellar lipid vesicles formed by the formation of closed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. Multilamellar liposomes form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components self-reorganize before forming a closed structure in which water and dissolved solutes are trapped between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have structures in solution that differ from the normal vesicular structure are also included. For example, lipids may adopt a micellar structure or simply exist as heterogeneous lipid molecular aggregates. Lipofectamine-nucleic acid complexes are also contemplated.

[0276] Regardless of the method used to introduce exogenous nucleic acid into a host cell or to expose other cells to the inhibitor of the present invention, a wide variety of assays may be carried out to confirm the presence of nucleic acid in the host cell. Such assays include, for example, "molecular biological" assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as detection of the presence or absence of a particular peptide, for example by immunological means (ELISA and Western blot) or by the assays described herein to identify agents that fall within the scope of the present invention.

[0277] Furthermore, the nucleic acids may be introduced by any means, such as transducing the expanded T cells, transfecting the expanded T cells, and electroporating the expanded T cells. Some nucleic acids may be introduced by one method and other nucleic acids may be introduced into the T cells by a different method.

[0278] RNA In one embodiment, the nucleic acid introduced into the host cell is RNA. In another embodiment, the RNA is mRNA, including in vitro transcribed RNA or synthetic RNA. RNA is produced by in vitro transcription using a template generated by polymerase chain reaction (PCR). The DNA of interest from any source can be directly converted into a template for in vitro mRNA synthesis by PCR using appropriate primers and RNA polymerase. The source of DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other suitable DNA source.

[0279] PCR can be used to generate a template for in vitro transcription of mRNA, which is then introduced into cells. Methods for carrying out PCR are well known in the art. Primers for use in PCR are designed to have a region that is substantially complementary to the region of DNA to be used as a template for PCR. "Substantially complementary" as used herein refers to a sequence of nucleotides in which most or all of the bases in the primer sequence are complementary, or one or more bases are non-complementary or mismatched. A substantially complementary sequence can anneal or hybridize with the intended 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 the part of a gene that is normally transcribed in cells (open reading frame), including the 5'UTR and 3'UTR. Primers can also be designed to amplify a part of a gene that codes for a particular domain of interest. In one embodiment, primers are designed to amplify the coding region of human cDNA, including all or part of the 5'UTR and 3'UTR. Primers useful for PCR are generated by synthetic methods well known in the art. A "forward primer" is a primer that contains a region of nucleotides that are substantially complementary to nucleotides on a DNA template that are upstream of the DNA sequence to be amplified. "Upstream" is used herein to refer to a position that is 5' of the DNA sequence to be amplified relative to the coding strand. A "reverse primer" is a primer that contains a region of nucleotides that are substantially complementary to a double-stranded DNA template that is downstream of the DNA sequence to be amplified. "Downstream" is used herein to refer to a position that is 3' of the DNA sequence to be amplified relative to the coding strand.

[0280] Chemical structures with the ability to promote RNA stability and / or translation efficiency may also be used. The RNA preferably has a 5'UTR and a 3'UTR. In one embodiment, the 5'UTR is 0-3000 nucleotides long. The length of the 5'UTR and 3'UTR sequences to be added to the coding region can be modified by different methods, including but not limited to designing primers for PCR that anneal to different regions of the UTR. Using this approach, one skilled in the art can modify the length of the 5'UTR and 3'UTR required to achieve optimal translation efficiency after transfection of the transcribed RNA.

[0281] 5'UTR and 3'UTR can be the naturally occurring endogenous 5'UTR and 3'UTR of the gene of interest. Alternatively, UTR sequence that is not endogenous to the gene of interest can be added by incorporating UTR sequence into forward and reverse primers or by any other modification of template. The use of UTR sequence that is not endogenous to the gene of interest can be useful for modifying RNA stability and / or translation efficiency. For example, it is known that AU-rich elements in 3'UTR sequence can reduce mRNA stability. Therefore, 3'UTR can be selected or designed to increase the stability of transcribed RNA based on the characteristics of UTR that are well known in the art.

[0282] In one embodiment, the 5'UTR can contain the Kozak sequence of the endogenous gene. Alternatively, when a 5'UTR that is not endogenous to the gene of interest is added by PCR as described above, the consensus Kozak sequence can be redesigned by adding a 5'UTR sequence. The Kozak sequence can increase the translation efficiency of some RNA transcripts, but is not required for all RNAs to allow efficient translation. The essential requirement of the Kozak sequence for many mRNAs is known in the art. In other embodiments, the 5'UTR can be derived from an RNA virus whose RNA genome is stable in the cell. In other embodiments, various nucleotide analogs can be used in the 3'UTR or 5'UTR to prevent exonuclease degradation of the mRNA.

[0283] To allow synthesis of RNA from a DNA template without the need for gene cloning, a promoter of transcription should be attached upstream of the sequence to be transcribed of the DNA template. When a sequence that functions as a promoter for RNA polymerase is added to the 5' end of the forward primer, the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame to be transcribed. In one embodiment, the promoter is the T7 polymerase promoter as described elsewhere herein. Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art.

[0284] In one embodiment, mRNA has both a cap on the 5' end and a 3' poly(A) tail, which determine ribosome binding, translation initiation and stability of mRNA in cells.On circular DNA templates, for example, on plasmid DNA, RNA polymerase produces long concatemeric products that are not suitable for expression in eukaryotic cells.Transcription of plasmid DNA linearized at the end of 3'UTR results in normal-sized mRNA that is not effective in eukaryotic transfection, even if it is polyadenylated after transcription.

[0285] On a linear DNA template, phage 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).

[0286] The conventional method for the integration of polyA / T stretches into DNA templates is molecular cloning. However, polyA / T sequences integrated into plasmid DNA can cause plasmid instability, since plasmid DNA templates obtained from bacterial cells are often highly contaminated with deletions and other abnormalities. This is because cloning procedures are not only laborious and time-consuming, but also often unreliable. That is why a method that allows the construction of DNA templates with polyA / T 3' stretches without cloning is highly desirable.

[0287] The polyA / T segment of the transcribed 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 after PCR by any other method, including but not limited to DNA ligation or in vitro recombination. The poly(A) tail also provides stability to the RNA, reducing RNA degradation. In general, 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 100-5000 adenosines.

[0288] The poly(A) tail of the RNA can be further extended after in vitro transcription by the use of a poly(A) polymerase, such as Escherichia coli (E. coli) polyA polymerase (E-PAP). In one embodiment, increasing the length of the poly(A) tail from 100 nucleotides to 300-400 nucleotides increases the translation efficiency of the RNA by approximately 2-fold. Additionally, attachment of different chemical groups to the 3' end can increase mRNA stability. Such attachments can contain modified / artificial nucleotides, aptamers and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. The ATP analogs can further increase the stability of the RNA.

[0289] The 5' cap also provides stability to the RNA molecule. In a preferred embodiment, the RNA produced by the method disclosed herein comprises 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)).

[0290] The RNA produced by the method disclosed herein can also include an internal ribosome entry site (IRES) sequence.The IRES sequence can be any viral, chromosomal or artificially designed sequence that initiates cap-independent ribosome binding to mRNA to facilitate the initiation of translation.Can include any solute suitable for cell electroporation, such as sugars, peptides, lipids, proteins, antioxidants and detergents, which can contain factors that promote cell permeability and viability.

[0291] In some embodiments, RNA, such as in vitro transcribed RNA, is electroporated into cells.

[0292] The methods of the present disclosure can be applied to modulating host cell activity in basic research and therapy in the areas of cancer, stem cells, acute and chronic infections, and autoimmune diseases, including assessing the ability of genetically modified host cells to kill target cancer cells.

[0293] The method also provides the ability to control the level of expression over a wide range, for example, by varying the amount of promoter or input RNA, thereby allowing expression levels to be individually regulated. Moreover, PCR-based mRNA production techniques greatly facilitate the design of mRNAs with different structures and combinations of these domains.

[0294] One advantage of the RNA transfection method of the present invention is that RNA transfection is essentially transient and vector-free.RNA transgenes can be delivered to lymphocytes and expressed therein after simple in vitro cell activation as minimal expression cassettes that do not require any additional viral sequences.Under these conditions, the integration of transgenes into host cell genome is unlikely.Due to the efficiency of RNA transfection and its ability to uniformly modify the entire lymphocyte population, cell cloning is not required.

[0295] Genetic modification of host cells with in vitro transcribed RNA (IVT-RNA) utilizes two different strategies, both of which have been successfully tested in various animal models. Cells are transfected with in vitro transcribed RNA using lipofection or electroporation. In order to achieve extended expression of the transferred IVT-RNA, it is desirable to stabilize the IVT-RNA using various modifications.

[0296] Several IVT vectors are known in the literature that are utilized in a standardized manner as templates for in vitro transcription and that have been genetically modified to produce stabilized RNA transcripts. Currently, protocols used in the art are based on plasmid vectors with the following structure: a 5' RNA polymerase promoter that allows RNA transcription followed by a gene of interest flanked either 3' and / or 5' by untranslated regions (UTRs) and a 3' polyadenylation cassette containing 50-70 A nucleotides. Prior to in vitro transcription, the circular plasmid is linearized downstream of the polyadenylation cassette by a type II restriction enzyme (the recognition sequence corresponds to the cleavage site). Thus, the polyadenylation cassette corresponds to the later poly(A) sequence in the transcript. As a result of this procedure, some nucleotides remain as part of the enzyme cleavage site after linearization, extending or masking the poly(A) sequence at the 3' end. It is unclear whether this non-physiological overhang affects the amount of protein produced in cells from such constructs.

[0297] RNA has several advantages over more classical plasmid or viral approaches. Gene expression from RNA sources does not require transcription, and protein products are rapidly produced after transfection. Furthermore, RNA only needs to enter the cytoplasm, not the nucleus, and therefore typical transfection methods result in extremely high transfection rates. In addition, plasmid-based approaches require that the promoter driving the expression of the gene of interest be active in the cells under study.

[0298] In another aspect, RNA construct is delivered to cell by electroporation.See, for example, the formulation and methodology of electroporation of nucleic acid construct into mammalian cell as taught in US 2004 / 0014645, US 2005 / 0052630A1, US 2005 / 0070841A1, US 2004 / 0059285A1, US 2004 / 0092907A1.Various parameters, including electric field strength, required for electroporation of any known cell type are generally known in related research literature and numerous patents and applications in the field.See, for example, US Patent No. 6,678,556, US Patent No. 7,171,264 and US Patent No. 7,173,116. Devices for therapeutic applications of electroporation are commercially available, such as, for example, MedPulser™ DNA Electroporation Therapy System (Inovio / Genetronics, San Diego, Calif.), and are described in patents such as U.S. Pat. No. 6,567,694; U.S. Pat. No. 6,516,223, U.S. Pat. No. 5,993,434, U.S. Pat. No. 6,181,964, U.S. Pat. No. 6,241,701, and U.S. Pat. No. 6,233,482; electroporation may also be used to transfect cells in vitro, as described, for example, in US20070128708A1. Electroporation may also be utilized to deliver nucleic acid to cells in vitro. Thus, electroporation-mediated administration of nucleic acid, including expression constructs, to cells using any of the many available devices and electroporation systems known to those skilled in the art represents an exciting new means to deliver RNA of interest to target cells.

[0299] Source of immune cells Prior to expansion, a source of immune cells is obtained from a subject for ex vivo manipulation. The source of target cells for ex vivo manipulation can also include, for example, autologous or heterologous donor blood, umbilical cord blood or bone marrow. For example, the source of immune cells can be derived from the subject to be treated with the modified immune cells of the present invention, for example, the subject's blood, the subject's umbilical cord blood or the subject's bone marrow. Non-limiting examples of subjects include humans, dogs, cats, mice, rats and their transgenic species. Preferably, the subject is a human.

[0300] Immune cells can be obtained from a number of sources, including blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, lymph, or lymphatic organs. Immune cells are cells of the immune system, such as cells of innate or adaptive immunity, such as bone marrow cells or lymphatic cells (including lymphocytes), typically T cells and / or NK cells. Other exemplary cells include stem cells, such as pluripotent stem cells, including multipotent stem cells and induced pluripotent stem cells (iPSCs). In some aspects, the cells are human cells. With respect to the subject to be treated, the cells can be allogeneic and / or autologous. The cells are typically primary cells, such as those directly isolated from the subject and / or those isolated from the subject and frozen.

[0301] In certain embodiments, the immune cells are T cells, e.g., CD8 + T cells (e.g., CD8 + naive T cells, central memory T cells, or effector memory T cells), CD4 +The target cells are T cells, natural killer T cells (NKT cells), regulatory T cells (Tregs), stem cell memory T cells, lymphoid progenitor cells, hematopoietic stem cells, natural killer cells (NK cells) or dendritic cells. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils and / or basophils. In some embodiments, the target cells are induced pluripotent stem (iPS) cells or cells derived from iPS cells, e.g., generated from a subject, engineered to alter (e.g., induce mutations) or manipulate expression of one or more target genes, and are, e.g., T cells, e.g., CD8 + T cells (e.g., CD8 + naive T cells, central memory T cells, or effector memory T cells), CD4 + These are iPS cells that have been differentiated into T cells, stem cell memory T cells, lymphoid progenitor cells, or hematopoietic stem cells.

[0302] In some embodiments, the cells are one or more subsets of T cells or other cell types, e.g., the total T cell population, CD4 + cells, CD8 + Cells and subpopulations thereof, including those defined by function, activation state, maturity, differentiation potential, expansion, recirculation, localization, and / or persistence, antigen specificity, type of antigen receptor, presence in specific organs or compartments, marker or cytokine secretion profile, and / or degree of differentiation, among others, T cells and / or CD4 + and / or CD8 +The subtypes and subpopulations of T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and their subtypes, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, innate and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, α / β T cells, and δ / γ T cells. In certain embodiments, a number of T cell lines are available in the art and may be used.

[0303] In some embodiments, the method includes isolating immune cells from a subject, preparing, treating, culturing, and / or manipulating them. In some embodiments, preparing the engineered cells includes one or more culturing and / or preparation steps. The cells for manipulation as described may be isolated from a sample, e.g., a biological sample, e.g., a sample obtained from or derived from a subject. In some embodiments, the subject from which the cells are isolated is a subject with a disease or condition, or a subject in need of cell therapy, or a subject to whom cell therapy will be administered. The subject is, in some embodiments, a human in need of a particular therapeutic intervention, e.g., adoptive cell therapy, from which the cells are isolated, treated, and / or manipulated. Thus, the cells are, in some embodiments, primary cells, e.g., primary human cells. Samples include tissues, fluids, and other samples taken directly from a subject, as well as samples obtained from one or more processing steps, such as separation, centrifugation, genetic manipulation (e.g., transduction with a viral vector), washing, and / or incubation. The biological sample can be a sample obtained directly from a biological source or a sample that has been processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples (including processed samples derived therefrom).

[0304] In some aspects, the sample from which the cells are derived or isolated is a blood or blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), white blood cells, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil or other organ, and / or cells derived therefrom. Samples include samples derived from autologous and allogeneic sources in the context of cell therapy, e.g., adoptive cell therapy.

[0305] In some embodiments, the cells are derived from a cell line, e.g., a T cell line. In some embodiments, the cells are obtained from heterologous sources, e.g., from mice, rats, non-human primates, and pigs. In some embodiments, the isolation of cells includes one or more preparation steps and / or non-affinity-based cell separation steps. In some examples, the cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, e.g., to remove unwanted components, enrich for desired components, lyse or remove cells that are sensitive to a particular reagent. In some examples, the cells are separated based on one or more properties, such as density, adhesiveness, size, sensitivity and / or resistance to a particular component.

[0306] In some examples, cells from the circulating blood of a subject are obtained, for example, by apheresis or leukapheresis. In some aspects, the sample contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells and / or platelets, and in some aspects, cells other than red blood cells and platelets. In some embodiments, blood cells collected from a subject are washed, for example, to remove the plasma fraction and place the cells in a suitable buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate-buffered saline (PBS). In some aspects, the washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in a wide variety of biocompatible buffers after washing. In certain embodiments, the components of the blood cell sample are removed and the cells are resuspended directly in culture medium. In some embodiments, the method includes a density-based cell separation method, such as preparing white blood cells from peripheral blood by lysing red blood cells and centrifuging through a Percoll or Ficoll gradient.

[0307] In one embodiment, immune cells are obtained from the circulating blood of an individual obtained by apheresis or leukapheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells and platelets. Cells collected by apheresis may be washed to remove the plasma fraction and place the cells in a suitable buffer or medium, such as phosphate-buffered saline (PBS) or a washing solution, for subsequent processing steps, which is calcium-free, may be magnesium-free, and may be many, but not all, divalent cations-free. After washing, the cells may be resuspended in a wide variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS. Alternatively, the undesirable components of the apheresis sample may be removed and the cells may be resuspended directly in culture medium.

[0308] In some embodiments, the isolation method involves the separation of different cell types based on the expression or presence in the cells of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acids. In some embodiments, any known separation method based on such markers may be used. In some embodiments, the separation is an affinity or immunoaffinity based separation. For example, the isolation involves in some aspects the separation of cells and cell populations based on the cellular expression or expression level of one or more markers, typically cell surface markers, e.g., by incubation with an antibody or binding partner that specifically binds such marker, typically followed by a washing step, and separating the cells that are bound to the antibody or binding partner from the cells that are not bound to the antibody or binding partner. Such a separation step can be based on positive selection, where the cells that are bound to the reagent are retained for further use, and / or negative selection, where the cells that are not bound to the antibody or binding partner are retained. In some instances, both fractions are retained for further use. In some aspects, negative selection can be particularly useful when the separation is best performed based on markers expressed by cells other than the desired population due to the unavailability of antibodies that specifically identify a cell type in a heterogeneous population. Separation does not necessarily result in the enrichment or removal of 100% of a particular cell population or cell that expresses a particular marker.For example, positive selection or enrichment of a particular type of cell, such as the cell that expresses a marker, refers to increasing the number or percentage of such cells, but does not necessarily result in the complete absence of cells that do not express the marker.Similarly, negative selection, removal or depletion of a particular type of cell, such as the cell that expresses a marker, refers to decreasing the number or percentage of such cells, but does not necessarily result in the complete removal of all such cells.

[0309] In some cases, multiple rounds of separation steps are carried out, where the fraction that has undergone positive or negative selection from one step is subjected to another separation step, such as subsequent positive or negative selection.In some cases, the cells that express multiple markers can be simultaneously depleted in a single separation step, for example, by incubating cells with multiple antibodies or binding partners, each specific to the marker targeted for negative selection.Similarly, positive selection can be carried out simultaneously for multiple cell types by incubating cells with multiple antibodies or binding partners that are expressed on various cell types.

[0310] In some embodiments, one or more of the T cell populations are identified as cells that are positive for one or more particular markers, such as surface markers (markers + ) or cells expressing the marker at high levels (marker high ), or cells that are negative for one or more markers ( - ) or cells expressing the marker at relatively lower levels (marker low For example, in some aspects, a particular subpopulation of T cells, such as cells that are positive for or express high levels of one or more surface markers, e.g., CD28 + , CD62L + , CCR7 + , CD27 + , CD127 + , CD4 + , CD8 + , CD45RA + and / or CD45RO + T cells are isolated by positive or negative selection techniques. In some cases, such markers are markers that are absent or expressed at relatively low levels on certain T cell populations (such as non-memory cells) but present or expressed at relatively higher levels on certain other T cell populations (such as memory cells). In one embodiment, cells (e.g., CD8 + Cells or T cells, e.g., CD3 +In some embodiments, the cells are enriched for cells that are positive for or express high surface levels of CD45RO, CCR7, CD28, CD27, CD44, CD127, and / or CD62L (i.e., have been subjected to positive selection) and / or depleted for cells that are positive for or express high surface levels of CD45RA (e.g., have been subjected to negative selection). In some embodiments, the cells are enriched or depleted for cells that are positive for or express high surface levels of CD122, CD95, CD25, CD27, and / or IL7-Ra (CD127). In some examples, the cells are enriched for cells that are positive for or express high surface levels of CD8 + T cells are enriched for cells that are CD45RO positive (or CD45RA negative) and CD62L positive. For example, CD3 / CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander) are used to enrich for CD3 + , CD28 + Positive selection can be performed on T cells.

[0311] In some embodiments, T cells are separated from the PBMC sample by negative selection of markers expressed on non-T cells (e.g., on B cells, monocytes, or other leukocytes such as CD14). + or CD8 + The selection process is + Helper and CD8 + It is used to separate cytotoxic T cells. Such CD4 + and CD8 + The population can be further sorted into subpopulations by positive or negative selection for markers expressed or expressed to a relatively greater extent on one or more naive, memory and / or effector T cell subpopulations. In some embodiments, CD8 +The cells are further enriched or depleted for naive, central memory, effector memory and / or central memory stem cells, for example, by positive or negative selection based on surface antigens associated with each subpopulation. In some embodiments, enrichment of central memory T (TCM) cells is performed to increase efficacy, such as improving long-term survival, expansion and / or engraftment after administration, which in some aspects is particularly robust in such subpopulations. In some embodiments, TCM enriched CD8 + T cells and CD4 + Combining T cells further enhances efficacy.

[0312] In some embodiments, the memory T cells are CD8 + CD62L on peripheral blood lymphocytes + Subsets and CD62L - PBMCs are then subjected to immunofluorescence assay using, for example, anti-CD8 and anti-CD62L antibodies to detect CD62L - CD8 + Fraction and / or CD62L + CD8 + The fraction can be enriched or depleted. In some embodiments, the CD4 + T cell populations and / or CD8 + The T population is enriched for central memory (TCM) cells. In some embodiments, enrichment for central memory T (TCM) cells is based on positivity or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127; in some aspects, it is based on negative selection of cells that express or highly express CD45RA and / or granzyme B. In some aspects, TCM cells are enriched for CD8 +Isolation of the population is performed by depletion of cells expressing CD4, CD14, CD45RA, and by positive selection or enrichment of cells expressing CD62L. In one aspect, enrichment for central memory T (TCM) cells is performed starting from a negative fraction of cells selected on the basis of CD4 expression, and the negative fraction is subjected to negative selection on the basis of expression of CD14 and CD45RA, and positive selection on the basis of CD62L. Such selections are performed simultaneously in some aspects, and sequentially in any order in other aspects. In some aspects, also enrichment for CD8 + The same CD4 expression-based selection process used in the preparation of the cell population or subpopulation may be used to select CD4 + By generating cell populations or subpopulations, both the positive and negative fractions from CD4-based separation are retained and used in subsequent steps of the method, optionally after one or more further positive or negative selection steps.

[0313] CD4 + Helper T cells are sorted into naive, central memory, and effector cells by identifying cell populations that bear cell surface antigens. CD4 + Lymphocytes can be obtained by standard methods. In some embodiments, naive CD4 + T lymphocytes are CD45RO - , CD45RA + , CD62L + , CD4 + In some embodiments, the T cells are central memory CD4 + The cells are CD62L + and CD45RO + In some embodiments, the effector CD4 + The cells are CD62L - and CD45RO. As an example, negative selection can result in the production of CD4 +To enrich for cells, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibodies or binding partners are bound to a solid support or matrix, such as magnetic or paramagnetic beads, to allow for separation of cells for positive and / or negative selection.

[0314] In some embodiments, the cells are incubated and / or cultured prior to or in conjunction with genetic manipulation. The incubation step can include cultivating, cultivating, stimulating, activating and / or propagating. In some embodiments, the composition or cells are incubated in the presence of stimulatory conditions or stimulants. Such conditions include conditions designed to induce proliferation, expansion, activation and / or survival of cells in the population, to mimic antigen exposure, and / or to prime cells for genetic manipulation, e.g., introduction of recombinant antigen receptors. The conditions can include one or more of a specific medium, temperature, oxygen content, carbon dioxide content, time, agents such as nutrients, amino acids, antibiotics, ions, and / or stimulatory factors such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate cells. In some embodiments, the stimulatory conditions or stimulants include one or more agents, e.g., ligands, that can activate the intracellular signaling domain of the TCR complex. In some aspects, the agent activates or initiates the TCR / CD3 intracellular signaling cascade in T cells. Such agents can include antibodies, e.g., antibodies specific for TCR components and / or costimulatory receptors, e.g., anti-CD3, anti-CD28, antibodies bound to a solid support, e.g., beads, and / or one or more cytokines. Optionally, the expansion method can further include adding anti-CD3 and / or anti-CD28 antibodies to the culture medium (e.g., at a concentration of at least about 0.5 ng / ml). In some embodiments, the stimulator includes IL-2 and / or IL-15, e.g., IL-2 at a concentration of at least about 10 units / mL.

[0315] In another embodiment, T cells are isolated from peripheral blood by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL™ gradient. Alternatively, T cells can be isolated from umbilical cord. In either case, specific subpopulations of T cells can be further isolated by positive or negative selection techniques.

[0316] The cord blood mononuclear cells so isolated can be depleted of cells expressing specific antigens, including but not limited to CD34, CD8, CD14, CD19, and CD56. Depletion of these cells can be accomplished using isolated antibodies, antibody-containing biological samples such as ascites fluid, antibodies bound to a physical support, and cell-bound antibodies.

[0317] Enrichment of a T cell population by negative selection can be achieved using a combination of antibodies that target surface markers unique to the cells undergoing negative selection. A preferred method is cell sorting and / or selection via negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies that target cell surface markers present on the cells undergoing negative selection. For example, negative selection can enrich for CD4 + Monoclonal antibody cocktails for enriching cells typically include antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

[0318] For the isolation of desired cell populations by positive or negative selection, the concentration of cells and surfaces (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly reduce the volume in which beads and cells are mixed together (i.e., increase the concentration of cells) to ensure maximum contact between cells and beads. For example, in one embodiment, a cell concentration of 2 billion cells / ml is used. In one embodiment, a cell concentration of 1 billion cells / ml is used. In a further embodiment, more than 100 million cells / ml are 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 further embodiments, a cell concentration of 125 or 150 million cells / ml is used. Higher concentrations can be used to provide increased cell yield, cell activation, and cell expansion growth.

[0319] T cells can also be frozen after a washing step, which does not require a monocyte removal step. Without being bound by theory, the freezing and subsequent thawing steps provide a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After a washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. As a non-limiting example, one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or other suitable cell freezing media, although many freezing solutions and parameters are known in the art and are useful in this context. The cells are then frozen to -80°C at a rate of 1°C per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other controlled freezing methods may be used, as well as uncontrolled methods of immediate freezing at -20°C or in liquid nitrogen.

[0320] In one embodiment, the population of T cells is contained within cells such as peripheral blood mononuclear cells, umbilical cord blood cells, purified T cell populations and T cell lines. In another embodiment, peripheral blood mononuclear cells comprise the population of T cells. In yet another embodiment, purified T cells comprise the population of T cells.

[0321] In certain embodiments, regulatory T cells (Treg) can be isolated from the sample. The sample can include, but is not limited to, umbilical cord blood or peripheral blood. In certain embodiments, Treg is isolated by flow cytometry sorting. The sample can be enriched for Treg before isolation by any means known in the art. The isolated Treg can be cryopreserved and / or expanded before use. Methods for isolating Treg are described in U.S. Patent Nos. 7,754,482, 8,722,400 and 9,555,105, and U.S. Patent Application No. 13 / 639,927, the contents of which are incorporated herein in their entirety.

[0322] Expansion of immune cells Either before or after modification of the cells to express a CAR of interest, the cells can be activated and expanded in number using methods such as those described in, for example, U.S. Patent Nos. 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. Publication No. 20060121005. For example, the immune cells of the present invention may be expanded by contacting with a surface attached with an agent that stimulates CD3 / TCR complex-associated signals and a ligand that stimulates costimulatory molecules on the surface of the immune cells.In particular, the immune cell population may be stimulated by contacting with an anti-CD3 antibody or its antigen-binding fragment, or an anti-CD2 antibody immobilized on a surface, or by contacting with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore.For costimulation of accessory molecules on the surface of the immune cells, a ligand that binds to the accessory molecules is used.For example, the immune cells may be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions suitable for stimulating the proliferation of the immune cells. Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France), which can be used in the present invention, as can other methods and reagents known in the art (see, e.g., ten Berge et al., Transplant Proc. (1998) 30(8): 3975-3977; Haanen et al., J. Exp. Med. (1999) 190(9): 1319-1328; and Garland et al., J. Immunol. Methods (1999) 227(1-2): 53-63).

[0323] Expansion of immune cells by the methods disclosed herein can be by about 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 200x, 300x, 400x, 500x, 600x, 700x, 800x, 900x, 1000x, 2000x, 3000x, 4000x, 5000x, 6000x, 7000x, 8000x, 9000x, 10,000x, 100,000x, 1,000,000x, 10,000,000x or more, and any whole or partial integer doublings therebetween. In one embodiment, immune cells are expanded in the range of about 20x to about 50x.

[0324] Following the culture, the immune cells may be incubated in the cell medium in the culture device for a period of time or until the cells reach confluency or high cell density for optimal passage, after which the cells may be transferred to another culture device. The culture device may be any culture device typically used for culturing cells in vitro. Preferably, the level of confluence is 70% or more before transferring the cells to another culture device. More preferably, the level of confluence is 90% or more. The period of time may be any time suitable for culturing the cells in vitro. The immune cell medium may be replaced at any time during the culture of the immune cells. Preferably, the immune cell medium is replaced about every 2-3 days. The immune cells are then collected from the culture device, where they may be used immediately or cryopreserved for storage for later use. In one embodiment, the invention includes cryopreserving the expanded immune cells. The cryopreserved immune cells are thawed before introducing the nucleic acid into the immune cells.

[0325] In another embodiment, the method comprises isolating the immune cells and expanding the immune cells. In another embodiment, the invention further comprises cryopreserving the immune cells prior to expansion. In yet another embodiment, the cryopreserved immune cells are thawed for electroporation with RNA encoding the chimeric membrane protein.

[0326] Another procedure for ex vivo expansion of cells is described in U.S. Patent No. 5,199,942 (incorporated herein by reference). Expansion as described in U.S. Patent No. 5,199,942 can be substituted or added to other expansion methods described herein. Briefly, ex vivo culture and expansion of immune cells includes the addition of cell growth factors as described in U.S. Patent No. 5,199,942, or other factors such as flt3-L, IL-1, IL-3 and c-kit ligand. In one embodiment, expanding immune cells includes culturing immune cells with factors selected from the group consisting of flt3-L, IL-1, IL-3 and c-kit ligand.

[0327] The culturing step as described herein (following contact with an agent as described herein or electroporation) can be very short, e.g., less than 24 hours, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours. The culturing step as further described herein (following contact with an agent as described herein) can be longer, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more.

[0328] Various terms are used to describe cells in culture. Cell culture generally refers to the growth of cells under controlled conditions taken from a living organism. A primary cell culture is the culture of cells, tissues, or organs taken directly from an organism prior to the first subculture. Cells expand in culture when placed in a growth medium and under conditions that facilitate cell growth and / or division, resulting in a larger population of cells. When cells expand in culture, the rate of cell growth is typically measured by the length of time required for the cells to double in number (otherwise known as "doubling time").

[0329] Each round of subculture is called a passage. When cells are subcultured, they are called passaged. A particular cell population, or cell line, is sometimes referred to or characterized by the number of times they have been passaged. For example, a cultured cell population that has been passaged 10 times may be called a P10 culture. A primary culture, i.e., the first culture after isolating cells from tissue, is called P0. After the first subculture, the cells are described as a second culture (P1 or passage 1). After the second subculture, the cells become (and continue in the same manner) a third culture (P2 or passage 2). It will be understood by those skilled in the art that there may be many population doublings during the passaging period; therefore, the number of population doublings of a culture is greater than the number of passages. The expansion growth of cells during the period between passaging (i.e., the increase in the number of population doublings) depends on many factors, including but not limited to the seeding density, substrate, medium, and time between passaging.

[0330] In one embodiment, the cells may be cultured for a few hours (about 3 hours) to about 14 days or any integer time unit therebetween. Conditions suitable for immune cell culture include an appropriate medium (e.g., Minimal Essential Media or RPMI Media 1640 or X-vivo 15 (Lonza)), which may contain factors required for growth and viability including serum (e.g., 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, TGF-β, and TNF-α, or any other additives for the growth of cells known to those skilled in the art. Other additives for the growth of cells include, but are not limited to, detergents, plasmanate, and reducing agents, such as N-acetyl-cysteine ​​and 2-mercaptoethanol. Culture media can include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or a fixed amount of cytokines sufficient for immune cell growth and expansion, supplemented with amino acids, sodium pyruvate, and vitamins. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells to be infused into subjects. Target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air+5% CO2).

[0331] The medium used to culture the immune cells may contain an agent capable of costimulating the immune cells. For example, an agent capable of stimulating CD3 is an antibody against CD3, and an agent capable of stimulating CD28 is an antibody against CD28. This is because, as demonstrated by the data disclosed herein, cells isolated by the methods disclosed herein can be expanded approximately 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 200x, 300x, 400x, 500x, 600x, 700x, 800x, 900x, 1000x, 2000x, 3000x, 4000x, 5000x, 6000x, 7000x, 8000x, 9000x, 10,000x, 100,000x, 1,000,000x, 10,000,000x, or more. In one embodiment, immune cells are expanded in the range of about 20x to about 50x, or more by culturing electroporated populations. In one embodiment, human regulatory T cells are expanded via anti-CD3 antibody-coated KT64.86 artificial antigen presenting cells (aAPCs). Methods for expanding and activating immune cells can be found in U.S. Patent Nos. 7,754,482, 8,722,400 and 9,555,105, the contents of which are incorporated herein in their entirety.

[0332] In one embodiment, the method of expanding immune cells can further include isolating the expanded immune cells for further application. In another embodiment, the method of expanding can further include subsequent electroporation of the expanded immune cells followed by culturing. Subsequent electroporation can include introducing a nucleic acid encoding an agent, e.g., transducing the expanded immune cells with a nucleic acid, transfecting the expanded immune cells, or electroporating the expanded immune cells into an expanded immune cell population, the agent further stimulating the immune cells. The agent can stimulate the immune cells, e.g., by stimulating further expansion, effector function, or another immune cell function.

[0333] Treatment method The modified immune cells (e.g., regulatory T cells) described herein may be included in a composition for immunotherapy, particularly for suppressive immunotherapy. The composition may include a pharmaceutical composition and may further include a pharma- ceutically acceptable carrier. A therapeutically effective amount of the pharmaceutical composition comprising the modified immune cells may be administered.

[0334] In one aspect, the invention includes a method for adoptive cell transfer therapy comprising administering an engineered immune cell (e.g., a regulatory T cell) of the invention to a subject in need thereof. In another aspect, the invention includes a method for treating a disease or condition in a subject comprising administering an engineered immune cell population to a subject in need thereof.

[0335] In one embodiment, a method of treating a disease or condition in a subject in need thereof comprises administering to the subject a therapeutically effective amount of engineered Tregs comprising a subject's CAR (e.g., an HLA-A2 CAR). In one embodiment, a method of treating a disease or condition in a subject in need thereof comprises administering to the subject a therapeutically effective amount of engineered Tregs comprising a subject's CAR (e.g., an HLA-A2 CAR), wherein the subject's CAR comprises an antigen binding domain capable of binding to HLA-A2, HLA-A28, and / or HLA-A68. In one embodiment, the HLA-A2 specific CAR comprises a CD8 signal peptide, an HLA-A2 V CAR ... H Domains, spacer sequences, HLA-A2 V L domain, the CD8 hinge region, the CD28 transmembrane domain, the CD28 costimulatory domain, and the CD3 zeta intracellular domain.

[0336] The HLA-A2 CAR of the present invention can redirect immune cells (e.g., regulatory T cells) to targets expressing HLA-A2 alloantigens. Thus, the subject CAR of the present invention is an alloantigen-specific CAR. Tregs expressing the HLA-A2 CAR of the present invention induce the proliferation of modified Tregs and enhance the suppressive function of modified Tregs upon activation by HLA-A2 binding.

[0337] When the modified immune cells comprising the subject CAR of the present invention are administered, the transplanted tissue is protected from rejection. In one embodiment, the modified immune cells comprising the subject CAR of the present invention (e.g., Tregs comprising an HLA-A2 CAR) can mediate HLA-A2-specific immune suppression. In one embodiment, the modified immune cells comprising the subject CAR of the present invention (e.g., Tregs comprising an HLA-A2 CAR) can suppress T cell proliferation in response to an alloantigen (e.g., HLA-A2 antigen). In some embodiments, upon transplantation of cells, tissues, and / or organs, HLA-A2 can be ubiquitously expressed on the transplanted cells, tissues, and / or organs. In such cases, substantial immune cell infiltration into the transplanted cells, tissues, and / or organs can occur, resulting in the destruction of the transplanted cells, tissues, and / or organs. Thus, in some embodiments, the modified immune cells comprising the subject CAR of the present invention (e.g., Tregs comprising an HLA-A2 CAR) can reduce immune cell infiltration and thus protect the transplanted cells, tissues, and / or organs from destruction. In some cases, transplanted cells, tissues, and / or organs may mediate toxicity. Thus, in some embodiments, modified immune cells comprising the subject CAR of the present invention (e.g., Tregs comprising HLA-A2 CAR) can reduce toxicity mediated by transplanted cells, tissues, and / or organs.

[0338] Thus, the present invention provides a method for achieving a prophylactic therapeutic effect in a subject in need thereof and / or a method for achieving an immunosuppressive effect in a subject in need thereof (e.g., a subject experiencing and / or suffering from an alloreaction or autoimmune reaction). In some embodiments, the method for achieving a prophylactic therapeutic effect in a subject in need thereof and / or a method for achieving an immunosuppressive effect in a subject in need thereof having an alloreaction or autoimmune reaction comprises administering to the subject an engineered immune cell comprising a CAR of the present invention. In one embodiment, the present invention provides a method for achieving an immunosuppressive effect in a subject in need thereof having an alloreaction or autoimmune reaction comprising administering to the subject an engineered regulatory T cell comprising a chimeric antigen receptor (CAR) having affinity for HLA-A2, wherein the CAR comprises an HLA-A2 binding domain, a CD8 hinge domain, a CD28 transmembrane domain, a CD28 costimulatory domain, and a CD3 zeta intracellular domain. In one aspect, the present invention provides a method for achieving a prophylactic therapeutic effect in a subject in need thereof comprising administering to the subject an engineered regulatory T cell comprising a chimeric antigen receptor (CAR) having affinity for HLA-A2, wherein the CAR comprises an HLA-A2 binding domain, a CD8 hinge domain, a CD28 transmembrane domain, a CD28 costimulatory domain, and a CD3 zeta intracellular domain.

[0339] Type 1 diabetes is a T cell-mediated autoimmune disease that leads to the destruction of pancreatic islet β cells, hypoinsulinemia and severely altered glucose homeostasis. The decline of regulatory T cells (Tregs) may play a role in the development of type 1 diabetes. During immune homeostasis, Tregs counteract the action of autoreactive effector T cells, thereby participating in peripheral tolerance. Thus, the imbalance between effector T cells and Tregs may contribute to the breakdown of peripheral tolerance and lead to the development of type 1 diabetes. In some embodiments, modified immune cells comprising the subject CAR of the present invention (e.g., Tregs comprising HLA-A2 CAR) can suppress T cell-mediated autoimmune diseases, such as type 1 diabetes. Thus, the present invention provides a method of treating diabetes in a subject in need thereof, comprising administering to the subject modified immune cells comprising the subject CAR of the present invention. In some embodiments, a method for treating diabetes in a subject in need thereof is provided, comprising administering to the subject a modified regulatory T cell comprising a chimeric antigen receptor (CAR) having affinity for HLA-A2, wherein the CAR comprises an HLA-A2 binding domain, a CD8 hinge domain, a CD28 transmembrane domain, a CD28 costimulatory domain, and a CD3 zeta intracellular domain. In some embodiments, the diabetes is type I diabetes.

[0340] In certain embodiments, the CAR is encoded by the nucleic acid sequence of SEQ ID NO: 24. In certain embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 23.

[0341] In certain embodiments, the modified immune cells are modified regulatory T cells (Tregs). In some embodiments, the modified immune cells are autologous cells. In some embodiments, the modified immune cells (e.g., modified regulatory T cells) are derived from humans.

[0342] CARs can redirect regulatory T cells to tissues expressing HLA-A2, HLA-A28, and / or HLA-A68, thus enhancing protection of transplanted tissues from rejection. T cells containing nucleic acids encoding specific HLA-A2 can be administered to a subject prior to, at, or immediately after tissue transplantation.

[0343] The methods of the invention should be construed to include protection against rejection of any type of transplanted organ, tissue or cell, including, but not limited to, lung, heart, heart valve, skin, liver, hand, kidney, pancreas, intestine, stomach, thymus, bone, tendon, cornea, testis, nerve, vein, blood, bone marrow, stem cells, islet cells of Langerhans and hematopoietic cells. The methods of the invention also include protection against graft-versus-host disease (GVHD).

[0344] In certain embodiments, the subject can be administered a secondary treatment in addition to the CAR, such as an immunosuppressant. Examples of immunosuppressants include, but are not limited to, prednisone, azathioprine, tacrolimus, and cyclosporine A.

[0345] Pharmaceutical Compositions The pharmaceutical composition of the present invention may comprise the modified immune cells as described herein combined with one or more pharma- ceutically or physiologically acceptable carriers, diluents or excipients.Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; amino acids such as polypeptides or glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.The composition of the present invention is preferably formulated for intravenous administration.

[0346] The pharmaceutical composition of the present invention may be administered in a manner appropriate to the disease to be treated (or prevented). The amount and frequency of administration are determined by factors such as the condition of the patient and the type and severity of the patient's disease, but the appropriate dosage may be determined by clinical trials.

[0347] The cells of the invention that are administered can be autologous, allogeneic, or xenogeneic with respect to the subject receiving treatment.

[0348] The cells of the present invention can be administered at dosages and routes and times to be determined in appropriate preclinical and clinical experiments and tests. The cell composition may be administered multiple times at dosages within these ranges. The administration of the cells of the present invention may be combined with other methods useful for treating the desired disease or condition as determined by those skilled in the art.

[0349] Also provided are immune cell populations of the invention, compositions containing such cells and / or enriched in such cells, for example compositions in which the cells expressing the recombinant receptor constitute at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the total cells in the composition, or constitute a particular type of cell, such as regulatory T cells. Among other things, the compositions are pharmaceutical compositions and formulations for administration, for example, adoptive cell therapy. Also provided are therapeutic methods for administering the cells and compositions to a subject, for example, a patient.

[0350] Also provided is a composition comprising cells for administration, including pharmaceutical compositions and formulations, for example, a unit dose form composition comprising a number of cells for administration at a given dose or a fraction thereof.Pharmaceutical compositions and formulations generally comprise one or more optional pharmacologic acceptable carriers or excipients.In some embodiments, the composition comprises at least one additional therapeutic agent.

[0351] The term "pharmaceutical formulation" refers to a preparation in such a form that the biological activity of the active ingredient contained therein is effective and does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than the active ingredient, that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives. In some aspects, the choice of carrier is determined, in part, by the particular cell and / or by the method of administration. Thus, there is a wide variety of suitable formulations. For example, the pharmaceutical composition can contain a preservative. Suitable preservatives can include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, for example, in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0352] Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; These include, but are not limited to, proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0353] A buffering agent is included in the composition in some aspects. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixture is typically present in an amount of about 0.001% to about 4% based on the total weight of the composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are detailed, for example, by Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

[0354] The formulation may comprise an aqueous solution. The formulation or composition may also contain two or more active ingredients useful for the particular indication, disease, or condition being treated with the cells, preferably those with complementary activities, where each activity does not adversely affect the other. Such active ingredients are preferably present in combination in an amount effective for the intended purpose. Thus, in some embodiments, the pharmaceutical composition further comprises other pharma- ceutical active agents or drugs, such as chemotherapeutic agents, such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and / or vincristine. The pharmaceutical composition, in some embodiments, contains the cells in an amount effective to treat or prevent the disease or condition, such as a therapeutically or prophylactically effective amount. The therapeutic or prophylactic effectiveness is, in some embodiments, monitored by periodically assessing the subject being treated. The desired dosage can be delivered by administration of a single bolus of cells, by administration of multiple boluses of cells, or by continuous infusion of cells.

[0355] Formulations include formulations for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell population is administered parenterally. The term "parenteral" as used herein includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cells are administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection. The composition is provided in some embodiments as a sterile liquid preparation, such as an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, which in some aspects may be buffered to a selected pH. Liquid preparations are usually easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially for administration by injection. On the other hand, viscous compositions can be formulated within an appropriate viscosity range to provide a longer contact period with a particular tissue. Liquid or viscous compositions can include a carrier, which can be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.

[0356] Sterile injectable solutions can be prepared by incorporating the cells in a solvent, for example, in a solvent mixed with a suitable carrier, diluent or excipient, such as sterile water, physiological saline, glucose, dextrose, etc. The composition can contain auxiliary substances, such as wetting agents, dispersing or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents and / or coloring agents, depending on the desired route of administration and preparation. In some aspects, standard textbooks may be consulted for preparing suitable preparations.

[0357] Various additives can be added to enhance the stability and sterility of the composition, including antibacterial preservatives, antioxidants, chelating agents, and buffers.Prevention of microbial action can be ensured by various antibacterial and antifungal agents, such as paraben, chlorobutanol, phenol, and sorbic acid.Prolonged absorption of injectable pharmaceutical dosage forms can be achieved by using agents that delay absorption, such as aluminum monostearate and gelatin.

[0358] Formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0359] Generally, pharmaceutical compositions comprising the modified immune cells described herein are administered in a dose-dependent manner. 4 ~10 9 cells / kg body weight, in some cases 10 5 ~10 6 It is understood that the immune cell composition may be administered at a dosage of up to 1000 cells / kg body weight, including all integer values ​​within these ranges. The immune cell composition may also be administered multiple times at these dosages. The cells can be administered using infusion techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regimen for a particular patient can be easily determined by those skilled in the medical arts by monitoring the patient for symptoms of disease and adjusting the treatment accordingly.

[0360] Administration of the modified immune cells of the invention may be performed in any convenient manner known to those of skill in the art. The cells of the invention may be administered to a subject by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient intraarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscular, intravenous (iv) injection, or intraperitoneally. In other cases, the cells of the invention are injected directly into a site of inflammation in a subject, a site of local disease in a subject, a lymph node, an organ, a tumor, etc.

[0361] It should be understood that the methods and compositions useful in the present invention are not limited to the particular formulations shown in the examples. The following examples are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the cells of the present invention, how to expand and culture them, and how to treat them, and are not intended to limit the scope of what the inventors regard as their invention.

[0362] The practice of the present invention employs conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology that are well within the scope of the skill of the art, unless otherwise specified. Such techniques are fully described in such publications as "Molecular Cloning: A Laboratory Manual", fourth edition (Sambrook, 2012); "Oligonucleotide Synthesis" (Gait, 1984); "Culture of Animal Cells" (Freshney, 2010); "Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1997); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Short Protocols in Molecular Biology" (Ausubel, 2002); "Polymerase Chain Reaction: Principles, Applications and Troubleshooting", (Babar, 2011); "Current Protocols in Immunology" (Coligan, 2002). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention and therefore may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments are discussed in the following sections.

[0363] The contents of the articles, patents and patent applications, and all other documents and electronically available information mentioned or cited in this specification are incorporated herein by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

[0364] Although the present invention has been described with reference to specific embodiments thereof, it should be understood by those skilled in the art that various modifications and equivalent substitutions may be made without departing from the true spirit and scope of the present invention. It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein may be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. In addition, many modifications may be made to adapt a particular situation, material, composition, process, process step or steps to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the appended claims. Although specific embodiments have been described in detail herein, the same will be more clearly understood by reference to the following examples, which are included for illustrative purposes only and are not intended to be limiting. EXAMPLES

[0365] Experimental Example The invention will now be described with reference to the following examples, which are provided for illustrative purposes only, and the invention is not limited to these examples, but rather encompasses all variations that become evident as a result of the teachings provided herein.

[0366] We now describe the materials and methods used in these experiments.

[0367] The 3PF12 antibody was derived from: Watkins et al., The isolation and characterization of human monoclonal HLA-A2 antibodies from an immune V gene phage display library. (2000). doi:10.1034 / j.1399-0039.2000.550305.

[0368] HLA-A2-specific CAR nucleotide sequence TIFF0007618192000013.tif136144

[0369] HLA-A2-specific CAR amino acid sequence TIFF0007618192000014.tif50144

[0370] The results of the experiment are now described.

[0371] Herein, we have generated an HLA-A2-specific chimeric antigen receptor (CAR). The HLA-A2-specific CAR contains an antigen-binding domain that can bind to HLA-A2, HLA-A28, and / or HLA-A68. When expressed on human regulatory cells (Treg), the CAR can mediate antigen-specific suppression. This novel CAR can redirect Treg to tissues that express HLA-A2, HLA-A28, and / or HLA-A68 and mediate tolerance.

[0372] In one embodiment, the CAR comprises a CD8 signal peptide, an HLA-A2 V H Domain, flexible GS spacer sequence, HLA-A2 V L The CAR comprises a CD8 signal peptide, an extracellular domain including a CD8 hinge region, a CD28 transmembrane / intracellular domain, and a CD3 zeta domain (Figure 1). In another embodiment, the CAR is encoded by the nucleotide sequence of SEQ ID NO:24. In yet another embodiment, the CAR comprises the amino acid sequence of SEQ ID NO:23. The CAR was assembled from the primary amino acid sequence of 3PF12 scFv detailed in Watkins et al. (2000) doi:10.1034 / j.1399-0039.2000.550305. The heavy and light chains were separated by a flexible GS linker and placed into the lentiviral vector pTRPE. The vector contains a CD8 signal peptide to direct the CAR to the cell surface, a CD8 hinge region for CAR flexibility, and a CD28 transmembrane / intracellular domain + CD3 zeta domain for signal transduction.

[0373] The CARs of the present invention are distinguishable from the CARs of MacDonald et al. (J Clin Invest. 2016;126(4):1413-142; and J Immunol May 1, 2016, 196 (1 Supplement) 140.6), i.e., CARs derived from the BB7.2 hybridoma rather than 3PF12, contain a myc tag after the scFv domain, and lack the CD8 hinge domain. Similarly, in contrast to the CARs of the present invention, the CAR from Boardman et al. (American Journal of Transplantation. 2016 Dec 1]) is derived from the 3PB2 sequence in Watkins et al. ((2000) doi:10.1034 / j.1399-0039.2000.550305) rather than 3PF12, contains a myc tag after the scFv domain, lacks the CD8 hinge domain, and contains eGFP following the signaling domain.

[0374] Example 1: HLA-A2-specific T cells Primary human CD8 + T cells were isolated from normal donor apheresis products by incubating with RosetteSep reagent for 20 min at room temperature, followed by layering the cells on top of Lymphoprep and centrifugation at 400 × g for 30 min at room temperature. Cells were then washed three times with OPTI-MEM reduced serum medium (Thermo Fisher Scientific) and isolated at 100 × 10 6The cells were resuspended in OPTI-MEM at 10 cells / mL, mixed with 10 μg of in vitro transcribed 3PF12-28z RNA, and electroporated for 700 μs at 500 V in a 0.2 cm electroporation cuvette using a BTX830 (Harvard Apparatus BTX). In vitro RNA transcription was performed using the mMessage mMachine T7 Transcription Kit (ThermoFisher Scientific) according to the manufacturer's instructions and cleaned up with RNeasy MinElute Clean Up Kit (Qiagen). After 16 h incubation in a 37°C / 5% CO2 incubator, cells were stained with F(ab')2 fragment-specific anti-biotin-SP (long spacer) AffiniPure goat anti-human IgG antibody (Jackson Labs) for 30 min on ice, followed by 3 washes with PBS and further incubation with 1 μL streptavidin-PE and 1 μL anti-human CD8-APC-H7 (BD Biosciences) diluted in PBS for 30 min. Cells were fixed with 2% paraformaldehyde and analyzed on an LSRII flow cytometer (BD Biosciences) (Figure 2). Cells electroporated with 3PF12-28z RNA showed detectable CAR expression on the cell surface.

[0375] Cells electroporated as in Figure 2 were mixed with thawed, HLA-haplotype-distinct allogeneic human donor PBMCs in a 3:1 ratio in the presence of GolgiPlug Protein Transport Inhibitor (BD Biosciences) for 6 hours in a 37°C / 5% CO2 incubator. Cells were then fixed and permeabilized by incubating with 100 μL Fixation Medium A (ThermoFisher Scientific) for 30 minutes at room temperature, washed, and then stained with 3 μL α-IL-2-APC and 2 μL α-TNF-α-PE-Cy7 antibodies (BD Biosciences) diluted in 100 μL Fixation Medium B (ThermoFisher Scientific). Cells were then washed with PBS and analyzed on an LSR II flow cytometer (Figure 3). CAR + CD8 + T cells were activated by HLA-A2 and HLA-A68 molecules on the surface of target PBMCs, leading to the production of IL-2 and TNF-α. + CD8 + T cells are HLA-A2 specific to three different individuals. - And HLA-A68 - The CAR was not activated by PBMCs derived from any of the donors, demonstrating the specificity of the CAR for certain, but not all, HLA molecules.

[0376] Example 2: HLA-A2-specific regulatory T cells From human umbilical cord blood donors, CD4 +Regulatory T cells were isolated by incubation with RosetteSep reagent (Stem Cell), layering on Lymphoprep, and centrifugation at 400×g for 30 min followed by CD25 magnetic positive selection (StemCell Technologies). Tregs were stimulated with α-CD3 / α-CD28 beads (Gibco) and expanded in XVIVO15 (Invitrogen) with 5% human AB serum containing 1× GlutaMAX and 300 IU / mL IL-2 in a 37°C / 5% CO2 incubator. 48 hours after the first stimulation, Tregs were transduced with lentiviral vectors to express the 3PF12-28z CAR or an irrelevant CAR. On day 4, α-CD3 / α-CD28 beads were removed and cells were fed with complete XVIVO15 medium as above on days 4, 6, 9, and 12, with IL-2 replaced as needed assuming consumption. Cells were left to rest until day 14, at which point Tregs were washed three times to remove IL-2 and antigen-specific suppression was assessed by mixing them with allogeneic T cells (electroporated with RNA to express SL9 WT TCR and labeled with 2.5 μM CFSE 16 h prior) and K562 cells (transgenically expressing HLA-A2 and SL9 antigens) at a ratio of 8:1:0.5 (Teff:Treg:K562). To examine nonspecific suppressive function, Tregs were mixed with CFSE-labeled PBMCs and α-CD3 stimulator beads (Gibco) at a ratio of 8:1:3 (Teff:Treg:beads). After 5 days of incubation in a 37°C / 5% CO2 incubator, the cells were stained with 1 μL of CD8-APC-H7 and 0.5 μL of CD4-BV421 antibodies diluted in 100 μL of PBS for 15 min at 4°C, then washed and resuspended in 2% PFA for fixation. The cells were then analyzed on an LSR II flow cytometer (Figure 4). Without any other intervention, the CFSE-labeled target cells divide due to the interaction of the SL9 WT TCR with the SL9 peptide presented by the MHC class I expressed on K562 cells, resulting in the dilution of the CFSE signal. HLA-A2 +Co-culturing CAR Tregs with target cells suppressed cell division as evidenced by less dilution of the CFSE signal (blue), whereas CFSE target cells suppressed cell division by irrelevant CAR Tregs (green) or non-Treg CD4 + To demonstrate that both sets of Tregs had equal suppressive potential, the right panel of Figure 4 shows that polyclonal stimulation of CFSE-labeled PBMCs and Tregs in coculture with α-CD3 stimulator beads suppressed suppression of Tregs with irrelevant CAR Tregs (green) and HLA-A2 Tregs (red). + Both CAR Tregs (blue) and non-Treg CD4 + This shows that the level of inhibition is much higher than in cells (red).

[0377] Example 3: HLA-A2-specific T cells are HLA-A2 + Targeting pancreatic islets HLA-A2 from human donors + Pancreatic islets were transplanted under the left kidney capsule of NSG mice. Three days later, 10 × 10 islets transduced with lentiviral vectors carrying either the 3PF12-28z CAR or the irrelevant CD19-28z CAR were transplanted under the left kidney capsule of NSG mice. 6 T cells were injected intravenously. Non-transduced T cells were used as a negative control. Urine samples were collected periodically and stored at -80°C until human c-peptide levels were assessed by ELISA (Figure 5). As shown in Figure 5, 3PF12-28z CAR-transduced T cells were able to deplete human C-peptide levels, indicating that 3PF12-28z CAR-transduced T cells could deplete human C-peptide levels by targeting the transplanted HLA-A2 + This indicates that the pancreatic islets were targeted.

[0378] Example 4: HLA-A2-specific Treg adoptive immunotherapy for the treatment of type 1 diabetes Type 1 diabetes is a T cell-mediated autoimmune disease that leads to pancreatic islet β cell destruction, hypoinsulinemia and severely altered glucose homeostasis. The decline of regulatory T cells (Treg) may play a role in the development of type 1 diabetes. During immune homeostasis, Treg counterbalances the action of autoreactive effector T cells, thereby participating in peripheral tolerance. Thus, the imbalance between effector T cells and Treg may contribute to the breakdown of peripheral tolerance, leading to the development of type 1 diabetes.

[0379] Autologous Tregs are isolated from subjects and stimulated ex vivo to expand. The Tregs are transduced with HLA-A2 CAR by lentiviral vector to produce HLA-A2-specific Tregs. HLA-A2-specific Tregs, such as those produced in Example 2 (Tregs transduced with 3PF12-28z CAR), are administered to subjects with type 1 diabetes.

[0380] Other Aspects The recitation of a list of elements in any definition of a variable herein includes a definition of that variable as any single element or combination (or subcombination) of the listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0381] The disclosures of any and all patents, patent applications and publications cited herein are incorporated herein by reference in their entirety. Although the present invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of the present invention may be devised by those skilled in the art without departing from the true spirit and scope of the present invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

1. 1. An engineered immune cell or a precursor thereof comprising a chimeric antigen receptor (CAR) having affinity for HLA-A2, the CAR comprising a CD8 signal peptide, an HLA-A2 binding domain comprising a heavy chain variable region comprising three complementarity determining regions (CDRs) as set forth in SEQ ID NOs:5-7 and a light chain variable region comprising three CDRs as set forth in SEQ ID NOs:10-12, a CD8 hinge domain, a CD28 transmembrane domain, a CD28 intracellular domain, and a CD3 zeta intracellular domain.

2. the HLA-A2 binding domain is selected from the group consisting of an antibody, a Fab, and an scFv; the HLA-A2 binding domain comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:3; the HLA-A2 binding domain comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:8; the HLA-A2 binding domain comprises a spacer sequence; the HLA-A2 binding domain comprises the amino acid sequence shown in SEQ ID NO: 1; the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:17; the CD3 zeta intracellular domain comprises the amino acid sequence set forth in SEQ ID NO:21; the CD8 hinge domain comprises the amino acid sequence shown in SEQ ID NO:15; and / or the CD8 signal peptide comprises the amino acid sequence shown in SEQ ID NO: 13; The modified immune cell or precursor thereof of claim 1.

3. A modified immune cell or its precursor cell described in claim 1 or 2, wherein the CAR comprises the amino acid sequence of SEQ ID NO:

1.

4. the CAR comprises the amino acid sequence shown in SEQ ID NO: 23, the HLA-A2 binding domain cross-reacts with HLA-A28, and / or the HLA-A2 binding domain cross-reacts with HLA-A68; The modified immune cell or a precursor thereof according to any one of claims 1 to 3.

5. an engineered regulatory T cell, are autologous, and / or Of human origin, The modified immune cell or its precursor cell according to any one of claims 1 to 4.

6. 1. An isolated nucleic acid comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) having affinity for HLA-A2, the CAR comprising a CD8 signal peptide, an HLA-A2 binding domain comprising a heavy chain variable region comprising three complementarity determining regions (CDRs) as set forth in SEQ ID NOs:5-7 and a light chain variable region comprising three CDRs as set forth in SEQ ID NOs:10-12, a CD8 hinge domain, a CD28 transmembrane domain, a CD28 intracellular domain, and a CD3 zeta intracellular domain.

7. The isolated nucleic acid of claim 6, wherein the nucleic acid comprises the sequence shown in SEQ ID NO:

24.

8. An expression construct comprising the isolated nucleic acid of claim 6 or 7, The expression construct is a viral vector selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector; The expression construct is a lentiviral vector. The lentiviral vector is a self-inactivating lentiviral vector; The expression construct comprises an EF-1α promoter, The expression construct comprises a rev response element (RRE), The expression construct comprises a Woodchuck Hepatitis Virus post-transcriptional regulatory element (WPRE), The expression construct comprises a cPPT sequence, or The expression construct comprises an EF-1α promoter, a rev response element (RRE), a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), and a cPPT sequence. Expression constructs.

9. 10. A method for generating a modified immune cell or a precursor thereof according to any one of claims 1 to 5, comprising introducing into said immune cell or a precursor thereof a nucleic acid according to claim 6 or 7, or an expression construct according to claim 8.

10. 10. A pharmaceutical composition comprising the modified immune cells or precursor cells thereof according to any one of claims 1 to 5 for achieving an immunosuppressive effect in a subject in need thereof, wherein the subject is suffering from an alloreaction and / or an autoimmune reaction.

11. 10. A pharmaceutical composition comprising the modified immune cells or precursor cells thereof according to any one of claims 1 to 5 for achieving a prophylactic therapeutic effect in a subject in need thereof, characterized in that the pharmaceutical composition is administered to the subject before an alloreaction or an autoimmune reaction develops.

12. 10. A pharmaceutical composition for achieving an immunosuppressive effect in a subject in need thereof, the subject having an alloreaction or an autoimmune reaction, the pharmaceutical composition comprising the modified immune cell or a precursor thereof according to any one of claims 1 to 5, the modified immune cell being a modified regulatory T cell.

13. the allogeneic or autoimmune response occurs following a tissue transplant and the pharmaceutical composition suppresses, blocks, or inhibits graft-versus-host disease in the subject; the modified cells are autologous cells; and / or the modified cells are of human origin; 13. The pharmaceutical composition according to any one of claims 10 to 12.

14. 10. A pharmaceutical composition for treating diabetes in a subject in need thereof comprising the modified immune cells or precursor cells thereof according to any one of claims 1 to 5.

15. A pharmaceutical composition for treating diabetes in a subject in need thereof, comprising a modified immune cell or a precursor cell thereof described in any one of claims 1 to 5, which is a modified regulatory T cell.

16. The diabetes is type 1 diabetes; the modified cells are autologous cells; and / or the modified cells are of human origin; 16. The pharmaceutical composition of claim 14 or 15.

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