# Patent

## Title
Recombinant human progenitor cells, engineered human thymocytes, and engineered human T cells

## Abstract

Disclosed herein are recombinant human progenitor cells, engineered human thymocytes, and engineered human T cells. The recombinant human progenitor cells are made by transducing a human hematopoietic stem cell with a vector having a nucleic acid molecule which encodes a human T cell receptor specific to a virus, such as Human Immunodeficiency Virus, or an epitope thereof. The recombinant human progenitor cells differentiate and mature into the engineered human thymocytes and the engineered human T cells. Also disclosed herein are methods of inhibiting, reducing or treating a viral infection in a subject, such as a human subject, which comprises administering recombinant human progenitor cells, engineered human thymocytes, and/or engineered human T cells to the subject.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Patent Application Ser. No. 61/312,736, filed 11 Mar. 2010, which is herein incorporated by reference in its entirety.


REFERENCE TO A SEQUENCE LISTING SUBMITTED VIA EFS-WEB
The content of the ASCII text file of the sequence listing named “20110309—034044—073_ST25” which is 80.7 kb in size was created on 9 Mar. 2011 and electronically submitted via EFS-Web herewith the application is incorporated herein by reference in its entirety.
THE BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention generally relates to recombinant human progenitor cells, engineered human thymocytes, and engineered human T cells, and methods of treating subjects therewith.
2. Description of the Related Art
There are currently no known therapeutic cures for a variety of chronic viral infections. Many viruses, including human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), various herpes family viruses (herpes simplex virus type 1 and 2, Varicella-Zoster virus, Epstein-Bar virus, etc), human papillomavirus, and many others establish a persistent, often lifelong infection with the host organism. Such chronic viral infections are often accompanied with significant morbidity and a lower quality of life. The persistence of these chronic viral infections is due in part to the inability of the human immune system to adequately control and ultimately clear the virus from the body and the lack of effective therapies and medicines that can clear the virus from the body.
In many of these viral infections, the cytotoxic T lymphocyte (CTL) response is important in controlling viral replication and the failure of this response may significantly contribute to the inability of the body to fully control or clear the virus. See Berzofsky, et al. (2004) J Clin Invest. 114(4): 450-462. For example, the CD8+ T cell CTL response plays an important role in controlling the amount of Human Immunodeficiency Virus type 1 (HIV-1) in the body of an infected individual. See Benito, et al. (2004) AIDS Rev. 6(2): 79-88; Borrow, et al. (1994) J Virol. 68(9): 6103-6110; and Rowland-Jones, et al. (2001) Immunol Lett. 79(1-2): 15-20. CTLs specific for various HIV-1 antigenic epitopes are primarily responsible for the initial control and lowering of the viral load in the body shortly after infection with HIV and are responsible for controlling viral loads throughout infection. See Koup, et al. (1994) J Virol. 68(7): 4650-4655. Inevitably, the CTL response in HIV infected individuals fails during the natural course of infection. The loss of the HIV-specific immune response, particularly the CTL response, is associated with an increase in the HIV viral load and a more rapid progression to AIDS and death. See Goulder, et al. (1997) Nat Med. 3(2): 212-217; and Huynen & Neumann (1986) Science. 272(5270): 1962. The virus itself, placed under selective pressure by the CTL response, mutates to avoid the CTL response. See Wolinsky, et al. (1996) Science. 272(5261): 537-542. This results in the virus escaping immune surveillance and is usually followed by the generation of new CTLs to different antigenic epitopes.
One method of augmenting CTL responses is to generate homologous antigen-specific CTLs ex vivo and then administer the ex vivo generated cells into the subject to be treated. This treatment has been effective for treating cytomegalovirus (CMV) and Epstein-Barr virus (EBV) chronic infections, however, this treatment has not been shown to be effective in treating HIV infected individuals. See Lieberman, et al. (1997) Blood. 90(6): 2196-2206; Brodie, et al. (1999) Nat Med. 5(1): 34-41; Bollard, et al. (2004) Biol Blood Marrow Transplant. 10(3): 143-55; and Joseph, et al. (2008) J Virol. 82(6): 3078-3089. In HIV infected individuals, the ex vivo generated CTLs are likely to be dysfunctional as the autologous CTLs are ineffective at clearing or controlling the viral infection as a direct result of the HIV infection and ongoing viral-induced pathology.
Several studies have demonstrated the ability of cloned, antigen specific TCR α-chains and β-chains to be genetically transferred into autologous, stimulated CD8+ T lymphocytes and generate antigen-specific cells. See Hughes, et al. (2005) Hum Gene Ther. 16(4): 457-472; Johnson, et al. (2006) J Immunol. 177(9): 6548-6559; Miles, et al. (2006) Curr Med Chem. 13(23): 2725-2736; and Morgan, et al. (2006) Science. 314(5796): 126-129. Genetic transfer of a cloned human TCR to the melanoma antigen MART-1 into autologous CD8+ T lymphocytes followed by re-infusion of the cells into cancer patients with metastatic melanoma resulted in tumor cell regression in treated individuals. Unfortunately, these autologous cells taken from the treated patient have to undergo extensive ex vivo manipulation to express the transgenic TCR following re-infusion, which could at least partially explain the large amount of MART-1 TCR specific cells that were functionally deficient in this study. In addition, while the cells in this study were maintained for a relatively long period of time, long-term regeneration of antigen-specific cells was limited and the methodology of the study does not allow the generation of antigen-specific cells of a “naïve”, or non-exhausted or unmanipulated, phenotype and thus lack the robust ability to respond and function.
With all the advances in stem cell technology today, the prior art has yet to provide recombinant human progenitor cells, engineered human thymocytes (which may be naïve cells), and engineered human T cells which express a human TCR specific for a target antigen, such as an HIV antigen, that may be used to effectively treat a human subject against a disease or infection involving the expression of the target antigen. Thus, a need still exists for such compositions and methods, especially for treating chronic viral infections where the virus inhibits or impairs the native CTL response.
SUMMARY OF THE INVENTION
In some embodiments, the present invention provides a recombinant human progenitor cell which comprises a hematopoietic stem cell transduced with a vector containing a nucleic acid molecule which encodes a T cell receptor specific for a virus or an epitope thereof. In some embodiments, the epitope comprises, consists essentially of, or consists of SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; or SEQ ID NO:6. In some embodiments, the hematopoietic stem cell, the nucleic acid molecule, and/or the T cell receptor is human or of human origin. In some embodiments, the T cell receptor is a functional T cell receptor when expressed. In some embodiments, the nucleic acid molecule encodes a according to the present invention. In some embodiments, the virus is a human immunodeficiency virus, such as HIV-1, or Orthomyxoviruses, such as Influenza virus. In some embodiments, the T cell receptor was cloned by a spectratyping-based cloning method. In some embodiments, the vector is pCCL.PPT.hPGK.tcr.IRES.eGFP vector, wherein the TCR segment encodes the T cell receptor.
In some embodiments, the present invention provides an isolated or purified polypeptide a polypeptide comprising, consisting essentially of, or consisting of a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:7 and a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:8; a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:9 and a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:10; a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:11 or SEQ ID NO:12 and a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:13; a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:14 and a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:15 or SEQ ID NO:16; a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:17 and a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:18; a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:19 and a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:20; a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:21 and a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:22; a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:23 and a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:24; a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:25 or SEQ ID NO:26 and a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:27; or a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:28; SEQ ID NO:29; or SEQ ID NO:30 and a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:31; SEQ ID NO:32; SEQ ID NO:33; or SEQ ID NO:34.
In some embodiments, the present invention provides an isolated or purified nucleic acid molecule which encodes a polypeptide comprising, consisting essentially of, or consisting of a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:7 and a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:8; a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:9 and a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:10; a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:11 or SEQ ID NO:12 and a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:13; a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:14 and a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:15 or SEQ ID NO:16; a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:17 and a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:18; a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:19 and a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:20; a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:21 and a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:22; a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:23 and a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:24; a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:25 or SEQ ID NO:26 and a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:27; or a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:28; SEQ ID NO:29; or SEQ ID NO:30 and a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:31; SEQ ID NO:32; SEQ ID NO:33; or SEQ ID NO:34.
In some embodiments, the present invention provides a recombinant cell and/or a vector which comprises one or more nucleic acid molecules according to the present invention.
In some embodiments, the present invention provides a method of producing an engineered human thymocyte or an engineered human T cell which comprises differentiating a genetically modified human progenitor cell as disclosed herein into the engineered human thymocyte, and maturing the engineered human thymocyte into the engineered human T cell. In some embodiments, the recombinant human progenitor cell is subjected to a thymus tissue which may be human thymus tissue. In some embodiments, the recombinant human progenitor cell is implanted in the thymus tissue of a subject or intravenously administered to the subject. In some embodiments, the engineered human T cell is activated by subjecting it to an HLA molecule specific for the T cell receptor. In some embodiments, the HLA molecule is HLA-A*0201.
In some embodiments, the present invention provides an engineered human thymocyte and/or an engineered human T cell made by the methods disclosed herein. In some embodiments, the engineered human thymocyte and/or the engineered human T cell express a functional T cell receptor, preferably a functional human T cell receptor. In some embodiments, the human T cell receptor is functional in vivo. In some embodiments, the engineered human T cell is a cytotoxic T cell.
In some embodiments, the present invention provides a method of inhibiting, reducing or treating a viral infection in a subject which comprises administering a recombinant human progenitor cell, an engineered human thymocyte and/or an engineered human T cell as described herein to the subject. In some embodiments, the hematopoietic stem cell, the nucleic acid molecule, the thymus tissue, or a combination thereof is obtained from the subject to be treated. In some embodiments, the hematopoietic stem cell, the nucleic acid molecule, the thymus tissue, or a combination thereof is obtained from a donor who is immunologically compatible with the subject to be treated. In some embodiments, the subject to be treated is determined to be in need thereof as the subject has the viral infection, has been exposed to the virus, or will be exposed to the virus.
In some embodiments, the present invention provides kits which comprise a recombinant human progenitor cell, an engineered human thymocyte and/or an engineered human T cell as described herein packaged together with a reagent and/or a device for administering the recombinant human progenitor cell, the engineered human thymocyte and/or the engineered human T cell to a subject.
In the embodiments disclosed herein, the subject is mammalian, preferably human.
Both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the invention as claimed. The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute part of this specification, illustrate several embodiments of the invention, and together with the description serve to explain the principles of the invention.



DESCRIPTION OF THE DRAWINGS
This invention is further understood by reference to the drawings wherein:
FIG. 1 provides the nucleic acid sequence for the HIV specific human TCR. The 1.9 sequence (SEQ ID NO:35) is the original sequence of the cloned TCR specific for the SL9 epitope from an infected individual. The 1.9 cys sequence (SEQ ID NO:36) is the original 1.9 sequence having modifications which introduce cysteine residues in the constant region to allow greater pairing of the alpha and beta chains. The codon optimized 1.9 sequence (SEQ ID NO:37) is the original 1.9 sequence with several codon modifications to allow greater expression in genetically modified cells. The codon optimized 1.9 cys sequence (SEQ ID NO:38) is the original 1.9 sequence having modifications which introduce cysteine residues in the constant region to allow for greater pairing of the alpha and beta chains and several codon modifications to allow for greater expression in genetically modified cells.
FIG. 2A schematically shows the pCCL.PPT.hPGK.tcr1.9.IRES.eGFP.Wpre vector exemplified herein. The backbone of this vector is described by Dull et al. (1998) J Virol. 72(11): 8463-8471, which is herein incorporated by reference in its entirety.
FIG. 2B shows a detail of the lentiviral vector contained in the pCCL.PPT.hPGK.tcr1.9.IRES.eGFP.Wpre plasmid.
FIG. 3 schematically shows the spectratyping-based TCR cloning strategy according to the present invention.
FIG. 4 shows a comparison of the spectratyping-based TCR cloning strategy to a prior art cloning strategy.
FIG. 5A provides the results which demonstrate that molecules, in this case siRNA, which can interact with cellular molecules to alter or enhance cellular function, can be introduced into and included in the TCR-containing vector.
FIG. 5B schematically shows the construct having the siRNA inserted therein.
FIG. 6 schematically shows the use of the SCID-hu mouse system to assess human TCR development and functionality.
FIG. 7 provides results which demonstrate that human hematopoietic progenitor cells transduced with a human TCR can become immature and mature human thymocytes 4 weeks following transduction and transplantation into human tissue in the SCID-hu mouse, mimicking that of human tissue in the body. At four weeks following implantation with TCR transduced stem cells, human thymic tissue was biopsied and cells were analyzed by flow cytometry for cell size (forward scatter—denoted “Size”) and SL9 specific tetramer staining Mock treated mice (top panels) and mice receiving TCR transduced cells (bottom panels) are indicated. The numbers on the left panels illustrate total SL9 tetramer, or HIV-specific TCR, staining or expressing cells. SL9-tetramer+ cells were gated and the frequency of cells expressing CD4 and/or CD8 are provided in the right panels.
FIG. 8 provides results which demonstrate that human hematopoietic progenitor cells transduced with a human TCR can develop into mature human thymocytes, predominantly CD8+ T cells, 7 weeks following transduction and transplantation into human tissue in the SCID-hu mouse, mimicking that of human tissue in the body. At seven weeks following implantation with TCR transduced stem cells, human thymic tissue was biopsied and cells were analyzed by flow cytometry for cell size (forward scatter—denoted “Size”) and SL9 specific tetramer staining Mock treated mice (top panels) and mice receiving TCR transduced cells (bottom panels) are indicated. The numbers on the left panels illustrate total SL9 tetramer, or HIV-specific TCR, staining or expressing cells. SL9-tetramer+ cells were gated and the frequency of cells expressing CD4 and/or CD8 are provided in the right panels.
FIG. 9 provides results which demonstrate that a specific human leukocyte antigen (HLA) is required for transgenic TCR containing T cell development into mature CD8+ single positive thymocytes. For the HIV-1 SL9 peptide-specific TCR, HLA-A*201 is a suitable HLA. Fetal liver derived CD34+ HSCs transduced with the SL9-TCR containing lentiviral vector were implanted into mice containing either HLA-A*0201+ thymic tissue (top panels) or into mice containing HLA-A*0201-thymic tissue (bottom panels) and the frequency of SL-9 tetramer+ cells assessed 6 weeks following implantation. Size (forward scatter) versus tetramer staining is presented in the left panels and the values inside the parentheses correspond to the percentage of tetramer positive cells. Tetramer expressing cells in the indicated gate were assessed for CD4 and CD8 expression (right panels) and the frequencies of cells expressing each marker are provided.
FIG. 10 provides a schematic representation of the strategy used to analyze the functionality of naïve, viral antigen specific T cells displaying the transgenic human TCR.
FIG. 11 provides the results which demonstrate the use of ELISPOT in the analysis of transgenic, virus-specific human TCRs that are derived from retroviral transduced human stem cells. Thymic tissues from 2 mice receiving SL9-specific TCR transduced stem cells (mouse #s 25 and 27) and 1 mock-treated mouse (mouse #17) were biopsied 7 weeks following introduction of stem cells and placed into culture with SL9 peptide coated antigen presenting cells for 1 week to allow differentiation from antigen naïve to effector cells. Effector cells were then stimulated with SL9 peptide or medium alone (no peptide) and IFN-γ production was measured by ELISPOT.
FIG. 12 provides results which demonstrate that newly originated, antigen specific T cells derived from transduced human stem cells can functionally respond to the TCR-specific peptide by producing the cytokine Interferon-gamma. Cells from SCID-hu mice receiving SL9-specific TCR transduced stem cells were obtained by biopsy following differentiation into thymocytes and activated in culture in the presence of an irradiated SL9-peptide coated HLA-A*0201+ B cell line and allogeneic feeder cells. Cells from mouse numbers Y09-13 and Y17-7 were then placed in a standard 51chromium release assay utilizing SL9 peptide coated T2 cells or untreated T2 cells as a control. Graph shows the specific lytic activity of cells at an effector to target cell ratio of 10:1.
FIG. 13 schematically shows the phenotypic developmental changes that newly stimulated, viral antigen specific TCRs undergo to become functional effector cells in an antigen specific manner.
FIG. 14 provides results which demonstrate that human TCR containing T cells are exported from the thymus into the peripheral lymphoid compartments. Mock treated mice (upper row) and mice receiving stem cells transduced with the HIV SL9-specific TCR (lower row) were analyzed 7 weeks following transplantation for CD3 and SL9-specific TCR expression by tetramer staining of cells from the thymus (left panels) or spleen (right panels). The frequency of CD3+ and SL9-tetramer+ cells is provided and the values inside the parentheses correspond to the percentage of tetramer positive cells in the human T cell (CD3+) populations. The presence of cells within these areas indicates that mature cells that express the transgenic TCR can undergo normal developmental mechanisms and are found in the peripheral organs.
FIG. 15 provides the results which demonstrate that cells expressing the SL-9 TCR are found in multiple organs of a different strain of immunodeficient mice implanted with genetically-modified stem cells. Non-obese diabetic, severe combined immunodeficient, common gamma chain knockout (NSG) mice were implanted with SL9 TCR genetically modified stem cells along with human fetal thymus and liver, known as the Bone marrow, thymus, liver (BLT) humanized mouse. These cells were allowed to engraft and develop in the mouse. Six weeks following implantation, the bone marrow, thymus, spleen, liver, and peripheral blood of the mice were assessed for human cells. This demonstrates the presence of human CD8+ T cells expressing the transgenic TCR in the peripheral organs of the mouse following development from genetically modified stem cells.
FIG. 16 provides the results which demonstrate the reduction in amount of HIV in mice containing human cells expressing a HIV-specific TCR as compared to uninfected mice or mice containing cells expressing a nonspecific control human TCR two weeks and six weeks following infection. Virally expressing cells were identified by the expression of the marker gene Heat Stable Antigen-Hemagglutinin (HSA-HA) that had previously been cloned into infectious HIV. Humanized BLT mice containing SL9-TCR specific cells or, separately, cells expressing a control non-specific TCR were infected with HIVNL-HSA-HA, or left uninfected, following human cell reconstitution. Virally expressing cells were analyzed in the peripheral blood of these mice 2 and 6 weeks following infection by flow cytometry. The figure represents the % of cells expressing HIV at the indicated time in each population of mice. The results demonstrate that mice that express the HIV-specific TCR have lower levels of virally infected cells in the periphery, indicating killing of virally infected cells by the cells expressing the SL9-specific TCR.
FIG. 17 provides the results which demonstrate the protection of CD4+ T cells in HIV infected mice containing human cells expressing HIV-specific TCR as compared to uninfected mice or HIV infected mice containing cells expressing a nonspecific control human TCR two weeks and six weeks following infection. The same BLT mice infected in FIG. 17 were assessed for CD4 cell percentages two weeks and 6 weeks following infection with HIV by flow cytometry. The results demonstrate that mice the express the HIV-specific TCR have greater percentages of CD4+ T cells in the periphery, indicating protection of these cells from infection with HIV.
FIG. 18 provides the results which demonstrate the molecular cloning and expression of a human TCR specific to a different viral antigen. Peripheral blood mononuclear cells from an HLA-A*0201, previously influenza infected individual were removed and stimulated with influenza peptide antigen (the GILGFVFTL matrix peptide (SEQ ID NO:39)). The TCR responding to this peptide was molecularly cloned by the spectratyping process and expressed by genetically modified CD8+ T cells from another individual. The figure represents MHC tetramer staining of the expressed TCR, indicating successful cloning and expression of a TCR specific to influenza.



DETAILED DESCRIPTION OF THE INVENTION
The present invention provides recombinant human progenitor cells, engineered human thymocytes, and engineered human antigen specific T cells, including cytotoxic T cells (CTLs), which are functional in vivo, and methods of making thereof. The recombinant progenitor cells are genetically engineered from human stem cells, preferably human hematopoietic stem cells, and result in the engineered human thymocytes and/or the engineered human T cells of the present invention.
The engineered human thymocytes and engineered human T cells express anti-viral specific human T cell receptors (TCRs) and are functional in vivo. As used herein, the term “T cell receptor” includes a complex of polypeptides comprising at least a T cell receptor α subunit and a T cell receptor β subunit. T cell receptors (TCRs) are able to bind a given antigen when expressed on the surface of a cell, such as a T lymphocyte. The α and β chains, or subunits, form a dimer that is independently capable of antigen binding. The α and β subunits typically comprise a constant domain and a variable domain and may be native, full-length polypeptides, or may be modified in some way, provided that the T cell receptor retains the ability to bind the given antigen. The complementarity-determining regions (CDR) of the α and β subunits are the antigen binding domain loops and are regions of sequence hypervariability. The specific sequences/structures in this region of a given TCR provides the ability of the TCR to recognize a specific antigen. In the context of a specific HLA molecule.
In some embodiments, the engineered human thymocytes and/or engineered human T cells express functional human TCRs which are functional in vivo. As used herein, a “functional TCR” is one that binds the specific antigen to which it is directed as determined by, for example, using an ELISA assay and/or mediates an immune response against the specific antigen. For example, a “functional HIV TCR” is one that binds an HIV antigen and/or mediates an immune response against HIV. In some embodiments of the present invention, the recombinant human progenitor cells, the engineered human thymocytes, and/or the engineered human T cells are functional in human subjects (as determined from tests in human subjects and/or human animal models). As used herein, a “receptor specific for” refers to the character of a receptor which recognizes and interacts with a given ligand, e.g. target antigen, but does not substantially recognize and interact with other molecules in a sample under given conditions.
The recombinant human progenitor cells, the engineered human thymocytes, and/or the engineered human T cells of the present invention may be used to reconstitute immune function and control replication of a virus, such as human immunodeficiency virus (HIV), in subjects, e.g. human subjects. Therefore, the present invention also provides methods of inhibiting, reducing and treating viral infections, such as an HIV infection, in a subject, such as a human subject. The treatment method according to the present invention may be therapeutic or prophylactic and need not completely eliminate the infection or completely prevent a subject from becoming infected. The present invention also provides methods for enhancing the antigen specific CTL response against a chronic viral infection, such as an HIV infection, in a subject, such as a human subject.
The methods of the present invention provide naïve human cells bearing a transgenic human TCR that is antigen specific, e.g. antigen specific CD8+ CTLs, that allow longer-term engraftment, continuous generation of new effector cells and a more efficient immune response through natural immune mechanisms in human subjects and/or humanized animal models as compared to genetic modification of mature, peripheral blood mononuclear cells.
Some of the human TCR clones, recombinant human progenitor cells, the engineered human thymocytes, the engineered human T cells, and methods of making and using thereof as disclosed herein are also described in the journal article by the inventors, i.e. Kitchen et al. (2009) PLoS ONE 4(12):e8208, all of which is herein incorporated by reference in its entirety.
Unlike prior art efforts which merely differentiate transgenic CTLs in vitro, the present invention provides recombinant human progenitor cells and engineered human naïve thymocytes which are capable of developing in vivo in human thymus tissue and mature into functional CD8+ T cells.
TCR Cloning
The present invention also provides methods of making the recombinant human progenitor cells, engineered human thymocytes, and engineered human T cells of the present invention. Generally, the recombinant human progenitor cells are made from human stem cells, preferably hematopoietic stem cells, by transducing the stem cells with a vector which is capable of expressing a TCR of interest. Briefly, the TCR specific for a virus of interest, e.g. HIV, or an immunogenic epitope thereof, e.g. SL9, is cloned and then the human stem cell is transduced with a vector containing the TCR clone to give the recombinant human progenitor cell. The recombinant human progenitor cell is allowed to develop into an engineered human thymocyte which then matures into the corresponding engineered human T cell, i.e. a functional human T cell which expresses the cloned T cell receptor. In some embodiments, the engineered human T cell is a CTL.
The TCR of interest may be cloned from a pool of samples obtained from a plurality of subjects infected with the given virus to obtain a “consensus” TCR clone. Alternatively, the TCR of interest may be cloned from a sample obtained from the subject to be treated in order to obtain a “personalized” TCR clone for providing the subject with a personalized therapeutic treatment. Methods known in the art may be used to obtain a consensus TCR clone and/or a personalized TCR clone.
For example, peripheral blood mononuclear cells (PBMCs) are removed from a subject having a viral infection, such as an HIV infection. Part of the PBMCs is then cultured with the antigen, i.e. the virus of interest or the immunogenic epitope thereof, and the other part of the PMBCs is cultured without the antigen. Fingerprints which break down all TCRs into over 200 populations for each of the TCR α- and β-genes are obtained for the PBMCs cultured with the antigen and the PMBCs cultured without the antigen by spectratyping. The differences between the fingerprints indicate the TCR sequences of interest. Then the TCR sequences of interest are cloned using methods known in the art.
The TCR clone, as exemplified herein, was isolated from an HIV-positive subject via peptide stimulation and limiting dilution. In particular, PBMCs were obtained from a HLA-A*0201+, HIV infected individual. Part of the PBMCs were cultured with allogeneic, irradiated PBMCs from a different HIV− donor and part of the PBMCs were cultured with a HLA-A*0201+ cell line pretreated with the epitope of interest, i.e. SL9. The cell cultures were fed with fresh medium every 3 to 4 days, and were split and placed on fresh, irradiated PBMCs once every 10 to 14 days.
Spectratyping was used to identify and isolate the TCR sequences specific for the SL9 epitope using methods known in the art. See Balamurugan, et al. (2010) J Immunol. 185(10): 5935-5942; and Frohman (1994) PCR Methods Appl. 4(1): S40-S58, which are herein incorporated by reference. Specifically, the TCR specific for the SL9 epitope was identified following isolation of total RNA, reverse transcription of total cDNA, and quantitative PCR using primers specific to each TCR family to determine the relative concentration of each family. Capillary electrophoresis was then performed, the size distribution of DNA fragments amplified within each TCR family was resolved, and individual peak concentrations were calculated. A comparison was made between the antigen stimulated and unstimulated cells and differences between the spectratypes of the TCR families were noted. The PCR reaction products of the TCRs that displayed differences following antigen stimulation were then directly sequenced.
The TCR sequences were then synthesized and molecularly cloned into a vector, preferably a viral gene therapy vector. Specifically, the cloned TCRα and TCRβ nucleotide sequences were then joined by a sequence encoding picornavirus-like 2A “self-cleaving” peptide. See Yang, et al. (2008) Gene Ther. 15(21): 1411-1423, which is herein incorporated by reference. FIG. 1 sets forth the exemplified TCR sequence of interest, i.e. 1.9 TCR. The short 18 amino acid 2A sequence which separates the TCRα and TCRβ results in equimolar expression of the TCRα and TCRβ via a “ribosomal skip” mechanism. See Szymczak, et al. (2004) Nat Biotechnol. 22(5): 589-594; and Szymczak & Vignali (2005) Expert Opin Biol Ther. 5(5): 627-638, which are herein incorporated by reference. The TCRα-2A-TCRβ nucleotide sequence was cloned into a lentiviral vector under control of the human phosphoglycerate kinase promoter (hPGK), followed by an internal ribosomal elongation site (IRES) which allows further enhancement of expression of a marker gene, i.e. enhanced green fluorescent protein (eGFP). IRES elements and 2A elements are known in the art. See U.S. Pat. No. 4,937,190; de Felipe, et al. (2004) Traffic 5: 616-626, which are incorporated herein by reference. Other expression control elements, viral vectors, and reporter genes known in the art may be used to further enhance or direct expression of the TCR to various cell types. See e.g. US 20080199424, which is herein incorporated by reference. As exemplified herein, the vector employed is the lentiviral pCCL.PPT.hPGK.tcr.IRES.eGFP vector. FIG. 2 schematically shows the vector containing the 1.9 TCR sequence.
SL9-specificity of the cloned TCR was confirmed by transfecting Jurkat cells with the lentiviral vector containing the TCRα-2A-TCRβ construct and staining with SL9 tetramer using methods known in the art. The lentiviral vector containing the TCRα-2A-TCRβ construct was then codon optimized for optimal expression in human cells using methods known in the art. See Scholten, et al. (2006) Clin Immunol. 119(2): 135-145, which is herein incorporated by reference. The cloned TCR of the codon optimized vector was also shown to retain SL9 specificity by transfecting Jurkat cells with the codon optimized vector containing the TCRα-2A-TCRβ construct and staining with SL9 tetramer using methods known in the art.
FIG. 3 schematically shows the TCR spectratyping-based cloning as exemplified herein and FIG. 4 compares this TCR spectratyping-based cloning with a conventional method known in the art.
The methods and vectors according to the present invention may be readily modified using methods known in the art to include clinically relevant reporters or selection markers that may be used to identify and/or isolate successfully transduced stem cells. For example, a recombinant progenitor cell could be engineered to express a receptor, such as a truncated human nerve growth factor receptor, on its surface. Preferably, the receptor would not have signaling properties that would have a detrimental effect on the desired function of the progenitor cell. Preferably, the receptor does not induce an immunogenic response in the subject to be treated. The receptor could then be used to sort or select recombinant progenitor cells prior to their introduction into a subject.
The cloned TCRs can be combined with small interfering (si), or short hairpin (sh) RNAs against molecules that modulate T cell development and T cell function to modify the activity of the cell expressing the transgenic TCR. For example, a siRNA specific against the Programmed Death-1 (PD-1) gene was designed and cloned into the gene therapy vector that also expresses the SL-9 specific TCR. See FIG. 5. Programmed Death-1 (PD-1) is associated with decreased development and deceased function of antigen-specific T cells. See Simone, et al. (2009) Curr HIV Res. 7(3): 266-272; Trautmann, et al. (2007) Curr Opin HIV AIDS, 2(3): 219-227; and Petrovas, et al. (2006) J Exp Med. 203(10): 2281-2292, which are herein incorporated by reference. The expression of this TCR-siRNA vector down-regulated PD-1 expression in transduced cells. Therefore, siRNA and/or shRNA may be used to further modify the activity of the cloned TCRs according to the present invention. For example, siRNA and/or shRNA molecules against the programmed death-1 (PD-1) molecule, the T-cell immunoglobulin domain and/or mucin domain 3 (Tim-3) molecule may be included in the vectors described herein.
Recombinant Human Progenitor Cells
Human hematopoietic stem cells were then transduced with the codon optimized vector containing the TCRα-2A-TCRβ construct to give recombinant human progenitor cells according to the present invention using methods known in the art. See e.g. Arnold, et al. (2004) J Immunol. 173(5): 3103-3111, which is herein incorporated by reference. In particular, as exemplified herein, human CD34+ hematopoietic stem cells were taken from hematopoietic tissue (e.g. fetal liver) and transduced with the codon optimized vector containing the TCRα-2A-TCRβ construct to give recombinant human progenitor cells. See FIG. 6.
In order to determine whether the recombinant human progenitor cells exhibit CTL activity, the recombinant human progenitor cells and irradiated peripheral blood mononuclear cells (PBMCs) were resuspended in media that contains irradiated HLA-A*0201 cells and 1 μg/ml of antigenic peptide (e.g. the SL9 peptide from HIV) and incubated overnight at 37° C. to prestimulate the cells. After overnight incubation, media containing 50 units/ml of recombinant interleukin (IL)-2 was added. Cells were fed with fresh media every 3 to 4 days, and passaged once every 10 to 14 days. The resulting recombinant human progenitor cells were tested for CTL activity 7 days following passaging by assessing their ability to lyse 51Chromium-labeled target cells in a standard chromium release assay known in the art. As exemplified herein, the target cells were either HIV infected cells that were matched with HLA-A*0201 or were HLA-A*0201 cells that were pretreated with the SL9 peptide. A recombinant human progenitor cell was designated as having CTL activity if it lysed a target cell.
Engineered Human Thymocytes
The recombinant human progenitor cells were allowed or induced to differentiate and mature into engineered human thymocytes and human T cells that express the transgenic anti-viral TCR. Specifically, the recombinant human progenitor cells were injected directly into the human thymic tissue in sub-lethally irradiated SCID-hu mice. See Amado, et al. (1999) Front Biosci. 4: D468-D475; Kitchen, et al. (2000) J Virol. 74(6): 2943-2948; McCune, J. M. (1992) Bone Marrow Transplant. 9 Suppl 1: 74-76; McCune, et al. (1998) Science. 241(4873): 1632-1639; and Withers-Ward, et al. (1997) Nat Med. 3(10): 1102-1109, which are herein incorporated by reference.
The irradiation was performed to clear niches for the newly implanted recombinant human progenitor cells through riddance of endogenous thymocytes. The implanted recombinant human progenitor cells were allowed to develop into engineered human thymocytes for a period of weeks following implantation. Subsequent analysis for markers of developing and mature engineered human thymocytes containing the transgenic SL9-specific TCR was performed following biopsy of the thymic tissue. Within 4 weeks following transplantation of the recombinant human progenitor cells, immature and mature engineered human thymocytes expressing the transgenic SL9-specific TCR were observed through flow cytometric analysis for phenotypic marker expression and through staining the cells with SL9-specific MHC Class 1 tetramer molecules. See FIG. 7. Within 7 weeks following implantation of the recombinant human progenitor cells, significant accumulation of CD8+ thymocytes expressing the transgenic SL9-specific TCR and the exclusion of mature CD4+ cells, thereby indicating correct TCR induced lineage commitment, was observed. See FIG. 8.
Functional Human Thymocytes
To demonstrate the ability of the recombinant human progenitor cells to develop in the presence or absence of the specific SL9 peptide-restricted HLA molecule (HLA-A*0201), the SL9-specific TCR retrovirally transduced CD34+ cells were injected into mice that contained human thymic tissue that was HLA-A*0201+ and mice that contained human thymic tissue that was HLA-A*0201-. HLA-A*0201+ mice developed mature CD8+ T cells that expressed the transgenic SL9-specific TCR, whereas HLA-A*0201− mice did not. See FIG. 9. This evidences that the correct HLA recipient tissue is required for the proper development of the recombinant human progenitor cells into mature human thymocytes and T cells, a process known as positive selection. This data clearly indicates that the HLA-A*0201 molecule is required for stem cells transduced with the SL9-specific TCR to develop properly in the thymus. Thus, engineered human thymocytes resulting from these recombinant human progenitor cells undergo appropriate positive and negative selection. Consequently, mature functional T cells would only be produced in a subject that expresses HLA-A*0201, and would only exercise an antigen-specific immune response if the subject was infected with HIV-1.
To demonstrate that the engineered human thymocytes are functional in responding to viral antigen, an assay which pre-stimulates naïve antigen specific thymocytes and assesses functional responses subsequent to cellular activation was conducted. The assay is schematically shown in FIG. 10.
Specifically, thymic tissue from the subject that previously received the recombinant human progenitor cells was biopsied. The naïve engineered human thymocytes obtained therefrom were placed in tissue culture with irradiated antigen presenting cells of a known HLA type, the specific peptide that the transgenic TCR recognizes, and interleukin 2 (IL-2). In particular, naïve engineered human thymocytes from the biopsied thymic tissue were cultured with the HLA-A*0201+ T1 cell line, the SL9 peptide, and IL-2. Following a sufficient period of time in culture which allows the naïve engineered human thymocytes to recognize the specific peptide in the context of the proper HLA molecule and become functionally activated, e.g. 1 week, the engineered human thymocytes were then subjected to further stimulation with the SL9 peptide and assayed for functional cellular responses using methods known in the art.
In particular, the engineered human thymocytes were assessed for cytokine production by ELISPOT for interferon gamma (IFN-γ). The results shown in FIG. 11 demonstrate that the recombinant human progenitor cells of the present invention result in engineered human thymocytes that express functional transgenic TCRs. In FIG. 11, the spots on the membranes, which were identified and quantitated, represent engineered human thymocytes that functionally responded specifically to stimulation with the SL9 peptide and produced IFN-γ. FIG. 12 graphically provides the amount of cells per 10,000 total thymocytes that reacted to the SL9 peptide in the ELISPOT assay.
To further demonstrate that the functionally responding engineered human thymocytes phenotypically represent effector cells, the engineered human thymocytes were stained with antibodies specific for molecules that are indicative of different stages of CD8+ T cell differentiation following antigen stimulation and analyzed by flow cytometry using methods known in the art. See FIG. 13. It was found that these previously naïve engineered human thymocytes acquired a phenotype that represents effector memory type cells 1 week following stimulation with irradiated T1 cells, SL9 peptide, and IL-2.
Functional Human T Cells
Engineered human T cells expressing the transgenic SL9-specific TCR were found in the peripheral organs (e.g. the mouse spleen) of the sub-lethally irradiated SCID-hu mice having the recombinant human progenitor cells directly implanted into the human thymic tissue. See FIG. 14. It was also found that functional engineered human T cells can develop and be exported to the periphery of another type of immunodeficient mouse model, the Non-obese diabetic, Severe combined immunodeficient, common Gamma chain knockout, humanized Bone marrow, fetal Liver, and fetal Thymus (NSG-BLT) mouse that allows the examination of immune responses within the mouse. See FIG. 15. In these studies, recombinant human progenitor cells (i.e. SL9-specific TCR retrovirally transduced human CD34+ cells) were injected intravenously into irradiated immunodeficient mice previously implanted with human fetal thymus and liver. See Melkus, et al. (2006) Nat Med. 12(11): 1316-1322; and Brainard, et al. (2009) J. Virol. 83(14): 7305-7321, which are herein incorporated by reference. In the current experiment, human HLA-A*0201+ fetal liver, containing CD34+ cells modified with the SL9-specific TCR, and thymus tissue were implanted into a NSG strain mouse. Three weeks following this, the mouse was irradiated and intravenously injected with additional, CD34+ cells from the same donor also modified with the SL9-specific TCR. The CD34+ progenitor cells become engrafted in the mouse bone marrow and human liver/thymus tissue and allow human progenitor cells to develop. 6 weeks following injection, engineered human T cells, i.e. SL-9 specific TCR expressing CD8+ T cells, were found in the peripheral blood and other organs following necropsy of the mice by flow cytometry analysis for cell markers and TCR expression. This demonstrates that, when intravenously administered, the recombinant human progenitor cells according to the present invention engraft and result in engineered human T cells that are efficiently exported to the periphery of subjects. Therefore, in the methods of the present invention, the recombinant human progenitor cells may be directly implanted in the thymic tissue of a subject or intravenously administered to the subject in order to result in engraftment and the development of engineered human T cells in the subject.
The engineered human T cells expressing the SL9-specific TCR were found to be functional in vivo as they were capable of reducing the amount of virally infected cells (FIG. 16) and preventing CD4 T cell loss (FIG. 17) in treated mice following infection with HIV. In these studies, NSG-BLT mice containing human cells genetically modified with the HIV TCR or mice containing cells genetically modified with a non-specific control TCR (as a negative control) were infected with HIV (in this case the HIV-1NL4-3-HSA-HA viral variant. Two weeks and 6 weeks following infection, human cells in the peripheral blood of these mice were analyzed for CD4, CD8 and HSA-HA (viral) expression by flow cytometry. Suppression of virally infected cells (FIG. 16) and protection of CD4+ T cell levels (FIG. 17) was found following infection. These results demonstrate that engineered human T cells according to the present invention that express an HIV-specific TCR can reduce or inhibit HIV replication and reduce or inhibit the loss of target CD4+ T cells in vivo. Therefore, the present invention provides methods for inhibiting, reducing or treating viral infections in a subject which comprise administering recombinant human progenitor cells, engineered human thymocytes, and/or engineered human T cells as described herein. In some embodiments, the amount administered is a therapeutically effective amount, which is an amount that inhibits or reduces viral replication and/or loss of target CD4+ T cells in the subject.
Since a given TCR for an antigen of interest resulting from a given recombinant progenitor cell cannot recognize the antigen of interest when presented by a different HLA molecule, a plurality of recombinant human progenitor cells having different TCRs specific for other peptides presented by different HLA molecules may be made and used in combination in the methods described herein.
In some embodiments, an antigen-specific TCR could be cloned from a subject to be treated, and then it could be introduced into the subject's hematopoietic stem cells. Alternatively, a bank of vectors could be generated following the cloning of many TCRs from a variety of individuals, each of which would be specific for a particular antigen presented in the context of one of the many HLA molecules in the population. These banked TCR gene vectors are preferably stable, and following tissue typing of an individual patient, may be cross-matched for the ability to react with the subject's HLA molecules, and subsequently introduced into the subject's stem cells.
The experiments provided herein evidence that human viral antigen-specific TCRs can be cloned out of immune cells from an infected individual. These TCR clones can then be placed into a gene therapy vector. Human stem cells can then be transduced with the vector and allowed to express the TCR following differentiation and development into mature cells in the presence of the appropriate HLA molecule. The experiments also evidence that the engineered human thymocytes and engineered human T cells expressing a cloned TCR are functional in vivo and are capable of mounting a cellular response against viruses having the antigen to which the cloned TCR is specific against. Therefore, the present invention provides methods for treating a viral infection, such as an HIV infection, in a subject.
For example, in some embodiments, peripheral blood is removed from a human subject having the viral infection and one or more viral antigen specific cells are identified. A T cell receptor (TCR) from one of the viral antigen specific cells is cloned through spectratyping-based cloning. The α- and β-subunits of the virus specific TCR is cloned into a vector that allows its concurrent expression in human cells. A viral gene therapy vector, which may be the same or different from the cloning vector, containing the cloned virus-specific TCR is obtained. In some embodiments, the vector is one that expresses a virus-specific TCR restricted to one of the class I HLA molecules of the human subject to be treated.
Human autologous or histocompatible stem cells are then transduced with the vector containing the cloned virus antigen specific TCR to give a recombinant human progenitor cell. The transduction efficiency may validated and the recombinant human progenitor cells can be analyzed. The recombinant human progenitor cells are transplanted into the human subject to be treated where the recombinant human progenitor cells differentiate and mature into engineered human thymocytes and engineered human T cells that express the cloned TCR. Engraftment of the transplanted recombinant human progenitor cells may be determined. The functional responses of the recombinant human progenitor cells, the engineered human thymocytes and/or the engineered human T cells may be monitored. Virus-specific immune responses and the clearance of the virus from the body may be monitored. Virus epitope mutation, especially the transgenic TCR-specific epitope, may be determined. In some embodiments, if “virologic failure” or “immune failure” is detected, the process may be repeated with the same or different viral antigen-specific TCR. In embodiments, where no virologic or immune failure is detected, the subject may undergo further monitoring until, for example, the infection is controlled or cleared.
In some embodiments, the gene therapy vector containing the TCR clone may be administered to the subject. Alternatively, engineered human thymocytes and/or engineered human T cells expressing the TCR clone may be administered to the subject. In some embodiments, a plurality of different recombinant human progenitor cells, a plurality of different engineered human thymocytes and/or a plurality of different engineered human T cells may be employed.
Additional Embodiments
In addition to the recombinant progenitor cells, engineered thymocytes, and engineered T cells which express TCRs specific to the SL9 peptide as exemplified herein, recombinant human progenitor cells, engineered human thymocytes, and engineered human T cells which express other TCRs, i.e. TCRs which are specific for other antigens, are contemplated herein. For example, a TCR specific for an influenza antigen (in this case the influenza A matrix protein 58-68 or GI-9) was cloned and recombinant human progenitor cells, engineered human thymocytes, and engineered human T cells which express the cloned TCR were created using the methods described herein. See FIG. 18. Briefly, PBMCs were taken from an HLA-A*0201+ subject who was previously exposed to influenza. Cells were then cultured in the presence and absence of the GI-9 peptide, to allow selection for the cells expressing the antigen specific TCR. Spectratyping-based cloning was then used to rapidly identify, sequence, and clone a TCR specific to the influenza GI-9 peptide.
Therefore, some embodiments, the target antigen comprises, consists essentially of, or consists of an epitope selected from the group consisting of SL9 (SLYNTVATL (SEQ ID NO:1)), GE11 (GHQAAMQMLKE (SEQ ID NO:2)), AL9-Vpr (AIIRILQQL (SEQ ID NO:3)), RI9-Vpr (RILQQLLFI (SEQ ID NO:4)), AL9-Nef (AFHHMAREL (SEQ ID NO:5)), and QL10-GP160 (QELKNSAVSL (SEQ ID NO:6)).
In some embodiments, the present invention provides a polypeptide, which may be isolated or purified, comprising, consisting essentially of, or consisting of
a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to







MMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDR


 


GSQSFFWYRQYSGKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRD


 


SQPSDSATYLCAVISNSGNTPLVFGKGTRLSVIANIQNPDPAVYQLRDSK


 


SSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWS


 


NKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLS


 


VIGFRILLLKVAGFNLLMTLRLWSS






(a TCR α subunit for HIV, SEQ ID NO:7, variable region underlined) and

a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to







MGSRLLCWVLLCLLGAGPVKAGVTQTPRYLIKTRGQQVTLSCSPISGHRS


 


VSWYQQTPGQGLQFLFEYFSETQRNKGNFPGRFSGRQFSNSRSEMNVSTL


 


ELGDSALYLCASSFDSEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEI


 


SHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALND


 


SRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIV


 


SAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAM


 


VKRKDSRG






(a TCR β subunit for HIV, SEQ ID NO:8, variable region underlined).

In some embodiments, the present invention provides an isolated or purified polypeptide comprising, consisting essentially of, or consisting of
a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to







MQLLEQSPQFLSIQEGENLTVYCNSSSVFSSLQWYRQEPGEGPVLLVTVV


 


TGGEVKKLKRLTFQFGDARKDSSLHITAAQPGDTGLYLCAGAGSQGNLIF


 


GKGTKLSVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDS


 


DVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPS


 


PESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWS


 


S






(a TCR α subunit for an influenza epitope, SEQ ID NO:9, variable region underlined); and

a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to







MVDGGITQSPKYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYY


 


SQIVNDFQKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLCASSSRSS


 


YEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGF


 


YPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYSLSSRLRVSATF


 


WQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSE


 


SYQQGVLSATILYEILLGKATLYAVLVSALVLMAMQEKGFQR






(TCR β subunit for an influenza epitope, SEQ ID NO:10, variable region underlined).

Other TCR clones obtained by the methods described herein include a polypeptide comprising, consisting essentially of, or consisting of:
a) a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to







MVLKFSVSILWIQLAWVSTQLLEQSPQFLSIQEGENLTVYCNSSSVFSSL


 


QWYRQEPGEGPVLLVTVVTGGEVKKLKRLTFQFGDARKDSSLHITAAQPG


 


DTGLYLCAGAGWRDDKIIFGKGTRLHILPNIQNPDPAVYQLRDSKSSDKS


 


VCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDF


 


ACANAFNNSIIPADTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFR


 


ILLLKVAGFNLLMTLRLWSS






(a TCR α subunit for GE11 in HIV Gag, SEQ ID NO:11, variable region underlined) or








MAMLLGASVLILWLQPDWVNSQQKNDDQQVKQNSPSLSVQEGRISILNCD


 


YTNSMFDYFLWYKKYPAEGPTFLISISSIKDKNEDGRFTVFLNKSAKHLS


 


LHIVPSQPGDSAVYFCAANSLDRDDKIIFGKGTRLHILPNIQNPDPAVYQ


 


LRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNS


 


AVAWSNKSDFACANAFNNSIIPADTFFPSPESSCDVKLVEKSFETDTNLN


 


FQNLSVIGFRILLLKVAGFNLLMTLRLWSS






(a TCR α subunit for GE11 in HIV Gag, SEQ ID NO:12, variable region underlined) and

a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to







MLSLLLLLLGLSVFSAVISQKPSRDICQRGTSLTIQCQVDSQVTMMFWYR


 


QQPGQSLTLIATANQGSEATYESGFVIDKFPISRPNLTFSTLTVSNMSPE


 


DSSIYLCSVGPRQGGEQYFGPGTRLTVTEDLNKVFPPEVAVFEPSEAEIS


 


HTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDS


 


RYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVS


 


AEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMV


 


KRKDF






(a TCR β subunit for GE11 in HIV Gag, SEQ ID NO:13, variable region underlined);

b) a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to







MKLVTSITVLLSLGIMGDAKTTQPNSMESNEEEPVHLPCNHSTISGTDYI


 


HWYRQLPSQGPEYVIHGLTSNVNNRMASLAIAEDRKSSTLILHRATLRDA


 


AVYYCILIPPPYSGAGSYQLTFGKGTKLSVIPNIQNPDPAVYQLRDSKSS


 


DKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNK


 


SDFACANAFNNSIIPADTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVI


 


GFRILLLKVAGFNLLMTLRLWSS






(a TCR α subunit for the AL9 epitope of HIV Vpr, SEQ ID NO:14, variable region underlined) and

a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to







MLSPDLPDSAWNTRLLCHVMLCLLGAVSVAAGVIQSPRHLIKEKRETATL


 


KCYPIPRHDTVYWYQQGPGQDPQFLISFYEKMQSDKGSIPDRFSAQQFSD


 


YHSELNMSSLELGDSALYFCASSSLRAASYGYTFGSGTRLTVVEDLNKVF


 


PPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVS


 


TDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSEND


 


EWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKAT


 


LYAVLVSALVLMAMVKRKDF






(a TCR β subunit for the AL9 epitope of HIV Vpr, SEQ ID NO:15, variable region underlined); or








MGTSLLCWMALCLLGADHADTGVSQDPRHKITKRGQNVTFRCDPISEHNR


 


LYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQR


 


TEQGDSAMYLCASSSQAVSTDTQYFGPGTRLTVLEDLNKVFPPEVAVFEP


 


SEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQ


 


PALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKP


 


VTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSAL


 


VLMAMVKRKDF






(a TCR β subunit for the AL9 epitope of HIV Vpr, SEQ ID NO:16, variable region underlined);

c) a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to







MLLLLIPVLGMIFALRDARAQSVSQHNHHVILSEAASLELGCNYSYGGTV


 


NLFWYVQYPGQHLQLLLKYFSGDPLVKGIKGFEAEFIKSKFSFNLRKPSV


 


QWSDTAEYFCAVIEDSSYKLIFGSGTRLLVRPDIQNPDPAVYQLRDSKSS


 


DKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNK


 


SDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVI


 


GFRILLLKVAGFNLLMTLRLWSS






(a TCR α subunit for the SL9 epitope of HIV Gag, SEQ ID NO:17, variable region underlined) and

a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to







MGTRLLCWAALCLLGAELTEAGVAQSPRYKIIEKRQSVAFWCNPISGHAT


 


LYWYQQILGQGPKLLIQFQNNGVVDDSQLPKDRFSAERLKGVDSTLKIQP


 


AKLEDSAVYLCASSLEHEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAE


 


ISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALN


 


DSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQI


 


VSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMA


 


MQEKGFQR






(a TCR β subunit for the SL9 epitope of HIV Gag, SEQ ID NO:18, variable region underlined);

d) a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to







MLLLLVPVLEVIFTLGGTRAQSVTQLGSHVSVSEGALVLLRCNYSSSVPP


 


YLFWYVQYPNQGLQLLLKYTTGATLVKGINGFEAEFKKSETSFHLTKPSA


 


HMSDAAEYFCAVSEIEFGNEKLTFGTGTRLTIIPNIQNPDPAVYQLRDSK


 


SSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWS


 


NKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLS


 


VIGFRILLLKVAGFNLLMTLRLWSS






(a TCR α subunit for the AL9 epitope of HIV Nef, SEQ ID NO:19, variable region underlined) and

a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to







MGTRLLCWVVLGFLGTDHTGAGVSQSPRYKVAKRGQDVALRCDPISGHVS


 


LFWYQQALGQGPEFLTYFQNEAQLDKSGLPSDRFFAERPEGSVSTLKIQR


 


TQQEDSAVYLCASSAGLGTGTSYEQYFGPGTRLTVTEDLKNVFPPEVAVF


 


EPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLK


 


EQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRA


 


KPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVS


 


ALVLMAMVKRKDSRG






(a TCR β subunit for the AL9 epitope of HIV Nef, SEQ ID NO:20, variable region underlined);

e) a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to







MAMLLGASVLILWLQPDWVNSQQKNDDQQVKQNSPSLSVQEGRISILNCD


 


YTNSMFDYFLWYKKYPAEGPTFLISISSIKDKNEDGRFTVFLNKSAKHLS


 


LHIVPSQPGDSAVYFCAASPFLSTGANSKLTFGKGITLSRPDIQNPDPAV


 


YQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKS


 


NSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTN


 


LNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS






(a TCR α subunit for the RI9 epitope of HIV Vpr, SEQ ID NO:21, variable region underlined) and

a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to







MGTRLLCWAALCLLGAELTEAGVAQSPRYKIIEKRQSVAFWCNPISGHAT


 


LYWYQQILGQGPKLLIQFQNNGVVDDSQLPKDRFSAERLKGVDSTLKIQP


 


AKLEDSAVYLCASSLEHEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAE


 


ISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALN


 


DSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQI


 


VSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMA


 


MVKRKDSRG






(a TCR β subunit for the RI9 epitope of HIV Vpr, SEQ ID NO:22, variable region underlined);

f) a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to







MAGIRALFMYLWLQLDWVSRGESVGLHLPTLSVQEGDNSIINCAYSNSAS


 


DYFIWYKQESGKGPQFIIDIRSNMDKRQGQRVTVLLNKTVKHLSLQIAAT


 


QPGDSAVYFCAERAGNQFYFGTGTSLTVIPNIQNPDPAVYQLRDSKSSDK


 


SVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSD


 


FACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGF


 


RILLLKVAGFNLLMTLRLWSS






(a TCR α subunit for the SL9 epitope of HIV p17, SEQ ID NO:23, variable region underlined) and

a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to







MSNQVLCCVVLCFLGANTVDGGITQSPKYLFRKEGQNVTLSCEQNLNHDA


 


MYWYRQDPGQGLRLIYYSQIVNDFQKGDIAEGYSVSREKKESFPLTVTSA


 


QKNPTAFYLCASKAGGMTEAFFGQGTRLTVVEDLKNVFPPEVAVFEPSEA


 


EISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPAL


 


NDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQ


 


IVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLM


 


AMVKRKDSRG






(a TCR β subunit for the SL9 epitope of HIV p17, SEQ ID NO:24, variable region underlined);

g) a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to







MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRG


 


SQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDS


 


QPSDSATYLCAVIGNAGNMLTFGGGTRLMVKPHIQNPDPAVYQLRDSKSS


 


DKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNK


 


SDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVI


 


GFRILLLKVAGFNLLMTLRLWSS






(a TCR α subunit for the QL10 epitope of HIV GP160, SEQ ID NO:25, variable region underlined) or








MWGVFLLYVSMKMGGTTGQNIDQPTEMTATEGAIVQINCTYQTSGFNGLF


 


WYQQHAGEAPTFLSYNVLDGLEEKGRFSSFLSRSKGYSYLLLKELQMKDS


 


ASYLCAVSDGGLNTDKLIFGTGTRLQVFPNIQNPDPAVYQLRDSKSSDKS


 


VCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDF


 


ACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFR


 


ILLLKVAGFNLLMTLRLWSS






(a TCR α subunit for the QL10 epitope of HIV GP160, SEQ ID NO:26, variable region underlined) and

a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to







MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLS


 


VYWYQQSLDQGLQFLIHYYNGEERAKGNILERFSAQQFPDLHSELNLSSL


 


ELGDSALYFCASSVALETPYILEREVGSQDLKNVFPPEVAVFEPSEAEIS


 


HTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDS


 


RYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVS


 


AEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMV


 


KRKDSRG






(a TCR β subunit for the QL10 epitope of HIV GP160, SEQ ID NO:27, variable region underlined);

h) a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to







LMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDR


 


GSQSFFWYRQYSGKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRD


 


SQPSDSATYLCAVISNSGNTPLVFGKGTRLSVIANIQNPDPAVYQLRDSK


 


SSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWS


 


NKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLS


 


VIGFRILLLKVAGFNLLMTLRLWSS






(a TCR α subunit for the SL9 epitope of HIV Gag, SEQ ID NO:28, variable region underlined),








WMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDR


 


GSQSFFWYRQYSGKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRD


 


SQPSDSATYLCAVISNSGNTPLVFGKGTRLSVIANIQNPDPAVYQLRDSK


 


SSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWS


 


NKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLS


 


VIGFRILLLKVAGFNLLMTLRLWSS






(a TCR α subunit for the SL9 epitope of HIV Gag, SEQ ID NO:29, variable region underlined) or








WMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDR


 


GSQSFFWYRQYSGKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRD


 


SQPSDSATYLCAVISNSGNTPLVFGKGTRLSVIANIQNPDPAVYQLRDSK


 


SSDKSVCLFTDFDSQTNVSQSKDSDVYITDKSVLDMRSMDFKSNSAVAWS


 


NKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLS


 


VIGFRILLLKVAGFNLLMTLRLWSS






(a TCR α subunit for the SL9 epitope of HIV Gag, SEQ ID NO:30, variable region underlined) and

a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to







MGSRLLCWVLLCLLGAGPVKAGVTQTPRYLIKTRGQQVTLSCSPISGHRS


 


VSWYQQTPGQGLQFLFEYFSETQRNKGNFPGRFSGRQFSNSRSEMNVSTL


 


ELGDSALYLCASSFDSEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEI


 


SHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALND


 


SRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIV


 


SAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAM


 


VKRKDSRG






(a TCR β subunit for the SL9 epitope of HIV Gag, SEQ ID NO:31, variable region underlined),








MGSRLLCWVLLCLLGAGPVKAGVTQTPRYLIKTRGQQVTLSCSPISGHRS


 


VSWYQQTPGQGLQFLFEYFSETQRNKGNFPGRFSGRQFSNSRSEMNVSTL


 


ELGDSALYLCASSFDSEQYFGPGTRLTVTEGLKNVFPPEVAVFEPSEAEI


 


SHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALND


 


SRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIV


 


SAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAM


 


VKRKDSRG






(a TCR β subunit for the SL9 epitope of HIV Gag, SEQ ID NO:32, variable region underlined),








MGSRLLCWVLLCLLGAGPVKAGVTQTPRYLIKTRGQQVTLSCSPISGHRS


 


VSWYQQTPGQGLQFLFEYFSETQRNKGNFPGRFSGRQFSNSRSEMNVSTL


 


ELGDSALYLCASSFDSEQYFGPGTRLTVTEGLKNVFPPEVAVFEPSEAEI


 


SHTQKATLVCLATGFYPDYVELSWWVNGKEVHSGVCTDPQPLKEQPALND


 


SRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIV


 


SAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAM


 


VKRKDSRG






(a TCR β subunit for the SL9 epitope of HIV Gag, SEQ ID NO:33, variable region underlined), or








MGSRLLCWVLLCLLGAGPVKAGVTQTPRYLIKTRGQQVTLSCSPISGHRS


 


VSWYQQTPGQGLQFLFEYFSETQRNKGNFPGRFSGRQFSNSRSEMNVSTL


 


ELGDSALYLCASSFDSEQYFGPGTRLTVTEGLKNVFPPEVAVFEPSEAEI


 


SHTQKATLVCLATGFYPDYVELSWWVNGKEVHSGVSTDPQPLKEQPALND


 


SRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIV


 


SAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAM


 


VKRKDSRG






(a TCR β subunit for the SL9 epitope of HIV Gag, SEQ ID NO:34, variable region underlined).

In preferred embodiments of the present invention, the polypeptide comprises a first sequence that has 98-100%, preferably 99-100%, identity to the variable region (underlined) of one of the first sequences indicated above and a second sequence that has 98-100%, preferably 99-100%, identity to the variable region (underlined) of one of the second sequences indicated above. For example, a polypeptide according to the present invention may comprise a first sequence having 100% identity to the variable region of SEQ ID NO:7, meaning that portions of the first sequence which correspond to the non-underlined portions of SEQ ID NO:7 can have less than 90% identity thereto. In some embodiments, the first sequence, the second sequence, or both, may have 98-100%, preferably 99-100%, identity to the variable regions provided above.
In some embodiments, the present invention provides a nucleic acid molecule (or its complement) which encodes a polypeptide comprising, consisting essentially of, or consisting of a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:7 and a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:8;
a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:9 and a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:10;
a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:11 or SEQ ID NO:12 and a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:13;
a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:14 and a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:15 or SEQ ID NO:16;
a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:17 and a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:18;
a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:19 and a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:20;
a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:21 and a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:22;
a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:23 and a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:24;
a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:25 or SEQ ID NO:26 and a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:27; or
a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:28; SEQ ID NO:29; or SEQ ID NO:30 and a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:31; SEQ ID NO:32; SEQ ID NO:33; or SEQ ID NO:34.
In some embodiments, the first sequence (amino acid sequence or nucleotide sequence) and the second sequence (amino acid sequence or nucleotide sequence) need not be directly linked to each other and/or in any particular order. For example, (1) one or more intervening molecules (e.g. amino acid residues or nucleotides) may be located between the first sequence (amino acid sequence or nucleotide sequence) and the second sequence (amino acid sequence or nucleotide sequence), and/or (2) first sequence (amino acid sequence or nucleotide sequence) may be located before or the second sequence (amino acid sequence or nucleotide sequence).
A first sequence having a given percent (%) sequence identity with respect to a second sequence is defined as the percentage of amino acid residues (or nucleotide bases) in the first sequence that are identical with the amino acid residues (or nucleotide bases) in the second sequence, after aligning the first and second sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as ALIGN, ALIGN-2, Megalign (DNASTAR) or BLAST (e.g., Blast, Blast-2, WU-Blast-2) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, the % identity values used herein are generated using WU-BLAST-2 (Altschul et al., Methods in Enzymology 266: 460-480 (1996). Most of the WU-BLAST-2 search parameters are set to the default values. For purposes herein, the default parameters of the BLAST alignment tools available online at blast.ncbi.nlm.nih.gov/Blast.cgi were used.
In some embodiments, the polypeptides and/or the nucleic acid molecules according to the present invention are isolated and/or purified. An “isolated” nucleic acid molecule or polypeptide refers to a nucleic acid molecule or polypeptide that is in an environment that is different from its native environment in which the nucleic acid molecule or polypeptide naturally occurs. Isolated nucleic acid molecules or polypeptides includes those having nucleotides or amino acids flanking at least one end that is not native to the given nucleic acid molecule or polypeptide. For example, a promoter P for a protein X is inserted at the 5′ end of a protein Y which does not natively have P at its 5′ end. Protein Y is thus considered to be “isolated”. As used herein, a “purified” polypeptide or nucleic acid molecule means that some or all of the components in the composition from which the polypeptide or the nucleic acid molecule was obtained have been removed.
In some embodiments, the present invention provides recombinant human progenitor cells, engineered human thymocytes, and engineered human T cells which express one or more TCRs clones as disclosed herein.
To the extent necessary to understand or complete the disclosure of the present invention, all publications, patents, and patent applications mentioned herein are expressly incorporated by reference therein to the same extent as though each were individually so incorporated.
Having thus described exemplary embodiments of the present invention, it should be noted by those skilled in the art that the within disclosures are exemplary only and that various other alternatives, adaptations, and modifications may be made within the scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments as illustrated herein, but is only limited by the following claims.

## Claims

1. A method of producing an engineered thymocyte or an engineered T cell which comprises
spectratyping-based cloning to obtain a nucleic acid molecule which encodes a human T cell receptor specific for a virus or an epitope thereof;
transducing a human hematopoietic stem cell with a vector containing the nucleic acid molecule to give a recombinant progenitor cell; and
differentiating or developing the recombinant progenitor cell into the engineered thymocyte by subjecting the recombinant progenitor cell to a thymus tissue, and then optionally maturing the engineered thymocyte into the engineered T cell, and
wherein the spectratyping-based cloning comprises
obtaining peripheral blood mononuclear cells from a subject infected with the virus and dividing the peripheral blood mononuclear cells into a first portion and a second portion;
culturing the first portion with the virus or the epitope thereof;
spectratyping the TCR α-genes and TCR β-genes in the first portion to obtain a first fingerprint;
spectratyping the TCR α-genes and TCR β-genes in the second portion to obtain a second fingerprint;
selecting a TCR α-gene and a TCR β-gene in the first portion which are not present in the second portion; and
recombinantly joining the selected TCR α-gene and TCR β-gene to give the nucleic acid molecule.

2. The method of claim 1, wherein the thymus tissue is human thymus tissue.

3. The method of claim 2, wherein the recombinant progenitor cell is implanted in the human thymus tissue of a subject or intravenously administered to the subject having the human thymus tissue.

4. The method of claim 1, and further comprising activating the engineered T cell by subjecting the engineered T cell to an HLA molecule specific for the human T cell receptor.

5. The method of claim 4, wherein the HLA molecule is HLA-A*0201, HLA-B*39, HLA-A*02, or HLA-B*40.

6. An engineered thymocyte or the engineered T cell made by the method of claim 1.

7. The engineered thymocyte or the engineered T cell of claim 6, which expresses a functional human T cell receptor.

8. The engineered T cell made by the method of claim 1, wherein the engineered T cell is a cytotoxic T cell.

9. The method of claim 1, wherein the nucleic acid molecule encodes a polypeptide comprising
a) a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the variable region of SEQ ID NO:7 and a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the variable region of SEQ ID NO:8;
b) a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the variable region of SEQ ID NO:9 and a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the variable region of SEQ ID NO:10;
c) a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the variable region of SEQ ID NO:11 or SEQ ID NO:12 and a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the variable region of SEQ ID NO:13;
d) a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the variable region of SEQ ID NO:14 and a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the variable region of SEQ ID NO:15 or SEQ ID NO:16;
e) a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the variable region of SEQ ID NO:17 and a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the variable region of SEQ ID NO:18;
f) a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the variable region of SEQ ID NO:19 and a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the variable region of SEQ ID NO:20;
g) a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the variable region of SEQ ID NO:21 and a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the variable region of SEQ ID NO:22;
h) a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the variable region of SEQ ID NO:23 and a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the variable region of SEQ ID NO:24;
i) a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the variable region of SEQ ID NO:25 or SEQ ID NO:26 and a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the variable region of SEQ ID NO:27; or
j) a first sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the variable region of SEQ ID NO:28; SEQ ID NO:29; or SEQ ID NO:30 and a second sequence having a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the variable region of SEQ ID NO:31; SEQ ID NO:32; SEQ ID NO:33; or SEQ ID NO:34.

10. The method of claim 1, wherein the virus is human immunodeficiency virus or influenza virus.

11. The method of claim 1, wherein the epitope comprises SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; or SEQ ID NO:6.

12. The method of claim 1, wherein the step of subjecting the recombinant progenitor cell to the thymus tissue is by administering the recombinant progenitor cell to a subject having the thymus tissue.