Dual car expressing t cells individually linked to CD28 and 4-1bb

Abstract

To provide modified immune cells or precursors thereof comprising dual (a first and a second) chimeric receptors (e.g., CARs), which are essential for the development of effective HIV therapy.SOLUTION: An immune cell or precursor thereof comprises a first CAR including a distinct costimulatory domain and a 4-1BB intracellular domain and a second CAR comprising a CD28 intracellular domain. In another aspect, a method is provided for treating an HIV-infected mammal using a modified T cell comprising a first CD4 CAR including a 4-1BB intracellular domain and a second CD4 CAR including a CD28 intracellular domain.SELECTED DRAWING: Figure 1

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 / 858,506, filed June 7, 2019, which is incorporated herein by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under grants AI117950 and AI126620 awarded by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0003] Background of the Invention Chimeric antigen receptor (CAR) T-cell immunotherapy has induced durable remissions in cases of refractory malignancies by injecting engineered cancer-specific effector T cells. Meanwhile, mechanisms by which HIV weakens host immunity, such as epitope escape due to rapid evolution, T-cell exhaustion, and CD4 + Despite the fact that next-generation CAR T cells may be uniquely equipped to overcome many of the attenuations of T cell help, little progress has been made in developing successful CAR T cell therapies for HIV infection. Indeed, the kind of potent and durable T cell responses that CAR T cells can confer are likely essential for the development of effective HIV treatments.

[0004] CARs confer new immune specificity to patient T cells through the expression of an intracellular T cell costimulatory domain and an extracellular antigen recognition domain linked to the CD3ζ chain. Typical costimulatory domains for second-generation CARs are CD28 and 4-1BB, both of which have been incorporated into approved CD19-targeted CAR T cell therapies. Preclinical cancer models have demonstrated that CD28-costimulated CAR T cells exhibit significant effector function, resulting in rapid tumor clearance, but have limited in vivo persistence. In contrast, 4-1BB-costimulated CAR T cells exhibit slower antitumor response kinetics but sustained cell division and longer-term survival. Importantly, the distinct signaling pathways used by CD28 and 4-1BB promote unique metabolic, phenotypic, and functional T cell profiles that appear to generate optimal CAR T cell activity for specific diseases. Therefore, significant emphasis has been placed on discovering costimulatory signals that sufficiently enhance CAR T cell function.

[0005] The earliest clinical trials of CAR T cell therapy utilized first-generation HIV-specific CD4-based CAR T cells expressing the CD3ζ endodomain, which were ineffective in treating chronically infected or antiretroviral therapy (ART)-suppressed individuals. However, the field of cancer immunotherapy has since driven significant advances in CAR technology, and there has been renewed interest in applying these advances to the treatment of HIV. Indeed, several recent studies have evaluated the utility of CAR T cells in this disease setting. However, critical knowledge gaps remain in understanding the mechanistic underpinnings of successful and unsuccessful CAR T cell therapies, particularly in model systems that recapitulate HIV pathogenesis, which will serve to accelerate the development of this strategy for therapeutic initiatives.

[0006] There is a need in the art for improved CAR T cell design. The present invention fulfills this need. Summary of the Invention

[0007] As described herein, the present invention relates to compositions and methods relating to T cells expressing dual CARs, each linked to a distinct costimulatory domain.

[0008] In one aspect, a nucleic acid is provided comprising: a first polynucleotide sequence encoding a first chimeric receptor comprising a first binding domain, a first transmembrane domain, a first costimulatory domain that confers enhanced survival-promoting function, and a CD3z intracellular signaling domain; and a second polynucleotide sequence encoding a second chimeric receptor comprising a second binding domain, a second transmembrane domain, a second costimulatory domain that confers enhanced effector function, and a CD3z intracellular signaling domain.

[0009] In another aspect, a nucleic acid is provided comprising: a first polynucleotide sequence encoding a first chimeric receptor comprising the extracellular domain of a CD4 molecule, the CD8α transmembrane domain, the 4-1BB costimulatory domain, and the CD3z intracellular signaling domain; and a second polynucleotide sequence encoding a second chimeric receptor comprising the extracellular domain of a CD4 molecule, the CD28 transmembrane domain, the CD28 costimulatory domain, and the CD3z intracellular signaling domain.

[0010] In another aspect, an engineered immune cell or progenitor thereof is provided, comprising: a first chimeric receptor comprising a first binding domain, a first transmembrane domain, a first costimulatory domain that confers enhanced pro-survival function, and a CD3z intracellular signaling domain; and a second chimeric receptor comprising a second binding domain, a second transmembrane domain, a second costimulatory domain that confers enhanced effector function, and a CD3z intracellular signaling domain.

[0011] In certain embodiments, the first costimulatory domain is a 4-1BB costimulatory domain, hi certain embodiments, the second costimulatory domain is a CD28 costimulatory domain.

[0012] In certain embodiments, the first transmembrane domain and / or the second transmembrane domain is selected from the group consisting of an artificial hydrophobic sequence and a transmembrane domain of a type I transmembrane protein, the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, OX40 (CD134), 4-1BB (CD137), and CD154. In certain embodiments, the first transmembrane domain is a 4-1BB transmembrane domain. In certain embodiments, the first transmembrane domain is a CD8α transmembrane domain. In certain embodiments, the second transmembrane domain is a CD28 transmembrane domain.

[0013] In certain embodiments, the first chimeric receptor and / or the second chimeric receptor further comprises a hinge domain selected from the group consisting of an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial hinge domain, a hinge comprising the amino acid sequence of CD8, or any combination thereof.

[0014] In certain embodiments, the first binding domain binds to a first target, and the second binding domain binds to a second target. In certain embodiments, the first target and the second target are the same. In certain embodiments, the first target and the second target are different epitopes of the same molecule. In certain embodiments, the first target and the second target are different.

[0015] In certain embodiments, the first target and / or the second target is human immunodeficiency virus type 1 (HIV-1). In certain embodiments, the first target and the second target are human immunodeficiency virus type 1 (HIV-1). In certain embodiments, the first target and / or the second target is envelope glycoprotein gp120. In certain embodiments, the first target and the second target are envelope glycoprotein gp120.

[0016] In certain embodiments, the first binding domain and / or the second binding domain comprises the extracellular domain of a CD4 molecule. In certain embodiments, the first binding domain and the second binding domain comprise the extracellular domain of a CD4 molecule.

[0017] In certain embodiments, the first target and / or the second target is a tumor-associated antigen. In certain embodiments, the tumor-associated antigen is a liquid tumor antigen. In certain embodiments, the liquid tumor antigen is CD19 or CD22. In certain embodiments, the tumor-associated antigen is a solid tumor antigen.

[0018] In certain embodiments, the first polynucleotide sequence and the second polynucleotide sequence are separated by a linker. In certain embodiments, the linker comprises an internal ribosome entry site (IRES), a furin cleavage site, a self-cleaving peptide, or any combination thereof. In certain embodiments, the linker comprises a furin cleavage site and a self-cleaving peptide. In certain embodiments, the self-cleaving peptide is a 2A peptide. In certain embodiments, the 2A peptide is selected from the group consisting of porcine teschovirus-1 2A (P2A), Thosea asigna virus 2A (T2A), equine rhinitis A virus 2A (E2A), and foot-and-mouth disease virus 2A (F2A).

[0019] In certain embodiments, the nucleic acid comprises, in a 5' to 3' direction, a first polynucleotide sequence, a linker, and a second polynucleotide sequence. In certain embodiments, the nucleic acid comprises, in a 5' to 3' direction, a second polynucleotide sequence, a linker, and the first polynucleotide sequence.

[0020] In certain embodiments, the nucleic acid further comprises a polynucleotide sequence encoding an HIV fusion inhibitor. Examples of fusion inhibitors include, but are not limited to, enfuvirtide, maraviroc, BMS-488043, PRO-542, leronlimab, aplaviroc, ibalizumab, temsavir, etc. In certain embodiments, the nucleic acid further comprises a polynucleotide sequence encoding an HIV fusion inhibitor, wherein the HIV fusion inhibitor is a cell surface-expressed HIV fusion inhibitor. In certain embodiments, the nucleic acid further comprises a polynucleotide sequence encoding an HIV fusion inhibitor, wherein the HIV fusion inhibitor is C34-CXCR4.

[0021] In certain embodiments, cells expressing an HIV fusion inhibitor exhibit increased resistance to infection by HIV compared to control cells that do not express the HIV fusion inhibitor.

[0022] In another aspect, there is provided an expression construct comprising any one of the nucleic acids disclosed herein.

[0023] In certain embodiments, the expression construct further comprises an EF-1α promoter. In certain embodiments, the expression construct further comprises a rev response element (RRE). In certain embodiments, the expression construct further comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). In certain embodiments, the expression construct further comprises a cPPT sequence.

[0024] In certain aspects, 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.

[0025] In certain aspects, the expression construct is a lentiviral vector. In certain aspects, the lentiviral vector is a self-inactivating lentiviral vector.

[0026] In another aspect, an engineered immune cell or a precursor thereof is provided, comprising: a first chimeric receptor comprising the extracellular domain of a CD4 molecule, a CD8α transmembrane domain, a 4-1BB costimulatory domain, and a CD3z intracellular signaling domain; and a second chimeric receptor comprising the extracellular domain of a CD4 molecule, a CD28 transmembrane domain, a CD28 costimulatory domain, and a CD3z intracellular signaling domain.

[0027] In certain embodiments, the modified cells are modified immune cells. In certain embodiments, the modified cells are modified T cells. In certain embodiments, the modified cells are autologous cells. In certain embodiments, the modified cells are autologous cells obtained from a human subject.

[0028] In another aspect, a pharmaceutical composition is provided comprising a therapeutically effective amount of modified immune cells or progenitor cells thereof, wherein the modified cells comprise a first chimeric receptor comprising a first binding domain, a first transmembrane domain, a first costimulatory domain that confers enhanced survival-promoting function, and a CD3z intracellular signaling domain; and a second chimeric receptor comprising a second binding domain, a second transmembrane domain, a second costimulatory domain that confers enhanced effector function, and a CD3z intracellular signaling domain.

[0029] In another aspect, a pharmaceutical composition is provided comprising a therapeutically effective amount of modified immune cells or precursor cells thereof, wherein the modified cells comprise: a first chimeric receptor comprising the extracellular domain of a CD4 molecule, the CD8α transmembrane domain, a 4-1BB costimulatory domain, and a CD3z intracellular signaling domain; and a second chimeric receptor comprising the extracellular domain of a CD4 molecule, the CD28 transmembrane domain, the CD28 costimulatory domain, and a CD3z intracellular signaling domain.

[0030] In another aspect, there is provided a method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject any one of the modified immune cells disclosed herein or any one of the pharmaceutical compositions disclosed herein.

[0031] In another aspect, provided is a method of treating a disease or disorder in a subject in need thereof, comprising administering an engineered immune cell, or a precursor thereof, comprising: a first chimeric receptor comprising a first binding domain, a first transmembrane domain, a first costimulatory domain that confers enhanced pro-survival function, and a CD3z intracellular signaling domain; and a second chimeric receptor comprising a second binding domain, a second transmembrane domain, a second costimulatory domain that confers enhanced effector function, and a CD3z intracellular signaling domain.

[0032] In certain embodiments, the disease or disorder is a viral disease, hi certain embodiments, the viral disease is HIV-1 infection.

[0033] In certain embodiments, the disease or disorder is cancer. In certain embodiments, the cancer is a liquid tumor. In certain embodiments, the cancer is a hematological malignancy. In certain embodiments, the cancer is a solid tumor.

[0034] In another aspect, a method of treating HIV-1 infection in a subject in need thereof is provided, comprising administering modified immune cells, or precursor cells thereof, comprising: a first chimeric receptor comprising the extracellular domain of a CD4 molecule, the CD8α transmembrane domain, the 4-1BB costimulatory domain, and the CD3z intracellular signaling domain; and a second chimeric receptor comprising the extracellular domain of a CD4 molecule, the CD28 transmembrane domain, the CD28 costimulatory domain, and the CD3z intracellular signaling domain.

[0035] In another aspect, a method of treating cancer in a subject in need thereof is provided, comprising administering modified T cells comprising a first chimeric receptor comprising a first binding domain, a first transmembrane domain, a first costimulatory domain that confers enhanced pro-survival function, and a CD3z intracellular signaling domain; and a second chimeric receptor comprising a second binding domain, a second transmembrane domain, a second costimulatory domain that confers enhanced effector function, and a CD3z intracellular signaling domain.

[0036] In another aspect, a method of treating HIV-1 infection in a subject in need thereof is provided, comprising administering modified T cells comprising a first chimeric receptor comprising the extracellular domain of a CD4 molecule, the CD8α transmembrane domain, a 4-1BB costimulatory domain, and a CD3z intracellular signaling domain; and a second chimeric receptor comprising the extracellular domain of a CD4 molecule, the CD28 transmembrane domain, the CD28 costimulatory domain, and a CD3z intracellular signaling domain.

[0037] In certain embodiments, the modified cells are modified immune cells. In certain embodiments, the modified cells are modified T cells. In certain embodiments, the modified cells are autologous cells. In certain embodiments, the modified cells are autologous cells obtained from a human subject.

[0038] In certain embodiments, the subject is a human.

[0039] In certain embodiments, administration of the modified cells results in an increase in the following cells compared to subjects who do not receive the modified cells: CD4 - T cells, CD4 - T cells, CD8 + T cells, CD8 - T cells, memory CD4 + T cells and CD14 +Reduce one or more HIV-induced losses of macrophages, hi certain embodiments, the reduction is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0040] In certain embodiments, administration of the modified cells results in an increase in the following cells compared to subjects who do not receive the modified cells: CD4 + T cells, CD4 - T cells, CD8 + T cells, CD8 - T cells, central memory CD4 + T cells and CD14 + In certain embodiments, administration reduces the occurrence of HIV-infected cells in one or more of the macrophages. In certain embodiments, administration reduces the occurrence of HIV-infected cells by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In certain embodiments, administration reduces the occurrence of HIV-infected CD4 + Reduce cell occurrence by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%.

[0041] In certain embodiments, the subject's blood comprises at least about 100 modified cells per μL of blood for at least three weeks after a single administration of modified T cells.

[0042] In certain embodiments, the modified cells bind to the first and second targets on cells expressing the first and second targets and kill the cells via granule-mediated cell lysis.

[0043] In certain embodiments, the method further comprises administering one or more antiretroviral therapeutic agents.

[0044] In another aspect, there is provided a method of producing a modified immune cell comprising introducing any of the nucleic acids disclosed herein into an immune cell.

[0045] In certain embodiments, the immune cells are obtained from the group consisting of T cells, dendritic cells, and stem cells. In certain embodiments, the immune cells are CD8 - T cells, CD4 + The T cells are selected from the group consisting of T cells, naive T cells, central memory T cells, stem cell memory T cells, effector memory T cells, natural killer T cells, and regulatory T cells.

[0046] In certain embodiments, the method further comprises expanding the T cells. In certain embodiments, the method further comprises expanding the T cells, wherein the T cells are expanded in the range of about 150-fold to about 500-fold. In certain embodiments, the method further comprises expanding the T cells, wherein the T cells are expanded at least about 150-fold. In certain embodiments, the method further comprises expanding the T cells, wherein the T cells are expanded at least about 300-fold. In certain embodiments, the method further comprises expanding the T cells, wherein the T cell expansion is in vivo. [The present invention 1001] a first polynucleotide sequence encoding a first chimeric receptor comprising a first binding domain, a first transmembrane domain, a first costimulatory domain that confers an enhanced pro-survival function, and a CD3z intracellular signaling domain; and a second polynucleotide sequence encoding a second chimeric receptor comprising a second binding domain, a second transmembrane domain, a second costimulatory domain that confers enhanced effector function, and a CD3z intracellular signaling domain; A nucleic acid comprising: [The present invention 1002] 1001. The nucleic acid of claim 1001, wherein the first costimulatory domain is a 4-1BB costimulatory domain. [The present invention 1003] The nucleic acid of claim 1001 or 1002, wherein the second costimulatory domain is a CD28 costimulatory domain. [The present invention 1004] Any of the aforementioned nucleic acids of the invention, wherein the first transmembrane domain and / or the second transmembrane domain is selected from the group consisting of an artificial hydrophobic sequence and the transmembrane domains of type I transmembrane proteins, the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, OX40 (CD134), 4-1BB (CD137), and CD154. [The present invention 1005] Any of the aforementioned nucleic acids of the invention, wherein the first transmembrane domain is a 4-1BB or CD8α transmembrane domain. [The present invention 1006] Any of the aforementioned nucleic acids of the invention, wherein the second transmembrane domain is a CD28 transmembrane domain. [The present invention 1007] Any of the preceding nucleic acids of the invention, wherein the first chimeric receptor and / or the second chimeric receptor further comprises a hinge domain. [The present invention 1008] 1007. The nucleic acid of the present invention, wherein the hinge domain is selected from the group consisting of an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial hinge domain, a hinge comprising the amino acid sequence of CD8, or any combination thereof. [The present invention 1009] Any of the aforementioned nucleic acids of the invention, wherein the first binding domain binds to a first target and the second binding domain binds to a second target. [The present invention 1010] 1009. The nucleic acid of claim 10, wherein the first target and the second target are the same. [The present invention 1011] The nucleic acid of claim 1009 or 1010, wherein the first target and the second target are distinct epitopes of the same molecule. [The present invention 1012] The nucleic acid of the present invention 1009, wherein the first target and the second target are different. [The present invention 1013] The nucleic acid of any one of claims 1009 to 1012, wherein the first target and / or the second target is human immunodeficiency virus type 1 (HIV-1). [The present invention 1014] 1013. The nucleic acid of the invention, wherein the first target and the second target are human immunodeficiency virus type 1 (HIV-1). [The present invention 1015] The nucleic acid of claim 1013 or 1014, wherein the first target and / or the second target is the envelope glycoprotein gp120. [The present invention 1016] 1015. The nucleic acid of the invention, wherein the first target and the second target are envelope glycoprotein gp120. [The present invention 1017] 1017. The nucleic acid of any one of claims 1009 to 1016, wherein the first binding domain and / or the second binding domain comprises the extracellular domain of a CD4 molecule. [The present invention 1018] 1017. The nucleic acid of the invention, wherein the first binding domain and the second binding domain comprise the extracellular domain of the CD4 molecule. [The present invention 1019] The nucleic acid of any of claims 1009 to 1012, wherein the first target and / or the second target is a tumor-associated antigen. [The present invention 1020] The nucleic acid of the present invention, wherein the tumor-associated antigen is a liquid tumor antigen. [The present invention 1021] 1020. The nucleic acid of the present invention, wherein the liquid tumor antigen is CD19 or CD22. [The present invention 1022] The nucleic acid of the present invention, wherein the tumor-associated antigen is a solid tumor antigen. [The present invention 1023] a first polynucleotide sequence encoding a first chimeric receptor comprising the extracellular domain of a CD4 molecule, the CD8α transmembrane domain, the 4-1BB costimulatory domain, and the CD3z intracellular signaling domain; and a second polynucleotide sequence encoding a second chimeric receptor comprising the extracellular domain of a CD4 molecule, the CD28 transmembrane domain, the CD28 costimulatory domain, and the CD3z intracellular signaling domain; A nucleic acid comprising: [The present invention 1024] Any of the aforementioned nucleic acids of the invention, wherein the first polynucleotide sequence and the second polynucleotide sequence are separated by a linker. [The present invention 1025] 1024. The nucleic acid of the present invention, wherein the linker comprises an internal ribosome entry site (IRES), a furin cleavage site, a self-cleaving peptide, or any combination thereof. [The present invention 1026] The nucleic acid of claim 1024 or 1025, wherein the linker comprises a furin cleavage site and a self-cleaving peptide. [The present invention 1027] 1026. The nucleic acid of claim 1026, wherein the self-cleaving peptide is a 2A peptide. [The present invention 1028] 1027. The nucleic acid of the present invention, wherein the 2A peptide is selected from the group consisting of Porcine Teschovirus-1 2A (P2A), Thosea asigna virus 2A (T2A), Equine rhinitis A virus 2A (E2A), and Foot and Mouth Disease virus 2A (F2A). [The present invention 1029] The nucleic acid of any of claims 1023 to 1028, comprising, in the 5' to 3' direction, a first polynucleotide sequence, a linker, and a second polynucleotide sequence. [The present invention 1030] The nucleic acid of any of claims 1023 to 1028, comprising, in the 5' to 3' direction, a second polynucleotide sequence, a linker, and a first polynucleotide sequence. [The present invention 1031] An expression construct comprising any of the nucleic acids of the present invention. [The present invention 1032] 1031. An expression construct of the present invention further comprising an EF-1α promoter. [The present invention 1033] The expression construct of 1031 or 1032 of the present invention, further comprising a rev response element (RRE). [The present invention 1034] The expression construct of any of claims 1031 to 1033, further comprising a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). [This invention 1035] An expression construct according to any one of claims 1031 to 1034, further comprising a cPPT sequence. [The present invention 1036] The expression construct of any one of claims 1031 to 1035, 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. [This invention 1037] An expression construct according to any one of claims 1031 to 1036, which is a lentiviral vector. [The present invention 1038] 1037. The expression construct of the present invention, wherein the lentiviral vector is a self-inactivating lentiviral vector. [This invention 1039] A modified immune cell or a precursor thereof, comprising any one of the nucleic acids of the present inventions 1001 to 1030 or the expression construct of the present inventions 1031 to 1038. [The present invention 1040] a first chimeric receptor comprising a first binding domain, a first transmembrane domain, a first costimulatory domain that confers enhanced pro-survival function, and a CD3z intracellular signaling domain; and a second chimeric receptor comprising a second binding domain, a second transmembrane domain, a second costimulatory domain that confers enhanced effector function, and a CD3z intracellular signaling domain. A modified immune cell or a precursor thereof, comprising: [The present invention 1041] 1040. The modified cell of the present invention, wherein the first costimulatory domain is a 4-1BB costimulatory domain. [The present invention 1042] The modified cell of invention 1040 or 1041, wherein the second costimulatory domain is a CD28 costimulatory domain. [This invention 1043] The modified cell of any of claims 1040 to 1042, wherein the first transmembrane domain and / or the second transmembrane domain is selected from the group consisting of an artificial hydrophobic sequence and a transmembrane domain of a type I transmembrane protein, the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, OX40 (CD134), 4-1BB (CD137), and CD154. [This invention 1044] The modified cell of any of claims 1040 to 1043, wherein the first transmembrane domain is a 4-1BB or CD8α transmembrane domain. [This invention 1045] The modified cell of any of claims 1040 to 1044, wherein the second transmembrane domain is a CD28 transmembrane domain. [The present invention 1046] 1046. The modified cell of any of claims 1040 to 1045, wherein the first chimeric receptor and / or the second chimeric receptor further comprises a hinge domain. [This invention 1047] The modified cell of the present invention 1046, wherein the hinge domain is selected from the group consisting of an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial hinge domain, a hinge comprising the amino acid sequence of CD8, or any combination thereof. [This invention 1048] 8. The modified cell of any of claims 1040 to 1047, wherein the first binding domain binds to a first target and the second binding domain binds to a second target. [This invention 1049] The modified cell of the present invention 1048, wherein the first target and the second target are the same. [The present invention 1050] The modified cell of invention 1048 or 1049, wherein the first target and second target are distinct epitopes of the same molecule. [This invention 1051] The modified cell of the present invention 1048, wherein the first target and the second target are different. [This invention 1052] The modified cell of any of claims 1048 to 1051, wherein the first target and / or the second target is human immunodeficiency virus type 1 (HIV-1). [This invention 1053] The modified cell of the present invention 1052, wherein the first target and the second target are human immunodeficiency virus type 1 (HIV-1). [This invention 1054] The modified cell of invention 1050 or 1051, wherein the first target and / or the second target is the envelope glycoprotein gp120. [This invention 1055] 1054. The modified cell of the present invention, wherein the first target and the second target are envelope glycoprotein gp120. [This invention 1056] 1056. The modified cell of any of claims 1048 to 1055, wherein the first binding domain and / or the second binding domain comprises the extracellular domain of a CD4 molecule. [This invention 1057] 1056. The modified cell of claim 1056, wherein the first binding domain and the second binding domain comprise an extracellular domain of a CD4 molecule. [This invention 1058] The modified cell of any of claims 1048 to 1051, wherein the first target and / or the second target is a tumor-associated antigen. [This invention 1059] The modified cell of the present invention 1058, wherein the tumor-associated antigen is a liquid tumor antigen. [The present invention 1060] The modified cell of the present invention 1059, wherein the liquid tumor antigen is CD19 or CD22. [This invention 1061] The modified cell of the present invention 1058, wherein the tumor-associated antigen is a solid tumor antigen. [The present invention 1062] a first chimeric receptor comprising the extracellular domain of a CD4 molecule, the CD8α transmembrane domain, the 4-1BB costimulatory domain, and the CD3z intracellular signaling domain; and a second chimeric receptor comprising the extracellular domain of the CD4 molecule, the CD28 transmembrane domain, the CD28 costimulatory domain, and the CD3z intracellular signaling domain; A modified immune cell or a precursor thereof, comprising: [This invention 1063] The modified cell of any one of 1040 to 1062 of the present invention, which is a modified immune cell. [This invention 1064] The modified cell of any one of 1040 to 1063 of the present invention, which is a modified T cell. [This invention 1065] The modified cell of any one of 1040 to 1064 of the present invention, which is an autologous cell. [The present invention 1066] The modified cell of any of claims 1040 to 1065, which is an autologous cell obtained from a human subject. [This invention 1067] A pharmaceutical composition comprising a therapeutically effective amount of the modified cells of any one of the present inventions 1038 to 1064. [The present invention 1068] 1. A pharmaceutical composition comprising a therapeutically effective amount of modified immune cells or precursor cells thereof, The modified cells are a first chimeric receptor comprising a first binding domain, a first transmembrane domain, a first costimulatory domain that confers enhanced pro-survival function, and a CD3z intracellular signaling domain; and a second chimeric receptor comprising a second binding domain, a second transmembrane domain, a second costimulatory domain that confers enhanced effector function, and a CD3z intracellular signaling domain. Including, Pharmaceutical compositions. [The present invention 1069] 1. A pharmaceutical composition comprising a therapeutically effective amount of modified immune cells or precursor cells thereof, The modified cells are a first chimeric receptor comprising the extracellular domain of a CD4 molecule, the CD8α transmembrane domain, the 4-1BB costimulatory domain, and the CD3z intracellular signaling domain; and a second chimeric receptor comprising the extracellular domain of the CD4 molecule, the CD28 transmembrane domain, the CD28 costimulatory domain, and the CD3z intracellular signaling domain; Including, Pharmaceutical compositions. [The present invention 1070] 1. A method of treating a disease or disorder in a subject in need thereof, comprising: The modified cell of any one of claims 1040 to 1066 of the present invention, or Any of the pharmaceutical compositions of the present invention Nos. 1067 to 1069 to said subject. [This invention 1071] 1. A method of treating a disease or disorder in a subject in need thereof, comprising: a first chimeric receptor comprising a first binding domain, a first transmembrane domain, a first costimulatory domain that confers enhanced pro-survival function, and a CD3z intracellular signaling domain; and a second chimeric receptor comprising a second binding domain, a second transmembrane domain, a second costimulatory domain that confers enhanced effector function, and a CD3z intracellular signaling domain. Modified immune cells or their precursor cells, comprising: The method of claim 1, further comprising administering [This invention 1072] The method of any one of claims 1070 to 1071, wherein the disease or disorder is a viral disease. [This invention 1073] The method of claim 1072, wherein the viral disease is HIV-1 infection. [This invention 1074] The method of claim 1071, wherein the disease or disorder is cancer. [This invention 1075] The method of claim 1074, wherein the cancer is a liquid tumor. [This invention 1076] The method of claim 1074, wherein the cancer is a hematological malignancy. [This invention 1077] The method of claim 1074, wherein the cancer is a solid tumor. [This invention 1078] 1. A method of treating an HIV-1 infection in a subject in need thereof, comprising: a first chimeric receptor comprising the extracellular domain of a CD4 molecule, the CD8α transmembrane domain, the 4-1BB costimulatory domain, and the CD3z intracellular signaling domain; and a second chimeric receptor comprising the extracellular domain of the CD4 molecule, the CD28 transmembrane domain, the CD28 costimulatory domain, and the CD3z intracellular signaling domain; Modified immune cells or their precursor cells, comprising: The method of claim 1, further comprising administering [This invention 1079] 1079. The method of any of claims 1070 to 1078, wherein the modified cell is a modified immune cell. [The present invention 1080] 1079. The method of any of claims 1070 to 1079, wherein the modified cell is a modified T cell. [This invention 1081] 1. A method of treating cancer in a subject in need thereof, comprising: a first chimeric receptor comprising a first binding domain, a first transmembrane domain, a first costimulatory domain that confers enhanced pro-survival function, and a CD3z intracellular signaling domain; and a second chimeric receptor comprising a second binding domain, a second transmembrane domain, a second costimulatory domain that confers enhanced effector function, and a CD3z intracellular signaling domain. modified T cells, including The method of claim 1, further comprising administering [This invention 1082] 1. A method of treating an HIV-1 infection in a subject in need thereof, comprising: a first chimeric receptor comprising the extracellular domain of a CD4 molecule, the CD8α transmembrane domain, the 4-1BB costimulatory domain, and the CD3z intracellular signaling domain; and a second chimeric receptor comprising the extracellular domain of the CD4 molecule, the CD28 transmembrane domain, the CD28 costimulatory domain, and the CD3z intracellular signaling domain; modified T cells, including The method of claim 1, further comprising administering [This invention 1083] The method of any of claims 1070 to 1082, wherein the modified cells are autologous cells. [This invention 1084] The method of any of claims 1070 to 1083, wherein the modified cells are autologous cells obtained from the human subject. [This invention 1085] The method of any one of claims 1070 to 1084, wherein the subject is a human. [Brief explanation of the drawings]

[0047] The following detailed description of preferred 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 embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0048] [Figure 1]Figure 1A is a schematic diagram showing CD4 CAR T cell infusion products containing T cells expressing an intracellular 4-1BB costimulatory domain and either an active signaling (left) or inactive signaling (right) CD3ζ domain. Inactive signaling CAR T cells (right) do not induce T cell activation after recognition of HIV-infected cells. Figure 1B is a schematic diagram of the experimental design used herein, in which CAR T cells are infused into humanized BLT mice 48 hours after HIV challenge. Mice were bled at the indicated time points to measure 1) viral levels and 2) the number of CAR T cells in the peripheral blood. Figure 1C shows quantification of HIV in peripheral blood, demonstrating that activated CAR T cells (red) were unable to prevent early viral replication compared to inactive CAR T cells (blue). Figure 1D shows the proliferation of activated CAR T cells (red) in peripheral blood compared to inactive CAR T cells (blue). These data demonstrate that signaling-competent CAR T cells expressing the 4-1BB signaling domain are capable of robust cell proliferation and survival after encountering HIV-infected cells. [Figure 2-1]Figure 2A is a schematic diagram of T cells expressing HIV-specific CARs: 4-1BBζ CD4 CAR (left), CD28ζ CD4 CAR (center), and dual CD4 CAR (4-1BBζ and CD28ζ CAR, right). Figure 2B shows the results of in vitro HIV suppression experiments in which HIV-infected CD4+ T cells were mixed with the indicated T cell populations. These data demonstrate that while both CD4 CAR T cell populations are able to suppress HIV replication compared to untransduced T cells (UTD), CD28ζ CAR T cells exert greater control over HIV than 4-1BBζ CAR T cells at the indicated time points. This demonstrates that CD28 CAR T cells exert greater effector function than 4-1BB CAR T cells. Figure 2C illustrates a CAR T cell product that combines the functional attributes of 4-1BB (pro-survival) and CD28 (effector function) signaling. T cells were co-transduced with viruses expressing the 4-1BB CAR and the CD28 CAR separately. This generated dual-transduced CD4 CAR T cell products, in which a fraction of cells expressed the 4-1BB CAR (top left), CD28 CAR T (bottom right), or both the 4-1BB CAR and CD28 CAR (top right). Figure 2D illustrates the experimental design in which the dual-transduced CAR T cell products were infused into humanized BLT mice 48 hours after infection with one of two HIV strains: JR-CSF and MJ4. Mice were bled at the indicated time points to measure 1) virus levels and 2) the number of CAR T cells in the peripheral blood. Figure 2E illustrates the expansion of the total CAR T cell population in the peripheral blood over time. Dual-transduced CAR T cells expressing both the 4-1BB CAR and the CD28 CAR expand to a greater extent than single-transduced CAR T cells. In HIV JRCSF- and MJ4-infected mice, dual-transduced CAR T cells accounted for over 60% and 14% of total T cells, respectively. Figure 2F shows results demonstrating that expression of dual CARs on T cells confers greater proliferative capacity than single-transduced CAR T cells.In dual-transduced CAR T cells, nearly 500-fold and 150-fold changes in cell concentration in HIV JRCSF and MJ4 infected mice are detected, whereas single-transduced CAR T cells demonstrate an average of only 125-fold (JRCSF) and 50-fold (MJ4) expansion. [Figure 2-2] See description of Figure 2-1. [Figure 3-1] Figure 3A illustrates the cytotoxic potential of individual CAR T cell populations by measuring the co-expression of perforin and granzyme B, two critical molecules that mediate T cell killing of target cells. Figure 3B shows results indicating that 4-1BB CAR T cells of both CD8+ and CD4+ T cell lineages express low levels of both perforin and granzyme B, but dual-transduced CAR T cells co-express substantially more, and to nearly the same extent as CD28 CAR T cells. Figure 3C shows results from CAR T cells isolated from tissues of HIV-infected mice and stimulated with HIV antigens to detect the production of MIP-1b, an antiviral chemokine. Figure 3D shows data showing that 4-1BB CAR T cells produce relatively few molecules associated with effector function, such as MIP-1b, an antiviral chemokine, CD107a, a marker of cytotoxicity, and TNF, an inflammatory cytokine, whereas dual-transduced CAR T cells upregulate MIP-1b, CD107a, and TNF to levels comparable to CD28 CAR T cells. [Figure 3-2] See description of Figure 3-1. [Figure 4-1]Figure 4A illustrates the discovery that HIV-specific CAR T cells derived from BLT mice are polyfunctional in vitro. Figure 4B shows a schematic diagram for the production of CAR T cells derived from BLT mice. Figure 4B shows representative proliferation rates of CAR.ζ T cells derived from BLT mice and adult human PBMCs after activation with anti-CD3 / CD28 Dynabeads. Figure 4C shows a series of FACS plots of CD4+ CAR.ζ T cells expressing MIP-1β, TNF, IL-2, and GM-CSF after in vitro stimulation with HIVyu2 GP160+ K562 cells (K.Env). Figure 4D shows quantification of intracellular expression of the indicated effector molecules by CD8+ CAR.ζ T cells. Figure 4E shows quantification of intracellular expression of the indicated effector molecules. Data show expression of each molecule from three separate donors per source. Figure 4F shows a schematic of the gating strategy used to identify active caspase-3+HIVGAG+ target cells for analysis in the HIV clearance assay. Figure 4G shows the FACS plot, and Figure 4H shows cumulative data demonstrating coordinated upregulation of granzyme B and perforin in CAR.ζ T cells from BLT mice and human donors after in vitro stimulation with K.Env (stimulated) or K.WT (unstimulated) cells. Data represent expression from three separate donors per source. For the data in Figure 4C, lines and error bars represent the mean ± SEM. [Figure 4-2] See description of Figure 4-1. [Figure 4-3] See description of Figure 4-1. [Figure 4-4] See description of Figure 4-1. [Figure 4-5] See description of Figure 4-1. [Figure 4-6] See description of Figure 4-1. [Figure 5-1]We demonstrate that HIV-specific CAR T cells derived from BLT mice are functionally indistinguishable from human-derived CAR T cells in vitro. Purified human T cells from BLT mice and PBMCs from healthy human donors were activated with αCD3 / CD28 Dynabeads and transduced with a CD4-based CAR.ζ construct co-expressing GFP. Figure 5A is a series of FACS plots identifying CAR.ζ T cells from each T cell source as GFP+ and CD4+. Figure 5B shows the results of mixing CD8+ CAR.ζ T cells with HIVYU2GP160+ K562 cells (K.Env) after 10 days of culture and measuring the upregulation of human cytokines. Figure 5C shows the polyfunctional profile of combined subsets of CD4+ and CD8+ CAR.ζ T cells producing zero to five of the human cytokines GM-CSF, IFN-γ, IL-2, MIP-1β, and TNF-α. An average of three unique donors per T cell source. Figures 5D-5F show the results of an HIV suppression assay as described in "Materials and Methods." Figure 5D shows a FACS plot showing the frequency of HIV-infected T cells after 6 days of coculture with CAR.ζ T cells from BLT mice or humans at the indicated effector:target (E:T) ratios. Figures 5E-5F show a summary of the frequency of HIV-infected target cells (viable CAR-CD8- T cells) after 2, 4, and 6 days of coculture with CAR.ζ T cells from BLT mice (Figure 5E) or humans and untransduced (UTD) T cells (Figure 5F) at the indicated E:T ratios. Figures 5G-5H show the results of an HIV elimination assay as described in "Materials and Methods." Figure 5G is a series of FACS plots, and Figure 5H summarizes the data for the frequency of active caspase-3 in live target cells (CTV+HIVgag+ T cells) after 24 hours of coculture with CAR.ζ and UTD T cells from BLT mice or humans at a 1:1 E:T ratio. Each symbol represents the mean value of replicates per donor (n=3). For Figures 5E-5F, each donor was run in triplicate. Symbols and lines indicate the mean value, and error bars indicate ±SEM. [Figure 5-2] See description of Figure 5-1. [Figure 5-3] See description of Figure 5-1. [Figure 5-4] See description of Figure 5-1. [Figure 5-5] See description of Figure 5-1. [Figure 5-6] See description of Figure 5-1. [Figure 6-1]We demonstrate that CAR T cells expressing the 4-1BB costimulatory domain exhibit a growth advantage and induce B cell hypoplasia in vivo. Figures 6A-6E show the results of transducing T cells from BLT mice with either mCherry.T2A.CAR.ζ, iRFP670.T2A.CAR.BBζ, or GFP.T2A.CAR.28ζ. 5 x 106 CAR-transduced T cells of each type were mixed prior to infusion into syngenic mice (n = 8). Figure 6A shows the frequency of each CAR T cell type within the pre-infusion T cell product. Figure 6B shows the frequency of peripheral CAR T cells within the same mice 5 weeks post-infusion. Figure 6C shows peripheral concentrations, and Figure 6D shows the cumulative persistence of each CAR T cell type over 5 weeks. Figure 6E shows the relative tissue frequency of each CAR T cell type 7 weeks post-infusion. Figure 6F shows the results of a separate study in which BLT mice were administered 10 irradiated wild-type K562 (K.WT; n = 8) or HIVYU2GP160+K562 (K.Env; n = 8) cells 2 weeks after infusion of the CAR T cell mixture described in Figure 6A. Peripheral concentrations of each CAR T cell type after K.WT or K.Env stimulation. Figure 6G is a series of FACS plots showing the frequency of MIP-1β+ and TNF-CAR.BBζ and CAR.28ζ T cells in the same mice after ex vivo stimulation. CAR.ζ T cells were too low in frequency for analysis. Figure 6H shows the frequency of granzyme B and perforin in CD8+ CAR T cells in the same mice ex vivo. Figures 6I-6K show the results of an experiment in which mice were infused with 5 x 10 CD19-specific CAR.BBζ (n = 4) or control CD4-based CAR.BBζ T cells (n = 3). Figure 6I shows the concentration of peripheral CD19+ cells after infusion. Figure 6J is a series of FACS plots showing the frequency of CD19+ cells among all huCD45+ cells. Figure 6K shows the number of CD19+ cells in tissues 7 weeks after infusion. Symbols in Figures 6C, 6F, and 6I indicate the mean, with error bars indicating ±SEM. Symbols in Figures 6D and 6K represent individual mice, with bars indicating the mean, and error bars indicating ±SEM.Figure 6D, Friedman test with Dunn's multiple correction test; Figures 6F and 6H, Wilcoxon matched-pairs signed-rank test performed to calculate significance (*P<0.05, **P<0.01). [Figure 6-2] See description of Figure 6-1. [Figure 6-3] See description of Figure 6-1. [Figure 6-4] See description of Figure 6-1. [Figure 6-5] See description of Figure 6-1. [Figure 6-6] See description of Figure 6-1. [Figure 7] This figure illustrates that CD28 costimulation enhances the ex vivo effector function of CAR T cells. Equal mixtures of CD4-based CAR T cells expressing either CD3-ζ, 4-1BB / CD3-ζ, or CD28 / CD3-ζ costimulatory domains linked to unique fluorescent proteins to facilitate in vivo identification, as described in Figures 6A-6K, were infused into HIV-uninfected mice. Cumulative data show the frequencies of TNF+, IL-2+, and MIP-1β+ CAR.BBζ and CAR.28ζ T cells within the same mouse after ex vivo stimulation with K.Env (stimulated) or K.WT (unstimulated) cells. Data represent the total number of cytokine-producing cells from the liver and terminal blood (n=8). CAR.ζ T cells were too infrequent for analysis. Data are shown as boxplots, with bars indicating minimum and maximum values. Significance was calculated using the Wilcoxon matched-pairs signed-rank test (**P<0.07). Symbols represent individual mice. [Figure 8-1]We illustrate that HIV-specific CAR.BBζ T cells display characteristics of T cell exhaustion following failure to suppress viral rebound. Figure 8A shows the mean log plasma viral RNA (copies mL) in HIVJRCSF-infected mice treated with ART from weeks 3 to 5 (G1 and G2 mice, gray boxes) or from weeks 3 to 8 (G3 and G4 mice). Five weeks after infection, G1 (n = 6) and G3 (n = 10) mice were administered 10 CAR.BBζ T cells, while G2 (n = 6) and G4 (n = 9) mice were administered 10 inactive control CAR.BBΔζ T cells. The thin dotted line indicates the limit of quantification. Figure 8B shows FACS plots, and Figure 8C shows summary data showing the frequency of total memory CD4+ T cells (CAR-) after ART withdrawal in CAR.BBζ and control CAR.BBΔζ T cell-treated mice. Figures 8D-8E show the concentration of peripheral CAR T cells in G1 / G2 and G3 / G4. Figure 8F shows the frequency of CAR T cells in tissues 12 weeks after CAR T cell infusion in G1 / G3 and G2 / G4. Figure 8G shows PD-1 and TIGIT expression in peripheral CAR.BBζ or CAR.BBΔζ T cells from G1 / G2 after ART cessation. Figures 8H-8L show FACS analysis of spleen tissue from BLT mice 12 weeks after ART cessation. Figure 8H shows coexpression of TOX with 2B4, PD-1, or TIGIT in peripheral CAR.BBζ or CAR.BBΔζ T cells. Figure 8I shows the frequency of TOX- and TOX+ CAR.BBΔζ T cells positive for the indicated inhibitory receptors. Figure 8J shows the frequency of T-bet and Eomes expressing CAR.BBζ and CAR.BBΔζ T cells. Figure 8K shows the frequency of TOX expression in T-bet+ and Eomes+ CAR.BBζ and CAR.BBΔζ T cells. Figure 8L shows the memory distribution of 2B4+PD-1+TIGIT+ and EomeshiT-betdimCAR.BBζ T cells. Figures 8F, 8I, 8J, and 8K: Wilcoxon rank sum test was used to calculate significance (**P<0.01, ***P<0.001, ****P<0.0001). Bars and symbols indicate the mean, and error bars indicate ±SEM.Figures 8I, 8J and 8K: symbols represent individual mice. [Figure 8-2] See description of Figure 8-1. [Figure 8-3] See description of Figure 8-1. [Figure 8-4] See description of Figure 8-1. [Figure 8-5] See description of Figure 8-1. [Figure 8-6] See description of Figure 8-1. [Figure 8-7] See description of Figure 8-1. [Figure 9A] Figure 9A shows that CAR.BBζ T cells are unable to prevent CD4+ T cell loss after ART cessation. Figure 9A is a schematic representation of the gating strategy used to identify total memory CD4+ T cells (CAR-). [Figure 9B] Figure 9B shows that CAR.BBζ T cells cannot prevent CD4+ T cell loss after ART cessation. Figure 9B shows the percentage of CD4+ T cells (CAR-) among total CD3+ cells from the indicated tissues in BLT mice treated with CAR.BBζ T cells (G1) or control CAR.BBΔζ (G2) T cells 12 weeks after ART cessation. Symbols represent individual mice. Bars indicate the mean, and error bars indicate ±SEM. [Figure 9C] Figure 9C shows that CAR.BBζ T cells are unable to prevent CD4+ T cell loss after ART cessation. Figure 9C shows results 9 weeks after ART cessation for G3 / 4. Symbols represent individual mice. Bars indicate mean values, and error bars indicate ±SEM. N / A indicates tissue samples with too low a frequency to analyze viable human cells. [Figure 10]We illustrate the finding that HIV infection preferentially depletes memory CD4+ T cells in BLT mice. Figure 10A shows the mean plasma viral RNA (copies mL) of mice in G1 (bold line; right axis) and the frequency of peripheral memory (CD45RA-) CD4+ T cells (left y-axis) postchallenge in HIV- mice (open circles) and HIV+ mice (G1; filled circles). The thin dotted line indicates the limit of viral load quantification. The shaded box indicates the ART window. Symbols indicate the mean, and error bars indicate ± SEM. Figure 10B shows the frequency of CCR5 expression in the indicated populations of CD4+ T cells from the peripheral blood of BLT mice. [Figure 11-1] Figure 11 illustrates that CAR.BBζ T cells accumulate multiple inhibitory receptors as the disease progresses. Figure 11A shows the frequency of CD4+ and Figure 11B shows the frequency of CD8+ CAR.BBζ T cells (G1) and control CAR.BBΔζ T cells (G2) that co-express TIGIT and PD-1 after infusion. The shaded box indicates the ART window. Symbols indicate the mean, and error bars indicate ±SEM. Figure 11C shows the frequency of CD4+ and Figure 11B shows the frequency of CD8+ CAR.BBζ T cells (G1) and control CAR.BBΔζ T cells (G2) that co-express TIGIT, PD-1, and 2B4 in tissues 12 weeks after infusion. Figure 11W shows cumulative data showing the frequency of 2B4+, PD-1+, and TIGIT+ CD4+ CAR.BBζ T cells (G1) compared with CAR-CD4+ T cells (G1) in the spleen of the same mice, and (Figure 11F) CD8+ CAR.BBζ T cells (G1) compared with CAR-CD8+ T cells (G1) in the spleen of the same mice. Figures 11C-11F: Bars indicate mean values, error bars indicate ±SEM, and symbols represent individual mice. Significance was calculated using the Wilcoxon rank-sum test (*P<0.05 and **P<0.01). [Figure 11-2] See description of Figure 11-1. [Figure 11-3] See description of Figure 11-1. [Figure 12-1]Figure 12 illustrates the accumulation of EomeshiT-betdimCAR.BBζ T cells from the acute to chronic phase of infection. Mice were infected with HIV JRCSF and, 48 hours later, infused with 2 x 107 of either CAR.BBζ T cells (n = 5) or inactive control CAR.BBΔζ T cells (n = 3). Figure 12A is a series of FACS plots showing the change in EomeshiT-betdim and T-bet expression over time within different CAR T cell types. Figure 12B shows summary data showing the longitudinal frequency (left y-axis) of EomeshiT-betdimCD8+ (left panel) and CD4+ (right panel) CAR T cells as well as the mean log plasma viral RNA (copies mL) (right y-axis). The thin dotted line indicates the limit of viral load quantification. Symbols indicate the mean, and error bars indicate ± SEM. Figure 12C shows a Spearman correlation analysis of the frequency of EomeshiT-betdimCD8 + CAR.BB ζ T cells compared with the viral load, measured as the frequency of HIV GAG + CD8 − T cells, in various tissues 10 weeks post-infection. [Figure 12-2] See description of Figure 12-1. [Figure 13A] Figure 13 illustrates that CAR.BBζ T cells from chronic infection exhibit attenuated ex vivo function compared with the CAR T cell product. 12 weeks after infusion, CAR.BBζ T cells (n = 14) and inactive control CAR.BBAζ T cells (n = 10) were isolated from the livers of chronically infected mice and stimulated ex vivo with the pre-infusion CAR.BBζ T cell product (TCP). Figure 13A shows FACS plots, and Figure 13B shows cumulative data for MIP-1β, CD107a, and granzyme B expression in CD8+ CAR.BBζ or CAR.BBΔζ T cells. Dotted lines indicate the frequency of CD8+ CAR.BBζ T cells from pre-infusion TCPs expressing the indicated proteins. Bars indicate the mean, and error bars indicate ±SEM. Significance was calculated using the Wilcoxon rank-sum test (*P<0.05 and ***P<0.001). [Figure 13B] See legend to Figure 13A. [Figure 14-1]We illustrate the finding that dual CAR T cell products attenuate CD4+ T cell loss and exhibit superior proliferation potential. Figures 14A-14J show the results of an experiment in which mice were challenged with HIVJRCSF (n = 12) or HIVMJ4 (n = 12). 48 hours later, six mice from each group were infused with the dual CAR T cell product (TCP) or untreated (Untx). Figure 14A illustrates that the dual CAR TCP contains CAR.BBζ, CAR.28ζ, and dual CAR T cells. Figures 14B and 14D show the concentration of total peripheral CAR T cells in individual mice (dotted line; left y-axis) and the mean log plasma viral RNA (copies mL) (solid line; right y-axis) of HIVJRCSF- and HIVMJ4-infected mice, respectively. The thin black dotted line indicates the limit of quantification. Figures 14C and 14E show the frequency of peripheral memory CD4+ T cells (CAR-). Figure 14F shows the frequency of CD4+ T cell (CAR-) memory subsets in tissues from HIVMJ4- and (Figure 14G) HIVJRCSF-infected mice 8 weeks after CAR T cell infusion. Figure 14H shows the longitudinal frequency of each CAR T cell type present in the dual-CAR TCP. Figure 14I shows the peak peripheral frequency, and Figure 14J shows the cumulative survival of CAR T cells. Figures 14K-14N show that the dual-CAR TCP was combined with third-generation (3G) CD4-based CAR T cells prior to infusion into HIVMJ4-infected mice (n=6) to equalize the frequency of dual-CAR and 3G-CAR T cells (Figure 19C). Figure 14K shows overlay FACS plots showing the frequency of peripheral dual-CAR (iRFP670+NGFR+) and 3G-CAR (GFP+) T cells within the same mouse. Figure 14L shows the concentration of peripheral CAR T cells. Figure 14M shows the total number of splenic CAR T cells, and Figure 14N shows the cumulative CAR T cell survival 5 weeks post-infection. For all data, bars and symbols represent the mean, and error bars represent ±SEM, except in Figures 14M-14N, where symbols represent individual mice. Significance was calculated using the Wilcoxon rank sum test (*P<0.05, **P<0.01, ****P<0.0001). [Figure 14-2] See description of Figure 14-1. [Figure 14-3] See description of Figure 14-1. [Figure 14-4] See description of Figure 14-1. [Figure 14-5] See description of Figure 14-1. [Figure 15] This figure illustrates the finding that dual CAR T cells exhibit similar in vitro effector function as CAR.28ζ T cells. The dual CAR T cell product includes CAR.BBζ, CAR.28ζ, and dual CAR T cells, with each population identified by a unique fluorescent protein. Cytokine upregulation was measured after in vitro stimulation with K.Env and K.WT cells. Each symbol represents a unique donor. [Figure 16-1] Figure 16 illustrates that the dual CAR T cell product temporarily delays CD4+ T cell loss despite persistent HIVJRCSF infection. Forty-eight hours after HIVJRCSF challenge, mice were administered the dual CAR T cell product (TCP) (n = 6), whereas control mice were untreated (Untx) (n = 5). Figure 16A shows the concentration of peripheral total memory CD4+ T cells (CAR-). Figure 16B shows the concentration of peripheral central memory (CD45RA-CD27+CCR7+; left panel), transitional memory (CD45RA-CD27+CCR7; center panel), and effector memory (CD45RA-CD27+CCR7-; right panel) CD4+ T cells (CAR-). Significance was calculated using the Wilcoxon rank-sum test (*P<0.05, **P<0.01). Figure 16C shows the frequency of memory CD4+ T cell (CAR-) subsets in tissues 8 weeks after infection. Symbols and bars indicate mean values, error bars indicate ±SEM. [Figure 16-2] See description of Figure 16-1. [Figure 17]The findings that HIVJRCSF and HIVMJ4 exhibit different replication kinetics in vitro and in vivo are illustrated. Figure 17A shows the results of an in vitro replication assay comparing the replication kinetics of HIVJRCSF and HIVMJ4 in human PBMCs stimulated with PHA and infected at a matched multiplicity of infection of 0.002. Viral replication was assessed by measuring p24 antigen in culture supernatants. Figure 17B shows the mean log plasma viral RNA (copies mL) in BLT mice challenged with HIVJRCSF (n = 3) or HIVMJ4 (n = 4). Thin dotted lines indicate the limits of quantification. Symbols indicate the mean, and error bars indicate ± SEM. Significance was calculated using the Wilcoxon rank-sum test (*P < 0.05). [Figure 18-1] This figure illustrates the finding that dual CAR T cell products prevent CD4+ T cell loss despite persistent HIVMJ4 infection. Forty-eight hours after HIVMJ4 challenge, mice were infused with the dual CAR T cell product (TCP) (n=6), whereas control mice were untreated (Untx) (n=6). Figure 18A shows the concentration of peripheral total memory CD4+ T cells (CAR-). Figure 18B shows the concentration of peripheral central memory (CD45RA-CD27+CCR7+; right panel), transitional memory (CD45RA-CD27+CCR7-; center panel), and effector memory (CD45RA-CD27-CCR7-; left panel) CD4+ T cells (CAR-). Significance was calculated using the Wilcoxon rank-sum test (**P<0.07). Figure 18C shows the frequency of memory CD4+ T cell (CAR-) subsets in tissues 8 weeks after infection. Symbols and bars indicate mean values, error bars indicate ±SEM. [Figure 18-2] See description of Figure 18-1. [Figure 19-1]These results illustrate the finding that dual CAR T cells exhibit superior in vivo expansion compared to 4-1BB, CD28, and third-generation CAR T cells. Figure 19A shows the results of an experiment in which BLT mice were challenged with either HIVJRCSF (n=6) or HIVMJ4 (n=6) and infused with 2x107 dual CAR T-cell products (TCP). The fold change in CAR T-cell concentration from baseline to peak levels in peripheral blood is shown. Data are aggregated across both infected cohorts. Figure 19B is a schematic diagram depicting the components of a third-generation (3G) CD4-based CAR construct. Figures 19C-19E show the results of combining the dual CAR T-cell product with 3G-CAR T cells prior to infusion into uninfected mice (n=9) to equalize the frequency of dual CAR and 3G-CAR T cells. Figure 19C shows a FACS plot depicting the frequency of dual CAR and 3G-CAR T cells present within the T-cell product prior to infusion. Figure 19D shows longitudinal concentrations of peripheral CAR T cells after adoptive transfer into HIV-negative mice. Symbols indicate mean values, and error bars indicate ±SEM. Figure 19E shows the results of administering either 10 irradiated K.Env cells (n=6) or 10 irradiated K.WT cells (n=3) to mice 2 weeks after infusion. Fold change in peripheral CAR T cell concentrations from baseline concentrations before K562 infusion to 1 week after K562 boost. Bars indicate mean values, error bars indicate ±SEM, and symbols represent individual mice. Figures 19A, 19D, and 19E: Significance was calculated using the Wilcoxon rank sum test (*P<0.05, **P<0.01). [Figure 19-2] See description of Figure 19-1. [Figure 20-1]Figure 20 illustrates the finding that CD4-based CAR T cells are susceptible to infection in vivo. Figure 20A shows a FACS plot, and Figure 20B shows cumulative data for the frequency of HIV GAG + T-cell populations sampled within the same mice (n=5) 10 weeks after HIVJRCSF infection. Data in Figure 20B represent the total tissues from five mice: bone marrow, liver, lung, lymph node, terminal blood, and spleen. Figure 20C shows a FACS plot, and Figure 20D shows cumulative data showing the expression of granzyme B and perforin within HIV GAG + and HIV GAG - CAR T-cell populations from HIVJRCSF-infected mice after ex vivo stimulation with K.Env (stimulated) or K.WT (unstimulated) cells. Data in Figure 20D are presented as the mean of three different CAR T-cell populations. Significance was calculated using a paired t-test (*P<0.05). Symbols and bars represent the mean, and error bars represent ±SEM. [Figure 20-2] See description of Figure 20-1. [Figure 21-1]We illustrate the finding that HIV-resistant dual CAR T cells mediate superior virus-specific immune responses. Figure 21A is a schematic diagram of HIV-resistant (C34-CXCR4+) dual CAR T cells. Figure 21B illustrates an experiment in which 107 CAR T cells were administered to HIVJRCSF-infected BLT mice 48 hours after challenge. HIV DNA load in sorted CAR T cells from individual mouse spleen tissue (n = 8) is shown. Figures 21C-21D illustrate an experiment in which 106 C34-CXCR4+, CAR.BBζ (n = 6), CAR.28ζ (n = 5), or purified dual CAR (n = 4) T cells were injected into HIVMJ4-infected mice 48 hours after challenge. Figure 21C shows longitudinal peripheral concentrations, and Figure 21D shows peak peripheral CAR T cell concentrations. Figures 21E-21N illustrate an experiment in which 48 hours after challenge, HIVMJ4-infected mice were infused with 10 purified C34-CXCR4+, CAR.BBζ.BBζ (n=5), CAR.28ζ.28ζ (n=5), or dual-CAR (n=5) T cells or were not treated (n=4). The purification strategy is described in Figures 25A-25D. Figure 21E shows the frequency of CAR T cell populations among total human CD45+ cells at 2 and 3 weeks postinfection. Figure 21F shows longitudinal concentration, and Figure 21G shows cumulative peripheral CAR T cell survival. Figure 21H is a series of FACS plots showing CCR5 expression in peripheral memory CD4+ T cells (CAR-). Figure 21I shows total memory concentration, and Figure 21J shows CCR5+ CD4+ T cells (CAR-) 6 weeks postinfection. Figure 21K is a series of FACS plots, and Figure 21L shows the frequency of MIP-1β+ and CD107a+CD8+ CAR T cells from tissues 8 weeks post-infection after ex vivo stimulation. Figure 21M shows the distribution, and Figure 21N shows the frequency of granzyme BB+ perforin+ cells with CD107a+ CAR T cells from tissues after ex vivo stimulation. Figure 21B: Wilcoxon matched-pairs signed-rank test used to calculate significance. For the remaining analyses, significance was calculated using the Wilcoxon rank sum test (*P<0.05, **P<0.01, ***P<0.001).Bars indicate mean values, error bars indicate ±SEM, and symbols represent individual mice, except for Figure 21C, which shows mean values. [Figure 21-2] See description of Figure 21-1. [Figure 21-3] See description of Figure 21-1. [Figure 21-4] See description of Figure 21-1. [Figure 21-5] See description of Figure 21-1. [Figure 21-6] See description of Figure 21-1. [Figure 22A] This figure illustrates the finding that HIV-resistant dual CAR T cell products cannot suppress acute HIV replication. Figure 22A shows that dual CAR T cell products (TCPs) were co-transduced with C34-CXCR4 linked to mCherry by an intervening T2A sequence. FACS plots show the frequency of C34-CXCR4+ cells within each cell population containing the dual CAR TCPs prior to infusion. [Figure 22B] The finding that HIV-resistant dual CAR T cell products cannot suppress acute HIV replication is illustrated in Figure 22B. Figure 22B shows the logarithmic plasma viral RNA (copy number mL) in individual BLT mice challenged with HIVJRCSF and 48 hours later infused with HIV-resistant (C34-CXCR4+) dual CAR TCP (n=7) or untreated (Untx; n=7). The thin dotted line indicates the limit of quantification. [Figure 23-1]Figure 23 illustrates that C34-CXCR4+ CAR T cells are selected during chronic infection and exhibit superior ex vivo effector function. Figure 23A shows mice were infected with HIVJRCSF and, 48 hours later, injected with 107 C34-CXCR4+ dual CAR T cell products (TCPs). FACS plots show the frequency of C34-CXCR4+ during infection. Figure 23B shows mice were infected with HIVMJ4 and, 48 hours later, injected with 106 C34-CXCR4+ CAR.BBζ (n=5), CAR.28ζ (n=5), or purified dual CAR (n=4) T cells. Frequency of C34-CXCR4+ CAR T cells in tissues 8 weeks post-infection. Thin dotted lines indicate the frequency of C34-CXCR4+ CAR T cells in the pre-infusion TCP for the indicated CAR T cell type. Figures 23C-23D show mice were infected with HIVMJ4 and, 48 hours later, administered 10 C34-CXCR4+, purified CAR.BBζ.BBζ (n=3), CAR.28ζ.28ζ (n=4), or dual-CAR (n=3) T cells. Figure 23C shows FACS plots, and Figure 23D shows cumulative data for the frequency of each CD8+ CAR T cell population expressing MIP-1β and CD107a, as well as the frequency of cytotoxic CAR T cells (granzyme B+perforin+CD107a+). Eight weeks after infection, CAR T cells were isolated from the spleen and bone marrow of mice and stimulated ex vivo. Significance was calculated using the Wilcoxon matched-pairs signed-rank test (**P<0.01). For all data, symbols represent individual mice. [Figure 23-2] See description of Figure 23-1. [Figure 23-3] See description of Figure 23-1. [Figure 24]Figure 1 illustrates the finding that low-dose dual CAR T cells attenuate CD4+ T cell loss during HIVMJ4 infection. 48 hours after challenge, HIVMJ4-infected mice were infused with 106 C34-CXCR4+, CAR.BBζ (n=6), CAR.28ζ (n=6), or purified dual CAR (n=4) T cells. For each group of mice, the change in peripheral concentration of CCR5+ CD4+ T cells (CAR-) was measured from the indicated time post-infection to pre-infection levels. Bars indicate mean values, and error bars indicate ±SEM. Symbols represent individual mice. [Figure 25-1] Figure 25A illustrates the two-step immunomagnetic selection process for generating purified T cells expressing two independent CARs. Figure 25A shows a schematic of the lentiviral constructs used to generate dual CD4-based CAR-transduced T cells. CD4-based CARs with 4-1BB / CD3-ζ or CD28 / CD3-ζ endodomains were linked to NGFR or truncated EGFR (EGFRt) to allow for two stages of positive magnetic selection during the T cell manufacturing process. Figure 25B illustrates the timeline for CAR T cell manufacturing. One day after T cell activation with αCD3 / CD28 Dynabeads, cells were transduced with an equivalent MOI of the lentivirus shown in Figure 25A. Four and seven days after activation, CAR T cells were positively selected using anti-EGFR and anti-NGFR coated magnetic beads, respectively, as described in "Materials and Methods." Figure 25C shows representative FACS plots illustrating the purity of dual CAR-transduced T cells after EGFR and NGFR selection. Figure 25D shows FACS plots demonstrating the frequency of CAR.BBζ.BBζ, CAR.28ζ.28ζ, and dual CAR T cells after selection in each pre-infusion T cell product prior to adoptive transfer into the mice described in Figures 21E-21N. [Figure 25-2] See description of Figure 25-1. [Figure 26-1]We illustrate that dual CAR T cells mediate superior CD4+ T cell expansion and protection during HIV infection in vivo. BLT mice were infected with HIVMJ4 and, 48 hours later, administered 106 purified C34-CXCR4+, CAR.BBζ.BBζ (n=5), CAR.28ζ.28ζ (n=5), or dual CAR (n=5) T cells, or were untreated (Untx; n=4). Figure 26A shows the fold change in CAR T cell concentrations in peripheral blood at 2–3 weeks post-infection. Numbers above the bars indicate the mean fold change. Symbols represent individual mice. Figure 26B shows the absolute numbers of each CAR T cell population in tissues 8 weeks post-infection. Figure 26C shows the concentration of peripheral total memory (CD45RA-), and Figure 26D shows the concentration of CD45RA-CCR5+CD4+ T cells (CAR-). Symbols represent the mean. Figure 26E shows the correlation between the fold change in CAR T cell concentration in peripheral blood and the change in total memory (CD45RA-) CD4+ T cells (CAR-) at 2-3 weeks post-infection. Symbols represent individual mice. Significance was calculated using the Spearman correlation test. Figures 26B-26D: Error bars indicate ± SEM, and significance was calculated using the Wilcoxon rank sum test (*P<0.05 and **P<0.01). [Figure 26-2] See description of Figure 26-1. [Figure 27A]

[0023] Figure 27 illustrates that CAR T cells from HIV-infected mice exhibit ex vivo cytotoxic function. Dual-CAR TCP-treated HIVJRCSF-infected mice (n=3) were euthanized, and bone marrow cells were stimulated ex vivo with K.Env or K.WT cells at the indicated E:T ratios for 24 hours. Figure 27A shows representative FACS plots. Symbols indicate mean values, and error bars indicate ±SEM. [Figure 27B] Figure 27B illustrates that CAR T cells from HIV-infected mice exhibit ex vivo cytotoxic function. Dual-CAR TCP-treated HIVJRCSF-infected mice (n=3) were euthanized, and bone marrow cells were stimulated ex vivo with K.Env or K.WT cells at the indicated E:T ratios for 24 hours. Figure 27B shows cumulative data demonstrating the induction of active caspase-3 in target cells. Symbols indicate mean values, and error bars indicate ±SEM. [Figure 28-1] Figure 28 illustrates that dual CAR and CAR.28ζ T cells exhibit similar ex vivo functional profiles. Mice were challenged with HIVJRCSF (n=5) and 48 hours postinfection were infused with 2x107 dual CAR T cell products (TCP). Figure 28A shows the frequency of CD8+ and Figure 28B shows the CD4+ CAR T cell population from tissues at necropsy (8 weeks postinfection) within the same mice expressing CD107a, MIP-1β, IL-2, and TNF after ex vivo stimulation. Bars indicate mean values, error bars indicate ±SEM, and symbols represent individual mice. Significance was calculated using the Wilcoxon rank-sum test (**P<0.07). Figure 28C shows principal component analysis (PCA) of IL-2, TNF, MIP-1β, and CD107a expression in ex vivo stimulated CD8+ and CD4+ CAR T cells from PBMCs of HIVJRCSF-infected mice (n=5). [Figure 28-2] See description of Figure 28-1. [Figure 29-1]We illustrate the finding that attenuation of CAR T cell infection improves control of HIV replication. Figure 29A shows the mean log plasma viral RNA (copies mL) in non-protective active CAR T cell-treated mice (n = 38) and untreated / inactive CAR T cell-treated mice (n = 36). Data are aggregated across six independent experiments. The thin dotted line indicates the limit of quantification. Figure 29B shows the mean log plasma viral RNA (copies mL) in mice injected with 107 fully protective (>98% C34-CXCR4) dual-CAR TCPs (n = 12) or untreated mice (n = 12) 48 hours after HIVMJ4 challenge. Figure 29C shows the frequency of splenic HIV GAG+ CD8- T cells (CAR-), and Figure 29D shows the cell-associated HIV DNA load in lymph nodes at 6–8 weeks post-infection. Figures 29E-29K show results from an experiment in which HIVJRCSF-infected mice were treated with ART and simultaneously infused with 107 HIV-resistant dual-CAR TCPs (n=12), inactive dual-ΔCAR TCPs (n=5), or no treatment (n=7). Figure 29E shows the mean log plasma HIV RNA (copies mL). The shaded box indicates ART, and the arrow indicates TCP infusion. Figure 29F shows the log percent reduction in plasma HIV RNA from pre-ART (week 3) to 1 and 1.5 weeks after ART. Figures 29G-29H show data compiled from HIVJRCSF- and HIVBAL-infected cohorts. Figure 29G shows the correlation between percent reduction in viral load at the first post-ART time point and peripheral CAR T cell concentrations over the same period. Figure 29H shows Kaplan-Meier curves of the time to viral suppression from treatment initiation for dual-CAR TCPs versus control mice. Figure 29I shows the frequency of HIV GAG + CD8- T cells (CAR-), and Figure 29J shows HIV GAG + CD14- macrophages counted from various tissues of plasma viremia-suppressed mice. Figure 29K shows cell-associated HIV DNA load in sorted central memory (CAR-CD45RA-CCR7+) CD4+ T cells. Statistical significance was calculated by Wilcoxon rank-sum test in Figures 29A-29E and 29I-29K, by Spearman correlation in Figure 29G, and by log-rank test in Figure 29H.For all data, *P<0.05, **P<0.01, and ***P<0.001. Bars indicate mean values, error bars indicate ±SEM. Symbols represent individual mice. [Figure 29-2] See description of Figure 29-1. [Figure 29-3] See description of Figure 29-1. [Figure 30-1] We illustrate that HIV-resistant dual-CAR TCP reduces viral replication in vivo. Figure 30A shows the frequency of HIV GAG + CD8- T cells (CAR-) in the bone marrow and spleen of HIV JRCSF-infected mice, and Figure 30B shows HIVMJ4-infected mice treated (TCP) or not (Untx) with the dual-CAR T cell product 48 hours after challenge. Figure 30C shows the mean log plasma HIVMJ4 RNA (copies mL-1) after ART discontinuation in mice injected with 107 C34-CXCR4+ cells (n=5) that were fully protected at ART initiation, or mice injected with 107 C34-CXCR4+ cells (n=7) or not treated (n=9) that were partially protected (<20% C34-CXCR4+ cells). Figures 30D-30E show the results of an experiment in which HIVBAL-infected mice were treated with ART and simultaneously injected with 107 HIV-resistant dual-CAR TCP (n=6) or not treated (n=6). Figure 30D shows the mean log plasma HIV RNA (copies mL). The shaded box indicates ART, and the arrow indicates TCP infusion. Figure 30E shows the log reduction in plasma viral RNA from pre-ART (week 3) to 0.5 and 1 week after ART. For all data, bars indicate the mean, error bars indicate ±SEM, and symbols represent individual mice. Significance was calculated using the Wilcoxon rank sum test (*P<0.05, **P<0.01, ****P<0.0001). [Figure 30-2] See description of Figure 30-1. [Figure 30-3] See description of Figure 30-1. [Figure 31A]The gating strategy for FACS sorting of CAR T cells and endogenous central memory CD4+ T cells is illustrated. Figure 31A: For the experiment described in Figure 21B, C34-CXCR4+ and C34-CXCR4-CAR T cells were bulk sorted by FACS according to the gating strategy shown. For all data, cell-associated HIV DNA load was quantified in the sorted cell populations by droplet digital PCR. [Figure 31B] The gating strategy for FACS sorting of CAR T cells and endogenous central memory CD4+ T cells is illustrated. Figure 31B: For the experiment described in Figure 29E, endogenous central memory CD4+ T cells (CAR-) were sorted from splenocytes harvested at necropsy (7 weeks post-infection) according to the illustrated gating strategy. For all data, cell-associated HIV DNA load was quantified by droplet digital PCR in the sorted cell populations. [Figure 32] 1 illustrates that CD19 and CD22 antigens are highly expressed on B-ALL. [Figure 33-1] Illustrates the CD19 and CD22 CAR constructs and the high yield of purified T cells expressing two independent CARs after a two-step immunomagnetic selection process. [Figure 33-2] See description of Figure 33-1. [Figure 34A] 1 illustrates that anti-CD19 / anti-CD22 transduced T cells exhibit cytokine production in co-culture with double-positive and CD19 knockout targets. [Figure 34B] 1 illustrates that anti-CD19 / anti-CD22 transduced T cells exhibit cytokine production in co-culture with double-positive and CD19 knockout targets. [Figure 35-1] 1 illustrates that anti-CD19 / anti-CD22 transduced T cells kill double-positive and CD19 knockout targets. [Figure 35-2] See description of Figure 35-1. [Figure 36]1 illustrates that anti-CD19 / anti-CD22 transduced T cells demonstrate anti-leukemic activity in vivo against CD19+Ve and CD19-VeB-ALL. [Figure 37] Schematic of the dual CD19T2ACD22 CAR structure and expression of anti-CD19 and anti-CD22 CARs in T2A CAR-transduced T cells. [Figure 38] 1 illustrates that dual CD19T2ACD22 CAR T cells demonstrate anti-leukemic activity in vitro and in vivo against CD19+Ve and CD19-VeB- ALL. [Figure 39] Illustrates anti-CD19 and anti-CD22 CAR expression in CD4 and CD8 T cells. [Figure 40] 1 illustrates that dual anti-CD19 and anti-CD22 CAR T cells enhance cytokine responses in CD4 and CD8 T cells after co-culture with NALM6. [Figure 41A] 1 illustrates that dual anti-CD19 and anti-CD22 CAR T cells demonstrate anti-leukemic activity in vitro against NALM6. [Figure 41B] 1 illustrates that dual anti-CD19 and anti-CD22 CAR T cells demonstrate anti-leukemic activity in vitro against NALM6. [Figure 42] 10 illustrates that dual CD19T2ACD22 CAR T cells enhance cytokine responses in CD4 T cells after co-culture with NALM6. [Figure 43] 10 illustrates that dual CD19T2ACD22 CAR T cells enhance cytokine responses in CD8 T cells after co-culture with NALM6. DETAILED DESCRIPTION OF THE INVENTION

[0049] Detailed Description A. Definition Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

[0050] 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.

[0051] 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.

[0052] As used herein, "about" when referring to a measurable value such as an amount, a temporal duration, and the like, is intended to encompass a variation 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.

[0053] As used herein, "activation" 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" particularly refers to T cells undergoing cell division.

[0054] As used herein, the term "adapter molecule" refers to a polypeptide having a sequence that allows two or more molecules to interact, in certain cases facilitating the activation or inactivation of cytotoxic cells.

[0055] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody can be an intact immunoglobulin derived from natural or recombinant sources, or can be an immunoreactive portion of an intact immunoglobulin. An antibody is typically a tetramer of immunoglobulin molecules. Antibodies in the present invention can exist in various 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).

[0056] 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.

[0057] As used herein, "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.

[0058] As used herein, "antibody light chain" refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations. The α and β light chains refer to the two major antibody light chain isotypes.

[0059] As used herein, the term "synthetic antibody" refers to an antibody produced using recombinant DNA techniques, such as, for example, the bacteriophage-expressed antibodies described herein. The term should also be taken to mean an antibody produced by synthesis of an antibody-encoding DNA molecule that expresses an antibody protein or an amino acid sequence defining that antibody, where the DNA or amino acid sequence is available and well known in the art, and obtained using DNA or amino acid sequence synthesis techniques.

[0060] As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an immune response. This immune response may include either or both antibody production and activation of specific immunocompetent cells. Those skilled in the art will understand that virtually any macromolecule, including all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an "antigen" as that term is used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by the 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 more than one gene, and that these nucleotide sequences may be arranged in various combinations to elicit a desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It will be readily apparent that an antigen can be generated, synthesized, or derived from a biological sample. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0061] As used herein, the term "anti-tumor effect" refers to a biological effect that can be manifested by a reduction in tumor volume, a reduction in tumor cell number, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with a cancerous condition. An "anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells, and antibodies of the present invention to prevent the development of tumors in the first place.

[0062] The term "autoantigen" according to the present invention means any self-antigen that is recognized as foreign by the immune system. Autoantigens include, but are not limited to, cellular proteins, phosphoproteins, cell surface proteins, cellular lipids, nucleic acids, glycoproteins, e.g., cell surface receptors.

[0063] The term " autoimmune disease " used herein is defined as the disorder resulting from autoimmune response.Autoimmune disease is the result of inappropriate and excessive response to autoantigen.Examples of autoimmune disease include but are not limited to Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes mellitus (type I), epididymitis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitritis, myxedema, pernicious anemia, ulcerative colitis, among others.

[0064] As used herein, the term "autologous" is intended to refer to any material derived from the same individual that is later reintroduced into that individual.

[0065] "Allogeneic" refers to a graft derived from a different animal of the same species.

[0066] "Xenogeneic" refers to a graft derived from an animal of a different species.

[0067] The term "broadly neutralizing antibody (bnAb)" refers to an antibody that protects cells from multiple strains of a particular virus by neutralizing its effect. In some embodiments, a broadly neutralizing HIV-1 antibody (bnAb) is a neutralizing antibody that neutralizes multiple HIV-1 viral strains.

[0068] The term "cancer" as used herein is defined as a disease characterized by the rapid and uncontrolled growth of abnormal cells.Cancer cells can spread locally or to other parts of the body through bloodstream and lymphatic system.Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc.

[0069] As used herein, the term "CD4" refers to any amino acid sequence that specifies CD4 from any source, including amino acid sequences of CD4 generated by codon optimization of a nucleic acid sequence encoding CD4. Codon optimization can be achieved using any available techniques and algorithms designed to optimize codons in amino acid sequences.

[0070] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificial T cell receptor that is expressed on immune effector cells and engineered to specifically bind to an antigen. CARs can be used as a therapy involving adoptive cell transfer. T cells are removed from a patient and modified to express a receptor specific for a particular form of antigen. In some embodiments, a CAR is expressed with specificity for, for example, a tumor-associated antigen. CARs can also include an intracellular activation domain, a transmembrane domain, and an extracellular domain containing a tumor-associated antigen-binding region. In some aspects, a CAR comprises a fusion of a monoclonal antibody derived from a single-chain variable fragment (scFv) fused to the transmembrane and intracellular domains. The specificity of the CAR design can be derived from the receptor's ligand (e.g., a peptide). In some embodiments, a CAR can target HIV-infected cells by redirecting the specificity of T cells expressing a CAR specific for an HIV-associated antigen.

[0071] The term "chimeric intracellular signaling molecule" refers to a recombinant receptor that contains one or more intracellular domains of one or more costimulatory molecules. A chimeric intracellular signaling molecule is substantially free of extracellular domains. In some embodiments, a chimeric intracellular signaling molecule contains additional domains, such as a transmembrane domain, a detectable tag, and a spacer domain.

[0072] As used herein, the term "conservative sequence modifications" is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies 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 having a similar side chain. Families of amino acid residues having similar side chains have been 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), beta-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 substituted with other amino acid residues from the same side chain family, and the modified antibodies can be tested for their ability to bind to antigen using the functional assays described herein.

[0073] "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 Toll ligand receptors, 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.

[0074] "Costimulatory molecule" refers to a 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 TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc epsilon R1b), CD79a, CD79b, Fc gamma RIIa, DAP10, DAP12, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, C Ligands that specifically bind to D83 include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD 11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(C D244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6(NTB-A, Ly10 8), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, other costimulatory molecules described herein, any derivative, variant, or fragment thereof, any synthetic sequence of a costimulatory molecule having the same functional capability, and any combination thereof.

[0075] 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.

[0076] The term "cytotoxicity" or "cytotoxicity" means killing or damaging a cell. In one aspect, the modified cells have improved cytotoxicity, e.g., increased cytolytic activity of T cells.

[0077] A "disease" is a state of health in an animal in which the animal is unable to maintain homeostasis and in which the animal's health will continue to deteriorate unless the disease is ameliorated. In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's health is less favorable than it would be without the disorder. If left untreated, a disorder does not necessarily cause a further decline in the animal's health.

[0078] "Effective amount" or "therapeutically effective amount," used interchangeably herein, refer to the amount of a compound, formulation, material, or composition described herein that is effective to achieve a particular biological result or provide a therapeutic or prophylactic benefit. Such results may include, but are not limited to, anti-tumor activity, as determined by any means suitable in the art.

[0079] "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, having either a defined nucleotide (i.e., rRNA, tRNA, and mRNA) sequence or a defined amino acid sequence. Thus, a gene encodes a protein when the protein is produced in a cell or other biological system by transcription and translation of the mRNA corresponding to that gene. Both the coding strand, which is the nucleotide sequence identical to the mRNA sequence and usually shown 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 encode the protein or other product of that gene or cDNA.

[0080] As used herein, "endogenous" refers to any material that originates or is produced within an organism, cell, tissue, or system.

[0081] As used herein, the term "envelope glycoprotein gp120" refers to a 120 kDa glycoprotein on the surface of the HIV envelope. gp120 binds to the CD4 receptor on host cells, such as CD4 T lymphocytes. This initiates the process by which HIV fuses its viral membrane with the host cell membrane and enters the host cell.

[0082] As used herein, the term "exogenous" refers to any material that is introduced from or produced outside an organism, cell, tissue or system.

[0083] As used herein, the term "expand" 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. As used herein, the term "ex vivo" refers to cells removed from an organism (e.g., a human) and propagated outside the organism (e.g., in a culture dish, test tube, or bioreactor).

[0084] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0085] "Expression vector" refers to a vector containing a recombinant polynucleotide comprising an expression control sequence operably linked to a 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., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating a recombinant polynucleotide.

[0086] As used herein, the term "human immunodeficiency virus" or "HIV" refers to any HIV strain or variant known in the art or previously unknown, including, but not limited to, HIV-1 and HIV-2.

[0087] As used herein, "homology" refers to the identity of subunit sequences between two polymer molecules, for example, between two nucleic acid molecules, such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. If a subunit position in both molecules is occupied by the same monomer subunit; for example, if a position in each of two DNA molecules is occupied by adenine, then 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 two sequences (e.g., five positions in a polymer 10 subunits long) are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 out of 10) are matching or homologous, the two sequences are 90% homologous. When applied to nucleic acids or proteins, "homology" as used herein refers to sequences with approximately 50% sequence identity. More preferably, the homologous sequences have about 75% sequence identity, and even more preferably at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity.

[0088] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from 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 instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise 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 human immunoglobulin sequences. A humanized antibody may also optionally 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.

[0089] "Fully human" refers to an immunoglobulin, such as an antibody, where the entire molecule is of human origin or consists of an amino acid sequence identical to that of a human form of the antibody.

[0090] As used herein, "identity" refers to the identity of the subunit sequence between two amino acid molecules, such as between two polymer molecules, particularly between two polypeptide molecules. If two amino acid sequences have the same residue at the same position; for example, if a position in each of 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 positions that match or are identical; 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.

[0091] By "substantially identical" is meant that a polypeptide or nucleic acid molecule exhibits at least 50% identity with a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, more preferably 90%, 95%, or 99% identical at the amino acid or nucleic acid level to the sequence used for comparison.

[0092] The guide nucleic acid sequence can be complementary to one strand (nucleotide sequence) of a double-stranded DNA target site. The percentage of complementarity between the guide nucleic acid sequence and the target sequence can be at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 63%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. The guide nucleic acid sequence can be at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or more nucleotides in length. In some embodiments, the guide nucleic acid sequence comprises a contiguous stretch of 10-40 nucleotides. The variable targeting domain can be composed of a DNA sequence, an RNA sequence, a modified DNA sequence, a modified RNA sequence (see, e.g., the modifications described herein), or any combination thereof.

[0093] Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions generally include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary method for measuring the degree of identity, the BLAST program can be used, e.g., -3 ~e -100 A probability score between indicates a closely related sequence.

[0094] As used herein, the term "immunoglobulin" or "Ig" is defined as a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes referred to as BCRs (B cell receptors) or antigen receptors. The five members of this protein class are IgA, IgG, IgM, IgD, and IgE. IgA is the major antibody present in bodily secretions such as saliva, tears, milk, gastrointestinal secretions, and mucus secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the major 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 can act 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.

[0095] As used herein, the term "immune response" is defined as a cellular response to an antigen that occurs when lymphocytes identify the antigen molecule as foreign and induce the formation of antibodies and / or activate lymphocytes to eliminate the antigen.

[0096] As used herein, "instruction material" includes publications, records, drawings, or any other medium of expression that can be used to communicate the utility of the compositions and methods of the invention. The instruction material of the kits of the invention may, for example, be affixed to a container that holds the nucleic acids, peptides, and / or compositions of the invention, or may be shipped together with a container that holds the nucleic acids, peptides, and / or compositions. Alternatively, the instruction material may be shipped separately from the container, with the intention that the instruction material and the compound will be used cooperatively by the recipient.

[0097] "Isolated" means changed or removed from the natural state. For example, a nucleic acid or peptide naturally occurring in a living animal is not "isolated," but the same nucleic acid or peptide 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, for example, a host cell.

[0098] " Lentivirus " as used herein refers to a genus of Retroviridae family.Lentivirus is unique among retroviruses in that it can infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of host cells, so they are one of the most efficient gene delivery vectors.HIV, SIV and FIV are all examples of lentivirus.Vector derived from lentivirus provides a means to achieve significant levels of gene transfer in vivo.

[0099] As used herein, the term "modified" refers to an altered state or structure of a molecule or cell of the present invention. Molecules can be modified in many ways, including chemically, structurally, and functionally. Cells can be modified by the introduction of nucleic acids.

[0100] As used herein, the term "modulate" refers to mediating a detectable increase or decrease in the level of a response in a subject compared to the level of the response in the subject in the absence of a treatment or compound, and / or compared to the level of the response in an otherwise identical but untreated subject. This term encompasses perturbing and / or affecting a natural signal or response in a subject, preferably a human, thereby mediating a beneficial therapeutic response.

[0101] In the context of the present invention, the following abbreviations for commonly occurring nucleobases are used: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0102] 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 a nucleotide sequence encoding a protein, depending on its form, may contain introns.

[0103] The term "operably linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, if necessary to link two protein-coding regions, in the same reading frame.

[0104] The term "overexpressed" tumor antigen or "overexpression" of a tumor antigen is intended to refer to the expression of a tumor antigen in cells from a diseased area, such as a solid tumor, within a particular tissue or organ of a patient to an abnormal level compared to the level of expression in normal cells from that tissue or organ. Patients with solid tumors or hematological malignancies characterized by overexpression of tumor antigens can be determined by standard assays known in the art.

[0105] "Parenteral" administration of the immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im) or intrasternal injection or infusion techniques.

[0106] The term "polynucleotide" as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Therefore, as used herein, nucleic acids and polynucleotides are interchangeable. Those skilled in the art have the general knowledge that nucleic acids are polynucleotides and can be hydrolyzed into monomeric "nucleotides." Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including recombinant means, i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques and PCR, etc., as well as synthetic means.

[0107] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can comprise a protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids joined together by peptide bonds. As used herein, the term refers to both short chains, commonly referred to in the art as peptides, oligopeptides, and oligomers, and longer chains, commonly referred to in the art as proteins, of which there are many types. "Polypeptide" includes, inter alia, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins. A polypeptide can be a natural peptide, a recombinant peptide, a synthetic peptide, or a combination thereof.

[0108] The term "promoter" as used herein is defined as a DNA sequence recognized by the synthetic machinery of a cell or introduced synthetic machinery required to initiate the specific transcription of a polynucleotide sequence.

[0109] As used herein, the term "promoter / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operably linked to the promoter / regulatory sequence. In some instances, this sequence may be a core promoter sequence, and in other instances, this sequence may also include enhancer sequences and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence may be, for example, a sequence that expresses a gene product in a tissue-specific manner.

[0110] A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0111] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell substantially only when an inducer corresponding to the promoter is present in the cell.

[0112] A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene, causes a gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0113] The term "resistance to immunosuppression" refers to a lack of or reduced suppression of immune system activity or activation.

[0114] A "signal transduction pathway" refers to the biochemical relationships between various signaling molecules that play a role in transmitting a signal from one part of a cell to another part of the cell. The phrase "cell surface receptor" includes molecules and complexes of molecules that can send and receive signals across the plasma membrane of a cell.

[0115] "Single-chain antibody" refers to an antibody formed by recombinant DNA technology in which immunoglobulin heavy and light chain fragments are linked to the Fv region via an engineered span of amino acids. Various methods for producing single-chain antibodies are known, including those described in U.S. Patent No. 4,694,778; Bird (1988) Science 242:423-442; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; Ward et al. (1989) Nature 334:54454; Skerra et al. (1988) Science 242:1038-1041.

[0116] As used herein, the term "specifically binds" with respect to an antibody refers to an antibody that recognizes a specific antigen but does not substantially recognize or bind to other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen 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 terms "specific binding" or "specifically binds" can be used in reference to the interaction of an antibody, protein, or peptide with a second chemical species to mean that the interaction depends on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than the entire protein. If an antibody is specific for epitope "A," then the presence of a molecule containing epitope A (or free, unlabeled A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A bound to the antibody.

[0117] The term "stimulation" refers to a primary response induced by the binding of a stimulatory molecule (e.g., a TCR / CD3 complex) to its cognate ligand, thereby mediating a signal transduction event, such as, but not limited to, signal transduction through the TCR / CD3 complex. Stimulation can mediate changes in the expression of certain molecules, such as downregulation of TGF-beta and / or rearrangement of cytoskeletal structures.

[0118] "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.

[0119] As used herein, a "stimulatory ligand" refers to a ligand that, when present on an antigen-presenting cell (e.g., an aAPC, a dendritic cell, a B cell, etc.), is capable of specifically binding to a cognate binding partner (referred to herein 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, inter alia, peptide-loaded MHC class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0120] The term "subject" is intended to include organisms (e.g., mammals) in which an immune response can be elicited. As used herein, a "subject" or "patient" can be a human or non-human mammal. Non-human mammals include livestock and pets, such as, for example, ovine, bovine, porcine, canine, feline, and murine mammals. Preferably, the subject is a human.

[0121] As used herein, the term "substantially lacking an extracellular domain" refers to a molecule that essentially does not contain a domain that is extruded outside the cell. In one aspect, the chimeric intracellular signaling molecule lacks any function performed by an extracellular domain, such as antigen binding. In another aspect, the chimeric intracellular signaling molecule comprises a transmembrane domain but lacks a functional extracellular domain.

[0122] 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 has been separated from other cell types with which it is normally associated in its natural state. In some instances, a population of substantially purified cells refers to a homogeneous cell population. In other instances, the term simply refers to cells that have been separated from the cells with which they are naturally associated in their natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0123] "Target site" or "target sequence" refers to a genomic nucleic acid sequence that defines a portion of nucleic acid to which a binding molecule can specifically bind under conditions sufficient for binding to occur.

[0124] As used herein, the term "T cell receptor" or "TCR" refers to a complex of membrane proteins that participates in the activation of T cells in response to antigen presentation. TCRs are responsible for recognizing antigens bound to major histocompatibility complex molecules. TCRs are composed of a heterodimer of an alpha (α) chain and a beta (β) chain, although in some cells, TCRs consist of gamma and delta (γ / δ) chains. TCRs can also exist in alpha / beta and gamma / delta forms, which are structurally similar but have different 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 gamma delta T cells.

[0125] As used herein, the term "therapeutic" means treatment and / or prophylaxis. A therapeutic effect is achieved by suppression, amelioration, or eradication of a disease state.

[0126] As used herein, the terms "transfected" or "transformed" or "transduced" 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 one that has been transfected, transformed, or transduced with exogenous nucleic acid. This cell includes the primary subject cell and its progeny.

[0127] "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 from which a subject suffers.

[0128] As used herein, the term "tumor" refers to an abnormal growth of tissue, which may be benign, pre-cancerous, malignant, or metastatic.

[0129] As used herein, the phrases "under transcriptional control" or "operably linked" mean that the promoter is in the correct location and orientation relative to the polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.

[0130] A "vector" is a material composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides linked to ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. This term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, etc.

[0131] Ranges: Throughout this disclosure, various aspects of the invention may 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. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges and individual numerical values ​​within that range. For example, description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0132] B. Modified Immune Cells The present invention provides modified immune cells or their precursors (e.g., T cells) comprising dual (first and second) chimeric receptors (e.g., chimeric antigen receptors (CARs)). Also provided are modified immune cells or their precursors comprising nucleic acids encoding the first and second chimeric receptors. Each chimeric receptor (e.g., CAR) comprises affinity for an antigen on a target cell. Thus, such modified cells have specificity dictated by the chimeric receptors expressed therein. For example, modified cells of the present disclosure comprising an HIV-1 chimeric receptor have specificity for HIV-1 on target cells.

[0133] In certain embodiments, the engineered immune cell or progenitor thereof comprises a first chimeric receptor comprising a first binding domain, a first transmembrane domain, a first costimulatory domain that confers enhanced survival-promoting function, and a CD3z intracellular signaling domain. The cell also comprises a second chimeric receptor comprising a second binding domain, a second transmembrane domain, a second costimulatory domain that confers enhanced effector function, and a CD3z intracellular signaling domain.

[0134] Thus, in certain embodiments, the first and second costimulatory domains are different costimulatory domains. Accordingly, in certain embodiments, the present invention provides modified immune cells or precursor cells thereof comprising first and second chimeric receptors, each comprising a distinct costimulatory domain. In certain embodiments, the first costimulatory domain is a 4-1BB costimulatory domain and / or the second costimulatory domain is a CD28 costimulatory domain.

[0135] In certain embodiments, the first transmembrane domain and / or the second transmembrane domain is selected from the group consisting of an artificial hydrophobic sequence and the transmembrane domains of type I transmembrane proteins, the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, OX40 (CD134), 4-1BB (CD137), and CD154.

[0136] In certain embodiments, the first transmembrane domain is a 4-1BB or CD8α transmembrane domain, and / or the second transmembrane domain is a CD28 transmembrane domain.

[0137] In certain embodiments, the first chimeric receptor and / or the second chimeric receptor further comprises a hinge domain selected from the group consisting of an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial hinge domain, a hinge comprising the amino acid sequence of CD8, or any combination thereof.

[0138] In certain embodiments, the first binding domain binds to a first target (e.g., an antigen) and the second binding domain binds to a second target. The first and second targets may be the same or different. The first and second targets may be distinct epitopes of the same molecule.

[0139] In certain embodiments, the first target and the second target are human immunodeficiency virus type 1 (HIV-1). In certain embodiments, the first target and the second target are envelope glycoprotein gp120. In certain embodiments, the first binding domain and / or the second binding domain comprise the extracellular domain of the CD4 molecule.

[0140] In certain embodiments, the first target and / or the second target is a tumor-associated antigen. Tumor-associated antigens are described in detail elsewhere herein. The tumor-associated antigen is a liquid tumor antigen (e.g., CD19 or CD22) or a solid tumor antigen.

[0141] In a particular embodiment, the first target is a tumor-associated antigen and the second target is human immunodeficiency virus type 1 (HIV-1).

[0142] In certain aspects, the present invention provides modified immune cells, or precursor cells thereof, comprising a first chimeric receptor comprising the extracellular domain of a CD4 molecule, a CD8α transmembrane domain, a 4-1BB costimulatory domain, and a CD3z intracellular signaling domain; and a second chimeric receptor comprising the extracellular domain of a CD4 molecule, a CD28 transmembrane domain, a CD28 costimulatory domain, and a CD3z intracellular signaling domain. In certain embodiments, the present invention provides modified immune cells, or precursor cells thereof, comprising a first chimeric receptor comprising a first binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a 4-1BB costimulatory domain, and a CD3z intracellular signaling domain; and a second chimeric receptor comprising a second binding domain, a CD8α hinge domain, a CD28 transmembrane domain, a CD28 costimulatory domain, and a CD3z intracellular signaling domain.

[0143] In certain aspects, the present invention provides modified immune cells or precursors thereof comprising a first chimeric receptor comprising the amino acid sequence set forth in SEQ ID NO: 1 and / or a second chimeric receptor comprising the amino acid sequence set forth in SEQ ID NO: 7.

[0144] In certain embodiments, the modified immune cells are modified T cells. In certain embodiments, the modified immune cells are autologous cells. In certain embodiments, the modified immune cells are autologous cells obtained from a human subject.

[0145] In certain embodiments, the cell further comprises an HIV fusion inhibitor. In certain embodiments, the cell further comprises a polynucleotide sequence encoding the HIV fusion inhibitor. In certain embodiments, the HIV fusion inhibitor is a cell surface-expressed HIV fusion inhibitor. In certain embodiments, the HIV fusion inhibitor is C34-CXCR4.

[0146] In certain embodiments, cells expressing an HIV fusion inhibitor exhibit increased resistance to infection by HIV compared to control cells that do not express the HIV fusion inhibitor.

[0147] One aspect of the present invention includes an engineered immune cell, or a precursor thereof, comprising: (a) any of the nucleic acids disclosed herein or any of the expression constructs disclosed herein; or (b) (i) a first chimeric receptor comprising a first binding domain, a first transmembrane domain, a first costimulatory domain that confers enhanced pro-survival function, and a CD3z intracellular signaling domain; and (ii) a second chimeric receptor comprising a second binding domain, a second transmembrane domain, a second costimulatory domain that confers enhanced effector function, and a CD3z intracellular signaling domain.

[0148] In certain embodiments of the modified cells, (a) the first costimulatory domain is a 4-1BB costimulatory domain; and / or (b) the second costimulatory domain is a CD28 costimulatory domain; and / or (c) the first transmembrane domain and / or the second transmembrane domain is selected from the group consisting of an artificial hydrophobic sequence, a type I transmembrane protein, the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, OX40 (CD134), 4-1BB (CD137), and CD154 transmembrane domain; and / or (d) the first transmembrane domain is a 4-1BB or CD8α transmembrane domain; and / or (e) the second transmembrane domain is a CD28 transmembrane domain; and / or (f) the first chimeric receptor and / or the second chimeric receptor further comprises a hinge domain; and / or (g) the first chimeric receptor and / or the second chimeric receptor further comprises a hinge domain, wherein the hinge domain is selected from the group consisting of an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial hinge domain, a hinge comprising the amino acid sequence of CD8, or any combination thereof; and / or (h) the first binding domain binds to a first target and the second binding domain binds to a second target; and / or (i) the first binding domain binds to a first target and the second binding domain binds to a second target, and the first target and the second target are the same; and / or (j) the first binding domain binds to a first target and the second binding domain binds to a second target, and the first target and the second target are distinct epitopes of the same molecule; and / or (k) the first binding domain binds to a first target and the second binding domain binds to a second target, the first target and the second target being different; and / or (l) the first binding domain binds to a first target and the second binding domain binds to a second target, and the first target and / or the second target is human immunodeficiency virus type 1 (HIV-1); and / or (m) the first binding domain binds to a first target and the second binding domain binds to a second target, and the first target and the second target are human immunodeficiency virus type 1 (HIV-1); and / or (n) the first binding domain binds to a first target and the second binding domain binds to a second target, and the first target and / or the second target is the envelope glycoprotein gp120 of human immunodeficiency virus type 1 (HIV-1); and / or (o) the first binding domain binds to a first target and the second binding domain binds to a second target, and the first target and the second target are envelope glycoprotein gp120 of human immunodeficiency virus type 1 (HIV-1); and / or (p) the first binding domain and / or the second binding domain comprises the extracellular domain of a CD4 molecule; and / or (q) the first binding domain and the second binding domain comprise the extracellular domain of a CD4 molecule; and / or (r) the first binding domain binds to a first target and the second binding domain binds to a second target, and the first target and / or the second target is a tumor-associated antigen; and / or (s) the first binding domain binds to a first target and the second binding domain binds to a second target, the first target and / or the second target is a tumor-associated antigen, the tumor-associated antigen is a liquid tumor antigen, and optionally, the liquid tumor antigen is CD19 or CD22; and / or (t) the first binding domain binds to a first target, the second binding domain binds to a second target, the first target and / or the second target is a tumor-associated antigen, and the tumor-associated antigen is a solid tumor antigen; and / or (u) Cells expressing an HIV fusion inhibitor exhibit increased resistance to infection by HIV compared to control cells that do not express an HIV fusion inhibitor.

[0149] Another aspect of the present invention includes an engineered immune cell or a precursor thereof comprising: (a) a first chimeric receptor comprising the extracellular domain of a CD4 molecule, the CD8α transmembrane domain, the 4-1BB costimulatory domain, and the CD3z intracellular signaling domain; and (b) a second chimeric receptor comprising the extracellular domain of a CD4 molecule, the CD28 transmembrane domain, the CD28 costimulatory domain, and the CD3z intracellular signaling domain.

[0150] In particular embodiments of the modified cells, (a) the modified cells are modified immune cells; and / or (b) the modified cells are modified T cells; and / or (c) the modified cells are autologous cells; and / or (d) the modified cells are autologous cells obtained from a human subject.

[0151] C. Chimeric Receptor The present invention provides compositions and methods relating to modified immune cells or their precursors, e.g., modified T cells, comprising dual (first and second) chimeric receptors (e.g., chimeric antigen receptors (CARs)). Thus, in some embodiments, the immune cells are genetically modified to express the first and second chimeric receptors. The chimeric receptors of the present invention comprise a binding domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain.

[0152] Binding domain The binding domain of a chimeric receptor is an extracellular domain of the chimeric receptor that binds to a specific target antigen, including proteins, carbohydrates, and glycolipids. In some embodiments, the chimeric receptor comprises an affinity for a target antigen on a target cell. The target antigen can include any type of protein or epitope associated with the target cell. For example, the chimeric receptor can comprise an affinity for a target antigen on a target cell that indicates a particular disease state of the target cell.

[0153] In certain embodiments, the binding domain of the chimeric receptor comprises a CD4 domain, particularly a CD4 extracellular domain that specifically binds to HIV virions or HIV-infected cells. CD4 is a member of the immunoglobulin superfamily and comprises four extracellular immunoglobulin domains (D1-D4). D1 and D3 resemble immunoglobulin variable domains, while D2 and D4 resemble immunoglobulin constant domains. D1 comprises the region of CD4 that interacts with beta-2 microglobulin of the major histocompatibility complex class II molecule. In one embodiment, the chimeric receptor comprises the extracellular domain of CD4 or a fragment thereof. In another embodiment, the membrane-bound chimeric receptor comprises at least one immunoglobulin domain of CD4. In another embodiment, the CD4 extracellular domain comprises SEQ ID NO: 2.

[0154] In certain aspects, the binding domain of the chimeric receptor comprises an antigen-binding domain that binds to a specific target antigen, e.g., a target antigen on a target cell that is indicative of a particular disease state of the target cell.

[0155] In one embodiment, target cell antigen is tumor-associated antigen (TAA).Examples of tumor-associated antigen (TAA) include but are not limited to differentiation antigen, such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific multi-lineage antigen, such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigen, such as CEA; overexpressed oncogene and mutated tumor suppressor gene, such as p53, Ras, HER-2 / neu; unique tumor antigen resulting from chromosomal translocation, such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigen, such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigen E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS. In preferred embodiments, the antigen-binding domain of the chimeric receptor targets antigens including, but not limited to, CD19, CD20, CD22, ROR1, mesothelin, CD33 / IL3Ra, c-Met, PSMA, PSCA, glycolipid F77, EGFRvIII, GD-2, MY-ESO-1 TCR, MAGE A3 TCR, and the like.

[0156] Depending on the desired antigen to be targeted, the chimeric receptors of the invention can be engineered to contain an appropriate antigen-binding domain that is specific for the desired antigen target. For example, if CD19 is the desired antigen to be targeted, an antibody to CD19 can be used as the antigen-binding portion for incorporation into the chimeric receptors of the invention.

[0157] In one embodiment, the target cell antigen is CD19. Thus, in one embodiment, the chimeric receptor of the present disclosure has affinity for CD19 on the target cell. This should not be construed as limiting in any way, as chimeric receptors with affinity for any target antigen are suitable for use in the compositions or methods of the present invention.

[0158] As described herein, the chimeric receptor of the present disclosure having affinity for a specific target antigen on a target cell can include 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 one embodiment, the chimeric receptor of the present disclosure having affinity for CD19 on a target cell can include a CD19 binding domain.

[0159] In some embodiments, the chimeric receptors of the present disclosure may have affinity for one or more target antigens on one or more target cells. In some embodiments, the chimeric receptors may have affinity for one or more target antigens on target cells. In such embodiments, the chimeric receptors are bispecific or multispecific chimeric receptors. In some embodiments, the chimeric receptors comprise one or more target-specific binding domains that confer affinity for one or more target antigens. In some embodiments, the chimeric receptors comprise one or more target-specific binding domains that confer affinity for the same target antigen. For example, a chimeric receptor comprising one or more target-specific binding domains with affinity for the same target antigen may also bind to distinct epitopes of the target antigen. When multiple target-specific binding domains are present in a chimeric receptor, the binding domains may be arranged in tandem or separated by a linker peptide. For example, in a chimeric receptor comprising two target-specific binding domains, the binding domains are covalently linked to each other in a single polypeptide chain via an oligopeptide or polypeptide linker, an Fc hinge region, or a membrane hinge region.

[0160] 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).

[0161] The antigen-binding domain can comprise any domain that binds to an antigen, and may include, but is 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. In some embodiments, the antigen-binding domain portion comprises a mammalian antibody or a fragment thereof. The choice of antigen-binding domain may depend on the type and number of antigens present on the surface of the target cell.

[0162] As used herein, the term "single-chain variable fragment" or "scFv" refers to a fusion protein in which the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin (e.g., murine or human) are covalently linked to form a VH::VL heterodimer. The heavy (VH) and light (VL) chains are either directly linked or linked by a peptide-encoded linker connecting the N-terminus of the VH to the C-terminus of the VL or the C-terminus of the VH to the N-terminus of the VL. In some embodiments, the antigen-binding domain (e.g., a CD19-binding domain) comprises an scFv having, from the N-terminus to the C-terminus, a VH-linker-VL configuration. In some embodiments, the antigen-binding domain comprises an scFv having, from the N-terminus to the C-terminus, a VL-linker-VH configuration. Those skilled in the art will be able to select a configuration suitable for use in the present invention.

[0163] The linker is usually rich in glycine for flexibility, and rich in serine or threonine for solubility. The linker can connect 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 International Publication No. 2014 / 087010, the contents of which are incorporated herein by reference in their entirety. (GS) n , (GSGGS) n (SEQ ID NO: 9), (GGGS) n (SEQ ID NO: 10) and (GGGGS) n A variety of linker sequences are known in the art, including, but not limited to, glycine serine (GS) linkers such as (SEQ ID NO: 11) [wherein n represents an integer of at least 1]. An exemplary linker sequence is: TIFF2025128172000002.tif21151. A person skilled in the art will be able to select a linker sequence suitable for use in the present invention. In one aspect, the antigen-binding domain of the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL are linked to the nucleic acid sequence TIFF2025128172000003.tif6151, This is the amino acid sequence It can be coded by TIFF2025128172000004.tif6154.

[0164] Despite the removal of the constant region and the introduction of the linker, the scFv protein retains the specificity of the original immunoglobulin. Single-chain Fv polypeptide antibodies can be expressed from nucleic acids containing VH and VL coding sequences as described by Huston, et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). See also U.S. Patent Nos. 5,091,513, 5,132,405, and 4,956,778; and U.S. Patent Application Publication Nos. 20050196754 and 20050196754. Antagonistic 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 2006116(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).

[0165] As used herein, "Fab" refers to the fragment of an antibody structure that binds to an antigen but is monovalent and does not have the Fc portion; for example, digestion of an antibody with the enzyme papain yields two Fab fragments and one Fc fragment (e.g., heavy (H) chain constant region; the Fc region that does not bind to antigen).

[0166] As used herein, "F(ab')2" refers to an antibody fragment produced by pepsin digestion of a whole IgG antibody, resulting in two antigen-binding (ab') (bivalent) regions, each of which contains two separate amino acid chains, a portion of a heavy chain for antigen binding and a light (L) chain, linked by an S-linkage, with the remaining heavy chain portions linked together. The "F(ab')2" fragment can be divided into two separate Fab' fragments.

[0167] In some embodiments, the antigen-binding domain may be derived from the same species as the species in which the chimeric receptor will ultimately be used. For example, for use in humans, the antigen-binding domain of the chimeric receptor may comprise a human antibody or a fragment thereof. In some embodiments, the antigen-binding domain may be derived from a different species than the species in which the chimeric receptor will ultimately be used. For example, for use in humans, the antigen-binding domain of the chimeric receptor may comprise a murine antibody or a fragment thereof.

[0168] The binding domains described herein can be combined with any of the transmembrane domains described herein, any of the costimulatory domains described herein, any of the intracellular signaling domains described herein, or any of the other domains described herein that can be included in the chimeric receptors of the present invention. The chimeric receptors of the present invention can also include a hinge domain as described herein. The chimeric receptors of the present invention can also include a spacer domain as described herein. In some embodiments, each of the binding domains, transmembrane domains, and intracellular signaling domains is separated by a linker.

[0169] Transmembrane domain The chimeric receptors of the present invention may comprise a transmembrane domain connecting the binding domain of the chimeric receptor with the intracellular domain (e.g., costimulatory domain) in the chimeric receptor. The transmembrane domain of a subject chimeric antigen receptor is a region capable of spanning the plasma membrane of a cell (e.g., an immune cell or its precursor cell). The transmembrane domain is for insertion into a cell membrane, e.g., a eukaryotic cell membrane. In some embodiments, the transmembrane domain is sandwiched between the antigen-binding domain and the intracellular domain of the chimeric receptor.

[0170] In some embodiments, the transmembrane domain is naturally associated with one or more domains in the chimeric receptor, hi some embodiments, the transmembrane domain can be selected or modified by one or more amino acid substitutions to avoid binding of such domain with the transmembrane domain of the same or a different surface membrane protein, minimizing interaction with other members of the receptor complex.

[0171] Transmembrane domain can be derived from either natural or synthetic source.When source is natural, domain can be derived from any membrane-binding protein or transmembrane protein, for example, type I transmembrane protein.When source is synthetic, transmembrane domain can be any artificial sequence, for example, artificial hydrophobic sequence, that facilitates the insertion of chimeric receptor into cell membrane. Examples of transmembrane domains 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 alpha, beta, or zeta chains of the T cell receptor, CD28, CD3 epsilon, 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, a phenylalanine, tryptophan, and valine triad will be found at each end of the synthetic transmembrane domain.

[0172] In certain embodiments, the transmembrane domain (of the first and / or second chimeric receptor) is selected from the group consisting of an artificial hydrophobic sequence and the transmembrane domains of type I transmembrane proteins, the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, OX40 (CD134), 4-1BB (CD137), and CD154.

[0173] In certain embodiments, the transmembrane domain is a 4-1BB transmembrane domain. In certain embodiments, the transmembrane domain is a CD8α transmembrane domain. In certain embodiments, the transmembrane domain comprises SEQ ID NO: 4. In certain embodiments, the transmembrane domain is a CD28 transmembrane domain.

[0174] 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 may be included in a subject chimeric receptor.

[0175] In some embodiments, the transmembrane domain further comprises a hinge region. The chimeric receptor of the present invention may also comprise a hinge region. The hinge region of a chimeric receptor is a hydrophilic region located between the antigen-binding domain and the transmembrane domain. In some embodiments, this domain facilitates proper protein folding of the chimeric receptor. The hinge region is an optional component of a chimeric receptor. The hinge region may comprise a domain selected from an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, 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 can be as small as three glycines (Gly), and the CH1 and CH3 domains of IgG (such as human IgG4).

[0176] In some embodiments, the chimeric receptors of the present disclosure include 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 preferably can assist the antigen-binding domain in recognizing and binding 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, 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 the hinge region to adopt many different conformations.

[0177] 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).

[0178] The hinge region can have a length of about 4 amino acids to about 50 amino acids, e.g., 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. In some embodiments, the hinge region can have a length of more than 5 aa, more than 10 aa, more than 15 aa, more than 20 aa, more than 25 aa, more than 30 aa, more than 35 aa, more than 40 aa, more than 45 aa, more than 50 aa, more than 55 aa, or more.

[0179] A suitable hinge region can be readily selected and can be any of several suitable lengths, for example, 1 amino acid (e.g., Gly) to 20 amino acids, 2 to 15 amino acids, 3 to 12 amino acids, including 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids. Suitable hinge regions can have a length of more than 20 amino acids (e.g., 30, 40, 50, 60 or more amino acids).

[0180] For example, the hinge region may be a glycine polymer (G) n , glycine-serine polymers (e.g., (GS) n , (GSGGS) n (SEQ ID NO: 9) and (GGGS) n(SEQ ID NO:10), 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 relatively conformationally undefined and can therefore serve as neutral tethers between components. Glycine polymers can be used; glycine has significantly more access to the φ-ψ space than 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 can comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 12), GGSGG (SEQ ID NO: 13), GSGSG (SEQ ID NO: 14), GSGGG (SEQ ID NO: 15), GGGSG (SEQ ID NO: 16), GSSSG (SEQ ID NO: 17), and the like.

[0181] In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. The amino acid sequences of immunoglobulin hinge regions 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 may have the following amino acid sequence: TIFF2025128172000005.tif5154 (see, e.g., Glaser et al., J. Biol. Chem. (2005) 280:41494-41503); It can contain one of the following: TIFF2025128172000006.tif36159.

[0182] 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 by Tyr, thereby causing the hinge region to have 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.

[0183] Intracellular domain The chimeric receptor of the present invention also comprises an intracellular domain. In certain embodiments, the intracellular domain comprises a costimulatory domain and an intracellular signaling domain. The intracellular domain of the chimeric receptor is responsible for activating at least one effector function of the cell (e.g., immune cell) in which the chimeric receptor is expressed. The intracellular domain transmits an effector function signal, instructing the cell (e.g., immune cell) to perform its specialized function, such as damaging and / or destroying a target cell.

[0184] 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 T cells, as well as any derivatives or variants of these elements and any synthetic sequences having the same functional capabilities.

[0185] Examples of intracellular 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, etc., 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 aspect, 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.

[0186] In one embodiment, the intracellular domain of the chimeric receptor comprises at least one signaling domain from any portion of one or more costimulatory molecules, e.g., CD2, 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.

[0187] In certain embodiments, the chimeric receptor comprises a costimulatory domain that confers enhanced pro-survival function. For example, members of the TNF family of receptors (4-1BB, OX40, CD27, GITR, etc.) are believed to contribute more to cell survival. In certain embodiments, the costimulatory domain is a 4-1BB costimulatory domain. In certain embodiments, the costimulatory domain comprises SEQ ID NO: 5.

[0188] In certain embodiments, the chimeric receptor comprises a costimulatory domain that confers enhanced effector function. For example, members of the CD28 family of receptors (CD28 and ICOS) are believed to contribute more to cellular effector function. In certain embodiments, the costimulatory domain is a CD28 costimulatory domain.

[0189] In certain embodiments, the chimeric receptor comprises a CD28 transmembrane and costimulatory domain. In certain embodiments, the CD28 transmembrane and costimulatory domain comprises SEQ ID NO:8.

[0190] Other examples of intracellular domains include TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc epsilon Rib), CD79a, CD79b, Fc gamma RiIa, DAP10, DAP12, 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, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD lib, ITGAX, CD11c, ITGBl, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT These 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, Ly108), 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 costimulatory molecules described herein, any derivative, variant, or fragment thereof, any synthetic sequence of a costimulatory molecule having the same functional capability, and any combination thereof.

[0191] Additional examples of intracellular domains include the intracellular signaling domains of several different 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, the intracellular signaling domain 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 DAP12 (see, e.g., Topfer et al., J. Immunol. (2015) 194(7): 3201-3212), NKG2D, NKp44, NKp46, DAP10, and CD3z.

[0192] Intracellular signaling domains suitable for use in the subject chimeric receptors 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 cellular behavior, e.g., cell death; cell proliferation; cell differentiation; cell survival; modulation of a cell signaling response, etc.) in response to activation of the chimeric receptor (i.e., activated by an antigen and a dimerizing 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 linked to the membrane-bound chimeric receptor, but instead is diffused in the cytoplasm.

[0193] Intracellular signaling domains suitable for use in the subject chimeric receptors of the present invention comprise immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptides. In some embodiments, the ITAM motif is repeated twice within the intracellular signaling domain, wherein the first and second instances of the ITAM motif are separated from each other by 6 to 8 amino acids. In one embodiment, the intracellular signaling domain of a subject chimeric receptor comprises three ITAM motifs.

[0194] 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, Fc gamma RI, Fc gamma RIIA, Fc gamma RIIC, Fc gamma RIIIA, FcRL5 (see, e.g., Gillis et al., Front. Immunol. (2014) 5:254).

[0195] 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 protein from which it is derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to, DAP12, FCER1G (Fc epsilon receptor I gamma chain), CD3D (CD3 delta), CD3E (CD3 epsilon), CD3G (CD3 gamma), CD3Z (CD3 zeta), and CD79A (antigen receptor complex-associated protein alpha chain).

[0196] In one aspect, 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 aspect, the intracellular signaling domain is derived from FCER1G (also known as FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma chain; fc-epsilon RI-gamma; fcR gamma; fceRl gamma; high-affinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin E receptor, high-affinity gamma chain, etc.). In one aspect, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3-DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain; T cell receptor T3 delta chain; T cell surface glycoprotein CD3 delta chain, etc.). In one aspect, 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 aspect, 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 aspect, 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 aspect, the intracellular signaling domain is derived from CD79A (B cell antigen receptor complex-associated protein alpha chain; also known as CD79a antigen (immunoglobulin-associated alpha); MB-1 membrane glycoprotein; ig-alpha; membrane-bound immunoglobulin-associated protein; surface IgM-associated protein, etc.).In one embodiment, an intracellular signaling domain suitable for use in an FN3 chimeric receptor of the present disclosure comprises a DAP10 / CD28-type signaling chain. In one embodiment, an intracellular signaling domain suitable for use in an FN3 chimeric receptor of the present disclosure comprises a ZAP70 polypeptide. In some embodiments, the intracellular signaling domain comprises the cytoplasmic signaling domain of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, or CD66d. In one embodiment, the intracellular signaling domain in the chimeric receptor comprises the cytoplasmic signaling domain of human CD3 zeta. In some embodiments, the intracellular signaling domain comprises SEQ ID NO:6.

[0197] Typically, the entire intracellular signaling domain can be used, although 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 transduces the effector function signal. The intracellular signaling domain includes any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

[0198] The intracellular signaling domains described herein can be combined with any of the 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 chimeric receptor.

[0199] In certain embodiments, the chimeric receptor comprises a CD4 binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a 4-1BB domain, and a CD3 zeta domain. In certain embodiments, the chimeric receptor comprises the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the chimeric receptor comprises a CD4 binding domain, a CD8α hinge domain, a CD28 transmembrane domain, a CD28 intracellular domain, and a CD3 zeta domain. In certain embodiments, the chimeric receptor comprises the amino acid sequence set forth in SEQ ID NO: 7.

[0200] Acceptable variations in chimeric receptor sequences will be known to those of skill in the art. For example, in some embodiments, the chimeric receptor 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%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or 7.

[0201] CD4 4-1BB CD3 zeta sequence: (SEQ ID NO: 1) TIFF2025128172000008.tif56161 Underlined - CD4 EC domain (SEQ ID NO: 2) Italics - CD8α hinge (SEQ ID NO: 3) Bold - CD8α™ (SEQ ID NO: 4) Double Underline-4-1BB ICD(SEQ ID NO: 5) Bold italics - CD3 zeta (SEQ ID NO: 6)

[0202] CD4 CD28 CD3 Zeta sequence: (SEQ ID NO: 7) TIFF2025128172000009.tif56161 Underlined - CD4 EC domain (SEQ ID NO: 2) Italics - CD8α hinge (SEQ ID NO: 3) Double underlined - CD8αTM and ICD (SEQ ID NO: 8) Bold italics - CD3 zeta (SEQ ID NO: 6)

[0203] D. Nucleic Acids and Expression Vectors The present disclosure provides nucleic acids comprising a first polynucleotide sequence encoding a first chimeric receptor and a second polynucleotide sequence encoding a second chimeric receptor. The first chimeric receptor comprises a first binding domain, a first transmembrane domain, a first costimulatory domain that confers enhanced survival-promoting function, and a CD3z intracellular signaling domain. The second chimeric receptor comprises a second binding domain, a second transmembrane domain, a second costimulatory domain that confers enhanced effector function, and a CD3z intracellular signaling domain.

[0204] In one aspect, the present invention provides a nucleic acid comprising a first polynucleotide sequence encoding a first chimeric receptor comprising the extracellular domain of a CD4 molecule, a CD8α transmembrane domain, a 4-1BB costimulatory domain, and a CD3z intracellular signaling domain; and a second polynucleotide sequence encoding a second chimeric receptor comprising the extracellular domain of a CD4 molecule, a CD28 transmembrane domain, a CD28 costimulatory domain, and a CD3z intracellular signaling domain. In a specific embodiment, the present invention provides a nucleic acid comprising a first polynucleotide sequence encoding a first chimeric receptor comprising a first binding domain, a CD8α transmembrane domain, a 4-1BB costimulatory domain, and a CD3z intracellular signaling domain; and a second polynucleotide sequence encoding a second chimeric receptor comprising a second binding domain, a CD28 transmembrane domain, a CD28 costimulatory domain, and a CD3z intracellular signaling domain.

[0205] In certain embodiments, the first and second binding domains bind to the same target. In certain embodiments, the first and second binding domains bind to distinct targets. In some embodiments, the first binding domain binds to a tumor-associated antigen and the second binding domain binds to HIV-1. In some embodiments, the first and second binding domains bind to HIV-1. In some embodiments, the first and second binding domains bind to a tumor-associated antigen. In some embodiments, the first and second binding domains bind to the same tumor-associated antigen. In some embodiments, the first and second binding domains bind to distinct tumor-associated antigens.

[0206] In certain embodiments, the nucleic acid further comprises a polynucleotide sequence encoding an HIV fusion inhibitor. In certain embodiments, the HIV fusion inhibitor is an HIV fusion inhibitor expressed on the cell surface. In certain embodiments, the HIV fusion inhibitor is C34-CXCR4. In certain embodiments, cells expressing the HIV fusion inhibitor exhibit increased resistance to infection by HIV compared to control cells that do not express the HIV fusion inhibitor.

[0207] In certain embodiments, the first polynucleotide sequence and the second polynucleotide sequence are separated by a linker.

[0208] In some embodiments, the linker comprises a nucleic acid sequence encoding an internal ribosome entry site (IRES). As used herein, "internal ribosome entry site" or "IRES" refers to an element that promotes direct internal ribosome entry into a start codon, such as ATG, of a protein-coding region, thereby resulting in cap-independent translation of the gene. A variety of internal ribosome entry sites are known to those skilled in the art, including, but not limited to, IRESs available from viral or cellular mRNA sources, such as immunoglobulin heavy chain binding protein (BiP); vascular endothelial growth factor (VEGF); fibroblast growth factor 2; insulin-like growth factor; translation initiation factor eIF4G; yeast transcription factors TFIID and HAP4; and IRESs available from, for example, cardioviruses, rhinoviruses, aphthoviruses, HCV, Friend murine leukemia virus (FrMLV), and Moloney murine leukemia virus (MoMLV). Those skilled in the art will be able to select a suitable IRES for use in the present invention.

[0209] In some embodiments, the linker comprises a nucleic acid sequence encoding a self-cleaving peptide. As used herein, a "self-cleaving peptide" or "2A peptide" refers to an oligopeptide that allows encoding of multiple proteins as a polyprotein that dissociates into component proteins upon translation. The use of the term "self-cleaving" is not intended to imply a proteolytic cleavage reaction. Various self-cleaving or 2A peptides are known to those of skill in the art, including, but not limited to, those found in members of the Picornaviridae virus family, such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV0), zosea siagnavirus (TaV), and porcine teschovirus-1 (PTV-1); and cardioviruses such as tylovirus and encephalomyocarditis virus. The 2A peptides derived from FMDV, ERAV, PTV-1, and TaV are referred to herein as "F2A," "E2A," "P2A," and "T2A," respectively. Those of skill in the art will be able to select a suitable self-cleaving peptide for use in the present invention.

[0210] In some embodiments, the linker further comprises a nucleic acid sequence encoding a furin cleavage site. Furin is a ubiquitously expressed protease that resides in the trans-Golgi and processes protein precursors prior to their secretion. Furin cleaves at the COOH-terminus of its consensus recognition sequence. Various furin consensus recognition sequences (or "furin cleavage sites") are known to those skilled in the art, including, but not limited to, Arg-X1-Lys-Arg (SEQ ID NO: 33) or Arg-X1-Arg-Arg (SEQ ID NO: 34), X2-Arg-X1-X3-Arg (SEQ ID NO: 35), and Arg-X1-X1-Arg (SEQ ID NO: 36), such as Arg-Gln-Lys-Arg (SEQ ID NO: 37), where X1 is any naturally occurring amino acid, X2 is Lys or Arg, and X3 is Lys or Arg. Those skilled in the art will be able to select a suitable furin cleavage site for use in the present invention.

[0211] In some embodiments, the linker comprises a nucleic acid sequence encoding a combination of a furin cleavage site and a 2A peptide. Examples include, but are not limited to, a linker comprising a nucleic acid sequence encoding furin and F2A, a linker comprising a nucleic acid sequence encoding furin and E2A, a linker comprising a nucleic acid sequence encoding furin and P2A, or a linker comprising a nucleic acid sequence encoding furin and T2A. Those skilled in the art will be able to select a suitable combination for use in the present invention. In such embodiments, the linker may further comprise a spacer sequence between the furin and the 2A peptide. Various spacer sequences are known in the art, including, but not limited to, glycine serine (GS) spacers such as (GS)n, (GSGGS)n (SEQ ID NO: 9), and (GGGS)n (SEQ ID NO: 10), where n represents an integer of at least 1. Exemplary spacer sequences can include amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 12), GGSGG (SEQ ID NO: 13), GSGSG (SEQ ID NO: 14), GSGGG (SEQ ID NO: 15), GGGSG (SEQ ID NO: 16), GSSSG (SEQ ID NO: 17), etc. One of skill in the art will be able to select suitable spacer sequences for use in the present invention.

[0212] In some aspects, the nucleic acids of the present disclosure provide for the production of chimeric receptors as described herein, e.g., in mammalian cells. In some aspects, the nucleic acids of the present disclosure provide for the amplification of nucleic acids encoding chimeric receptors.

[0213] In some aspects, the nucleic acids of the present disclosure can include a leader sequence. Suitable leader sequences are known to those of skill in the art.

[0214] In some aspects, the nucleic acids of the present disclosure may be operably linked to transcriptional control elements, such as promoters, enhancers, etc. Suitable promoter and enhancer elements are known to those of skill in the art.

[0215] In certain embodiments, the nucleic acid is operably linked to a promoter. In certain embodiments, the promoter is a phosphoglycerate kinase 1 (PGK) promoter.

[0216] Suitable promoters for expression in bacterial cells include, but are not limited to, lacI, lacZ, T3, T7, gpt, lambda P, and trc. Suitable promoters for expression in eukaryotic cells include, but are not limited to, the light and / or heavy chain immunoglobulin gene promoter and enhancer element; the cytomegalovirus immediate early promoter; the herpes simplex virus thymidine kinase promoter; the early and late SV40 promoter; the promoter present in the long terminal repeat of retrovirus; the mouse metallothionein-I promoter; and various tissue-specific promoters known in the art. Suitable reversible promoters, including reversibly inducible promoters, are known in the art. Such reversible promoters can be isolated and derived from many organisms, for example, eukaryotes and prokaryotes. The modification of a reversible promoter from a first organism for use in a second organism (e.g., a first prokaryote and a second eukaryote, a first eukaryote and a 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, but are not limited to, alcohol-regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, alcohol transactivator protein (A1cR) responsive promoter, etc.), tetracycline-regulated promoters (e.g., promoter systems including TetActivator, TetON, TetOFF, etc.), steroid-regulated promoters (e.g., rat glucocorticoid receptor promoter system, human estrogen receptor promoter system, retinoid promoter system, thyroid promoter system, ecdysone promoter system, mifepristone promoter system, etc.), metal-regulated promoters (e.g., metallothionein promoter system, etc.), pathogenesis-related regulated promoters (e.g., salicylic acid-regulated promoter, ethylene-regulated promoter, benzothiadiazole-regulated promoter, 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.

[0217] In some embodiments, the promoter is a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, or an 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.

[0218] Suitable promoters for expression in yeast cells are constitutive promoters such as the ADH1 promoter, PGK1 promoter, ENO promoter, PYK1 promoter, etc.; or regulatable promoters such as the GAL1 promoter, GAL10 promoter, ADH2 promoter, PHOS promoter, CUP1 promoter, GALT promoter, MET25 promoter, MET3 promoter, CYC1 promoter, HIS3 promoter, ADH1 promoter, PGK promoter, GAPDH promoter, ADC1 promoter, TRP1 promoter, URA3 promoter, LEU2 promoter, ENO promoter, TP1 promoter, and AOX1 (e.g., for use in Pichia). Selection of an 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, e.g., the lac / tac hybrid promoter, the tac / trc hybrid promoter, the trp / lac promoter, the T7 / lac promoter; the trc promoter; the tac promoter, etc.; the araBAD promoter; in vivo regulated promoters such as the ssaG promoter or related promoters (see, e.g., U.S. Patent Application 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., consensus sigma70 promoters (see, e.g., GenBank accession numbers AX798980, AX798961, and AX798183); stationary phase promoters, e.g., dps promoters, spv promoters, and the like; promoters from pathogenicity island SPI-2 (see, e.g., WO96 / 17951); actA promoters (see, e.g., Shetron-Rama et al., Infect. Immun. (2002) 70:1087-1096); rpsM promoters (see, e.g., Valdivia and Falkow Mol. Microbiol. (1996). 22:367); tet promoters (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). Strong promoters suitable 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 when contacted 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).

[0219] 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-level expression of any polynucleotide sequence operably linked to it. Other constitutive promoter sequences may also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) or 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-1 alpha promoter, and human gene promoters such as, but not limited to, the 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 contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence operably linked to it when such expression is desired, or turning off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0220] In some embodiments, a locus or construct or transgene containing a suitable promoter is irreversibly switched via induction of an inducible system. Suitable systems for inducing irreversible switches are well known in the art; for example, induction of irreversible switches may utilize Cre-lox-mediated recombination (see, e.g., 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, and the like known in the art may be used to generate irreversibly switchable promoters. The methods, mechanisms, and requirements for performing site-specific recombination described elsewhere herein are used to generate irreversibly switched promoters and are well known in the art. See, for example, 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.

[0221] In some embodiments, the nucleic acid of the present disclosure further comprises a nucleic acid sequence encoding a chimeric receptor-induced expression cassette. In one embodiment, the chimeric receptor-induced expression cassette is for production of a transgenic polypeptide product released upon chimeric receptor signaling. See, e.g., Chmielewski and Abken, Expert Opin. Biol. Ther. (2015) 15(8): 1145-1154; and Abken, Immunotherapy (2015) 7(5): 535-544. In some embodiments, the nucleic acid of the present disclosure further comprises a nucleic acid sequence encoding a cytokine operably linked to a T cell activation response promoter. In some embodiments, the cytokine operably linked to the T cell activation response promoter is present on a separate nucleic acid sequence. In one embodiment, the cytokine is IL-12.

[0222] The nucleic acids of the present disclosure can be present in an expression vector and / or a cloning vector. Expression vectors can include a selectable 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 and should not be construed as limiting in any way: 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). Prokaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).

[0223] 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 operative in the expression host may be present. Suitable expression vectors include viral vectors (e.g., vaccinia virus; poliovirus; adenovirus (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., 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., vectors derived from murine leukemia virus, spleen necrosis virus, and 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.

[0224] 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, herpesvirus vectors, engineered hybrid virus vectors, transposon-mediated vectors, etc. Viral vector techniques are well known in the art and are 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, herpesviruses, and lentiviruses.

[0225] 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. Pat. No. 6,326,193).

[0226] In some embodiments, an expression vector (e.g., a lentiviral vector) may be used to introduce a chimeric receptor into immune cells or their precursor cells (e.g., T cells). Thus, an expression vector (e.g., a lentiviral vector) of the present invention may comprise a nucleic acid encoding a chimeric receptor. In some embodiments, an expression vector (e.g., a lentiviral vector) will comprise additional elements that aid in the functional expression of the chimeric receptor encoded therein. In some embodiments, an expression vector comprising a nucleic acid encoding a chimeric receptor further comprises a mammalian promoter. In one embodiment, the vector further comprises an elongation factor-1-alpha promoter (EF-1α promoter). Use of the EF-1α promoter may increase the efficiency of expression of a downstream transgene (e.g., a nucleic acid sequence encoding a chimeric receptor). A physiological promoter (e.g., an 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 a non-essential cis-acting sequence that may improve titer and gene expression. A 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 transport. Other non-essential cis-acting sequences are known to those skilled in the art and may be incorporated into the vector (e.g., lentiviral vector) of the present invention.

[0227] In some embodiments, the vector further comprises a post-transcriptional regulatory element. Post-transcriptional regulatory elements may improve RNA translation, improve transgene expression, and stabilize RNA transcripts. One example of a post-transcriptional regulatory element is the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). Thus, in some embodiments, the vector of the present invention further comprises a WPRE sequence. Various post-transcriptional regulatory elements are known to those skilled in the art and may be incorporated into the vectors of the present invention (e.g., lentiviral vectors). The vectors 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 (LTRs). The term "long terminal repeat" or "LTR" refers to the base-pair domains located at the ends of retroviral DNA, including the U3, R, and U5 regions. LTRs generally provide functions necessary for retroviral gene expression (e.g., promotion, initiation, and polyadenylation of gene transcripts) and viral replication. In one embodiment, the vectors (e.g., lentiviral vectors) of the present invention comprise a 3' U3-deleted LTR. Thus, the vectors (e.g., lentiviral vectors) of the present invention may comprise any combination of the elements described herein to increase the efficiency of functional expression of the transgene. For example, the vectors (e.g., lentiviral vectors) of the present invention may comprise a WPRE sequence, a cPPT sequence, an RRE sequence, a 5' LTR, and a 3' U3-deleted LTR' in addition to the nucleic acid encoding the chimeric receptor.

[0228] The vector of the present invention may be a self-inactivating vector. As used herein, the term "self-inactivating vector" refers to a vector in which the 3' LTR enhancer promoter region (U3 region) has been modified (e.g., by deletion or substitution). A self-inactivating vector can prevent viral transcription beyond the first round of viral replication. As a result, a self-inactivating vector can infect and then be integrated into the host genome (e.g., a mammalian genome) only once, but cannot be further passaged. Therefore, a self-inactivating vector can greatly reduce the risk of generating a replication-competent virus.

[0229] 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 containing a sequence encoding the chimeric receptor of the present disclosure. Methods for introducing RNA into host cells are known in the art. See, for example, Zhao et al. Cancer Res. (2010) 15: 9053. Introducing RNA containing a nucleotide sequence encoding the chimeric receptor of the present disclosure into host cells can be performed in vitro, 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 containing a nucleotide sequence encoding the chimeric receptor of the present disclosure.

[0230] To evaluate the expression of a polypeptide or a portion thereof, the expression vector to be introduced into cells may also contain a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a cell population to be transfected or infected with a viral vector. In some embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be adjacent to appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, but are not limited to, antibiotic resistance genes.

[0231] Reporter genes are used to identify potentially transfected cells and evaluate the functionality of regulatory sequences.Generally, reporter genes are genes that are not present in or expressed by recipient organisms or tissues, and encode polypeptides whose expression is revealed by some easily detectable characteristics, such as enzymatic activity.The expression of reporter genes is evaluated at an appropriate time after DNA is introduced into recipient cells.Suitable reporter genes may include, but are not limited to, luciferase, beta-galactosidase, chloramphenicol acetyltransferase, genes encoding secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82).

[0232] One aspect of the present invention includes a nucleic acid comprising: (a) a first polynucleotide sequence encoding a first chimeric receptor comprising a first binding domain, a first transmembrane domain, a first costimulatory domain that confers enhanced survival-promoting function, and a CD3z intracellular signaling domain; and (b) a second polynucleotide sequence encoding a second chimeric receptor comprising a second binding domain, a second transmembrane domain, a second costimulatory domain that confers enhanced effector function, and a CD3z intracellular signaling domain.

[0233] In particular embodiments of the nucleic acid, (a) the first costimulatory domain is a 4-1BB costimulatory domain; and / or (b) the second costimulatory domain is a CD28 costimulatory domain; and / or (c) the first transmembrane domain and / or the second transmembrane domain is selected from the group consisting of an artificial hydrophobic sequence, a type I transmembrane protein, the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, OX40 (CD134), 4-1BB (CD137), and CD154 transmembrane domain; and / or (d) the first transmembrane domain is a 4-1BB or CD8α transmembrane domain; and / or (e) the second transmembrane domain is a CD28 transmembrane domain; and / or (f) the first chimeric receptor and / or the second chimeric receptor further comprises a hinge domain; and / or (g) the first chimeric receptor and / or the second chimeric receptor further comprises a hinge domain, wherein the hinge domain is selected from the group consisting of an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial hinge domain, a hinge comprising the amino acid sequence of CD8, and any combination thereof; and / or (h) the first binding domain binds to a first target and the second binding domain binds to a second target; and / or (i) the first binding domain binds to a first target and the second binding domain binds to a second target, and the first target and the second target are the same; and / or (j) the first binding domain binds to a first target and the second binding domain binds to a second target, and the first target and the second target are distinct epitopes of the same molecule; and / or (k) the first binding domain binds to a first target and the second binding domain binds to a second target, the first target and the second target being different; and / or (l) the first binding domain binds to a first target and the second binding domain binds to a second target, and the first target and / or the second target is human immunodeficiency virus type 1 (HIV-1); and / or (m) the first binding domain binds to a first target and the second binding domain binds to a second target, and the first target and the second target are human immunodeficiency virus type 1 (HIV-1); and / or (n) the first binding domain binds to a first target and the second binding domain binds to a second target, and the first target and / or the second target is the envelope glycoprotein gp120 of human immunodeficiency virus type 1 (HIV-1); and / or (o) the first binding domain binds to a first target and the second binding domain binds to a second target, and the first target and the second target are envelope glycoprotein gp120 of human immunodeficiency virus type 1 (HIV-1); and / or (p) the first binding domain binds to a first target, the second binding domain binds to a second target, and the first binding domain and / or the second binding domain comprise the extracellular domain of a CD4 molecule; and / or (q) the first binding domain binds to a first target, the second binding domain binds to a second target, and the first binding domain and the second binding domain comprise the extracellular domain of a CD4 molecule; and / or (r) the first binding domain binds to a first target and the second binding domain binds to a second target, and the first target and / or the second target is a tumor-associated antigen; and / or (s) the first binding domain binds to a first target and the second binding domain binds to a second target, the first target and / or the second target is a tumor-associated antigen, the tumor-associated antigen is a liquid tumor antigen, and optionally, the liquid tumor antigen is CD19 or CD22; and / or (t) the first binding domain binds to a first target, the second binding domain binds to a second target, the first target and / or the second target is a tumor-associated antigen, and the tumor-associated antigen is a solid tumor antigen.

[0234] Another aspect of the present invention includes a nucleic acid comprising: (a) a first polynucleotide sequence encoding a first chimeric receptor comprising the extracellular domain of a CD4 molecule, the CD8α transmembrane domain, the 4-1BB costimulatory domain, and the CD3z intracellular signaling domain; and (b) a second polynucleotide sequence encoding a second chimeric receptor comprising the extracellular domain of a CD4 molecule, the CD28 transmembrane domain, the CD28 costimulatory domain, and the CD3z intracellular signaling domain.

[0235] In particular embodiments of the nucleic acid, (a) the first polynucleotide sequence and the second polynucleotide sequence are separated by a linker; and / or (b) the first polynucleotide sequence and the second polynucleotide sequence are separated by a linker, the linker comprising an internal ribosome entry site (IRES), a furin cleavage site, a self-cleaving peptide, or any combination thereof; and / or (c) the first polynucleotide sequence and the second polynucleotide sequence are separated by a linker, the linker comprising a furin cleavage site and a self-cleaving peptide; and / or (d) the first polynucleotide sequence and the second polynucleotide sequence are separated by a linker, the linker comprising a furin cleavage site and a self-cleaving peptide, the self-cleaving peptide being a 2A peptide, and optionally, the 2A peptide is selected from the group consisting of porcine teschovirus-1 2A (P2A), zosea asignavirus 2A (T2A), equine rhinitis A virus 2A (E2A), and foot-and-mouth disease virus 2A (F2A); and / or (e) the nucleic acid comprises, in a 5' to 3' direction, a first polynucleotide sequence, a linker, and a second polynucleotide sequence; and / or (f) the nucleic acid comprises, in a 5' to 3' direction, a second polynucleotide sequence, a linker, and a first polynucleotide sequence; and / or (g) the nucleic acid further comprises a polynucleotide sequence encoding an HIV fusion inhibitor; and / or (h) the nucleic acid further comprises a polynucleotide sequence encoding an HIV fusion inhibitor, wherein the HIV fusion inhibitor is a cell surface expressed HIV fusion inhibitor; and / or (i) The nucleic acid further comprises a polynucleotide sequence encoding an HIV fusion inhibitor, wherein the HIV fusion inhibitor is C34-CXCR4.

[0236] Another aspect of the present invention is a method for producing a pharmaceutical composition comprising: (a) comprising any of the nucleic acids disclosed herein; and / or (b) comprising any of the nucleic acids disclosed herein, and further comprising an EF-1α promoter; and / or (c) comprises any of the nucleic acids disclosed herein, and further comprises a rev response element (RRE); and / or (d) comprising any of the nucleic acids disclosed herein, and further comprising a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE); and / or (e) comprises any of the nucleic acids disclosed herein, and further comprises a cPPT sequence; and / or (f) a viral vector comprising any of the nucleic acids disclosed herein and selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector; and / or (g) comprises any of the nucleic acids disclosed herein and is a lentiviral vector; and / or (h) a nucleic acid comprising any of the nucleic acids disclosed herein, wherein the nucleic acid is a lentiviral vector, and the lentiviral vector is a self-inactivating lentiviral vector; and an expression construct comprising:

[0237] E. Treatment Method The modified cells described herein (e.g., T cells comprising dual chimeric cell receptors) may be included in compositions for use in treating diseases or disorders. The compositions include pharmaceutical compositions and may further include a pharmaceutically acceptable carrier. A therapeutically effective amount of a pharmaceutical composition comprising the modified T cells may be administered.

[0238] In one aspect, the invention includes a method of treating a disease or disorder (e.g., cancer or HIV) in a subject, the method comprising administering to a subject in need thereof a population of modified T cells of the invention. In another aspect, the invention includes a method for adoptive cell transfer therapy, the method comprising administering to a subject in need thereof a modified T cell of the invention.

[0239] In one aspect, the invention includes a method of treating a disease or disorder in a subject in need thereof, comprising administering an engineered immune cell, or a precursor thereof, comprising: a first chimeric receptor comprising a first binding domain, a first transmembrane domain, a first costimulatory domain that confers enhanced pro-survival function, and a CD3z intracellular signaling domain; and a second chimeric receptor comprising a second binding domain, a second transmembrane domain, a second costimulatory domain that confers enhanced effector function, and a CD3z intracellular signaling domain.

[0240] The diseases or disorders that can be treated include, but are not limited to, cancer, infectious diseases, autoimmunity and transplantation.In certain embodiments, the disease or disorder is a viral disease.In certain embodiments, the viral disease is HIV-1 infection.In certain embodiments, the disease or disorder is cancer.

[0241] The method for administering immune cells for adoptive cell therapy is known, and can be used with the provided method and composition.For example, adoptive T cell therapy is described in, for example, Gruenberg et al., U.S. Patent Application Publication No. 2003 / 0170238; Rosenberg, U.S. Patent No. 4,690,915; Rosenberg (2011) Nat Rev Clin Oncol.8(10):577-85).See, for example, Themeli et al. (2013) Nat Biotechnol.31(10):928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1):84-9; Davila et al. (2013) PLoS ONE 8(4):e61338. In some embodiments, cell therapy, e.g., adoptive T cell therapy, is performed by autologous transfer, in which cells are isolated and / or otherwise prepared from the subject to be treated or a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject, e.g., a patient, in need of treatment, and the cells are administered to the same subject after isolation and processing.

[0242] In some embodiments, cell therapy, for example, adoptive T cell therapy, is carried out by allogeneic transfer, in which cells are isolated and / or otherwise prepared from a subject other than the subject who will or will ultimately receive cell therapy, for example, the first subject.In such embodiments, the cells are then administered to a different subject of the same species, for example, the second subject.In some embodiments, the first and second subjects are genetically identical.In some embodiments, the first and second subjects are genetically similar.In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.

[0243] In some embodiments, the subject has been treated with a disease or condition, e.g., a tumor-targeting therapeutic agent, prior to administration of the cells or cell-containing composition. In some aspects, the subject is refractory or non-responsive to other therapeutic agents. In some embodiments, the subject has persistent or recurrent disease after treatment with another therapeutic intervention, e.g., chemotherapy, radiation, and / or hematopoietic stem cell transplantation (HSCT), e.g., allogeneic HSCT. In some embodiments, administration effectively treats the subject despite the subject becoming resistant to another therapy.

[0244] In some embodiments, the subject is responsive to another therapeutic agent, and treatment with the therapeutic agent reduces the disease burden. In some aspects, the subject is initially responsive to the therapeutic agent, but exhibits a recurrence of the disease or condition over time. In some embodiments, the subject has not relapsed. In some such embodiments, the subject is determined to be at risk of relapse, e.g., at high risk of relapse, and therefore the cells are administered prophylactically, e.g., to reduce the likelihood of relapse or prevent relapse. In some aspects, the subject has not received prior treatment with another therapeutic agent.

[0245] In some embodiments, the subject has persistent or recurrent disease after treatment with another therapeutic intervention, including, for example, chemotherapy, radiation, and / or hematopoietic stem cell transplantation (HSCT), e.g., allogeneic HSCT. In some embodiments, administration effectively treats the subject despite the subject becoming refractory to another therapy.

[0246] The modified immune cells of the present invention can be administered to animals, preferably mammals, and even more preferably humans, to treat cancer. Additionally, the cells of the present invention can be used to treat any cancer-related condition, particularly cell-mediated immune responses against tumor cells, where it is desirable to treat or alleviate the disease. Cancer types to be treated with the modified cells or pharmaceutical compositions of the present invention include carcinomas, blastomas, and sarcomas, as well as certain leukemias or lymphoid tumors, benign and malignant tumors, and malignant diseases such as sarcomas, carcinomas, and melanomas. Other exemplary cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, thyroid cancer, and the like. The cancer can be a non-solid tumor (such as a blood tumor) or a solid tumor. Adult tumors / cancers and pediatric tumors / cancers are also included. In one embodiment, the cancer is a solid tumor or a blood tumor. In one embodiment, the cancer is a carcinoma. In one embodiment, the cancer is a sarcoma. In one aspect, the cancer is leukemia. In one aspect, the cancer is a solid tumor.

[0247] A solid tumor is an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them (such as sarcoma, carcinoma, and lymphoma). Examples of solid tumors, such as sarcoma and carcinoma, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphatic tumors, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, and renal cell carcinoma. Cancer types include: hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular cancer, seminoma, bladder cancer, melanoma, and CNS tumors such as gliomas (such as brain stem gliomas and mixed gliomas), glioblastomas (also known as glioblastoma multiforme), astrocytomas, CNS lymphomas, germinomas, medulloblastomas, schwannomas, craniopharyngiomas, ependymomas, pinealomas, hemangioblastomas, acoustic neuromas, oligodendroglioma, meningiomas, neuroblastomas, retinoblastomas, and brain metastases.

[0248] Carcinomas amenable to treatment by the methods disclosed herein include, but are not limited to, esophageal cancer, hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer), squamous cell carcinoma (various tissues), bladder cancer including transitional cell carcinoma (a malignant neoplasm of the bladder), bronchogenic carcinoma, colon cancer, colorectal cancer, gastric cancer, lung cancer including small cell lung cancer and non-small cell lung cancer, adrenocortical carcinoma, thyroid cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, osteogenic carcinoma, epithelial carcinoma, and nasopharyngeal carcinoma.

[0249] Sarcomas that may be treated by the methods disclosed herein include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.

[0250] In certain exemplary embodiments, the modified immune cells of the present invention are used to treat myeloma or a condition related to myeloma. Examples of myeloma or a condition related to myeloma include, but are not limited to, light chain myeloma, non-secretory myeloma, monoclonal gammopathy of undetermined significance (MGUS), plasmacytoma (e.g., solitary, multiple solitary, extramedullary plasmacytoma), amyloidosis, and multiple myeloma. In one embodiment, the method of the present disclosure is used to treat multiple myeloma. In one embodiment, the method of the present disclosure is used to treat refractory myeloma. In one embodiment, the method of the present disclosure is used to treat relapsed myeloma.

[0251] In certain exemplary embodiments, the modified immune cells of the present invention are used to treat melanoma or a condition associated with melanoma. Examples of melanoma or a condition associated with melanoma include, but are not limited to, superficial spreading melanoma, nodular melanoma, lentiginous melanoma, acral lentiginous melanoma, amelanotic melanoma, or cutaneous melanoma (e.g., cutaneous, ocular, vulvar, vaginal, or rectal melanoma). In one embodiment, the method of the present disclosure is used to treat cutaneous melanoma. In one embodiment, the method of the present disclosure is used to treat refractory melanoma. In one embodiment, the method of the present disclosure is used to treat recurrent melanoma.

[0252] In yet another exemplary embodiment, the modified immune cells of the present disclosure are used to treat sarcoma or a sarcoma-related condition. Examples of sarcoma or a sarcoma-related condition include, but are not limited to, angiosarcoma, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, gastrointestinal stromal tumor, leiomyosarcoma, liposarcoma, malignant peripheral nerve sheath tumor, osteosarcoma, pleomorphic sarcoma, rhabdomyosarcoma, and synovial sarcoma. In one embodiment, the method of the present disclosure is used to treat synovial sarcoma. In one embodiment, the method of the present disclosure is used to treat liposarcomas, such as myxoid / round cell liposarcoma, differentiated / dedifferentiated liposarcoma, and pleomorphic liposarcoma. In one embodiment, the method of the present disclosure is used to treat myxoid / round cell liposarcoma. In one embodiment, the method of the present disclosure is used to treat refractory sarcoma. In one embodiment, the method of the present disclosure is used to treat recurrent sarcoma.

[0253] The cells of the invention to be administered can be autologous to the subject being treated.

[0254] Administration of the cells of the present invention may be carried out by any convenient method known to those skilled in the art. The cells of the present invention may be administered to a subject by aerosol inhalation, injection, oral ingestion, transfusion, implantation, or transplantation. The compositions described herein may be administered to a patient intraarterially, subcutaneously, intradermally, intratumorally, intranodally, intramuscularly, by intravenous (iv) injection, or intraperitoneally. In another example, the cells of the present 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, or the like.

[0255] In some embodiments, the cells are administered at a desired dosage, which in some aspects includes a desired dose or number of cells or cell types and / or a desired ratio of cell types. Thus, the dosage of cells is in some embodiments based on the total number of cells (or number per kg body weight) and the desired ratio of individual populations or subtypes, such as the ratio of CD4+ to CD8+. In some embodiments, the dosage of cells is based on the desired total number of cells (or number per kg body weight) in an individual population or of an individual cell type. In some embodiments, the dosage is based on a combination of such characteristics, such as the desired total number of cells, the desired ratio, and the desired total number of cells in an individual population.

[0256] In some embodiments, CD8 + T cells and CD4 + A population or subtype of cells, such as T cells, is administered at or within an acceptable variance of a desired dose of total cells, e.g., a desired dose of T cells. In some aspects, the desired dose is a desired number of cells, or a desired number of cells per unit body weight of the subject to whom the cells are administered, e.g., cells / kg. In some aspects, the desired dose is at or exceeds a minimum number of cells or a minimum number of cells per unit body weight. In some aspects, among the total cells administered at a desired dose, individual populations or subtypes are administered at or exceed a desired output ratio (CD4 + Against CD8 + (e.g., ratio of ) or thereabouts, e.g., within a certain allowable difference or error in such ratio.

[0257] In some embodiments, cells are administered at or within a tolerable variance of a desired dose of one or more of the individual populations or subtypes of cells, e.g., a desired dose of CD4+ cells and / or a desired dose of CD8+ cells. In some aspects, the desired dose is the desired number of cells of a subtype or population, or the desired number of such cells per unit body weight of the subject to whom the cells are administered, e.g., cells / kg. In some aspects, the desired dose is or exceeds the minimum number of cells of a population or subtype, or the minimum number of cells of a population or subtype per unit body weight. Thus, in some embodiments, the dosage is based on a desired fixed dose and desired ratio of total cells, and / or on one or more, e.g., a desired fixed dose of each, of individual subtypes or subpopulations. Thus, in some embodiments, the dosage is based on a desired fixed dose or minimum dose of T cells and CD4 + Cells vs. CD8 + Based on the desired ratio of cells and / or CD4 + Cells and / or CD8 + Based on a desired fixed or minimum dose of cells.

[0258] In certain embodiments, a distinct population of cells, or subtypes of cells, is administered to a subject in the range of about 1 million to about 100 billion cells, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), e.g., about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and optionally about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells), or any value between these ranges.

[0259] In some embodiments, the total cell dose and / or the dose of individual subpopulations of cells is greater than or equal to 1 x 10 cells. 5 pieces or approximately 1 x 10 5 pieces / kg ~ approx. 1×10 11 cells / kg, 10 cells 4 ~10 pieces 11 pieces or about 10 11 Pieces / kilogram (kg) body weight, e.g., 10 cells 5 ~10 6 cells / kg body weight, e.g., 1 x 10 cells 5 pieces or approximately 1 x 10 5 cells / kg, cells 1.5×10 5 cells / kg, cells 2×10 5 cells / kg, or 1 x 10 cells 6 For example, in some embodiments, the cells are 10 T cells / kg body weight. 4 pieces or about 10 4 ~10 pieces 9 pieces or about 109 cells / kilogram (kg) body weight, e.g., 10 T cells 5 ~10 pieces 6 cells / kg body weight or within a certain margin of error, e.g., 1 x 10 T cells 5 or approximately 1 x 10 5 cells / kg, T cells 1.5×10 5 cells / kg, T cells 2×10 5 cells / kg, or 1 x 10 T cells 6 In other exemplary embodiments, the dosage range of modified cells suitable for use in the methods of the present disclosure is about 1 x 10 cells / kg body weight. 5 cells / kg~cells approx. 1×10 6 cells / kg, approximately 1×10 cells 6 cells / kg~cells approx. 1×10 7 cells / kg, approximately 1×10 cells 7 cells / kg~cells approx. 1×10 8 cells / kg, approximately 1×10 cells 8 cells / kg~cells approx. 1×10 9 cells / kg, approximately 1×10 cells 9 cells / kg~cells approx. 1×10 10 cells / kg, approximately 1×10 cells 10 cells / kg~cells approx. 1×10 11 In an exemplary embodiment, a dosage suitable for use in the methods of the present disclosure is about 1 x 10 cells / kg. 8 In an exemplary embodiment, a dosage suitable for use in the methods of the present disclosure is about 1 x 10 cells / kg. 7 In other embodiments, a suitable dosage is about 1 x 10 total cells / kg. 7 ~ Total cells: approx. 5 x 10 7 In some embodiments, a suitable dosage is about 1 x 10 total cells. 8 ~ Total cells: approx. 5 x 10 8 In some embodiments, a suitable dosage is about 1.4 x 10 total cells. 7 ~ Total cells: approx. 1.1 x 10 9 In an exemplary embodiment, a dosage suitable for use in the methods of the present disclosure is about 7 x 10 total cells. 9 There are individuals.

[0260] In some embodiments, the cells are 4 or about 10 4 ~10 9 or about 10 9 CD4 count / kilogram (kg) of body weight + and / or CD8 + cells, e.g., 10 5 ~10 6 CD4 count / kg body weight + and / or CD8 + in cells, or within a certain range of error, e.g., 1 x 10 5 Or about 1 x 10 5 CD4 count / kg + and / or CD8 + cells, 1.5 x 10 5 CD4 count / kg + and / or CD8 + cells, 2 x 10 5 CD4 count / kg + and / or CD8 + cells, or 1 x 10 6 CD4 count / kg body weight + and / or CD8 + In some embodiments, the cells are administered at a concentration of about 1 x 10 6 pieces, approximately 2.5×10 6 pieces, about 5×10 6 pieces, approximately 7.5×10 6 pieces, or approximately 9 x 10 6 More than or at least about 1 × 10 6 pieces, approximately 2.5×10 6 pieces, about 5×10 6 pieces, approximately 7.5×10 6 pieces, or approximately 9 x 10 6 CD4 + cells, and / or at least about 1 x 10 6 pieces, approximately 2.5×10 6 pieces, about 5×10 6 pieces, approximately 7.5×10 6 pieces, or approximately 9 x 10 6 CD8+ cells, and / or at least about 1 x 10 6 pieces, approximately 2.5×10 6 pieces, about 5×106 pieces, approximately 7.5×10 6 pieces, or approximately 9 x 10 6 In some embodiments, the cells are administered at about 10 T cells or within a certain margin of error. 8 ~10 pieces 12 pieces or about 10 10 ~10 pieces 11 T cells, approximately 10 8 ~10 pieces 12 pieces or about 10 10 ~10 pieces 11 CD4 - cells, and / or approximately 10 8 ~10 pieces 12 pieces or about 10 10 ~10 pieces 11 CD8 + It is administered in cells or within a certain range of error.

[0261] In some embodiments, cells are administered at a desired output ratio of multiple cell populations or subtypes, such as CD4+ and CD8+ cells or subtypes, or within a tolerated range thereof. In some aspects, the desired ratio can be a specific ratio or can be a range of ratios, e.g., in some embodiments, the desired ratio (e.g., CD4 + Cells vs. CD8 +The ratio of cells) is 5:1 or about 5:1 to 5:1 or about 5:1 (or greater than about 1:5 and less than about 5:1), or 1:3 or about 1:3 to 3:1 or about 3:1 (or greater than about 1:3 and less than about 3:1), for example, 2:1 or about 2:1 to 1:5 or about 1:5 (or greater than about 1:5 and less than about 2:1, for example, 5:1, 4.5:1, 4:1, 3.5:1, 3:1, 2.5:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 , or 1:5, or about 5:1, about 4.5:1, about 4:1, about 3.5:1, about 3:1, about 2.5:1, about 2:1, about 1.9:1, about 1.8:1, about 1.7:1, about 1.6:1, about 1.5:1, about 1.4:1, about 1.3:1, about 1.2:1, about 1.1:1, about 1:1, about 1:1.1, about 1:1.2, about 1:1.3, about 1:1.4, about 1:1.5, about 1:1.6, about 1:1.7, about The ratio is 1:1.8, about 1:1.9, about 1:2, about 1:2.5, about 1:3, about 1:3.5, about 1:4, about 1:4.5, or about 1:5. In some aspects, tolerated differences are within about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50% of the desired ratio, including any value between these ranges.

[0262] In some embodiments, the dose of modified cells is administered to a subject in need thereof in a single dose or multiple doses. In some embodiments, the dose of modified cells is administered in multiple doses, for example, once a week or every 7 days, once every 2 weeks or every 14 days, once every 3 weeks or every 21 days, once every 4 weeks or every 28 days. In exemplary embodiments, a single dose of modified cells is administered to a subject in need thereof. In exemplary embodiments, a single dose of modified cells is administered to a subject in need thereof by rapid intravenous infusion.

[0263] The dosage appropriate for disease prevention or treatment may depend on the type of disease being treated, the type of cells or recombinant receptor, the severity and course of the disease, whether the cells are administered for prophylactic or therapeutic purposes, prior therapy, the subject's clinical history and response to the cells, and the judgment of the attending physician. The compositions and cells, in some embodiments, are administered to the subject at one time or over a series of treatments, as appropriate.

[0264] In some embodiments, the cells are administered as part of a combination treatment, e.g., simultaneously with another therapeutic intervention, e.g., an antibody or engineered cell or receptor, or agent, e.g., a cytotoxic or therapeutic agent, or sequentially in any order. The cells are co-administered in some embodiments with one or more additional therapeutic agents or in conjunction with another therapeutic intervention, simultaneously or sequentially in any order. In some situations, the cells are co-administered with another therapy close enough in time that the cell population enhances the effect of the one or more additional therapeutic agents, or vice versa. In some embodiments, the cells are administered before the one or more additional therapeutic agents. In some embodiments, the cells are administered after the one or more additional therapeutic agents. In some embodiments, the one or more additional agents include a cytokine, such as IL-2, for example, to enhance persistence. In some embodiments, the method includes administration of a chemotherapeutic agent.

[0265] In certain embodiments, the modified cells of the present invention may be administered to a subject in combination with an immune checkpoint antibody (e.g., an anti-PD1, anti-CTA-4, or anti-PDL1 antibody). For example, the modified cells may be administered in combination with, for example, an antibody or antibody fragment that targets PD-1 (programmed cell death 1 protein). Examples of anti-PD-1 antibodies include, but are not limited to, pembrolizumab (KEYTRUDA®, formerly known as lambrolizumab and also known as MK-3475), and nivolumab (BMS-936558, MDX-1106, ONO-4538, OPDIVA®) or an antigen-binding fragment thereof. In certain embodiments, the modified cells may be administered in combination with an anti-PD-L1 antibody or an antigen-binding fragment thereof. Examples of anti-PD-L1 antibodies include, but are not limited to, BMS-936559, MPDL3280A (TECENTRIQ®, atezolizumab), and MEDI4736 (durvalumab, Imfinzi). In certain embodiments, the modified cells may be administered in combination with an anti-CTA-4 antibody or antigen-binding fragment thereof. An example of an anti-CTA-4 antibody includes, but is not limited to, ipilimumab (trade name Yervoy). Other types of immune checkpoint modulators may be used, including, but not limited to, small molecules, siRNA, miRNA, and CRISPR systems. The immune checkpoint modulator may be administered before, after, or concurrently with the modified cells comprising a CAR. In certain embodiments, a combination treatment including an immune checkpoint modulator can enhance the therapeutic efficacy of a treatment including the modified cells of the invention.

[0266] Following administration of the cells, the biological activity of the engineered cell population is measured, in some embodiments, by any of several known methods, for example. Parameters for evaluation include specific binding of engineered or natural T cells or other immune cells to an antigen in vivo, for example, by imaging, or ex vivo, for example, by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as the cytotoxicity assays described in Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009) and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004). In certain embodiments, the biological activity of the cells is measured by assaying the expression and / or secretion of one or more cytokines, for example, CD107a, IFNγ, IL-2, and TNF. In some aspects, biological activity is measured by assessing a clinical outcome, such as a reduction in tumor burden or tumor mass.

[0267] In certain embodiments, the subject is provided with a secondary treatment, including but not limited to chemotherapy, radiation therapy, surgery, and medication.

[0268] In some embodiments, a subject can be administered a conditioning therapy before CAR T-cell therapy. In some embodiments, the conditioning therapy comprises administering an effective amount of cyclophosphamide to the subject. In some embodiments, the conditioning therapy comprises administering an effective amount of fludarabine to the subject. In a preferred embodiment, the conditioning therapy comprises administering an effective amount of a combination of cyclophosphamide and fludarabine to the subject. The administration of a conditioning therapy before CAR T-cell therapy can enhance the efficacy of CAR T-cell therapy. A method for conditioning a patient for T-cell therapy is described in U.S. Patent No. 9,855,298, the entire contents of which are incorporated herein by reference.

[0269] In some embodiments, the specific dosing regimens of the present disclosure include a lymphodepletion step prior to administration of the engineered T cells. In exemplary embodiments, the lymphodepletion step includes administration of cyclophosphamide and / or fludarabine.

[0270] In some embodiments, the lymphodepletion step is about 200 mg / m 2 / day~about 2000mg / m 2 / day (e.g., 200 mg / m 2 / day, 300mg / m 2 / day, or 500 mg / m 2 / day). In an exemplary embodiment, the dose of cyclophosphamide is about 300 mg / m 2 In some embodiments, the lymphodepletion phase is about 20 mg / m 2 / day~about 900mg / m 2 / day (e.g., 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, or 60 mg / m 2 / day). In an exemplary embodiment, the dose of fludarabine is about 30 mg / m 2 / day.

[0271] In some embodiments, the lymphodepletion step is about 200 mg / m 2 / day~about 2000mg / m 2 / day (e.g., 200 mg / m 2 / day, 300mg / m 2 / day, or 500 mg / m 2 / day), and cyclophosphamide at a dose of approximately 20 mg / m 2 / day~about 900mg / m 2 / day (e.g., 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, or 60 mg / m 2 / day). In an exemplary embodiment, the lymphodepletion step comprises the administration of fludarabine at a dose of about 300 mg / m 2 / day dose of cyclophosphamide, and approximately 30 mg / m 2 This involves the administration of fludarabine at a dose of 100mg / day.

[0272] In an exemplary embodiment, the dosing of cyclophosphamide is 30 mg / m for 3 days. 2 / day and fludarabine dosing was 30 mg / m for 3 days. 2 / day.

[0273] Lymphodepleting chemotherapy dosing may be scheduled on days -6 to -4 relative to T cell (e.g., CAR-T, TCR-T, modified T cells, etc.) infusion on day 0 (dosing within a -1 day window, i.e., days -7 to -5).

[0274] In an exemplary embodiment, for a subject with cancer, the subject receives 300 mg / m 2 of ribozyme via intravenous infusion 3 days prior to administration of the engineered T cells. 2 In an exemplary embodiment, for subjects with cancer, the subject receives lymphodepleting chemotherapy including cyclophosphamide at 300 mg / m by intravenous infusion for three days prior to administration of the modified T cells. 2 undergo lymphodepleting chemotherapy, including cyclophosphamide.

[0275] In an exemplary embodiment, for a subject with cancer, the subject receives about 20 mg / m 2 / day~about 900mg / m 2 / day (e.g., 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, or 60 mg / m 2 / day). In an exemplary embodiment, for a subject with cancer, the subject receives lymphodepleting chemotherapy including fludarabine at a dose of 30 mg / m 2 patients will receive lymphodepleting chemotherapy containing fludarabine at a dose of 100 mg / kg for three days.

[0276] In an exemplary embodiment, for a subject with cancer, the subject receives about 200 mg / m 2 / day~about 2000mg / m 2 / day (e.g., 200 mg / m 2 / day, 300mg / m 2 / day, or 500 mg / m 2 / day), and cyclophosphamide at a dose of approximately 20 mg / m 2 / day~about 900mg / m 2 / day (e.g., 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, or 60 mg / m 2 / day). In an exemplary embodiment, for a subject with cancer, the subject receives lymphodepleting chemotherapy including fludarabine at a dose of about 300 mg / m 2 / day dose of cyclophosphamide, and 30 mg / m 2 patients will receive lymphodepleting chemotherapy containing fludarabine at a dose of 100 mg / kg for three days.

[0277] The cells of the present invention can be administered at dosages, 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. 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.

[0278] It is known in the art that one of the adverse effects after CAR T cell infusion is the onset of immune activation known as cytokine release syndrome (CRS). CRS is immune activation that leads to an increase in inflammatory cytokines. CRS is a known on-target toxicity, and its occurrence likely correlates with efficacy. Clinical and laboratory measures range from mild CRS (systemic symptoms and / or grade 2 organ toxicity) to severe CRS (sCRS; grade ≥ 3 organ toxicity, aggressive clinical intervention, and / or potentially life-threatening). Clinical features include high fever, malaise, fatigue, myalgia, nausea, anorexia, tachycardia / hypotension, capillary leak, cardiac dysfunction, renal dysfunction, liver failure, and disseminated intravascular coagulation. Dramatic increases in cytokines, including interferon-gamma, granulocyte-macrophage colony-stimulating factor, IL-10, and IL-6, have been shown after CAR T cell infusion. One CRS signature is elevated cytokines, including IL-6 (severe elevation), IFN-gamma, TNF-alpha (moderate elevation), and IL-2 (mild elevation). Elevated clinically available inflammatory markers, including ferritin and C-reactive protein (CRP), have also been shown to correlate with CRS syndrome. The presence of CRS generally correlates with expansion of adoptively transferred cells and progressive immune activation. It has been demonstrated that the severity of CRS is determined by the disease burden at the time of infusion, as patients with high tumor burden experience greater sCRS.

[0279] Therefore, the present invention provides a CRS management strategy suitable for alleviating the physiological symptoms of uncontrolled inflammation after the diagnosis of CRS without compromising the anti-tumor efficacy of engineered cells (e.g., CAR T cells). CRS management strategies are known in the art. For example, systemic corticosteroids may be administered to rapidly reverse the symptoms of sCRS (e.g., grade 3 CRS) without impairing the initial anti-tumor response.

[0280] In some embodiments, an anti-IL-6R antibody may be administered. An example of an anti-IL-6R antibody is the monoclonal antibody tocilizumab, also known as atlizumab, which has been approved by the US Food and Drug Administration (commercially available as Actemra or RoActemra). Tocilizumab is a humanized monoclonal antibody against interleukin-6 receptor (IL-6R). Administration of tocilizumab has demonstrated almost immediate regression of CRS.

[0281] CRS is generally managed based on the severity of the syndrome recognized, and interventions are tailored as such. CRS management decisions may also be based on clinical signs and symptoms and response to interventions, rather than solely on laboratory values.

[0282] Mild to moderate cases are generally treated with symptom management using fluid therapy, nonsteroidal anti-inflammatory drugs (NSAIDs), and antihistamines as needed for adequate symptom relief. More severe cases include patients with any degree of hemodynamic instability; in the presence of any hemodynamic instability, administration of tocilizumab is recommended. The first-line treatment for CRS can be tocilizumab, and in some embodiments, a labeled dose of 8 mg / kg IV is used over 60 minutes (not to exceed 800 mg / dose). Tocilizumab can be repeated every 8 hours. If the response to the initial dose of tocilizumab is suboptimal, additional doses of tocilizumab may be considered. Tocilizumab can be administered alone or in combination with corticosteroid therapy. Patients with persistent or progressive CRS symptoms, inadequate clinical improvement within 12–18 hours, or an inadequate response to tocilizumab may be treated with high-dose corticosteroid therapy, typically hydrocortisone 100 mg IV or methylprednisolone 1–2 mg / kg. For patients with more severe hemodynamic instability or respiratory symptoms, high-dose corticosteroid therapy may be administered earlier in the course of CRS. CRS management guidance can be based on published standards (Lee et al. (2019) Biol Blood Marrow Transplant, doi.org / 10.1016 / j.bbmt.2018.12.758; Neelapu et al. (2018) Nat Rev Clin Oncology, 15:47; Teachey et al. (2016) Cancer Discov, 6(6):664–679).

[0283] Features consistent with macrophage activation syndrome (MAS) or hemophagocytic lymphohistiocytosis (HLH) have been observed in patients treated with CAR-T therapy, coinciding with the clinical manifestations of CRS (Henter, 2007). MAS appears to be a response to immune activation resulting from CRS and should therefore be considered a manifestation of CRS. MAS resembles HLH (also a response to immune stimulation). The clinical syndrome of MAS is characterized by persistent high fever, cytopenias affecting at least two of three blood systems, and hepatosplenomegaly. This is associated with elevated serum ferritin, soluble interleukin-2 receptor, and triglycerides, as well as decreased circulating natural killer (NK) activity.

[0284] In certain embodiments, administration of the modified cells results in an increase in the following cells compared to subjects who do not receive the modified cells: CD4 - T cells, CD4 - T cells, CD8 + T cells, CD8 - T cells, memory CD4 + T cells and CD14 + Reduce one or more HIV-induced losses of macrophages.

[0285] In certain embodiments, administration of the modified cells results in an increase in the following cells compared to subjects who do not receive the modified cells: CD4 + T cells, CD4 - T cells, CD8 + T cells, CD8 - T cells, central memory CD4 + T cells and CD14 + Reduce the occurrence of HIV-infected cells in one or more of the macrophages.

[0286] In certain embodiments, the subject's blood comprises at least about 100 modified cells per μL of blood for at least three weeks after a single administration of modified T cells.

[0287] In certain embodiments, the subject's blood comprises at least about 100 modified cells per μL of blood for at least three weeks after a single administration of modified T cells.

[0288] In certain embodiments, the modified cells bind to the first and second targets of cells expressing the first and second targets and kill the cells via granule-mediated cell lysis.

[0289] In certain embodiments, the method further comprises administering one or more antiretroviral therapeutic agents. Examples of antiretroviral therapeutic agents include: a) nucleoside / nucleotide reverse transcriptase inhibitors (NRTIs), such as abacavir or ABC (Ziagen), didanosine or ddl (Videx), emtricitabine or FTC (Emtriva), lamivudine or 3TC (Epivir), stavudine or d4T (Zerit), tenofovir alafenamide or TAF (Vemlidy), tenofovir disoproxil fumarate, or TDF (Viread), zidovudine or ZDV (Retroviral). b) non-nucleoside reverse transcriptase inhibitors (NNRTIs), such as delavirdine or DLV (Rescriptor), doravirine or DOR (Pifeltro), efavirenz or EFV (Sustiva), etravirine or ETR (Intelence), nevirapine or NVP (Viramune), rilpivirine or RPV (Edurant); c) protease inhibitors (PIs), such as atazanavir or ATV (Reyataz), darunavir or DRV (Prezista), fosamprenavir or FPV (Lexiva), indinavir or IDV (Crixivan), lopinavir + ritonavir or LPV / r (Kaletra), nelfinavir or NFV (Viracept), ritonavir or RTV (Norvir), saquinavir or SQV (Invirase, Fortovase), tipranavir or TPV (Aptivs); d) integrase inhibitors, e.g., bictegravir or BIC (combined with other drugs such as Biktarvy), dolutegravir or DTG (Tivicay), elvitegravir or EVG (Vitekta), raltegravir or RAL (Isentress); e) fusion inhibitors, e.g., enfuvirtide or ENF or T-20 (Fuseon); f) CCR5 antagonists, e.g., maraviroc or MVC (CelCentri); f) post-attachment inhibitors or monoclonal antibodies; g) pharmacological enhancers or "drug boosters," etc., and any combination thereof.

[0290] In one aspect, the present invention includes a method of treating cancer in a subject in need thereof, comprising administering to the subject any one of the modified immune or progenitor cells disclosed herein.

[0291] In another aspect, the present invention includes a method of treating HIV in a subject in need thereof, comprising administering to the subject any one of the modified immune or progenitor cells disclosed herein.

[0292] In yet another aspect, the present invention includes a method of treating HIV-1 infection in a subject in need thereof, comprising administering modified immune cells, or precursor cells thereof, comprising a first chimeric receptor comprising the extracellular domain of a CD4 molecule, the CD8α transmembrane domain, the 4-1BB costimulatory domain, and the CD3z intracellular signaling domain, and a second chimeric receptor comprising the extracellular domain of a CD4 molecule, the CD28 transmembrane domain, the CD28 costimulatory domain, and the CD3z intracellular signaling domain.

[0293] In yet another aspect, the present invention includes a method of treating cancer in a subject in need thereof, comprising administering modified T cells comprising a first chimeric receptor comprising a first binding domain, a first transmembrane domain, a first costimulatory domain that confers enhanced survival-promoting function, and a CD3z intracellular signaling domain, and a second chimeric receptor comprising a second binding domain, a second transmembrane domain, a second costimulatory domain that confers enhanced effector function, and a CD3z intracellular signaling domain.

[0294] Another aspect of the present invention includes a method of treating HIV-1 infection in a subject in need thereof, comprising administering modified T cells comprising: a first chimeric receptor comprising the extracellular domain of a CD4 molecule, the CD8α transmembrane domain, a 4-1BB costimulatory domain, and a CD3z intracellular signaling domain; and a second chimeric receptor comprising the extracellular domain of a CD4 molecule, the CD28 transmembrane domain, the CD28 costimulatory domain, and the CD3z intracellular signaling domain.

[0295] In certain embodiments, the modified cells are autologous cells. In certain embodiments, the modified cells are autologous cells obtained from a human subject. In certain embodiments, the modified cells are modified T cells.

[0296] Another aspect of the present invention is a method of treating a disease or disorder in a subject in need thereof, comprising: (a) administering any of the modified immune cells disclosed herein or any of the pharmaceutical compositions disclosed herein; or (b)(i) a first chimeric receptor comprising a first binding domain, a first transmembrane domain, a first costimulatory domain that confers enhanced pro-survival function, and a CD3z intracellular signaling domain; and (ii) a second chimeric receptor comprising a second binding domain, a second transmembrane domain, a second costimulatory domain that confers enhanced effector function, and a CD3z intracellular signaling domain. administering the modified immune cells or precursor cells thereof, The present invention includes a method comprising the steps of:

[0297] In certain aspects of the method, (a) the disease or disorder is a viral disease; and / or (b) the disease or disorder is a viral disease, and the viral disease is HIV-1 infection; and / or (c) the disease or disorder is cancer; and / or (d) the disease or disorder is cancer and the cancer is a liquid tumor; and / or (e) the disease or disorder is cancer and the cancer is a hematological malignancy; and / or (f) The disease or disorder is cancer, and the cancer is a solid tumor.

[0298] In certain embodiments of the method, the method relates to treating an HIV-1 infection in a subject in need thereof, (a) a first chimeric receptor comprising the extracellular domain of a CD4 molecule, the CD8α transmembrane domain, the 4-1BB costimulatory domain, and the CD3z intracellular signaling domain; and (b) A second chimeric receptor comprising the extracellular domain of the CD4 molecule, the CD28 transmembrane domain, the CD28 costimulatory domain, and the CD3z intracellular signaling domain. The method comprises administering the modified immune cells or precursor cells thereof, comprising:

[0299] In certain method embodiments, the method is for treating cancer in a subject in need thereof, comprising administering engineered T cells comprising: (a) a first chimeric receptor comprising a first binding domain, a first transmembrane domain, a first costimulatory domain that confers enhanced pro-survival function, and a CD3z intracellular signaling domain; and (b) a second chimeric receptor comprising a second binding domain, a second transmembrane domain, a second costimulatory domain that confers enhanced effector function, and a CD3z intracellular signaling domain.

[0300] In certain embodiments, a method is provided for treating HIV-1 infection in a subject in need thereof, comprising administering engineered T cells comprising: (a) a first chimeric receptor comprising the extracellular domain of a CD4 molecule, the CD8α transmembrane domain, the 4-1BB costimulatory domain, and the CD3z intracellular signaling domain; and (b) a second chimeric receptor comprising the extracellular domain of a CD4 molecule, the CD28 transmembrane domain, the CD28 costimulatory domain, and the CD3z intracellular signaling domain.

[0301] In certain aspects of the method, (a) the modified cell is a modified immune cell; and / or (b) the modified cell is an modified T cell; and / or (c) the modified cells are autologous; and / or (d) the modified cells are autologous cells obtained from the human subject; and / or (e) the subject is a human; and / or (f) Administration of the modified cells increases the number of the following cells compared to subjects who do not receive the modified cells: CD4 + T cells, CD4 - T cells, CD8 + T cells, CD8 - T cells, memory CD4 + T cells and CD14 + reducing one or more HIV-induced losses of macrophages; and / or (g) Administration of the modified cells increases the number of the following cells compared to subjects who do not receive the modified cells: CD4 + T cells, CD4 - T cells, CD8 - T cells, CD8 - T cells, central memory CD4 + T cells and CD14 + reducing the occurrence of HIV-infected cells in one or more of the macrophages; and / or (h) the subject's blood contains at least about 100 modified cells per μL of blood for at least 3 weeks after the single administration of modified T cells; and / or (i) the modified cells bind to the first and second targets on cells expressing the first and second targets and kill the cells via granule-mediated cell lysis; and / or (j) The method further comprises administering one or more antiretroviral therapeutic agents.

[0302] F. Sources 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 may also include, for example, autologous or heterologous donor blood, umbilical cord blood, or bone marrow. For example, the source of immune cells may be derived from the subject to be treated with the modified immune cells of the present invention, such as 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 transgenic species thereof. Preferably, the subject is human.

[0303] Immune cells can be obtained from several sources, including blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, lymph, or lymphoid organs. Immune cells are cells of the immune system, such as cells of innate or adaptive immunity, for example, lymphocytes, typically myeloid or lymphoid cells, including T cells and / or NK cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including 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, for example, primary cells isolated directly from the subject and / or isolated and frozen from the subject.

[0304] In certain embodiments, the immune cell is a T cell, e.g., a CD8+ T cell (e.g., a CD8+ naive T cell, a central memory T cell, or an effector memory T cell), a CD4+ T cell, a natural killer T cell (NKT cell), a regulatory T cell (Treg), a stem cell memory T cell, a lymphoid progenitor cell, a hematopoietic stem cell, a natural killer cell (NK cell), or a dendritic cell. In some embodiments, the cell is a monocyte or granulocyte, e.g., a myeloid cell, a macrophage, a neutrophil, a dendritic cell, a mast cell, an eosinophil, and / or a basophil. In certain embodiments, the target cell is an induced pluripotent stem (iPS) cell or a cell derived from an iPS cell, e.g., an iPS cell that has been generated from a subject and engineered to alter (e.g., induce mutations in) or manipulate the expression of one or more target genes, and differentiated into, e.g., a T cell, e.g., a CD8+ T cell (e.g., a CD8+ naive T cell, a central memory T cell, or an effector memory T cell), a CD4+ T cell, a stem cell memory T cell, a lymphoid progenitor cell, or a hematopoietic stem cell.

[0305] In some embodiments, the cells include one or more subsets of T cells or other cell types, e.g., the entire T cell population, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturation, differentiation potential, expansion, recirculation, localization, and / or persistence capacity, antigen specificity, antigen receptor type, presence in specific organs or compartments, marker or cytokine secretion profile, and / or degree of differentiation. T cells and / or CD4+ and / or CD8+ T cell subtypes and subpopulations include, among others, 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, alpha / beta T cells, and delta / gamma T cells. In certain embodiments, any number of T cell lines available in the art may be used.

[0306] 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. Cells for manipulation as described may be isolated from a sample, such as a biological sample, e.g., a sample obtained 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 or to whom cell therapy will be administered. The subject, in some embodiments, is a human in need of a specific therapeutic intervention, such as adoptive cell therapy, for which the cells are isolated, treated, and / or manipulated. Thus, the cells, in some embodiments, are primary cells, e.g., primary human cells. Samples include tissues, fluids, and other samples taken directly from a subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic manipulation (e.g., transduction with a viral vector), washing, and / or incubation. Biological samples can be samples obtained directly from a biological source or processed samples. Biological samples include, but are not limited to, bodily fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue, and organ samples, including processed samples derived therefrom.

[0307] 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 from autologous and allogeneic sources in the context of cell therapy, e.g., adoptive cell therapy.

[0308] 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., mice, rats, non-human primates, and pigs. In some embodiments, the isolation of cells includes one or more preparative 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 undesired components, enrich for desired components, or lyse or remove cells sensitive to a particular reagent. In some examples, the cells are separated based on one or more properties, such as density, adhesive properties, size, sensitivity and / or resistance to a particular component.

[0309] In some examples, cells from a subject's circulating blood are obtained, for example, by apheresis or leukapheresis. In some aspects, the sample contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, 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, e.g., to remove the plasma fraction and place the cells in a buffer or medium suitable 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 various biocompatible buffers after washing. In certain embodiments, components of the blood cell sample are removed, and the cells are directly resuspended in culture medium. In some embodiments, the methods involve density-based cell separation methods, such as preparation of white blood cells from peripheral blood by lysing red blood cells and centrifugation through a Percoll or Ficoll gradient.

[0310] In one embodiment, the immune cells are cells obtained from an individual's circulating blood 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 an appropriate buffer or medium, such as phosphate-buffered saline (PBS). Alternatively, the wash solution may be calcium-free and magnesium-free, or may lack many, if not all, divalent cations, for subsequent processing steps. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as Ca-free and Mg-free PBS. Alternatively, undesirable components of the apheresis sample may be removed, and the cells may be resuspended directly in culture medium.

[0311] In some embodiments, the isolation method involves separating different cell types based on the expression or presence of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acids, in cells. In some embodiments, any known method can be used for such marker-based separation. In some embodiments, the separation is based on affinity or immunoaffinity. For example, in some aspects, the separation involves separating cells and cell populations based on the cellular expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such marker, typically followed by a washing step, and separating the cells that bind to the antibody or binding partner from the cells that do not bind to the antibody or binding partner.

[0312] Such separation steps can be based on positive selection, in which cells bound to the reagent are retained for further use, and / or negative selection, in which 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 antibodies that specifically identify cell types in a heterogeneous population are unavailable, so that separation is best performed based on markers expressed by cells other than the desired population. Separation does not necessarily result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection or enrichment of a particular type of cell, such as cells expressing 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 cells expressing a marker, refers to reducing the number or percentage of such cells, but does not necessarily result in the complete removal of all such cells.

[0313] In some instances, multiple rounds of separation steps are performed, with the positively or negatively selected fraction from one step being subjected to another separation step, such as a subsequent positive or negative selection. In some instances, cells co-expressing multiple markers can be depleted in a single separation step, such as by incubating cells with multiple antibodies or binding partners, each specific to a marker targeted for negative selection. Similarly, multiple cell types can be simultaneously positively selected by incubating cells with multiple antibodies or binding partners expressed on different cell types.

[0314] In some embodiments, one or more of the T cell populations are positive for or express high levels of one or more particular markers, e.g., surface markers (marker+). high ) cells, or cells that are negative for one or more markers ( -) or express it at a relatively low level (marker low ) cells are enriched or depleted. For example, in some aspects, specific subpopulations of T cells, e.g., cells positive for or expressing 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 absent or expressed at relatively low levels on certain populations of T cells (e.g., non-memory cells) but present or expressed at relatively high levels on certain other populations of T cells (e.g., memory cells). In one embodiment, the cells (CD8+ cells, or T cells, such as CD3+ 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., positively selected for), and / or are depleted for cells that are positive for or express high surface levels of CD45RA (e.g., negatively selected for). In some embodiments, the cells are enriched for 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 CD8+ T cells are enriched for cells that are positive for CD45RO (or negative for CD45RA) and positive for CD62L. For example, CD3+, CD28+ T cells can be positively selected using CD3 / CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander).

[0315] In some embodiments, T cells are separated from PBMC samples by negative selection for markers expressed on non-T cells, such as B cells, monocytes, or other leukocytes, e.g., CD14. In some aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper T cells and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into subpopulations by positive or negative selection for markers expressed on or expressed at a relatively high level on one or more naive, memory, and / or effector T cell subpopulations. In some embodiments, CD8+ cells are further enriched or depleted for naive, central memory, effector memory, and / or central memory stem cells, e.g., by positive or negative selection based on surface antigens associated with each subpopulation. In some embodiments, enrichment for central memory T (TCM) cells is performed to increase efficacy, e.g., to improve long-term survival, expansion, and / or engraftment after administration, which in some aspects is particularly robust in such subpopulations. In some embodiments, combining TCM-enriched CD8+ T cells and CD4+ T cells further enhances efficacy.

[0316] In some embodiments, memory T cells exist in both the CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMCs can be enriched for or depleted of CD62L-CD8+ and / or CD62L+CD8+ fractions using, for example, anti-CD8 and anti-CD62L antibodies. In some embodiments, the CD4+ T cell population and CD8+ T cell subpopulation, e.g., a subpopulation enriched for central memory (TCM) cells, are enriched for central memory T (TCM) cells. In some embodiments, enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and / or granzyme B. In some aspects, isolation of a CD8+ population enriched for TCM cells is performed by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment of central memory T (TCM) cells is performed starting with a negative fraction of cells selected based on CD4 expression, which is then subjected to negative selection based on CD14 and CD45RA expression, and positive selection based on CD62L. Such selections are performed simultaneously in some aspects, and sequentially in other aspects, in either order. In some aspects, the same selection step based on CD4 expression used to prepare a CD8+ cell population or subpopulation is also used to generate a CD4+ cell population or subpopulation, whereby both the positive and negative fractions from CD4-based separation are retained and used in subsequent steps of the method, optionally following one or more additional positive or negative selection steps.

[0317] CD4+ T helper cells are sorted into naive, central memory, and effector cells by identifying cell populations bearing cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+, CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L- and CD45RO. In one example, to enrich CD4+ cells by negative selection, a 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 enable separation of cells for positive and / or negative selection.

[0318] In some embodiments, cells are incubated and / or cultured prior to or in conjunction with genetic manipulation. Incubation steps can include culture, cultivation, stimulation, activation, and / or propagation. In some embodiments, the composition or cells are incubated under stimulatory conditions or in the presence of a stimulatory substance. Such conditions include conditions designed to induce proliferation, expansion, activation, and / or survival of cells in a population, to mimic antigen exposure, and / or to prime cells for genetic manipulation, e.g., introduction of a recombinant antigen receptor. 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, e.g., 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 stimulatory substances include one or more agents, e.g., ligands, capable of activating the intracellular signaling domain of a TCR complex. In some aspects, the agent triggers or initiates the TCR / CD3 intracellular signaling cascade in T cells. Such an agent can include, for example, an antibody bound to a solid support such as a bead, e.g., an antibody specific for a TCR component and / or a costimulatory receptor, e.g., anti-CD3, anti-CD28, 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., an IL-2 concentration of at least about 10 units / mL.

[0319] 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 cords. In any event, specific subpopulations of T cells can be further isolated by positive or negative selection techniques.

[0320] The cord blood mononuclear cells so isolated can be depleted of cells expressing certain 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, or antibodies bound to cells.

[0321] Enrichment of a T cell population by negative selection can be achieved using a combination of antibodies against surface markers unique to the negatively selected cells. A preferred method is cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on the negatively selected cells. For example, negative selection can enrich for CD4 + To enrich for cells, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

[0322] To isolate a desired population of cells by positive or negative selection, cell concentration and surface (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 cell concentration) 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 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million, or 50 million cells / ml is used. In yet another embodiment, a cell concentration of 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells / ml is used. In further embodiments, cell concentrations of 125 or 150 million / ml can be used. Using higher concentrations can result in increased cell yield, cell activation, and cell expansion.

[0323] T cells can also be frozen after a washing step that does not require a monocyte removal step. Without wishing to be bound by theory, the freezing and subsequent thawing step provides a more uniform product by removing granulocytes and some monocytes from the cell population. After a washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While numerous freezing solutions and parameters are known in the art and would be useful in this context, in a non-limiting example, one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or other suitable cell freezing medium. The cells are then frozen to -80°C at a rate of 1°C / min and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing, as well as immediate uncontrolled freezing at -20°C or in liquid nitrogen, may also be used.

[0324] In one embodiment, the T cell population is comprised in cells such as peripheral blood mononuclear cells, umbilical cord blood cells, purified populations of T cells, and T cell lines. In another embodiment, peripheral blood mononuclear cells comprise the T cell population. In yet another embodiment, purified T cells comprise the T cell population.

[0325] In certain embodiments, regulatory T cells (Tregs) can be isolated from a sample. The sample can include, but is not limited to, umbilical cord blood or peripheral blood. In certain embodiments, Tregs are isolated by flow cytometry sorting. The sample can be enriched for Tregs before isolation by any means known in the art. The isolated Tregs can be cryopreserved and / or expanded before use. Methods for isolating Tregs 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.

[0326] G. Increased immune cells Whether before or after modification of the cells, see, e.g., 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; 7,17 Cells can be activated and expanded in number using methods such as those described in US Patent Application Publication Nos. 2,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and US Patent Application Publication No. 20060121005. For example, the T cells of the present invention can be expanded by contact with a surface to which is attached an agent that stimulates CD3 / TCR complex-associated signals and a ligand that stimulates costimulatory molecules on the T cell surface. In particular, T cell populations can be stimulated by contact with a surface-immobilized anti-CD3 antibody, or an antigen-binding fragment thereof, or anti-CD2 antibody, or by contact with a protein kinase C activator (e.g., bryostatin) together with a calcium ionophore. For costimulation of accessory molecules on the T cell surface, a ligand that binds to the accessory molecule is used. For example, T cells can be contacted with anti-CD3 antibody and anti-CD28 antibody under conditions suitable for stimulating T cell proliferation. Examples of anti-CD28 antibodies include 9.3, B-T3, and XR-CD28 (Diaclone, Besancon, France), which can be used in the present invention, but other methods and reagents known in the art can also be used (see, for example, 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).

[0327] T cells can be expanded by the methods disclosed herein by about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, 10,000-fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold, or more, and any and all whole integers and partial integers therebetween. In one embodiment, T cells are expanded by about 20-fold to about 50-fold.

[0328] Following culturing, the T cells can be incubated in the cell culture medium in the culture device for a period of time, or until the cells reach confluence or a high cell density for optimal passage before passaging the cells to another culture device. The culture device can be any culture device commonly used for in vitro cell culture. Preferably, the level of confluence is 70% or greater before passaging the cells to another culture device. More preferably, the level of confluence is 90% or greater. The period can be any time suitable for in vitro cell culture. The T cell medium may be changed during the culture of the T cells at any time. Preferably, the T cell medium is changed approximately every 2-3 days. The T cells are then harvested from the culture device, from which they can be used immediately or cryopreserved and stored for later use. In one embodiment, the present invention includes cryopreserving the expanded T cells. The cryopreserved T cells are thawed before introducing a nucleic acid into the T cells.

[0329] In another embodiment, the method comprises isolating T cells and expanding the T cells. In another embodiment, the invention further comprises cryopreserving the T cells prior to expansion. In yet another embodiment, the cryopreserved T cells are thawed for electroporation with RNA encoding the chimeric membrane protein.

[0330] 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 an alternative to or in addition to other methods of expansion described herein. Briefly, ex vivo culture and expansion of T cells includes the addition of cell growth factors or other factors, such as those described in U.S. Patent No. 5,199,942, such as flt3-L, IL-1, IL-3, and c-kit ligand. In one embodiment, expanding T cells includes culturing the T cells with a factor selected from the group consisting of flt3-L, IL-1, IL-3, and c-kit ligand.

[0331] 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 (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, or more days.

[0332] Various terms are used to describe cultured cells. Cell culture generally refers to cells obtained from a living organism and grown under controlled conditions. Primary cell culture is a culture of cells, tissues, or organs obtained directly from an organism before the first subculture. Cells are expanded in culture when they are placed in a growth medium under conditions that facilitate cell growth and / or division, resulting in a larger cell population. When cells are expanded in culture, the growth rate of the cells is typically measured by the time required for the cells to double in number, otherwise known as the doubling time.

[0333] Each round of subculture is called a passage. When cells are subcultured, they are said to be passaged. A particular cell population or cell line is sometimes referred to or characterized by the number of times they have been subcultured. For example, a cultured cell population that has been subcultured 10 times may be referred to as a P10 culture. The 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 secondary culture (P1 or passage 1). After the second subculture, the cells become a tertiary culture (P2 or passage 2), and so on. It will be understood by those skilled in the art that there may be multiple population doublings during the subculture period; therefore, the population doubling number of a culture is greater than the passage number. The expansion of cells during the period between subcultures (i.e., the number of population doublings) depends on many factors, including, but not limited to, the seeding density, the substrate, the medium, and the subculture interval.

[0334] In one embodiment, cells may be cultured for a few hours (about 3 hours) to about 14 days, or any integer value therebetween. Suitable conditions for T cell culture include an appropriate medium (e.g., Minimum Essential Medium or RPMI Medium 1640 or X-vivo 15, (Lonza)) that may contain factors necessary for growth and survival, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-gamma, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF-beta, and TNF-α, or any other additive for cell growth known to those of skill in the art. Other additives for cell growth 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, supplemented with amino acids, sodium pyruvate, and vitamins, and serum-free or supplemented with an appropriate amount of serum (or plasma) or a predetermined set of hormones and / or cytokines in amounts sufficient for T cell growth and expansion. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and 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).

[0335] The medium used to culture T cells may contain an agent that can costimulate T cells. For example, an agent that can stimulate CD3 is an antibody against CD3, and an agent that can stimulate CD28 is an antibody against CD28. Cells isolated by the methods disclosed herein can be expanded 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. In one embodiment, T cells are expanded by about 20x to about 50x, or more. In one embodiment, human regulatory T cells are expanded via KT64.86 artificial antigen-presenting cells (aAPCs) coated with anti-CD3 antibodies. Methods for expanding and activating T 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 entireties.

[0336] In one embodiment, the method of expanding T cells can further include isolating the expanded T cells for further application. In another embodiment, the method of expansion can further include subsequent electroporation of the expanded T cells followed by culturing. The subsequent electroporation can include introducing a nucleic acid encoding an agent, e.g., transducing the expanded T cells, transfecting the expanded T cells, or electroporating the expanded T cells with a nucleic acid to create an expanded T cell population, where the agent further stimulates the T cells. The agent can stimulate T cells, e.g., by stimulating further expansion, effector function, or another T cell function.

[0337] H. Pharmaceutical Compositions and Formulations Also provided are modified immune cell populations of the invention, compositions containing such cells and / or enriched in such cells, for example, compositions in which cells expressing a chimeric receptor account for at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of the total cells or a particular type of cell, such as T cells, CD8+ cells, or CD4+ cells, in the composition. The compositions are, among others, pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy. Also provided are therapeutic methods for administering the cells and compositions to a subject, e.g., a patient.

[0338] Also provided are compositions comprising cells for administration, including pharmaceutical compositions and formulations, for example, unit dose compositions comprising a number of cells for administration at a given dose or fraction thereof.Pharmaceutical compositions and formulations generally comprise one or more optional pharmaceutically acceptable carriers or excipients.In some embodiments, the composition comprises at least one additional therapeutic agent.

[0339] The term "pharmaceutical formulation" refers to a preparation in a form that allows the biological activity of the active ingredient contained therein to be effective, without containing additional ingredients that are unacceptably toxic to the subject to which the formulation will be administered. A "pharmaceutically acceptable carrier" refers to ingredients in a pharmaceutical formulation, other than the active ingredient, that are 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 the method of administration. Thus, there is a wide variety of suitable formulations. For example, pharmaceutical compositions can contain preservatives. Suitable preservatives can include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, mixtures of two or more preservatives are 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).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 alcohol, or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polymers; 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).

[0340] In some aspects, a buffering agent is included in the composition. 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 thereof is typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail, for example, in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

[0341] The formulation may comprise an aqueous solution. The formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated by the cells, preferably with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts effective for the intended purpose. Thus, in some embodiments, the pharmaceutical composition further comprises other pharmaceutically active agents or drugs, such as chemotherapeutic agents, for example, asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and / or vincristine. In some embodiments, the pharmaceutical composition contains the cells in an amount effective to treat or prevent the disease or condition, e.g., a therapeutically or prophylactically effective amount. Therapeutic or prophylactic effectiveness is, in some embodiments, monitored by periodic evaluation of the treated subject. The desired dose can be delivered by administration of a single bolus of cells, by administration of multiple boluses of cells, or by administration of continuous infusions of cells.

[0342] Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, intrapulmonary, 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 a subject using peripheral systemic delivery via intravenous, intraperitoneal, or subcutaneous injection. In some embodiments, the composition is provided 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 generally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient for administration, particularly by injection. On the other hand, viscous compositions can be formulated within a viscosity range suitable for providing a longer contact period with specific tissues. Liquid or viscous compositions can contain 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.

[0343] Sterile injectable solutions can be prepared by incorporating the cells in a solvent, for example, mixed with a suitable carrier, diluent, or excipient, such as sterile water, 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, flavorings, and / or coloring agents, depending on the desired route of administration and preparation. In some aspects, standard textbooks may be consulted for preparing appropriate preparations.

[0344] Various additives can be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents and buffers.Prevention of microbial action can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol and sorbic acid.Prolonged absorption of injectable pharmaceutical dosage forms can be achieved by using agents that delay absorption, for example, aluminum monostearate and gelatin.

[0345] Formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0346] The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein are incorporated herein by reference in their entirety to the same extent as if each individual publication were 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.

[0347] In certain aspects, the present invention provides pharmaceutical compositions comprising a therapeutically effective amount of any of the modified cells disclosed herein.

[0348] In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of modified immune cells or precursor cells thereof, wherein the modified cells comprise a first chimeric receptor comprising a first binding domain, a first transmembrane domain, a first costimulatory domain that confers enhanced survival-promoting function, and a CD3z intracellular signaling domain; and a second chimeric receptor comprising a second binding domain, a second transmembrane domain, a second costimulatory domain that confers enhanced effector function, and a CD3z intracellular signaling domain.

[0349] In yet another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of modified immune cells or precursor cells thereof, wherein the modified cells comprise: a first chimeric receptor comprising the extracellular domain of a CD4 molecule, a 4-1BB transmembrane domain, a 4-1BB costimulatory domain, and a CD3z intracellular signaling domain; and a second chimeric receptor comprising the extracellular domain of a CD4 molecule, a CD28 transmembrane domain, a CD28 costimulatory domain, and a CD3z intracellular signaling domain.

[0350] In another aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: (a) a therapeutically effective amount of any of the modified cells disclosed herein; and / or (b)(i) a first chimeric receptor comprising a first binding domain, a first transmembrane domain, a first costimulatory domain that confers enhanced pro-survival function, and a CD3z intracellular signaling domain; and (ii) a second chimeric receptor comprising a second binding domain, a second transmembrane domain, a second costimulatory domain that confers enhanced effector function, and a CD3z intracellular signaling domain. a therapeutically effective amount of modified immune cells or precursor cells thereof, comprising: (c) (i) a first chimeric receptor comprising the extracellular domain of a CD4 molecule, the CD8α transmembrane domain, the 4-1BB costimulatory domain, and the CD3z intracellular signaling domain; and (ii) a second chimeric receptor comprising the extracellular domain of the CD4 molecule, the CD28 transmembrane domain, the CD28 costimulatory domain, and the CD3z intracellular signaling domain; a therapeutically effective amount of modified immune cells or precursor cells thereof, comprising The present invention provides a pharmaceutical composition comprising:

[0351] I. Methods for Producing Genetically Modified Immune Cells The present disclosure provides methods for producing or generating the modified immune cells of the invention or their precursors (e.g., T cells comprising dual chimeric receptors) for tumor immunotherapy, e.g., adoptive immunotherapy, or treatment of diseases, e.g., HIV.

[0352] One aspect of the invention includes a method of producing a modified immune cell comprising introducing any of the nucleic acids disclosed herein into an immune cell.

[0353] In certain embodiments, the immune cells are obtained from the group consisting of T cells, dendritic cells, and stem cells. In certain embodiments, the immune cells are CD8 - T cells, CD4 + The T cells are selected from the group consisting of T cells, naive T cells, central memory T cells, stem cell memory T cells, effector memory T cells, natural killer T cells, and regulatory T cells.

[0354] In certain embodiments, the method further comprises expanding the T cells. In certain embodiments, the method further comprises expanding the T cells, wherein the T cells are expanded in the range of about 150-fold to about 500-fold. In certain embodiments, the method further comprises expanding the T cells, wherein the T cells are expanded at least about 150-fold. In certain embodiments, the method further comprises expanding the T cells, wherein the T cells are expanded at least about 300-fold. In certain embodiments, the method further comprises expanding the T cells, wherein the T cell expansion is in vivo.

[0355] In some embodiments, the dual chimeric receptor is introduced into cells by an expression vector. The present specification provides an expression vector comprising a nucleic acid sequence encoding the dual chimeric receptor of the present invention. 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 Sleeping Beauty, Piggybak, and Phi31 integrase. Some other suitable expression vectors include herpes simplex virus (HSV) and retrovirus expression vectors.

[0356] Adenoviral expression vectors are based on adenovirus and have a low capacity for integration into genomic DNA, but a high efficiency for transfection into host cells. Adenoviral expression vectors contain sufficient adenoviral sequences to (a) support the packaging of the expression vector and (b) ultimately express the dual chimeric receptor 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., a nucleic acid encoding a dual chimeric receptor) can be inserted to replace a large piece of adenoviral DNA to create the expression vector of the present invention (see, for example, Danthinne and Imperiale, Gene Therapy (2000) 7(20): 1707-1714).

[0357] Another expression vector is based on adeno-associated virus (AAV) and utilizes an adenovirus coupled system. This AAV expression vector has a high integration rate into the host genome. This vector can infect non-dividing cells, making it useful for delivering genes into mammalian cells, for example, in tissue culture or in vivo. AAV vectors have a broad host range for infectivit...

Claims

1. a first polynucleotide sequence encoding a first chimeric receptor comprising a first binding domain, a first transmembrane domain, a first costimulatory domain that confers an enhanced pro-survival function, and a CD3z intracellular signaling domain; and a second polynucleotide sequence encoding a second chimeric receptor comprising a second binding domain, a second transmembrane domain, a second costimulatory domain that confers enhanced effector function, and a CD3z intracellular signaling domain; A nucleic acid comprising:

2. The nucleic acid of claim 1, wherein the first costimulatory domain is a 4-1BB costimulatory domain.

3. 3. The nucleic acid of claim 1 or 2, wherein the second costimulatory domain is a CD28 costimulatory domain.

4. 10. The nucleic acid of claim 1, wherein the first transmembrane domain and / or the second transmembrane domain is selected from the group consisting of an artificial hydrophobic sequence and the transmembrane domains of type I transmembrane proteins, the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, OX40 (CD134), 4-1BB (CD137), and CD154.

5. 10. The nucleic acid of claim 1, wherein the first transmembrane domain is a 4-1BB or CD8α transmembrane domain.

6. 10. The nucleic acid of claim 1, wherein the second transmembrane domain is a CD28 transmembrane domain.

7. The nucleic acid of any one of the preceding claims, wherein the first chimeric receptor and / or the second chimeric receptor further comprises a hinge domain.

8. 8. The nucleic acid of claim 7, wherein the hinge domain is selected from the group consisting of an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial hinge domain, a hinge comprising the amino acid sequence of CD8, or any combination thereof.

9. 10. The nucleic acid of claim 1, wherein the first binding domain binds to a first target and the second binding domain binds to a second target.

10. 10. The nucleic acid of claim 9, wherein the first target and the second target are the same.

11. 11. The nucleic acid of claim 9 or 10, wherein the first target and the second target are distinct epitopes of the same molecule.

12. 10. The nucleic acid of claim 9, wherein the first target and the second target are different.

13. 13. The nucleic acid of any one of claims 9 to 12, wherein the first target and / or the second target is human immunodeficiency virus type 1 (HIV-1).

14. 14. The nucleic acid of claim 13, wherein the first target and the second target are human immunodeficiency virus type 1 (HIV-1).

15. 15. The nucleic acid of claim 13 or 14, wherein the first target and / or the second target is the envelope glycoprotein gp120.

16. 16. The nucleic acid of claim 15, wherein the first target and the second target are envelope glycoprotein gp120.

17. 17. The nucleic acid of any one of claims 9 to 16, wherein the first binding domain and / or the second binding domain comprises the extracellular domain of the CD4 molecule.

18. 18. The nucleic acid of claim 17, wherein the first binding domain and the second binding domain comprise the extracellular domain of a CD4 molecule.

19. The nucleic acid of any one of claims 9 to 12, wherein the first target and / or the second target is a tumor-associated antigen.

20. 20. The nucleic acid of claim 19, wherein the tumor-associated antigen is a liquid tumor antigen.

21. 21. The nucleic acid of claim 20, wherein the liquid tumor antigen is CD19 or CD22.

22. 20. The nucleic acid of claim 19, wherein the tumor-associated antigen is a solid tumor antigen.

23. a first polynucleotide sequence encoding a first chimeric receptor comprising the extracellular domain of a CD4 molecule, the CD8α transmembrane domain, the 4-1BB costimulatory domain, and the CD3z intracellular signaling domain; and a second polynucleotide sequence encoding a second chimeric receptor comprising the extracellular domain of a CD4 molecule, the CD28 transmembrane domain, the CD28 costimulatory domain, and the CD3z intracellular signaling domain; A nucleic acid comprising:

24. 10. The nucleic acid of claim 1, wherein the first polynucleotide sequence and the second polynucleotide sequence are separated by a linker.

25. 25. The nucleic acid of claim 24, wherein the linker comprises an internal ribosome entry site (IRES), a furin cleavage site, a self-cleaving peptide, or any combination thereof.

26. 26. The nucleic acid of claim 24 or 25, wherein the linker comprises a furin cleavage site and a self-cleaving peptide.

27. 27. The nucleic acid of claim 26, wherein the self-cleaving peptide is a 2A peptide.

28. 28. The nucleic acid of claim 27, wherein the 2A peptide is selected from the group consisting of porcine teschovirus-1 2A (P2A), Thosea asigna virus 2A (T2A), equine rhinitis A virus 2A (E2A), and foot-and-mouth disease virus 2A (F2A).

29. 29. The nucleic acid of any one of claims 23 to 28, comprising, in the 5' to 3' direction, a first polynucleotide sequence, a linker, and a second polynucleotide sequence.

30. 29. The nucleic acid of any one of claims 23 to 28, comprising, in the 5' to 3' direction, a second polynucleotide sequence, a linker, and the first polynucleotide sequence.

31. An expression construct comprising a nucleic acid according to any one of the preceding claims.

32. 32. The expression construct of claim 31, further comprising an EF-1α promoter.

33. 33. The expression construct of claim 31 or 32, further comprising a rev response element (RRE).

34. 34. The expression construct of any one of claims 31 to 33, further comprising a Woodchuck Hepatitis Virus Post-Transcriptional Regulatory Element (WPRE).

35. 35. The expression construct of any one of claims 31 to 34, further comprising a cPPT sequence.

36. 36. The expression construct of any one of claims 31 to 35, 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.

37. 37. The expression construct of any one of claims 31 to 36, which is a lentiviral vector.

38. 38. The expression construct of claim 37, wherein the lentiviral vector is a self-inactivating lentiviral vector.

39. 39. A modified immune cell or precursor thereof comprising a nucleic acid according to any one of claims 1 to 30 or an expression construct according to claims 31 to 38.

40. a first chimeric receptor comprising a first binding domain, a first transmembrane domain, a first costimulatory domain that confers enhanced pro-survival function, and a CD3z intracellular signaling domain; and a second chimeric receptor comprising a second binding domain, a second transmembrane domain, a second costimulatory domain that confers enhanced effector function, and a CD3z intracellular signaling domain. A modified immune cell or a precursor thereof, comprising:

41. The modified cell of claim 40, wherein the first costimulatory domain is a 4-1BB costimulatory domain.

42. 42. The modified cell of claim 40 or 41, wherein the second costimulatory domain is a CD28 costimulatory domain.

43. 43. The modified cell of any one of claims 40 to 42, wherein the first transmembrane domain and / or the second transmembrane domain is selected from the group consisting of an artificial hydrophobic sequence and a transmembrane domain of a type I transmembrane protein, the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, OX40 (CD134), 4-1BB (CD137), and CD154.

44. 44. The modified cell of any one of claims 40 to 43, wherein the first transmembrane domain is a 4-1BB or CD8α transmembrane domain.

45. 45. The modified cell of any one of claims 40 to 44, wherein the second transmembrane domain is a CD28 transmembrane domain.

46. 46. ​​The modified cell of any one of claims 40 to 45, wherein the first chimeric receptor and / or the second chimeric receptor further comprises a hinge domain.

47. 47. The modified cell of claim 46, wherein the hinge domain is selected from the group consisting of an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial hinge domain, a hinge comprising the amino acid sequence of CD8, or any combination thereof.

48. 48. The modified cell of any one of claims 40-47, wherein the first binding domain binds to a first target and the second binding domain binds to a second target.

49. 49. The modified cell of claim 48, wherein the first target and the second target are the same.

50. 50. The modified cell of claim 48 or 49, wherein the first target and the second target are distinct epitopes of the same molecule.

51. 49. The modified cell of claim 48, wherein the first target and the second target are different.

52. 52. The modified cell of any one of claims 48 to 51, wherein the first target and / or the second target is human immunodeficiency virus type 1 (HIV-1).

53. 53. The modified cell of claim 52, wherein the first target and the second target are human immunodeficiency virus type 1 (HIV-1).

54. 52. The modified cell of claim 50 or 51, wherein the first target and / or the second target is the envelope glycoprotein gp120.

55. 55. The modified cell of claim 54, wherein the first target and the second target are envelope glycoprotein gp120.

56. 56. The modified cell of any one of claims 48 to 55, wherein the first binding domain and / or the second binding domain comprises the extracellular domain of a CD4 molecule.

57. 57. The modified cell of claim 56, wherein the first binding domain and the second binding domain comprise the extracellular domain of a CD4 molecule.

58. The modified cell of any one of claims 48 to 51, wherein the first target and / or the second target is a tumor-associated antigen.

59. 59. The modified cell of claim 58, wherein the tumor-associated antigen is a liquid tumor antigen.

60. 60. The modified cell of claim 59, wherein the liquid tumor antigen is CD19 or CD22.

61. 59. The modified cell of claim 58, wherein the tumor-associated antigen is a solid tumor antigen.

62. a first chimeric receptor comprising the extracellular domain of a CD4 molecule, the CD8α transmembrane domain, the 4-1BB costimulatory domain, and the CD3z intracellular signaling domain; and a second chimeric receptor comprising the extracellular domain of the CD4 molecule, the CD28 transmembrane domain, the CD28 costimulatory domain, and the CD3z intracellular signaling domain; A modified immune cell or a precursor thereof, comprising:

63. 63. The modified cell of any one of claims 40 to 62, which is a modified immune cell.

64. 64. The modified cell of any one of claims 40 to 63, which is a modified T cell.

65. 65. The modified cell of any one of claims 40 to 64, which is an autologous cell.

66. 66. The modified cell of any one of claims 40-65, which is an autologous cell obtained from a human subject.

67. 65. A pharmaceutical composition comprising a therapeutically effective amount of the modified cells of any one of claims 38 to 64.

68. 1. A pharmaceutical composition comprising a therapeutically effective amount of modified immune cells or precursor cells thereof, The modified cells are a first chimeric receptor comprising a first binding domain, a first transmembrane domain, a first costimulatory domain that confers enhanced pro-survival function, and a CD3z intracellular signaling domain; and a second chimeric receptor comprising a second binding domain, a second transmembrane domain, a second costimulatory domain that confers enhanced effector function, and a CD3z intracellular signaling domain. Including, Pharmaceutical compositions.

69. 1. A pharmaceutical composition comprising a therapeutically effective amount of modified immune cells or precursor cells thereof, The modified cells are a first chimeric receptor comprising the extracellular domain of a CD4 molecule, the CD8α transmembrane domain, the 4-1BB costimulatory domain, and the CD3z intracellular signaling domain; and a second chimeric receptor comprising the extracellular domain of the CD4 molecule, the CD28 transmembrane domain, the CD28 costimulatory domain, and the CD3z intracellular signaling domain; Including, Pharmaceutical compositions.

70. 1. A method of treating a disease or disorder in a subject in need thereof, comprising: A modified cell according to any one of claims 40 to 66, or The pharmaceutical composition of any one of claims 67 to 69. to said subject.

71. 1. A method of treating a disease or disorder in a subject in need thereof, comprising: a first chimeric receptor comprising a first binding domain, a first transmembrane domain, a first costimulatory domain that confers enhanced pro-survival function, and a CD3z intracellular signaling domain; and a second chimeric receptor comprising a second binding domain, a second transmembrane domain, a second costimulatory domain that confers enhanced effector function, and a CD3z intracellular signaling domain. Modified immune cells or their precursor cells, comprising: The method of claim 1, further comprising administering

72. 72. The method of claim 70 or 71, wherein the disease or disorder is a viral disease.

73. 73. The method of claim 72, wherein the viral disease is HIV-1 infection.

74. 72. The method of claim 71, wherein the disease or disorder is cancer.

75. 75. The method of claim 74, wherein the cancer is a liquid tumor.

76. 75. The method of claim 74, wherein the cancer is a hematological malignancy.

77. 75. The method of claim 74, wherein the cancer is a solid tumor.

78. 1. A method of treating an HIV-1 infection in a subject in need thereof, comprising: a first chimeric receptor comprising the extracellular domain of a CD4 molecule, the CD8α transmembrane domain, the 4-1BB costimulatory domain, and the CD3z intracellular signaling domain; and a second chimeric receptor comprising the extracellular domain of the CD4 molecule, the CD28 transmembrane domain, the CD28 costimulatory domain, and the CD3z intracellular signaling domain; Modified immune cells or their precursor cells, comprising: The method of claim 1, further comprising administering

79. The method of any one of claims 70-78, wherein the modified cell is a modified immune cell.

80. 80. The method of any one of claims 70-79, wherein the modified cell is a modified T cell.

81. 1. A method of treating cancer in a subject in need thereof, comprising: a first chimeric receptor comprising a first binding domain, a first transmembrane domain, a first costimulatory domain that confers enhanced pro-survival function, and a CD3z intracellular signaling domain; and a second chimeric receptor comprising a second binding domain, a second transmembrane domain, a second costimulatory domain that confers enhanced effector function, and a CD3z intracellular signaling domain. modified T cells, including The method of claim 1, further comprising administering

82. 1. A method of treating an HIV-1 infection in a subject in need thereof, comprising: a first chimeric receptor comprising the extracellular domain of a CD4 molecule, the CD8α transmembrane domain, the 4-1BB costimulatory domain, and the CD3z intracellular signaling domain; and a second chimeric receptor comprising the extracellular domain of the CD4 molecule, the CD28 transmembrane domain, the CD28 costimulatory domain, and the CD3z intracellular signaling domain; modified T cells, including The method of claim 1, further comprising administering

83. The method of any one of claims 70-82, wherein the modified cells are autologous cells.

84. 84. The method of any one of claims 70-83, wherein the modified cells are autologous cells obtained from the human subject.

85. The method of any one of claims 70 to 84, wherein the subject is a human.

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