# Patent

## Title
HIV vpr-specific T-cell receptors

## Abstract

The instant invention provides TCRs having one or more amino acid substitutions that bind to the AL9 epitope of the HIV protein vpr (AIIRILQQQL).

## Background
RELATED APPLICATIONS
This application is a divisional application of U.S. Ser. No. 12/731,485, filed Mar. 25, 2010, which claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 61/163,421, filed Mar. 25, 2009, the entire contents of each of which are incorporated herein by reference.


SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 28, 2010, is named 83717.txt and is 35,656 bytes in size.
BACKGROUND OF THE INVENTION
Current approaches for treating HIV infection are primarily aimed at limiting viral replication or infectivity but do not target latent virus or virally infected cells. As a result of HIV's high mutation rate, drug resistant variants emerge from the latent viral reservoir leading to disease progression in treated patients and infection of other individuals. Several studies have shown that viral escape from CTL responses commonly occurs during the course of the infection. Escape variants may arise through the selection of sequence variations in positions that are important for binding to MHC or in positions that are responsible for interaction between the TCR and the peptide-MHC complex. Circumventing these viral CTL escape mutations is important for eliciting an effective CTL response and for the development of vaccines against HIV-1.
Accordingly, the need exists for therapeutic agents that can effectively target not only the wide-type HIV but also variants that develop during the course of infection.
SUMMARY OF THE INVENTION
The instant inventors have discovered TCR molecules that bind to peptides derived from the vpr protein of HIV when presented in their cognate MHC complex. Specifically, the instant inventors have made mutations in these TCRs that allow the resulting TCR molecules to bind to variants of the vpr peptide epitope, while still binding to the consensus vpr peptide epitope. In a specific embodiment, the inventors have made mutant TCRs that bind to the AL9 consensus epitope of vpr (AIIRILQQQL (SEQ ID NO: 1), amino acids 59-67) with improved binding affinity, while also binding common variants of this epitope.
Accordingly, in one aspect, the invention provides an isolated T cell receptor (TCR) that specifically binds an HIV epitope from HIV-1 vpr, comprising at least one mutation in the wild type TCR Vα or Vβ sequence of set forth in FIG. 1. In one embodiment, the mutation increases the productivity, stability, specific binding activity, or functional activity of the TCR. In another embodiment, the mutation reduces the non-specific binding of the TCR mutant compared to the wild type TCR. In specific embodiments, the TCR is a single-chain TCR or a heterodimeric TCR. In one embodiment, the TCR comprises more than one TCR binding domain.
In one embodiment, the TCRs of the invention recognize the AL9 peptide (AIIRILQQL (SEQ ID NO: 1)) of vpr. In another embodiment, the AL9 peptide is presented in the context of HLA-A2.
In one embodiment, the mutation is at amino acid residue 39 in the TCR Vα chain of the TCR. In another embodiment, the mutation at amino acid residue 93 in the TCR Vα chain. In a specific embodiment, the mutation at amino acid residue 39 is Ser to Pro. In another specific embodiment, the mutation at amino acid residue 93 is Tyr to His, Leu, Lys, Gln, or Ala.
In another embodiment, the TCRs of the invention specifically bind to one or more HIV-1 vpr AL9 peptide variants. In one embodiment, the TCRs of the invention bind the AL9 peptide variants in the context of HLA-A2. In a related embodiment, the AL9 peptide variants comprise one or more amino acid changes within the AL9 peptide sequence.
In another embodiment, the TCRs of the invention have greater stability at elevated temperatures than the wild type TCR. In preferred embodiments of the invention, the TCRs are soluble.
In a specific embodiment, the invention provides an isolated single chain T cell receptor (TCR) that specifically binds to the AL9 peptide from HIV-1 vpr. In one embodiment the scTCR recognizes the AL9 epitope. In one embodiment, the scTCR has a one or more mutations in the Vα chain as compared to the wild-type sequence set forth in FIG. 1.
In another embodiment, the TCR further comprises a functional polypeptide domain, diagnostic/imaging agent, drug-containing nanoparticle, therapeutic agent, cytotoxic agent or an anti-viral agent. In exemplary embodiments, the functional polypeptide domain comprises a cytokine, immunoglobulin domain, receptor domain or polypeptide tag sequence. In another exemplary embodiment, the cytokine comprises IL-2, IL-15, GM-CSF, interferon, the immunoglobulin domain comprises IgG1 constant regions or Ig Fc domains, or a fragment thereof. In another exemplary embodiment, the polypeptide tag comprises the birA sequence.
In another embodiment, the TCR further comprises a transmembrane domain allowing cell surface expression of the TCR. In one embodiment, the TCR further comprises a cytoplasmic domain, wherein the cytoplasmic domain allows intracellular signaling in response to interactions between the TCR mutant and the HIV epitope. In one embodiment, the TCR comprises transmembrane and/or cytoplasmic signaling domains from CD3, CD28, CD8, 4-1BB, Ox-40, ICOS and/or Lck proteins. Alternatively, the TCR can comprise TCR transmembrane and/or TCR cytoplasmic signaling domains. Various soluble and membrane bound fusion proteins have been disclosed previously (Card et al. 2004 Cancer Immunol Immunother. 53:345; Mosquera et al. 2005 J. Immunol. 174:4781; Zhu X et al. 2006 J. Immunol. 176:3223; Belmont et al. 2006 Clin. Immunol. 121:29; Finney et al. 2004. J. Immunol. 172: 104; Brentjens et al. 2007 Clin Cancer Res. 13, 5426; Zhang et al. 2004. Cancer Gene Therapy 11, 487).
In another embodiment, the mutation further increases the activity of the TCR to detect cells presenting an HIV epitope compared to the wild type TCR. In another embodiment, the mutation increases the cytotoxic functional activity of the TCR against cells presenting an HIV epitope as compared to the wild type TCR.
In another embodiment, the invention provides nucleic acid molecules encoding the TCRs of the invention, e.g., such as those presented in FIG. 2. In one embodiment, the invention provides expression vectors comprising the nucleic acid molecules of the invention. In yet another related embodiment, the invention provides host cells comprising the expression vector of the invention. In one embodiment, the host cell is mammalian host cell, e.g., a human cell.
In one aspect, the invention provides methods for detecting cells infected with HIV by contacting a cell with the TCR of the invention and determining if the TCR binds to the cell, wherein TCR binding to the cell is indicative of HIV infection.
In another aspect, the invention provides methods of killing a cell infected with HIV by contacting the cell with a TCR of the invention, thereby killing the cell infected with HIV.
In another aspect, the invention provides methods for determining if a subject is infected with HIV by obtaining a biological sample from an individual and contacting the biological sample with a TCR of the invention, wherein binding of the TCR to the biological sample is indicative of the subject being infected with HIV.
In another aspect, the invention provides methods for treating a subject having HIV by administering to the individual a TCR of the invention or a nucleic acid, vector of the invention or host cell of the invention, thereby treating the subject.
In another aspect, the invention provides methods for inhibiting HIV infection in a subject by administering to the individual a TCR of the invention, the nucleic acid or vector of the invention or host cell of the invention—thereby inhibiting HIV infection in a subject.
In related embodiment, the methods further comprise identifying a subject with an increased risk of HIV infection.
In another aspect, the invention provides pharmaceutical compositions comprising a TCR of the invention and a pharmaceutically acceptable carrier.
In another aspect, the invention provides kits comprising a TCR of the invention and instructions for use.



DESCRIPTION OF THE DRAWINGS
FIG. 1A sets forth the AL9scTCR wild type protein sequence (CDRs italicized).
FIG. 1B sets forth the AL9scTCR single mutant (Y93H) protein sequence.
FIG. 1C sets forth the AL9scTCR double mutant (S39P, Y93H) protein sequence.
FIG. 2A sets forth the AL9scTCR wild type nucleic acid sequence (CDRs italicized).
FIG. 2B sets forth the AL9scTCR single mutant nucleic acid sequence.
FIG. 2C sets forth the AL9scTCR double mutant nucleic acid sequence.
FIG. 3A sets forth exemplary leader sequences.
FIG. 3B sets forth various protein domains.
FIG. 3C sets forth various linker sequences.
FIG. 4A depicts leader nucleic acid sequence.
FIG. 4B depicts fusion domain nucleic acid sequences.
FIG. 4C shows a variety of linker nucleic acid sequences.
FIG. 5 is a characterization of Y93 and A97 CDR3 Vα mutations in AL9scTCR produced in HEK-293 cells.
FIG. 6 is a characterization of Y93H AL9scTCR sm produced in CHO cells.
FIG. 7 is a characterization of S39P AL9scTCR produced in CHO cells.
FIG. 8 is a characterization of AL9scTCR sm and dm produced in CHO cells.
FIG. 9 is a characterization of purified AL9scTCR sm and dm proteins.
FIG. 10A depicts a titration of AL9scTCR wt and sm proteins to AL9-loaded T2 cells by flow cytometry.
FIG. 10B depicts the binding of wt, sm and dm AL9scTCR multimers to T2 cells loaded with AL9 or p53 (control) peptide.
FIG. 10C depicts the binding of wt, sm and dm AL9scTCR multimers to T2 or B cells loaded with AL9 or p53 (control) peptide.
FIG. 10D depicts the lack of binding of wt and sm AL9scTCR multimers to non-specific peptides loaded on T2 cells.
FIG. 11 depicts the competitive binding analysis by flow cytometry.
FIG. 12 depicts the binding of wt and sm AL9scTCR monomers to T2 cells loaded with different amounts of AL9 peptide.
FIG. 13 depicts the binding of wt and sm AL9scTCR multimers to T2 cells loaded with AL9 peptide mutants.
FIG. 14A depicts published analysis of the most frequently occurring vpr AL9 peptide variants (Altfeld et al. 2005. J. Virol. 79: 5000).
FIG. 14B depicts the flow cytometry analysis of the binding of wt, sm and dm AL9scTCR fusions to AL9 peptide variants compared to the AL9 consensus.
FIG. 14C depicts the AL9 I63M peptide titration.
FIG. 14D depicts a summary of the relative binding of wt and sm AL9scTCR fusions to AL9 peptide variants compared to the AL9 consensus.
FIG. 15 depicts the binding of wt and sm AL9scTCR multimers to AL9 peptide presented by HIV infected human CD4 T cells.
FIG. 16A depicts DM AL9scTCR-Ig fusion protein binds AL9 and AL9 variants.
FIG. 16B depicts DM AL9scTCR-Ig fusion protein binding to AL9 I63M variant
FIG. 16C depicts WT, SM and DM AL9scTCR-Ig fusion protein binds AL9 and AL9 variants
FIG. 17A depicts DM AL9scTCR-Ig fusion protein mediates cytolytic activity against AL9-loaded T2 cells
FIG. 17B depicts DM and SM AL9scTCR-Ig fusion proteins mediate cytolytic activity against AL9-loaded T2 cells.



DETAILED DESCRIPTION OF THE INVENTION
The instant inventors have discovered TCR molecules that bind to peptides derived from the vpr protein of HIV when presented in their cognate MHC complex. Specifically, the instant inventors have identified TCRs that bind to the AL9 epitope of vpr (AIIRILQQQL (SEQ ID NO: 1), amino acids 59-67). Moreover, the inventors have identified variants of these TCRs that recognize a number of common variants that arise in the AL9 epitope during the course of infection by the virus. The data disclosed herein elucidates sequences for HIV-1-specific TCRs for diagnostic and therapeutic use.
Presently, recombinant HIV-1-specific antibodies are available for the direct targeting of HIV-1 infected cells. One drawback of the antibody approach is that only the envelope of the HIV-1 virus is accessible for HIV-1 antibodies, while the functionally most important HIV proteins are hidden inside the infected cell and only accessible to the immune system after intracellular processing and presentation by MHC class I or II molecules. Once presented by MHC molecules, these HIV gene products are recognized by TCRs, but not by antibodies. HIV-1 antibodies therefore only allow for a very limited targeting of HIV-1 infected cells. The compositions described herein provide a solution to this problem.
The soluble TCRs which are specific for HIV 1 have significant advantages over existing approaches.
The TCR sequences are useful for the production of recombinant single chain TCR that are able to specifically recognize HIV-1 infected cells. These recombinant TCR are practically used for (i) the in vivo targeting of HIV-1 infected cells in immunotherapeutic, gene transfer of cell-based therapeutic approaches, (ii) the ex vivo assessment of HIV-1 antigen expression on lymphocytes or professional antigen presenting cells. The quantitative analysis of HIV-1 antigen expression is important in studies on HIV-1 immunopathogenesis and are useful for the ex vivo screening/monitoring for preventative and therapeutic approaches.
Currently, treatment of HIV-1 infected patients is based on the use of antiretroviral drugs. These drugs are very effective, but have cumulative toxicity, are associated with high pill burdens and can lead to viral resistance. Therefore, there is a continuing need for other treatment options for these patients. Immunotherapeutic treatment approaches with soluble TCRs represent an alternative treatment option for the HIV-1 infected patient population. In addition, the TCR are used for the ex vivo assessment of HIV-1 antigen expression.
The TCRs described herein recognize a number of variants of the vpr gene that commonly arise during HIV infection allowing for increased efficacy for the molecules of the invention as compared to TCRs previously identified.
The invention features methods and compositions for diagnosis and treatment of viral infections. The methods are based on the identification of T-cell receptors that are specific for HIV-1. Specifically, the instant invention provides TCR molecules that specifically bind to an epitope of the vpr protein from HIV. The epitope comprises or consists of a short, 8 to 11-mer peptide that is found in the vpr protein. Specifically, the vpr epitope is AIIRILQQL (SEQ ID NO: 1) (amino acids 59-67) of the vpr protein.
The T cell receptors of the invention can be heteromultimeric molecules or can be single chain TCR molecules. The scTCRs comprise a Vα domain linked to Vβ/Cβ domains either directly or via a flexible linker. Specific molecules of the invention further comprise an additional polypeptide. In certain embodiments that contain this additional polypeptide, the Cβ domain is truncated just prior to the final cysteine.
The invention provides TCRs comprising one or more mutation in the variable regions that allows for the recognition of epitopes that contain one or more amino acid substitution. For example, the invention provides TCRs that bind not only to the consensus AL9 epitope of the vpr protein, but also to epitopes that comprise one, two or three amino acid substitutions in the AL9 epitope.
In exemplary embodiments, the TCRs of the invention recognize the AL9 epitope comprising mutations in at the position corresponding to amino acid number 60, 61, 62, or 63 of the vpr protein. In exemplary embodiments, one or more mutations in the Vα chain of the TCR allow for the recognition of variant vpr sequences.
Methods of Diagnosis
Soluble TCRs are used to analyze HLA-mediated presentation of cytotoxic T cell epitope presentation on professional antigen presenting cells or HIV infected cells. For example, a sample of bodily fluid, e.g., blood, or bodily tissue, e.g., lymph node, is obtained from a subject. Leukocytes from the sample are contacted with single chain TCRs described herein. To increase sensitivity, four single chain TCR constructs linked together, e.g., with a central streptavidin to form a tetrameric complex. The construct is linked to a detectable marker, e.g., it is labeled with a fluorescence fluorophore. Detectable markers include fluorochromes such as Phycoerythrin (PE), Fluorescein isothiocyanate (FITC), and Allophycocyanin (APC). Detection is carried out by flow cytometry and/or tissue staining (fluorescence microscopy or immunohistochemistry). In another example, a plurality of TCR constructs are immobilized in a microarray, e.g., a chip or plate, and a patient-derived sample is allowed to contact the array, the array is washed, and bound cells detected. In this manner, the peptide expressed or presented on the antigen presenting cell or HIV infected cell of a patient is determined. Thus, soluble TCRs are also useful as a research tool for the ex vivo assessment and quantification of HIV-1 epitope presentation. They are useful tools for identifying patients who express specific HIV-1 epitopes, e.g., the AL9 epitope of vpr, and are therefore promising candidates for immunotherapeutic interventions described herein.
Methods of Therapy
To treat patients infected with HIV, one or a mixture of soluble single chain TCR constructs are administered. In one embodiment, the TCRs of the invention induce antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC provides an effective immune response to HIV infection. It has been associated with protection from simian immunodeficiency virus disease in macaques delayed progressive HIV infection in humans, protection of intravenous drug users from HIV-1infection, and lower genital HIV viral loads. The ability of, for example, the high affinity AL9 scTCR Ig to mediate ADCC-like activity demonstrates that they could be used in a passive immune therapy approach to stimulate targeted, potent, innate immune responses against HIV-infected cells.
In an alternative embodiment, the TCRs further comprise a second composition such as a functional polypeptide domain, diagnostic/imaging agent, drug-containing nanoparticle, therapeutic agent, cytotoxic agent or anti-viral agent. In exemplary embodiments, functional polypeptide domain comprises a cytokine, immunoglobulin domain, receptor domain or polypeptide tag sequence. Exemplary cytokines are IL-2, IL-15, GM-CSF, or interferon, and exemplary immunoglobulin domain comprises IgG1 constant regions or Ig Fc domains, or a fragment thereof.
One advantage of such a construct is increased half-life and the antigen-specific delivery of these reagents directly to infected cells. This therapeutic strategy reduces the overall drug dose, the dosing frequency, and the treatment-associated side effects.
In an alternative embodiment, nucleic acids encoding the TCRs of the invention or cell expressing the TCRs of the invention can be used in gene transfer or cell-based approaches to kill HIV infected cells or to prevent or treat HIV infection. In one example, genes encoding TCRs of the invention could be transferred into immune cells such that the immune cells now expressed TCRs of the invention on the cell surface. These cells could then be activated to express immune functions when the TCR interacts with HIV vpr peptide displayed in the HLA-A2 complex. These cells could be used in killing HIV-infected cells ex vivo or, following infusion into patients with HIV infection, kill HIV infected cells in the patient. Alternatively, nucleic acids encoding the TCRs of the invention could be directly administered to the patients, thereby allowing gene transfer into the patient's immune cells and expression of the TCRs of the invention on these cells in vivo. Such cells could provide protective or therapeutic activity against HIV infection.
The TCRs of the invention can be delivered by methods known to one of skill in the art. For example, parenteral administration, such as intravenous, subcutaneous, intramuscular, and intraperitoneal delivery routes, may be used to deliver the TCRs. For instance, soluble TCR fusion have been intravenously injected into mice and humans at a dose of 0.015 to 10 mg/kg. Determination of patient doses is carried using methods wells known in the art.
The compositions of the invention can be administered to prevent or treat HIV infection or reduce the number of HIV-infected cell. Determination of the proper dosage and administration regime for a particular situation is within the skill of the art. An effective amount of a therapeutic protein is preferably from about 0.01 mg/kg to about 150 mg/kg. Effective doses vary, as recognized by those skilled in the art, depending on the nature of the therapeutic treatment (i.e. biologic, nucleic acid or cell-based), route of administration, excipient usage, and coadministration with other therapeutic treatments including use of other agents or therapeutic agents. A therapeutic regimen is carried out by identifying a mammal, e.g., a human patient suffering from (or at risk of developing) infection by a viral pathogen, using standard methods. The pharmaceutical compound is administered to such an individual using methods known in the art. Preferably, the compound is administered orally, rectally, nasally, topically or parenterally, e.g., subcutaneously, intraperitoneally, intrathecally, intramuscularly, and intravenously.
In the case of polypeptide sequences which contain mutations or substitutions as compared to the wild-type TCR sequence, the mutated positions are preferably, but not necessarily, conservative substitutions. Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine. In peptides in which amino acids substitutions are made relative to the wild-type TCR sequence, the resulting TCR molecules recognize one or more HIV peptides comprising one or more amino acid mutations. In specific embodiments, the HIV protein is vpr. In peptides in which amino acids substitutions are made relative to the wild-type TCR sequence, the binding affinity of the T-cell receptor to its HLA-restricted epitope is increased. Alternatively, constructs containing substitutions have great stability, e.g., a longer half-life in a physiologically acceptable solution such as culture media or a bodily fluid such as blood, plasma, or serum. For example, the binding affinity and/or stability of such derivative peptides is at least 5%, 10%, 25%, 50%, 75%, 90%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, or more relative to that of the reference peptide sequence.
EXAMPLES
It should be appreciated that the invention should not be construed to be limited to the examples that are now described; rather, the invention should be construed to include any and all applications provided herein and all equivalent variations within the skill of the ordinary artisan.
Example 1
Generation of Single Chain TCR Constructs with Specific Mutations
CTLs from HLA-A2 Vpr, a 14 kD accessory protein of HIV, plays a crucial role in viral replication and suppression of the host immune response. It is one of the most frequently targeted proteins by CTL relative to the length of the protein. CD8+ T cell responses against Vpr were found in 45% of individuals (Altfeld et al. 2001, J. Immunol. 167: 2743). The HLA-A2.1-restricted Vpr epitope AIIRILQQL (SEQ ID NO: 1) (amino acids 59-67) (AL9 epitope) can represent a good early target for CD8+ T cells, in primary infection in a subset of HLA-A2 positive individuals. It is the most common current sequence within this epitope (consensus sequence) and is targeted with similar frequencies in individuals with acute (21%) and chronic (24%) infections (Altfeld et al. 2005. J. Virol. 79: 5000). Therapies targeted against vpr expressing cells could be potentially useful in eliminating HIV infected cells. However Altfeld et al also showed that individuals infected in the acute stage of infection with viruses containing the consensus sequence were unable to mount epitope-specific T cell responses, but subjects infected with the 160L variant all developed these responses. Variants of the AL9 epitope can arise frequently and need to be targeted by therapies designed to eliminate the virus.
Soluble T cell receptors could be used to effectively deliver therapeutic molecules to infected cells. The inherently low affinity (1-100 μM) of TCRs for cognate peptide MHC has been a major limitation for therapeutic and diagnostic applications. Contacts between TCR and peptide-MHC occur through the Vα CDR1, CDR2 and CDR3 and/or Vβ CDR1, CDR2 and CDR3 with the CDR3 domains making the most contacts with the peptide. Several studies have shown that mutation of residues within these regions can improve TCR affinity. High affinity variants of the 2C TCR, a mouse alloreactive TCR, were identified by yeast display following selection from a library of CDR3α mutants. Mutation of the CDR3 regions of TCRs specific for the NY-ESO-1 tumor associated antigen and the human T-cell lymphotropic virus type 1 (HTLV-1) tax peptide and their display on the surface of phage has also been used to improve TCR affinity.
The examples describe the generation and characterization of a single chain TCR that recognizes amino acids 59-67 of the vpr protein (AL9 epitope) in context with HLA-A2. The native TCR binds the AL9 consensus peptide with a KD of 1.8 μM, which is in the high affinity range of most TCRs. However it does not recognize some variants that are known to commonly arise during the course of infection, with good affinity. To overcome this limitation, and further increase its affinity, a mutant version of the protein was created by substitution of tyrosine (Y) to histidine (H) in the CDR3α loop region. The mutant binds with much higher affinity to the consensus AIIRILQQL (SEQ ID NO: 1) sequence, while retaining a high degree of peptide specificity. Importantly, variant epitopes which are known to frequently arise during HIV infection, could be recognized by the newly improved TCR with much higher affinity than the wild type TCR. Additionally, a multimeric form of the high affinity TCR could efficiently bind to CD4+ T cells infected with HIV-1. The AL9scTCR wild type and high affinity mutants were fused to the Ig heavy chain constant domain to deliver effector function. This fusion protein retains peptide specific binding activity, forms dimers and can mediate target cell killing by Fc-dependent cellular cytotoxicity.
CTLs specific to AL9 peptide (amino acids 59-67: AIIRILQQL (SEQ ID NO: 1)/HLA-A2 complexes were isolated from an HIV-1 infected patient, as described previously (Altfeld et al. 2005. J. Virol. 79: 5000) and served as the source for generating the TCR α and β cDNAs. The V regions of most frequently occurring TCR α and β chains obtained from these CTLs were TRAV5/TRAVJ36 and TRBV14/TRBJ2-1, respectively, as described previously (U.S. Ser. No. 11/784,277).
To generate high affinity TCRs, the TCR CDR regions and neighboring amino acid residues can be mutated and the resulting proteins screened. For the HLA-A2/AL9-specific TCR (AL9 TCR), the Vα and Vβ CDR sequences are shown in FIG. 1A with the corresponding nucleic acid coding regions shown in FIG. 2A. For this example, Vα CDR3 regions were mutated using a mutagenesis strategy where each of the amino acids in the CDR3 Vα region was systematically replaced by one of nine amino acids, chosen to represent the major side-chain chemistries provided by the 20 natural amino acids. Previous studies have shown that it is possible to generate high affinity TCRs by mutating the amino acids in the CDR3α loop (Holler et al. 2000 PNAS 10: 5387), which lie at the center of the peptide-MHC interface. Hence the gene sequence of AL9 TCR encoding 5 amino acids (Y-Q-T-G-A (SEQ ID NO: 2)) in the CDR3 Vα loop were mutated to the 9 amino acids representative of the major side chain chemistries. These amino acids were located between aa 93-97 of the TCR Vα chain, with changes at position 93 (tyr) showing improved (higher) affinity for HLA-A2/AL9 complex.
In the isolated Vα gene segment, a non-consensus serine codon was found at the position aa 39 in the second framework region. This codon was mutated to a consensus proline codon to assess the effects of this change on TCR productivity, stability and affinity.
To introduce sequences encoding different mutations in the Vα gene, the CDR3α sequence was modified by site-directed mutagenesis to contain a unique restriction enzyme site (AgeI). This change did not result in a change in the encoded amino acids. The mutated gene TCR Vα gene fragments were then generated by standard PCR methods using oligonucleotide primers encoding the desired sequence changes. These gene fragments were then cloned into the Vα gene replacing the wild-type sequence. The TCR α and β chains were cloned in the single-chain (sc) TCR format composed of a Vα domain linked to Vβ/Cβ domains via a flexible linker (Card et al. 2004 Cancer Immunol Immunother. 53:345; Mosquera et al. 2005 J. Immunol. 174:4781; Zhu X et al. 2006 J. Immunol. 176:3223). The Cβ domain was truncated just prior to the final cysteine.
To create the scTCR-birA fusion, the scTCR gene segment was cloned upstream of sequence encoding the birA tag sequence. The birA tag allows site-specific biotinylation of the protein for subsequent multimerization using streptavidin. To generate the scTCR IgG1 fusion, the scTCR gene segment was cloned upstream sequence encoding the human IgG1 constant chain region. Similarly scTCR-IL2, scTCR-INFα, scTCR-GMCSF, scTCR-IL15 and scTCR-IL15R fusions were generated by linking the scTCR gene segments with the appropriate cytokine or cytokine receptor gene sequences. For membrane expression, the scTCR gene was cloned upstream sequence encoding the HLA A2 transmembrane domain or the CD3zeta transmembrane domain. For membrane expression and intracellular signaling, the scTCR gene segments can be linked to sequences encoding CD3zeta, CD28, CD8, 4-1BB, Ox-40, ICOS and/or Lck domains. Alternatively, the TCR can be expressed on the cell surface as a heterodimeric α/β TCR containing TCR transmembrane and cytoplasmic signaling domains. Various soluble and membrane bound fusion proteins have been disclosed previously (Card et al. 2004 Cancer Immunol Immunother. 53:345; Mosquera et al. 2005 J. Immunol. 174:4781; Zhu X et al. 2006 J. Immunol. 176:3223; Belmont et al. 2006 Clin. Immunol. 121:29; Finney et al. 2004. J. Immunol. 172: 104; Brentjens et al. 2007 Clin Cancer Res. 13, 5426; Zhang et al. 2004. Cancer Gene Therapy 11, 487). Additional peptide linkers can be positioned between the scTCR and fusion protein domains and/or between different fusion protein domains to provide of optimal production, solubility, biological activity, protein-protein interactions, multimerization or positioning of the respective domains to avoid steric interference.
The scTCR fusion constructs were cloned into an expression vector downstream of a leader sequence to allow soluble or cell membrane expression. The vector contains promoter/enhancer regulatory sequences and poly A sequences to allow proper gene expression and genes encoding selectable markers to allow isolation of host cells containing the expression vector.
The AL9scTCR wild type (non-mutated) protein sequence, referred to as wild type or “wt”, is shown in FIG. 1A. The AL9scTCR protein sequence containing a Tyr to His mutation at position 93 of the TCR Vα chain, referred to as single mutant or “sm”, is shown in FIG. 1B. The AL9scTCR protein sequence containing a Tyr to His mutation at position 93 and a Ser to Pro mutation at position 39 of the TCR Vα chain, referred to as double mutant or “dm”, is shown in FIG. 1C. Corresponding AL9scTCR wt, sm and dm nucleic acids sequences are shown in FIG. 2A-C. The protein sequences for leader sequence, various soluble fusion protein domains and linker sequences are shown in FIG. 3A-C with corresponding nucleic acid sequences shown in FIG. 4A-C.
Example 2
Protein Production and Purification
For production of the fusion proteins in mammalian cells, HEK-294 or CHO cells were transfected with the expression vectors using lipofectamine 2000. Single cell clones were selected by limiting dilution cloning, in medium containing G418 (2 mg/ml).
The AL9scTCR-birA wild type and mutant fusion proteins were purified from cell culture supernatants by immunoaffinity chromatography, using the anti-human TCR antibody Cβ mAb (BF1) 8A3.31, coupled to a Sepharose 4 Fast Flow column (Amersham Biosciences). The purified fusion proteins were biotinylated with biotin-protein ligase (Avidity) in the presence of excess biotin, according to the manufacturer's instructions to create monomers AL9sm/birA. The biotinylated AL9scTCR birA protein was multimerized with R-PE-conjugated streptavidin (Jackson Immunoresearch) at a TCR/streptavidin molar ratio of 4:1 for at least 60 min at 4° C.
The purified AL9scTCR-birA wild type and mutant fusion proteins were analyzed SDS-PAGE and stained with Coomassie G-250. The SDS gel analysis of the proteins under reducing and non-reducing conditions showed that the proteins were monomeric and had an approximate molecular weight of 52 kD. This is larger than the calculated molecular weight of 44.3 kD, suggesting that these proteins are glycosylated.
The AL9scTCR birA fusion proteins were biotinylated using biotin protein ligase. The efficiency with which the wild type and mutant proteins were biotinylated was compared in an ELISA using the BF1 antibody for capture and SA-HRP for detection. The results showed that both the proteins were biotinylated to a similar extent.
The AL9scTCR-IgG1 fusions are comprised of TCRs directly linked to the constant domain of the human IgG1 chain. The cysteine residues in the IgG hinge region are intact, allowing dimer formation, which is observed when the proteins are analyzed on non-reducing SDS-PAGE.
Example 3
Characterization of AL9scTCR Mutant Fusion Proteins by Concentration and Peptide-MHC-Binding Activity ELISAs
The AL9scTCR wild type and mutant fusions produced in short-term cell cultures were assessed for TCR protein concentration and binding activity to peptide-MHC tetramers by ELISAs. In these assays, 96-well Maxisorb plates (Nunc, Rochester, N.Y.) were coated with the BF1 mAb (1 μg/ml). Cell culture supernatants containing the fusion proteins were added to blocked plates and incubated for 2 h at room temperature. After washing, the proteins were detected using AL9 peptide-HLA-A2 tetramer-HRP (0.5 μg/ml) for binding activity or the biotinylated anti-human TCR Cβ antibody mAb W4F (0.5 μg/ml) followed by streptavidin-HRP (0.5 μg/ml) for TCR protein concentration. The ABTS substrate was then added and absorbance was measured at 405 nm using a 96-well plate reader (Bio-Tek Instruments).
Various mutations at positions 93 and 97 of the TCR Vα chain were characterized in these assays as shown in FIG. 5. A majority of mutations at position 93 resulted in higher levels of protein in the short-term cell cultures, indicating higher expression levels and/or increased protein stability. Additionally several of the mutations also resulted in increased binding to AL9 peptide-HLA-A2 tetramer, indicating enhanced binding activity. In contrast, changes at position 97 (FIG. 5) or other positions in either the CDR3α or CDR3β regions failed to improve binding activity. When binding activity was normalized to protein levels, mutations at Y93 to L, K, Q or H showed significantly higher levels of AL9-HLA-A2 binding activity compared to the AL9scTCR wt protein. The Y93 to H substitution in the CDR3α loop yielded a TCR that had the best affinity compared to the wild type TCR and was further characterized. Previous studies showed that AL9scTCR wt protein was sensitive to temperature such that the protein was not observed at high levels in media following production in 37° C. cell culture conditions. Levels of the sm (Y93H) and wt AL9scTCR proteins produced by transfected CHO cells was examined in short-term cultures maintained at room temperature or 37° C. As shown in FIG. 6, the sm AL9scTCR exhibited much higher levels of expressed protein in 37° C. CHO cultures than the wt AL9scTCR protein, indicating improved production and/or protein stability. Again the sm AL9scTCR sm showed better binding to AL9-HLA-A2 complex than the AL9scTCR wt protein and when normalized to protein levels, indicating the Y93H AL9scTCR sm protein produced at either room temperature or 37° C. showed higher binding activity than the wt protein.
Similar methods were used to characterize the S39P mutation in the TCR Vα chain. As shown in FIG. 7, AL9scTCR S39P expressed by transfected CHO cells in short term cultures exhibited increased binding activity to AL9-HLA-A2 complex than the AL9scTCR wt protein. When the S39P and Y93H mutations were combined into a single AL9scTCR, the resulting double mutant exhibited better productivity, stability at 37° C. and binding activity to AL9-HLA-A2 complex than the single mutant or wt AL9scTCR proteins (FIG. 8). Similar results were observed with proteins purified from CHO supernatants by BF1 mAb affinity chromatography. Both the AL9scTCR sm and dm constructs showed better stability at 37° C. and increased binding activity to AL9-HLA-A2 complex than the AL9scTCR wt protein (FIG. 9).
Example 4
Characterization of AL9scTCR Mutant Fusion Proteins by Flow Cytometry
Functional binding of AL9scTCR wt and mutant fusion proteins to AL9 peptide loaded HLA-A2-positive antigen presenting cells (T2 cells) was measured by flow cytometry. T2 cells were loaded with 50 μM of the consensus AL9 peptide for 3 hrs, at 37° C. The biotinylated AL9scTCR-birA proteins that had been multimerized by the addition of SA-PE were added at different concentrations from 1 to 0.001 μg for 30 min at 4° C. Following a wash step, the samples were analyzed on a FACS scan flow cytometer using CellQuest software (BD Biosciences). As shown in FIG. 10A, sm AL9scTCR-birA multimers showed 2-3 fold higher binding activity to the AL9 peptide loaded cells than the wt AL9scTCR-birA multimers when the TCR concentration was <0.5 μg/test.
The monomeric forms of the wt and sm AL9scTCR-birA fusions were also used to detect peptide-MHC on the surface of target cells, in the same experiment. T2 cells were loaded with 50 μM AL9 peptide as described above. Biotinylated forms of AL9 wt and AL9sm TCRs were added at equivalent molar amounts as with the multimeric forms ranging from 1 to 0.001 μg. Following a wash step the bound monomers were detected with SA-PE. There was a 2-3 fold higher binding of the AL9scTCR sm monomer compared to the wild type monomer at 0.5-1 μg. This difference was increased to 4-8 fold at concentrations from 0.25 to 0.06 μg and 20-30 fold at concentrations from 0.03 to 0.001 μg. Thus the difference between the wild type and sm AL9scTCR was particularly evident when the monomeric form of the scTCR was used (FIG. 10A). There was little or no binding of either the wild type or mutant TCR monomers or tetramers to unpulsed cells even at the highest concentration. Similar studies conducted with the dm AL9scTCR-birA fusion showed that this protein also exhibited increased binding activity to AL9 peptide loaded T2 cells compared to the wt AL9scTCR (FIG. 10B). Additionally both the sm and dm AL9scTCR-birA fusions were able to bind AL9 peptide presented by HLA-A2+ B cells (FIG. 10C). In these studies the 264scTCR and p53 aa264-272 peptide were used as negative control reagents to assess TCR and peptide specificity and expected specificities were observed. Together these data confirm that the AL9scTCRs are biologically functional in the three-domain single chain format and that the AL9scTCR sm and dm fusion specifically binds to AL9 peptide loaded cells with higher affinity than the wild type AL9scTCR.
Specific interaction between the wild type or sm AL9scTCR birA proteins and cognate peptide-MHC was confirmed by analyzing their binding to a panel of irrelevant control peptides. T2 cells were loaded with the consensus AL9 peptide or peptides from HIV-1 gag (77-85), pol (464-472), CMV, HCV core, HBV env, p53 (264-272), MART-1 or were left unpulsed. Biotinylated wild type AL9scTCR, sm AL9scTCR or control MART1scTCR were then added, followed by incubation with streptavidin-PE. No binding of either the wild type or sm AL9scTCRs to any irrelevant peptides was observed (FIG. 10D). Thus the higher affinity of the sm AL9scTCR does not compromise its peptide binding specificity.
The ability of the wild type or sm AL9scTCR-birA fusion proteins to compete with each other for binding to AL9 peptide loaded T2 cells was analyzed. The binding of the biotinylated wild type or sm AL9scTCR was determined in the presence or absence of 2-100 fold excess of non-biotinylated competitor. The high affinity single mutant could compete with the wild type AL9scTCR very efficiently even at 2 fold excess, as shown in FIG. 11. Higher concentrations of 5-10 fold excess were only slightly better and there was almost complete abrogation of binding of the wt AL9scTCR with 25 fold excess of the mutant. In contrast, the wild type AL9scTCR could not compete efficiently with the sm AL9scTCR. With a 10 fold excess or less of the wild type there was little or no inhibition. At a 25-100 fold excess, some inhibition is observed, but it was not very significant. Thus the wild type AL9scTCR cannot efficiently block binding of the sm AL9scTCR even at high concentrations, but the mutant is a very good competitor of the wt AL9scTCR even at a 2 fold excess. These data support the conclusion that the sm AL9scTCR has higher binding activity to AL9-HLA-A2 complex and can inhibit binding of the wt AL9 TCR.
The sensitivity of the wt and sm AL9scTCR-birA fusions was evaluated by comparing their ability to detect varying amounts of peptide on the cell surface. T2 cells were loaded with AL9 peptide at concentrations ranging from 12 nM to 100 μM. 0.5 μg of monomeric wt or sm AL9scTCR-birA were then added and binding was evaluated by flow cytometry. To estimate the number of PE molecules bound per cell, the mean fluorescent intensity obtained at various concentrations of peptide was compared to a standard curve of PE-coupled calibration beads, which have known amounts of PE per bead. As shown in FIG. 12, there is a significant increase in the ability of the sm AL9scTCR compared to wt AL9scTCR to detect AL9 peptide loaded T2 cells, at all the peptide concentrations tested. The sm AL9scTCR was able to stain T2 loaded cells with as little as 12 nM AL9 peptide whereas the wild type TCR could only detect 195 nM peptide on the surface of T2 cells. Similar results were observed with the dm AL9scTCR fusion.
Example 5
Characterization of AL9scTCR Mutant Fusion Proteins Binding to AL9 Peptide Variants
Crystal structures of TCR-peptide/HLA-A2 complexes have suggested that amino acids in the middle of a 9-mer peptide make contacts with the CDR3 regions of the TCR. In order to determine which peptide amino acids are important in binding the wild type and mutant TCRs, alanine substitutions were made at positions 60, 62, 64, 65, 66 and 67 of the AL9 peptide (aa59-67: AIIRILQQL (SEQ ID NO: 1)). The relative binding (IC50) of these variants to HLA-A2 complex was assessed by competition analysis as described previously (Altfeld et al. 2005. J. Virol. 79: 5000) and is indicated for each peptide in FIG. 13. These peptides were loaded on T2 cells and the cells were stained with the wt and sm AL9scTCR-birA multimers as described above. Flow cytometric analysis was performed to evaluate the relative binding affinity of wt and sm AL9scTCR multimers for the consensus peptide and variant peptide loaded cells. The 264scTCR multimer served as a control reagent.
The results showed that replacement of amino acids at positions 60, 65, 66 and 67 did not result in any significant differences in the binding between the wt and sm AL9scTCRs (FIG. 13). However, substitution of the Leu at position 64 with Ala significantly diminished the binding of the wild type, but not the sm AL9scTCR. This suggests that position 64 may be a crucial contact for the native TCR, but not for the high affinity mutant.
Replacement of the Arg at position 62 to Ala resulted in an insoluble peptide whereas a Glu substitution at this position was soluble. This substitution completely abolished binding of both the wt and sm AL9scTCR multimers both in the monomer and tetramer formats, suggesting that this is a critical contact for both the wild type and mutant TCR-peptide interaction.
Different naturally occurring variants of the AL9 epitope in HIV have been reported in the Los Alamos database (http://www.hiv.lan1.gov). Analysis of the most frequently occurring variants, shows isoleucine to leucine variants at position 60 and threonine and methionine substitutions at positions 61 and 63 (FIG. 14A, Altfeld et al. 2005. J. Virol. 79: 5000). Peptides corresponding to these and other variants were synthesized and the binding of the wild type, sm or dm AL9scTCRs to variant peptide loaded cells was compared by flow cytometry in a series of studies shown in FIG. 14B. As above, T2 cells were loaded with AL9 consensus or variant peptides (50 μM each) for 3 hrs, at 37° C., and stained with the wt, sm or dm AL9scTCR multimers labeled with PE.
The wt AL9scTCR multimer showed reduced binding to the AL9 variants with Thr and Met substitutions at position 63. In contrast the sm and dm AL9scTCR multimers exhibited good binding to these variants. Substitution of positions 60 and 61 in the AL9 peptide did not affect binding of either the wt or sm AL9scTCR. Titration of the I63M peptide at concentrations from 50 μM-0 nM confirmed that the sm AL9scTCR, but not the wild type TCR tetramer, could recognize this variant as shown in FIG. 14C.
Since substitution at position 62 to Glu had been shown to abrogate binding of both the wild type and mutant AL9scTCR, it was of interest to determine what effect replacement of Arg with Lys would have on the binding of these TCRs. This substitution has been reported as a naturally occurring variant of HIV in the database. A Lys substitution at position 62 significantly decreased binding of the wild type but not the high affinity sm AL9scTCR multimer (FIG. 14B).
Thus the sm and dm AL9scTCRs not only binds the AL9 peptide consensus with higher affinity, these proteins can also recognize variants at position 62, 63 and 64 much better than the wild type AL9 TCR (FIG. 14B). FIG. 14D shows a summary of the relative binding activity of the wt and sm AL9scTCR multimers to common AL9 epitope variants relative to the AL9 consensus peptide. The sm AL9scTCR retains binding activity to these variants at >70% compared with that observed for the consensus peptide, indicating that this mutant TCR fusion protein should be able to recognize HIV-infected cells displaying these variant epitopes.
Example 6
Detection of AL9 Antigen Presentation on HIV-Infected Human T-Cells by AL9scTCR Fusion Proteins
The results shown above demonstrate that the wt and mutant AL9scTCR proteins were capable of binding AL9 peptide-HLA-A2 complex and endogenously added AL9 peptide presented on HLA-A2+ cells. However, in order to target HIV-infected cells, the TCR fusion proteins must be sensitive and specific enough to detect AL9 presented on the cell surface that is generated via the antigen presenting pathway for internally expressed HIV vpr protein. To examine this, CD4+ T cells from HLA-A*0201 donors were isolated by magnetic beads (Miltenyi Biotech) and were stimulated with media containing ant-CD3 (OKT3; Coulter), anti-CD28 (PharMingen), and IL-2 as described previously (Migueles et al. 2002 Nat Immunol 3: 1061). On day 3, 5×106 cells were infected with 5,000 TCID50 of HIVSF162 in 200 μl of media for 1 h at 37 C and then were maintained in culture at 106 cells/ml in 24-well plates for 6 days further. On day 6, CD8+ cells were depleted by using magnetic beads. The percent of infected cells was documented by intracellular staining for p24 with Kc57-FITC or Kc57-RD1 (Coulter). The infected cells then were stained with wt and sm AL9scTCR multimers and anti-CD4 mAb-APC. HIV infections results in down regulation of cell surface expression of CD4. Thus AL9 peptide presentation can be assessed in infected cells with low levels of CD4 staining. As shown in FIG. 15, wt and sm AL9scTCR-birA multimers were capable of detecting AL9 peptide presented by HIV infected T cells. There was a 15-fold increase in the percentage of wt AL9scTCR-positively staining cells and a 27-fold increase in percentage of sm AL9scTCR-positively staining cells when comparing infected cells to uninfected cells. Additionally increased AL9scTCR staining was observed in cells that have down-regulated CD4. In this study the sm AL9scTCR-birA multimer detected about 2-fold more HIV-infected cells than the wt AL9scTCR-birA multimer, consistent with the increased binding activity of the TCR mutant for the AL9 peptide.
Example 7
Characterization of AL9scTCR-Ig Fusion Proteins Binding to AL9 Peptide and Peptide Variants
As indicated above, AL9scTCR-Ig fusion proteins were produced and purified. The binding activity of the dm AL9scTCR-Ig fusion was compared to wt and sm AL9scTCR-birA fusions using AL9-loaded T2 cells. In this assay TCR binding was detected using a biotinylated BF1 mAb followed by SA-PE. As shown in FIG. 16A, the dm AL9scTCR-Ig fusion protein exhibited good binding to T2 cells loaded with either the AL9 consensus peptide or the AL9 I63M variant. In fact the dimeric dm AL9scTCR-Ig shows better binding to the AL9 I63M variant than the monomeric sm AL9scTCR-birA fusion. The binding activity of the dm and sm AL9scTCR-Ig fusions was compared to the wt AL9scTCR-birA multimers and wt AL9scTCR-Ig fusions using T2 cells loaded with the AL9 consensus and AL9 I63M variant (FIG. 16B-C). These studies verified that the dm and sm AL9scTCR-Ig fusions exhibited better binding to the AL9 and AL9 variants than the wt AL9scTCR protein. Interestingly the dm AL9scTCR-Ig fusion protein shows less background binding to unpulsed T2 cells than either the wt or sm AL9scTCR-Ig fusions (FIG. 16C). The higher background staining of the wt or sm AL9scTCR-Ig fusion is likely due to binding of the Ig Fc domain to receptors on T2 cells. The difference in background binding between the dm and sm AL9scTCR proteins was not observed with the birA fusion multimer format, suggesting that the TCRα FR2 mutation in the double mutant in the Ig format results in less non-specific binding activity. This effect is advantageous for targeting specific biological activity.
Example 8
AL9scTCR-Ig Fusion Proteins Mediate AL9 Peptide Specific Cytotoxic Activity
The ability of the wt, dm and sm AL9scTCR-Ig fusions to direct Fc-dependent cellular cytotoxicity against AL9 loaded targeted cells was examined. In these assays the AL9scTCR-Ig fusions act to conjugate the Fc receptor-bearing immune effector cells and the peptide-MHC presenting target cells and the effector cells mediate target cell lysis through an ADCC-like mechanism. Human PBMCs, which serve as immune effector cells in this example, were isolated from human blood buffy coats by gradient centrifugation through HistoPague (Sigma-Aldrich). T2 cells pulsed with peptide at 50 μg/ml at 37° C. for 2 hours were used as target cells. The target cells are labeled with Calcein-AM at 50 μg/ml at 37° C. for 1 hours and washed two times and added into 96-U-plate at 20000/well in RPMI-10 media. The dm, sm and wt AL9scTCR-Ig was added to the wells at different concentration. The effector cells were then added (100:1 E:T ratio) and the culture incubate at 37° C. for 2 hours. The fluorescent intensity (FI) of the Calcein released into the supernatant by cell lysis was measured with a fluorescent plate reader (excitation wavelength=485 nm; emission wavelength=538 nm; cutoff=530 nm). The specific cytotoxicity was calculated using the following formula: percentage of cytotoxicity=(FI of test sample−FI of target cells with medium)÷(FI of target cells treated with 0.04% Triton X-100−FI of target cells with medium)×100.
As shown in FIG. 17A, the dm AL9scTCR-Ig fusion protein effectively directs human PBMC cytolytic activity against T2 cells pulsed with AL9 peptide but not T2 cells pulsed with p53 264-272 peptide. A control 264scTCR-Ig fusion protein did not show the same activity against the AL9-loaded T2 cells demonstrating the specificity of this assay. Similar results were seen with different preparations of PBMCs. FIG. 17B shows additional cytotoxicity assays comparing the activity of the dm, sm and wt AL9scTCR-Ig fusion proteins against AL9 loaded T2 cells. The results indicated that dm and sm AL9scTCR-Ig fusion were effective at mediating ADCC-like activity against AL9-loaded cells whereas the wt AL9scTCR-Ig fusion was not. These results indicate that AL9scTCR mutants confer increased cytolytic bioactivity against cells displaying the HIV AL9 epitope that was not observed with the wt AL9 TCR.
Similarly, the ability of the mutant AL9scTCR-Ig fusion proteins to direct immune effector cell cytolytic activity against HIV infected cells will be assess. HIV infected cells will be generated as described in Example 6 and will be used as target cells in the Calcein release assays as described above. A specific increase in the lysis of HIV-infected cells due to the presence of the mutant AL9scTCR-Ig fusion in the culture media would demonstrate that the mutant AL9scTCR-Ig fusion can act as a bridging molecule directing immune cytolytic activity against HIV infected cells.
Example 9
Mutant AL9scTCR Fusion Proteins Exhibit Increased Affinity to AL9/HLA-A2 Complexes
The binding affinity parameters of AL9scTCR fusion protein:AL9/HLA-A2 complexes interactions were determined by surface plasmon resonance methods. Biotinylated AL9 or I63M pMHC complexes or irrelevant control pMHC complexes were bound to a streptavidin sensor chip surface. Soluble TCR-birA proteins were allowed to flow over the relevant cells and responses measuring protein-protein interactions were recorded in real time using BIAcore technology. Binding parameters were estimated using BIAevaluation software. The results of these analysis for AL9scTCR-birA fusion proteins are shown in Table 1 and for the AL9scTCR-Ig fusion proteins are shown in Table 2. The results clearly indicate that the sm AL9scTCR protein as a birA or Ig fusion has increase binding affinity to AL9/HLA-A2 and I63M AL9/HLA-A2 complexes. Additionally the dimeric sm AL9scTCR-Ig fusions display better binding to the AL9/HLA-A2 complexes than the monomeric sm AL9scTCR-birA molecules due to their increase avidity.







TABLE 1







Binding affinities for wt and sm AL9scTCR-birA fusion for AL9


peptide and I63M AL9 variants complexed to HLA-A2 molecules











Constants
Wt AL9scTCR birA
sm AL9scTCR-birA











AL9-HLA-A2











1.8 μM
120
nM











Kon (M−1s−1)
9440
30200



Koff (s−1)
1.75 × 10−2
3.6 × 10−3







I63M AL9-HLA-A2












KD
No binding
6
μM

















TABLE 2







Binding affinities for wt and sm AL9scTCR-Ig fusion for AL9


peptide and I63M AL9 variants complexed to HLA-A2 molecules











Constants
Wt AL9scTCR Ig
sm AL9scTCR Ig











AL9-HLA-A2












KD
83.4 nM
58.9
nM











Kon (M−1s−1)
2.62 × 104 
2.01 × 104



Koff (s−1)
2.18 × 10−3
 1.19 × 10−3







I63M AL9-HLA-A2












KD
No binding
224
nM











Kon (M−1s−1)

2.98 × 104



Koff (s−1)

 6.67 × 10−3










Example 10
Gene Transfer and Cell-Based Approaches Using Mutant AL9 TCRs to Kill HIV Infected Cells
The TCRs of the invention could be used in gene therapy or cell-based therapeutic approaches to kill HIV infected cells or prevent or treat HIV infection. For example, TCR genes encoding the mutant AL9 TCR and transmembrane and cytoplasmic signaling domains will be transferred into immune cells such that the immune cells now expressed the high affinity AL9 TCR on the cell surface. These cells will become activated to express immune functions when the AL9 TCR interacts with HIV vpr peptide displayed in the HLA-A2 complex. These cells will be used in killing HIV-infected cells ex vivo or, following infusion into patients with HIV infection, kill HIV infected cells in the patient. Alternatively, the TCR genes in the appropriate vectors will be directly administered to the patients, thereby allowing gene transfer into the patient's immune cells and expression of the high affinity AL9 TCR on the surface of these cells in vivo. Such cells could provide protective or therapeutic activity against HIV infection.
In one particular example, human PBMC will be isolated from an HIV-infected patient. TCR genes encoding the mutant AL9 TCR and transmembrane and cytoplasmic signaling domains will be transferred into these cells ex vivo allowing expression of the TCRs on the cell surface. The immune cells will be transferred back into the patient where they will mediate effector functions against HIV infected cells. The cytotoxic activity of the immune cells against HIV-infected cells will also be tested in vitro using the calcein release assays described above.
INCORPORATION BY REFERENCE
The contents of all references, patents, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference.
EQUIVALENTS
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

## Claims

1. An isolated nucleic acid molecule encoding an isolated T cell receptor (TCR) that specifically binds an HIV Vpr epitope, wherein said HIV Vpr epitope comprises a peptide sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NOs: 31-44, and wherein the isolated TCR comprises a Y93H or S39P mutation in the Vα chain of the polypeptide set forth in SEQ ID NO: 3.

2. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid comprises the nucleic acid sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 8.

3. An expression vector comprising the nucleic acid molecule of claim 1.

4. An isolated host cell comprising the expression vector of claim 3.

5. The host cell of claim 4, wherein the host cell is mammalian host cell.

6. The host cell of claim 5, wherein the mammalian cell is a human cell.

7. The host cell of claim 6, wherein the human cell is a peripheral blood mononuclear cell (PBMC).

8. The host cell of claim 4, wherein the host cell expresses an isolated TCR that specifically binds an HIV Vpr epitope, wherein said HIV Vpr epitope comprises a peptide sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NOs: 31-44, and wherein the isolated T cell receptor comprises a Y93 H or S39P mutation in the TCR Va chain of the polypeptide set forth in SEQ ID NO: 3.

9. The isolated nucleic acid molecule of claim 1, wherein the mutation increases the productivity, stability, specific binding activity, or functional activity of the TCR.

10. The isolated nucleic acid molecule of claim 1, wherein the mutation reduces the non-specific binding of the TCR compared to the wild type TCR.

11. The isolated nucleic acid molecule of claim 1, wherein the TCR is a single-chain TCR.

12. The isolated nucleic acid molecule of claim 1, wherein the TCR is a heterodimeric TCR.

13. The isolated nucleic acid molecule of claim 1, wherein the TCR comprises more than one TCR binding domain.

14. The isolated nucleic acid molecule of claim 1, wherein said TCR comprises Y93H and S39P mutations.

15. The isolated nucleic acid molecule of claim 1, wherein the peptide is presented in the context of HLA-A2.

16. The isolated nucleic acid molecule of claim 1, wherein the HIV epitope is presented in the context of HLA-A2.

17. The isolated nucleic acid molecule of claim 1, wherein the TCR has greater stability at elevated temperatures than the wild type TCR.

18. The isolated nucleic acid molecule of claim 1, wherein the TCR is soluble.

19. The isolated nucleic acid molecule of claim 1, wherein the TCR further comprises a fusion to a functional polypeptide domain, diagnostic/imaging agent, drug-containing nanoparticle, therapeutic agent, cytotoxic agent or anti-viral agent.

20. The isolated nucleic acid molecule of claim 1, wherein the functional polypeptide domain comprises a cytokine, immunoglobulin domain, receptor domain or polypeptide tag sequence.

21. The isolated nucleic acid molecule of claim 20, wherein the cytokine comprises IL-2, IL-15, GM-CSF, or interferon, the immunoglobulin domain comprises IgG1 constant regions or Ig Fc domain, or a fragment thereof.

22. The isolated nucleic acid molecule of claim 20, wherein the polypeptide tag comprises the BirA sequence.

23. The isolated nucleic acid molecule of claim 1, wherein the TCR further comprises a transmembrane domain allowing cell surface expression of the TCR.

24. The isolated nucleic acid molecule of claim 23, wherein the TCR further comprises a cytoplasmic domain, wherein the cytoplasmic domain allows intracellular signaling in response to interactions between the TCR and the HIV epitope.

25. The isolated nucleic acid molecule of claim 1, wherein the mutation further increases the activity of the TCR to detect cells presenting an HIV epitope compared to the wild type TCR.

26. The isolated nucleic acid molecule of claim 1, wherein the mutation further increases the cytotoxic functional activity of the TCR against cells presenting an HIV epitope compared to the wild type TCR.