# Patent

## Title
Single chain trimers and uses therefor

## Abstract

Single chain trimer (SCT) molecules are disclosed, comprising an MHC antigen peptide sequence, a β2-microglobulin sequence and a full-length MHC class I heavy chain sequence, joined by linker sequences. Further described are nucleic acids encoding single chain trimers. Methods for expansion of antigen-specific T cell populations using single chain trimer molecules are also disclosed. In some configurations, these methods comprise co-culturing, in a first stage, CD8+ T cells from a donor with antigen presenting cells comprising an MHC antigen peptide, and co-culturing, in a second stage, the CD8+ T cells with cells comprising an SCT which has an MHC antigen peptide sequence identical to the sequence of the antigen peptide in the first stage. The methods can provide 10,000-100,000 fold expansion of antigen-specific CD8+ T cells within about 28 days after establishing culture, and can yield over 1 billion antigen-specific CD8+ T cells expanded from an individual donor.

## Background
PRIORITY
This application is a Divisional of and claims the benefit of priority to U.S. Nonprovisional application Ser. No. 11/397,377, filed Apr. 4, 2006, which is pending, and which is incorporated herein by reference in its entirety.


GOVERNMENT RIGHTS
This invention was made with the support of Grants AI19687, AI42793 and AI46553 from the National Institutes of Health. The government of the United States of America has certain rights in this work.


REFERENCE TO A SEQUENCE LISTING
The Sequence Listing, which is a part of the present disclosure, includes a text file comprising primer nucleotide and/or amino acid sequences of the present invention on a compact disc. The subject matter of the Sequence Listing is incorporated herein by reference in its entirety.
INTRODUCTION
Responses of the immune system to foreign antigens involve, in part, presentation of antigenic peptides to antigen-specific T cells such as CD8 by antigen presenting cells (APCs). Antigenic peptides can be presented on APCs (which include many cell types, including “professional APC's” such as dendritic cells) by cell surface molecules comprising three non-covalently linked components: an antigenic peptide, a Class I major histocompatability complex (MHC) polypeptide, and a β2-microglobulin polypeptide (reviewed in Fundamental Immunology, 2d Ed., W. E. Paul, ed., Ravens Press N.Y. 1989).
CD8+ T cells have numerous clinical, research and therapeutic uses. In particular, cellular immunotherapy methods, such as adoptive T cell immunotherapy, can involve administering to a patient about one billion antigen-specific CD8+ T cells. However, presently practiced methods, such as the Rapid Expansion Method (REM) require at least 3 months following establishment of an ex vivo culture of a patient's T cells to generate sufficient numbers of antigen-specific CD8+ T cells (Riddell, S., et al., J. Immunol. Methods 128: 189-201, 1990; Blattman, J. N., et al., Science 305: 200-205, 2004).
Initial methods that attempted to selectively expand antigen-specific CD8+ T cells required single cell cloning. This approach requires a prolonged culture period of in vitro antigen-driven selection followed by a period of (non-specific) expansion with anti-CD3 monoclonal antibody to generate sufficient numbers of antigen-specific CD8+ T cells. As described by the rapid expansion method (REM), this typically requires 3-4 months to generate one billion antigen-specific T cells. A recent approach taken by NIH investigators uses a modified REM to treat patients with melanoma. Melanoma metastases are surgically resected from individual patients and T cells are isolated ex vivo from each tumor and cultured in the presence of IL-2 only in microtiter trays. Each well exhibiting growth is tested for antigen-specificity using standard cytokine release assays. Wells that exhibit melanoma reactivity are then expanded through multiple rounds of REM. The T cells are infused to the patient generally 2-4 months later.
Other methods employing anti-CD3 monoclonal antibody based strategies to activate and promote the in vitro expansion of T cells have been described. One approach employs paramagnetic beads coated with activating combinations of antibodies and/or proteins (such as anti-CD3/anti-CD28) which induce significant polyclonal activation of α/β T cells. A second variation involves using the anti-CD3/anti-CD28 combination in the presence of CD137 ligand (aka 4-IBBL) with K562 cells to propagate human CD8+ T cells. One limitation of these approaches using anti-CD3 monoclonal antibodies is the lack of enrichment for antigen-specific T cell responses. Long term cultures propagated using this technology (either REM or anti-CD3/anti-CD28 methods) require a prior enrichment step to obtain antigen-specific T cells in sufficient numbers for therapeutic use. Sufficient numbers for therapeutic use are generally thought to be a minimum of one billion T cells (1×109 cells). One variation of this approach is to employ HLA class I-immunoglobulin protein dimers coupled to paramagnetic beads in place of anti-CD3 monoclonal antibody. The HLA class I molecule is loaded with exogenous peptide. The peptide loaded magnetic beads (with anti-CD28 immobilized) are then added to purified CD8+ T cells every week to stimulate growth. This strategy results in selective enrichment and growth but generally requires >2 months of repeated stimulations done weekly.
One additional method describes an artificial antigen presenting cell (murine NIH 3T3) transfected with multiple (6 genes) constructs encoding HLA class I allele, human β2-microbulin, CD54, CD58, CD80 and antigen. Human CD8+ T cells can be selectively grown by this method and expanded up to 3×107 total cells after multiple weekly rounds of stimulation. Additional data presented using this method supports some degree of enrichment over an extended period (3 months) of culture.
An MHC heavy chain comprises about 350 amino acids; a β2-microglobulin comprises about 100 amino acids; and an antigen peptide having a length of from about 8 to about 15 amino acids. The class I heavy chains are encoded by genes of the major histocompatability complex, designated HLA-A, -B and -C in humans, and H-2K, D, and L in mice. The class I heavy chains and β2-microglobulin are separately encoded on different chromosomes. Antigen peptides are normally processed by APCs from protein sources such as, for example, viruses, bacteria, or cancer cells. Diverse variants have been identified for the polypeptides encoded by the HLA-A, -B and -C MHC genes in humans, as well as the murine H-2K, D, and L MHC genes.
Previously, covalently fused components of MHC molecules have been used to stimulate T cells. For example, Mottez et al. (J. Exp. Med. 181: 493-502, 1995) reported producing soluble Kd MHC-peptide fusions comprising a single polypeptide chain comprising, in amino-to-carboxyl terminal order, a Kd antigen peptide, an extracellular portion of a heavy chain of a murine Kd class I heavy chain, and a β2-microglobulin polypeptide. The peptide-Kd sequence was linked to the β2-microglobulin sequence by a 15 residue spacer. Cells expressing these constructs were shown to be immunogens capable of inducing specific murine cytotoxic T cells. However, the antigen peptide does not stably bind to the heavy chain (Madden et al. Cell 70: 1035-1048, 1992; Matsumura et al., Science 257: 927-934, 1992).
In one approach, β2-microglobulin has been coupled to the amino terminals of different class I MHC molecules with a linker (Mage et al., Proc. Natl. Acad. Sci. USA 89: 10658-10662, 1992; Toshitani et al, 1996; Chung et al, 1999). In addition, antigen peptide covalently attached to free β2-microglobulin has been reported (Uger and Barber, The Journal of Immunology 160: 1598-1605, 1998; Uger et al., The Journal of Immunology 162, pp. 6024-6028, 1999; White et al., J. Immunol. 162: 2671-2676, 1999).
PCT Patent application WO 96/04314 discloses “fusion complexes” of MHC molecules in which a presenting peptide is covalently bound to an MHC heavy chain such a class II heavy chain. In some embodiments, the MHC fusion complexes include a flexible linker sequence interposed between the MHC molecule and the presenting peptide. However, WO 96/04314 does not describe a single-chain fusion polypeptide comprising an antigen peptide, a β2-microglobulin and a class I heavy chain.
U.S. Pat. No. 6,011,146 to Mottez discloses an altered MHC class I determining comprising α1, α2, α3 β2-microglobulin polypeptide domains encoded by a mammalian MHC class I locus in which the α3 domain is covalently linked to the β2-microglobulin domain. This patent does not describe a single chain trimer comprising an antigen peptide sequence, a β2-microglobulin sequence and a class I heavy chain sequence as set forth below.
U.S. Pat. No. 4,478,823 discloses an immunologically active preparation comprising an antigenic material in combination with an MHC antigen, including an MHC-β2-microglobulin complex combined with further antigenic material. This patent does not describe a single chain trimer comprising an antigen peptide sequence, a β2-microglobulin sequence and a class I heavy chain sequence as set forth below.
SUMMARY
The present inventors have developed single chain trimer (SCT) molecules which are unbranched polypeptide chains comprising, in amino-to-carboxy terminal order, an antigen peptide sequence, a β2-microglobulin sequence, and a class I heavy chain sequence comprising sequences of extracellular, transmembrane, and intracellular domains of a class I heavy chain molecule. An SCT can further comprise a signal peptide sequence at the amino terminal, as well as first linker sequence extending between the peptide sequence and the β2-microglobulin sequence, and/or a second linker sequence extending between the β2-microglobulin sequence and the class I heavy chain sequence. In various aspects, the β2-microglobulin and the class I heavy chain sequences can be of human and/or murine origin.
In some aspects, an antigen peptide sequence can be that of a peptide which can be presented by an MHC class I molecule. In various configurations, an antigen peptide sequence can comprise from about 8 to about 15 contiguous amino acids. In some configurations, the antigen peptide sequence can comprise 9 contiguous amino acids. In various aspects, a peptide sequence can be that of a protein fragment, wherein the protein is a pathogen protein or a cellular protein, such as, for example, a protein expressed by a cancer cell, and wherein the peptide sequence can be bound by the MHC class I heavy chain sequence. In some configurations, the SCT can comprise any antigen peptide sequence that can bind the corresponding MHC class I heavy chain comprised by the SCT (discussed below). In some aspects, an antigen peptide sequence can be that of an HLA-A restricted peptide or HLA-B restricted peptide, such as an HLA-A*0201-restricted peptide. In some aspects, an antigen peptide can have a sequence as set forth in Table 1:







TABLE 1







Antigen Peptide Sequences*










Name
Source
Sequence
Identification





CMVpp65
Cytomegalovirus
NLVPMVATV
SEQ ID NO: 1


 


EBV BMLF
Ebstein-Barr virus
GLCTLVAML
SEQ ID NO: 2


I


 


fluM1
Influenza A virus
GILGFVFTL
SEQ ID NO: 3


 


G209-2M
human melanoma
IMDQVPFSV
SEQ ID NO: 4


 


G280-9V
human melanoma
YLEPGPVTV
SEQ ID NO: 5





*Sequences are presented using standard single-letter amino acid abbreviations as follows: A = alanine; C = cysteine; D = aspartic acid; E = glutamic acid; F = phenylalanine; G = glycine; H = histidine; I = isoleucine; K = lysine; L = leucine; M = methionine; N = asparagine; P = proline; Q = glutamine; R = arginine; S = serine; T = threonine; V = valine; W = tryptophan;Y = tyrosine.






In some aspects, a β2-microglobulin sequence comprised by an SCT can be a human β2-microglobulin sequence or a murine β2-microglobulin sequence. The β2-microglobulin sequence can a contiguous full-length β2-microglobulin sequence as expressed on the cell surface (i.e., without the leader peptide sequence). Accordingly, in some configurations, the β2-microglobulin sequence can comprise about 99 amino acids, and can be a human β2-microglobulin sequence as set forth in SEQ ID NO: 6.
In various aspects, an MHC class I heavy chain sequence comprised by an SCT can be a human MHC class I heavy chain sequence or a murine MHC class I heavy chain sequence. A human MHC class I heavy chain sequence can be any human class I heavy chain sequence, such as an HLA-A or an HLA-B class I heavy chain sequence. In some configurations, an HLA-A sequence can be an HLA-A*0201 sequence. In various aspects, an SCT can include all the domains of an MHC class I heavy chain, i.e., extracellular, transmembrane, and intracellular domains. In various aspects, the MHC class I heavy chain sequence can be the sequence set forth as SEQ ID NO: 7.
In some configurations, the MHC class I heavy chain sequence of an SCT can include single amino acid substitutions, additions, and/or deletions, such as a substitution of tyr-84 with a non-aromatic amino acid other than proline. In these configurations, the amino acid substitution can be an amino acid encoded by the standard genetic code such leucine, isoleucine, valine, serine, threonine, alanine, histidine, glutamine, asparagine, lysine, aspartic acid, glutamic acid, cysteine, arginine, serine or glycine, or can be a modified or unusual amino acid such as an amino acid recited in WIPO standard ST.25 (1998), Appendix 2, Table 4, which is incorporated by reference herein. In some aspects, the MHC class I heavy chain sequence can have the sequence set forth in SEQ ID NO: 8. In some aspects, tyr-84 can be substituted with an alanine, so that the MHC class I heavy chain sequence comprised by an SCT can have the sequence set forth as SEQ ID NO: 9.
In some aspects of the present teachings, the inventors disclose nucleic acid molecules comprising sequences encoding single chain trimers. In these aspects, a nucleic acid encoding a single chain trimer molecule can further comprise a sequence encoding a leader peptide sequence. In various aspects, the leader peptide sequence can be that of any secreted or cell surface protein that is expressed with a leader peptide sequence. In some embodiments, the leader peptide sequence can be that of secreted or cell surface polypeptide such as a human or murine β2-microglobulin, and can comprise about 20 amino acids. In some configurations, the leader peptide sequence can be that of the amino terminal sequence of a human β2-microglobulin or a murine β2-microglobulin, such as the 20 contiguous amino acid sequence from the amino terminal of murine β2-microglobulin, as set forth as SEQ ID NO: 10. Expression of a nucleic acid encoding an SCT including a leader peptide can, in some configurations, lead to expression of an SCT polypeptide which includes a complete leader peptide at the amino terminus, but in various configurations, an expressed SCT can omit the leader peptide sequence. Without being limited by theory, it is presumed that a cell expressing an SCT can proteolytically remove a leader peptide from an SCT post-translation.
In various aspects, an SCT can further comprise linker sequences, which can be sequences which impart flexibility between neighboring domains. In some aspects, a first linker sequence can extend between the antigen peptide sequence and the β2-microglobulin sequence. In some aspects, a first linker sequence can comprise at least about 10 amino acids up to about 15 amino acids, or up to about 20 amino acids. In other aspects, a second linker can extend between the β2-microglobulin sequence and the MHC class I heavy chain sequence, and can comprise, in some configurations, at least about 10 amino acids, or at least about 15 amino acids, up to about 20 amino acids. In various configurations, the first and second linkers can each contain at least about 80 percent glycine, alanine and/or serine residues, and in some aspects, about 80 percent glycine residues. In some configurations, the first linker can have a sequence (stated using standard single-letter amino acid abbreviations), of GGGGSGGGGS (SEQ ID NO: 11), GGGASGGGGSGGGGS (SEQ ID NO: 12), GGGGSGGGGSGGGGS (SEQ ID NO: 13), or GGGASGGGGS (SEQ ID NO: 27) and the second linker can have a sequence set forth as GGGGSGGGGSGGGGS (SEQ ID NO: 13) or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 14).
Accordingly, a full-length single chain trimer molecule of the present teachings comprises, in amino-to-carboxy terminal order, a leader peptide sequence, an antigen peptide sequence, a first linker sequence, a β2-microglobulin sequence, a second linker sequence, and an MHC class I heavy chain sequence. In various configurations, the MHC class I heavy chain sequence can be that of any vertebrate, such as an HLA-A, -B or -C human class I heavy chain sequence, or, among murine sequences, a class I heavy chain sequence, an H-2-L class I heavy chain sequence such as an lb sequence, an H-2-K class I heavy chain sequence, an H-2-D heavy chain sequence such as an H-2-Dd class I sequence or an H-2-L class I heavy chain sequence such as an H-2-Ld sequence. In some aspects, the MHC heavy chain sequence can be a human HLA-A or an HLA-B sequence, and the SCT can have a sequence set forth as SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.
Other aspects of the present teachings include cells comprising a single chain trimer. Such cells can be antigen presenting cells, such as dendritic cells, B cells or macrophages. In various embodiments, the cells can be mammalian cells such as cells of human or rodent origin, including murine cells.
In some aspects, the present teachings encompass nucleic acids comprising sequences encoding the single chain trimers described herein. In certain embodiments of these aspects, a nucleic acid comprising a sequence encoding an SCT can further comprise a promoter operably linked to the SCT-encoding sequence. A nucleic acid of these aspects can also be comprised by a vector, such as a virus or a plasmid. In addition, in some aspects a nucleic acid can be comprised by a cell. A cell of these aspects can be a eukaryotic or prokaryotic cell, such as a vertebrate cell, an E. coli, or an insect cell. A vertebrate cell can be a mammalian cell, including a human cell or a rodent cell, such as a murine cell. A vertebrate cell can be, in various aspects, an antigen presenting cell such as a dendritic cell, a B cell or a macrophage.
The present inventors also disclose herein methods for expanding populations of antigen-specific CD8+ T cells ex vivo using single chain trimers. The antigen-specific CD8+ T cells to be expanded can be, in various aspects, autologous to a patient in need of adoptive T cell immunotherapy, or can be allogeneic to such a patient. In various configurations, methods of expanding a population of antigen-specific CD8+ T cells ex vivo comprise first co-culturing, in a first culture, a population of antigen-specific CD8+ T cells with a first population of antigen presenting cells comprising an MHC antigen peptide sequence. The initial co-culturing is then followed by co-culturing, in a second culture, the population of antigen-specific CD8+ T cells with a population of cells comprising a single chain trimer (SCT) comprising the same MHC antigen peptide sequence as used in the first co-culture. These methods can be used to obtain about 1×109 antigen-specific CD8+ T cells autologous to a patient in less than about two months after initiating the co-culturing of the antigen-specific CD8− T cells with the first population of antigen presenting cells, and, in some aspects, the methods can be used to obtain at least about 1×109 antigen-specific CD8+ T cells autologous to a patient within about 28 days after initiating the co-culturing of the antigen-specific CD8+ T cells with the first population of antigen presenting cells. In various configurations, expansion of a population of antigen-specific CD8+ T cells can comprise an expansion of at least about 10,000 fold in less than about 2 months after initiating the co-culturing of the antigen-specific CD8+ T cells with the first population of antigen presenting cells, or, in some configurations, at least about 100,000 fold within about 28 days after initiating the co-culturing of the antigen-specific CD8+ T cells with the first population of antigen presenting cells. In various aspects, the population of antigen-specific CD8+ T cells following the expanding can be at least about 55% pure, at least about 75% pure, or at least about 95% pure. In addition, following the expanding, the population of antigen-specific CD8+ T cells can exhibit an increase of at least about 1.1×105 fold in Tetramer-positive cells (Altman, J. D. et al., Science 274: 94-96, 1996), or an increase of at least about 4.1×106 fold increase in Tetramer-positive cells. Furthermore, in some aspects, the population of antigen-specific CD8+ T cells following the expanding can comprise at least one antigen selected from the group consisting of CD62L+, CD27+, CCR7+ and CD28+, and in some aspects, the population of antigen-specific CD8+ T cells following the expanding can effect at least about 50% specific lysis at a 2:1 effector:target ratio.
In various aspects of these methods for expanding populations of antigen-specific CD8+ T cells ex vivo, the antigen presenting cells of the first co-culture can be dendritic cells, B cells, macrophages or a combination thereof. In some configurations, these antigen presenting cells can be cells autologous to a patient in need of adoptive T cell immunotherapy. In some configurations, the culturing of the antigen-specific CD8+ T cells with the antigen presenting cells comprising an MHC antigen peptide in the first culture can have a duration of about 7-21 days, such as about 14 days. In some aspects, the first culture can comprise a culture medium comprising interleukin-7 and/or interleukin-2. In various other aspects, in the second culture, the cells comprising a single chain trimer can include any cell type capable of harboring an SCT on the cell surface, such as a second population of antigen presenting cells. In various configurations, these cells can be cells expressing the SCT, and can be cells comprising a nucleic acid comprising a sequence encoding an SCT. These cells can be, in various aspects, hematopoietic cells including human hematopoietic cells, such as K562 human erythroleukemia cells. In some aspects, an SCT can be displayed by antigen presenting cells such as dendritic cells, B cells, macrophages or a combination thereof These cells can also be cells autologous to a patient in need of adoptive T cell immunotherapy. In some configurations, the culturing of the antigen-specific CD8+ T cells with the cells comprising an SCT in the second culture can have a duration of about 7-21 days, such as about 14 days.
In various aspects, a single chain trimer comprised by a population of antigen presenting cells in the second culture medium can comprise a human β2-microglobulin sequence, and can further comprise a human class I heavy chain sequence, such as an HLA-A or an HLA-B class I heavy chain sequence, including, in non-limiting example, the sequence of an HLA-A*0201 heavy chain. In addition, in some aspects, an antigen peptide portion of a single chain trimer can comprise at least about 8 contiguous amino acids up to about 13 contiguous amino acids, and, in some configurations, 9 contiguous amino acids, and can comprise a sequence of an MHC antigen. A sequence of an antigen peptide portion of a single chain trimer can be, in various configurations, identical to that of an MHC antigen comprised by a cell infected with a pathogen, such as a viral pathogen or a bacterial pathogen. In other configurations, a sequence of an antigen peptide portion of a single chain trimer can be, in various configurations, identical to that of an MHC antigen comprised by a cancer cell. Cancer cells comprising antigens for which single chain trimers can be produced include, without limitation, cells of a melanoma, a glioma, a lung carcinoma, a prostate carcinoma, a breast carcinoma, a colon carcinoma, a leukemia, a lymphoma, a myeloma, and a pancreatic carcinoma. An MHC antigen peptide portion comprised by a single chain trimer comprised by cells comprising the second culture medium the cells can be, in various configurations, identical to or have conservative substitutions in comparison to the MHC antigen peptide comprised by the antigen presenting cells comprised by the first culture. Sequences of antigen peptide portions of single cell trimers can be, for example, those set forth in Table 1, supra.
The present inventors also disclose herein methods for treating infection in a human subject. In various aspects, these methods comprise expanding a population of antigen-specific CD8− T cells autologous to a human subject in need of treatment of a pathogen infection in accordance with the methods disclosed herein, and administering the expanded population to the human subject.
The present inventors also disclose herein methods for treating cancer in a human subject. In various aspects, these methods comprise expanding a population of antigen-specific CD8− T cells autologous to a human subject in need of treatment of a cancer in accordance with the methods disclosed herein, and administering the expanded population to the human subject.
The present inventors also disclose herein nucleic acids which encode single chain trimers. A nucleic acid of these configurations can be, in various configurations, a vector comprising a promoter operably linked to a sequence encoding a single chain trimer, such as a plasmid or virus comprising a promoter operably linked to a sequence encoding an SCT.
The present inventors also disclose embodiments of the present teachings in which a vaccine comprises a single chain trimer, and/or a nucleic acid comprising a sequence encoding a single chain trimer. In various configurations, administration of an SCT to a mammal including a human can elicit a humoral antibody response and/or a cellular immunity response. The present inventors also contemplate a complex comprising a plurality of SCTs, such as for example a tetramer, a hexamer, or an octomer of an SCT. SCTs comprising such complexes can be attached covalently or non-covalently, such as through avidin-biotin or streptavidin-biotin complexes (see, e.g., Altman, J. D. et al., Science 274: 94-6, 1996) or with covalent cross-linkers such as, for example, those distributed by Pierce Chemical Co. (Rockford Ill.) or Sigma Chemical Co. (St. Louis Mo.). In some configurations, such complexes can be used in detection assays, such as, for example, flow cytometry analyses.
In various aspects, an SCT of the present teachings can also be used as probes to quantify the number of antigen/class I-specific complexes on the surface of cells such as tumor cells or pathogen-infected cells. Such complex-specific probes could be used to aid in the diagnosis of cancer or infection, or as laboratory research reagents.
In various aspects, an SCT of the present teachings can also be used as immunogen to raise antibodies peptides directed against MHC class I molecules comprising a bound peptide. The antibodies can be, in some configurations, monoclonal or polyclonal antibodies which recognize a complex comprising an MHC heavy chain and an antigen peptide. In some configurations, polyclonal antibodies raised against an SCT can be affinity purified using the SCT. Furthermore, in some configurations, an antibody reactive with MHC class I heavy chain but not specific for the peptide-heavy chain complex for a particular MHC peptide antigen can be removed by adsorption using methods well known in the art.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 illustrates flow cytometric analysis of cell surface expression and T cell recognition of Ld-derived murine SCTs. FIG. 1A illustrates flow cytometric analysis of DLD-1 cells expressing either QL9.β2mb.Ld single chain trimer OVA.or a single chain dimer of β2mb.Ld. FIG. 1B illustrates cytotoxic T cell recognition of QL9.β2mb.Ld expressed by DLD-1 cells.
FIG. 2 illustrates flow cytometric analysis of cell surface expression and T cell recognition of OVA. β2-microglobulin.Kb compositions. FIG. 2A illustrates flow cytometric analysis of LM1.8 cells expressing either OVA.β2mb.Kd single chain trimer or a single chain dimer of β2mb.Kd, with or without OVA peptide. OVA peptide is derived from ovalbumin and has the sequence Ser-Ile-Ile-Asn-Phe-Glu-Lys-Leu (SIINFEKL) (SEQ ID NO: 18). FIG. 2B illustrates cytotoxic T cell recognition of OVA.β2mb.Kd single chain trimer expressed by mouse L cells.
FIG. 3 illustrates accessibility of an SCT to loading with exogenous peptide, as indicated by resistance of an OVA.β2-microglobulin.Kb SCT to displacement by high affinity Kb binding peptide.
FIG. 4 flow cytometric analysis of cell surface expression and T cell recognition of varying OVA. β2-microglobulin.Kb SCTs. FIG. 4A illustrates expression of SCT's with varying linker lengths. FIG. 4B illustrates cytotoxic T cell recognition of the SCT's of varying length.
FIG. 5 illustrates biochemical stability of OVA.β2-microglobulin.Kb SCT. FIG. 5A illustrates immunoprecipitation of Kb, β2-microglobulin.Kb, and OVA.β2-microglobulin.Kb SCT. FIG. 5B illustrates Endo-H sensitivity of these molecules.
FIG. 6 illustrates structure of single chain trimers.
FIG. 7 illustrates alignment of sequences of selected single chain trimers.
FIG. 8 illustrates a representative experiment in which CD8+ T cells from a normal volunteer were activated with autologous dendritic cells pulse with MHC antigen peptide P495 (SEQ ID NO: 1). FIG. 8A illustrates flow cytometric analysis of CD8+ T cells showing increasing percentages of T cells specific for P495 as determined by tetramer staining (Altman, J. D. et al., Science 274: 94-96, 1996): 4.6% at start of culture; 61.1% positive at day 14 of culture, and 96.3% at day 28 of culture. FIG. 8B illustrates a growth curve during secondary stimulation, in which diamonds represent CD8+ cells purified on a cell sorter, compared to unpurified CD8+ cells (squares).
FIG. 9 illustrates ex vivo generation of large numbers of functional antigen-specific human CD8+ cells using K562 cells expressing a single chain trimer. FIG. 9A illustrates CD8+ T cell growth over a 28 day period, comparing stimulation using the present methods including dendritic cells expressing a single chain trimer comprising NLVPMVATV (SEQ ID NO: 1) antigen peptide sequence (closed circles) vs. stimulation using a rapid expansion method (REM) protocol (open circles). FIG. 9B illustrates flow cytometry analysis of CD8+ cells during the culture period.
FIG. 10 illustrates retention of CD28 expression and display of a phenotype consistent with central memory, effector memory and effector T cell subsets in CD8+ T cells expanded with K562-SCT.
FIG. 11 illustrates lytic activity of CD8+ T cells expanded with K562-SCT.
FIG. 12 illustrates that adoptive transfer of K562-SCT expanded CMV-specific human CD8+ T cells can result in tumor regression. Bioluminescence images are shown in FIG. 12A, and light emission quantification is shown in FIG. 12B.



DETAILED DESCRIPTION
The present inventors have developed single chain trimer (“SCT”) molecules. In various configurations, an SCT molecule comprises, in amino-to-carboxy terminal order, an antigen peptide segment, a β2-microglobulin segment, and a class I heavy chain segment. In various configurations, an SCT molecule can further comprise a first linker segment extending between the peptide ligand segment and the β2-microglobulin segment, and/or a second linker segment extending between the β2-microglobulin and the class I heavy chain segment. In some configurations, an SCT molecule can further comprise a leader peptide. Disclosed herein are SCT's using both human and murine sequences, prepared using MHC antigen peptides from both human and murine pathogens or cells. Some non-limiting examples of SCTs and nucleic acid vectors comprising sequences encoding the SCTs are disclosed in table 2, and in the sequence listings which are incorporated herein by reference:










TABLE 2








Identification





of vector





comprising DNA





sequence


SCT name
Source
Identification
encoding SCT







CMV SCT
Cytomegalovirus
SEQ ID NO: 21
SEQ ID NO: 28


EBV BMLF I
Epstein-Barr virus
SEQ ID NO: 22
SEQ ID NO: 29


SCT


fluM1 SCT
Influenza A virus
SEQ ID NO: 23


G209-2M SCT
Human melanoma
SEQ ID NO: 24
SEQ ID NO: 30


G280-9V SCT
Human melanoma
SEQ ID NO: 25
SEQ ID NO: 31


SCT consensus

SEQ ID NO: 26









Adoptive T cell immunotherapy is a promising modality for the treatment of infectious diseases and cancer. However, the major limitation toward further progress is the difficulty obtaining large numbers of autologous antigen-specific T cells (either CD3+CD4+ [helper subset] or CD3+CD8+ [cytoxic subset]) in a timely manner for therapeutic use. Implementation of this approach in clinical medicine will depend on manufacturing large numbers (>one billion) of antigen-specific T cells in a timeframe that is feasible and does not compromise the standards of patient care for any given disorder. The present disclosure describes a method using single chain trimer (SCT) to amplify, enrich and rapidly expand antigen peptide-specific CD8+ T cells in less than one month. This is accomplished using a two step culture process starting with purified CD8+ T cells co-cultured with autologous dendritic cells pulsed with the relevant peptide. The CD8+ T cell-DC cultures are maintained for no greater than 14 days and require addition of IL-7 (at time 0) and IL-2 (every 2-4 days). The responding T cells are then co-cultured with artificial APC (aAPC) expressing SCT for an additional 14 days in the presence of IL-2. Our experience to date shows that  ̃50 fold expansion of tetramer positive CD8+ T cells occurs during the initial culture period with peptide-pulsed DC. Amplification using aAPC with SCT results in significant expansion (2000-43,000 fold) with enrichment (55-95% positive by tetramer staining). After the start of the culture period, the percentage (0.07-4.5%, range) and number (18000-680,000 range) of antigen peptide-specific CD8+ T cells were low as expected in a group of healthy normal volunteers. After 28 days of culture, the CD8+ cells grown using SCT method as described exhibited features of enrichment (55-95% pure by tetramer staining), growth (1.1×105-4.1×106 ̃fold tetramer positive cells), central memory phenotype (CD62L+CD27+CD28+), and potent function (50% specific lysis at 2:1 effector to target ratio). Advantages of this new method include: no need for single cell cloning or cell sorting/selection, brief culture period of less than one month, and reproducibility.
The present teachings describe the use of a two step culture process to obtain large numbers of antigen-specific CD8+ T cells. The entire culture period can be 28 days and can result in from about 10,000-fold to about 100,000-fold expansion of antigen-specific CD8+ T cells. The resultant T cells can show potent cytolytic activity and can contain central memory cells as well as effector memory T cells. Adoptive transfer of the resultant T cells to tumor-bearing animals can result in rapid tumor clearance as monitored by whole body bioluminescence imaging.
The methods and compositions described herein utilize laboratory techniques well known to skilled artisans and can be found in laboratory manuals such as Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Spector, D. L. et al., Cells: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998; and Harlow, E., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1999.
SCTs disclosed herein i) can undergo expeditious heavy chain folding and ER to Golgi transport, ii) can remain covalently attached, iii) can be at least 1000 fold less accessible to exogenous peptide than class I molecules loaded with endogenous peptides, and iv) can be potent stimulators of peptide-specific cytotoxic T lymphocytes (“CTL”). Furthermore, these SCTs reduce or circumvent immune evasion by viruses and tumors. Nucleic acids comprising sequences encoding SCT molecules have application as DNA vaccines against virus infection or tumors, as well as probes of molecular mechanisms of class I assembly. Accordingly, in various embodiments, the present teachings include a vaccine comprising a single chain trimer, a nucleic acid encoding a single chain trimer, or a combination thereof.
The amino acid sequences of class I heavy chains that comprise the class I heavy chain segment, as well as nucleic acids encoding these proteins, are well known in the art and are available from numerous sources including GenBank.
The present invention also provides sequence variants of the class I heavy chain. In some embodiments, the heavy chain can comprise a substitution for tyrosine at position 84 in the natural sequence. The substitution can be any amino acid other than proline or an aromatic amino acid.
Sequences of the present teachings, including variants thereof, can be prepared by methods well known to skilled artisans, such as organic synthetic methods, molecular cloning of a nucleic acid into a vector such as plasmid, bacteriophage or virus, polymerase chain reaction, and/or site-directed mutagenesis (e.g., Wallace et al., 1981, Nucleic Acids Res. 9, 3647-3656; Zoller and Smith, 1982, Nucleic Acids Res. 10, 6487-6500; and Deng and Nickoloff, 1992, Anal. Biochem. 200, 81-88). Some examples of sequences of primers which can be used in the construction of nucleic acids comprising sequences encoding SCTs are set forth in table 3:









TABLE 3





Name of




primer
Sequence
Identification







G280-9V F1
CCGGCCGTAGACGGCATCGCAG
SEQ ID NO: 32



CTTGGATACACGCCGCCCACGT



GAAGGCTGCCGACCCCGGGGGT



GGACCATCCTCTAGACTGCCAT



GGCTCGCTCGGTGACCCTGG


 


SCT R1
CGCGCGGCGGCCGCTCACACTT
SEQ ID NO: 33



TACAAGCTGTG


 


CMV SCT-SDM-F
CTGACCGGTTTGTATGCTAACC
SEQ ID NO: 34



TGGTGCCAATGGTGGCTACCGT



GGGAGGAGGTGCTAGCGGTG


 


CMV SCT-SDM-R
CACCGCTAGCACCTCCTCCCAC
SEQ ID NO: 35



GGTAGCCACCATTGGCACCAGG



GTAGCATACAAACCGGTCAG


 


EBV SCT SDM-F
CTGACCGGTTTGTATGCTGGCC
SEQ ID NO: 36



TGTGCACCCTGGTGGCCATGCT



GGGAGGAGGTGCTAGCGGTG


 


EBV SCT SDM-R
CACCGCTAGCACCTCCTCCCAG
SEQ ID NO: 37



CATGGCCACCAGGGTGCACAGG



CCAGCATACAAACCGGTCAG


 


G209-2M SDM-F
CTGACCGGTTTGTATGCTATCA
SEQ ID NO: 38



TGGACCAGGTGCCTTTCTCCGT



GGGAGGAGGTGCTAGCGGTG


 


G209-2M SDG-R
ACCGCTAGCACCTCCTCCCACG
SEQ ID NO: 39



GAGAAAGGCACCTGGTCCATGA



TAGCATACAAACCGGTCAG









The peptide linkers which can comprise an SCT of the present teachings can predominantly comprise amino acids with small side chains, such as glycine, alanine and serine. In some aspects, at least about 80 percent of the linkers comprise glycine, alanine or serine residues, particularly glycine and serine residues. In various aspects, the linkers do not contain any proline residues. In various embodiments, linkers which can be used can comprise any of a number of flexible linker designs that have been used successfully to join antibody variable regions together (see M. Whitlow et al., Methods: A Companion to Methods in Enzymology, 2:97-105 (1991). Suitable linkers can be readily identified empirically. In non-limiting example, a DNA construct coding for an SCT which includes a linker can be cloned and expressed, and the SCT molecule can be tested to determine if it is capable of modulating the activity of a T cell receptor, either to induce T-cell proliferation or to inhibit or inactivate T cell development. Suitable size and sequences of linkers also can be determined by conventional computer modeling techniques based on the predicted size and shape of the SCT.
In various embodiments, an SCT can comprise a first flexible linker interposed between the peptide ligand segment and the β2-microglobulin segment. Such linkers can extend from and connect the carboxy terminal of the peptide ligand segment to the amino terminal of the β2-microglobulin segment. Without being limited by theory, it is believed that when an SCT is expressed, the linked peptide ligand can fold into the binding groove resulting in a functional SCT. In various embodiments, this linker can comprise at least about 10 amino acids, up to about 15 amino acids.
In various embodiments, an SCT can comprise a second flexible linker interposed between the β2-microglobulin segment and the heavy chain segment. Such linkers can extend from and connect the carboxy terminal of the β2-microglobulin segment to the amino terminal of the heavy chain segment. Without being limited by theory, it is believed that when an SCT is expressed, the β2-microglobulin and the heavy chain can fold into the binding groove resulting in a molecule which can function in promoting T cell expansion. In various embodiments, this linker can comprise at least about 15 amino acids, up to about 20 amino acids.
A polypeptide or peptide disclosed herein can be of a variety of lengths, either in a neutral (uncharged) form or in a salt form, and can include one or more modifications such as glycosylation, side chain oxidation, or phosphorylation, provided that the modification not destroy biological activity.
As used herein, the term “peptide” refers to a peptide, glycopeptide, glycolipid or any other compound associated with a ligand binding groove of various different molecules with an MHC class I or MHC class I-like structure (Fundamental Immunology, 2d Ed., W. E. Paul, ed., Ravens Press N.Y. 1989). Antigen peptides from a number of sources have been characterized in detail, including, in some non-limiting examples, antigen peptides from honey bee venom allergens, dust mite allergens, toxins produced by bacteria (such as tetanus toxin) and human tissue antigens involved in autoimmune diseases. Detailed discussions of such peptides are presented in U.S. Pat. Nos. 5,595,881, 5,468,481 and 5,284,935. Other non-limiting examples of antigen peptides include those identified in the pathogenesis of rheumatoid arthritis (type II collagen), myasthenia gravis (acetyl choline receptor), and multiple sclerosis (myelin basic protein). As an additional example, suitable peptides which induce Class I MHC-restricted CTL responses against HBV antigen are disclosed in U.S. Pat. No. 6,322,789.
As used herein, the term antigen peptide encompasses peptides derived from both non-self and self sources which can associate with the binding groove of an MHC molecule.
In various configurations, an antigen peptide sequence comprised by an SCT can comprise from about 8 contiguous amino acid residues to about 15 contiguous amino acid residues, and in some configurations, 9 contiguous amino acid residues.
In general, preparation of an SCT can be accomplished by procedures disclosed herein and by recognized recombinant DNA techniques, e.g., preparation of plasmid DNA, cleavage of DNA with restriction enzymes, ligation of DNA, transformation or transfection of a host, culturing of the host, and isolation and purification of the expressed fusion complex. Such procedures are generally known and disclosed in standard references such as in Sambrook et al., supra.
In some aspects, DNA encoding a class I heavy chain can be obtained from a suitable cell line such as, for example, human lymphoblastoid cells. In various configurations, a gene or cDNA encoding a class I heavy chain can be amplified by the polymerase chain reaction (PCR) or other means known in the art. In some aspects, a PCR product can also include sequences encoding linkers, and/or one or more restriction enzyme sites for ligation of such sequences.
In some configurations, a vector encoding an SCT can be prepared by ligation of sequences encoding the MHC heavy chain and the β2-microglobulin to a sequence encoding an antigen peptide. DNA encoding the antigen peptide can be obtained by isolating DNA from natural sources or by known synthetic methods, e.g., the phosphate triester method. See, e.g., Oligonucleotide Synthesis, IRL Press (M. Gait, ed., 1984). Synthetic oligonucleotides can also be prepared using commercially available automated oligonucleotide synthesizers. A DNA sequence encoding a linker as discussed supra can be interposed between a sequence encoding a β2-microglobulin segment and a sequence encoding an antigen peptide segment, and can be interposed between a β2-microglobulin segment and the heavy chain segment. In some configurations, the segments can be joined using a ligase.
Other nucleotide sequences also can be included in the gene construct. For example, a promoter sequence, which controls expression of the sequence coding for the β2-microglobulin segment covalently bound to the peptide ligand segment, and a sequence encoding a leader peptide (which can direct an SCT to the cell surface or the culture medium), can be included in the construct or present in the expression vector into which the construct is inserted. In non-limiting example, an immunoglobulin or CMV promoter can be used for expression of an SCT. A strong translation initiation sequence also can be included in the construct to enhance efficiency of translational initiation, such as, for example, the Kozak consensus sequence (CCACCATG), or an internal ribosome entry site (IRES). In some configurations, a nucleic acid encoding an SCT can further encode an amino terminal leader peptide. When expressed in a host cell, the primary translation product of such a nucleic acid can comprise a leader peptide which can be removed by the host cell post-translationally.
In some configurations, a leader sequence encoded by a DNA construct can contain one or more restriction sites so that an oligonucleotide encoding an antigen peptide segment of interest can be attached to the first linker. In some aspects, a restriction site can be incorporated into the 3′ end of the DNA sequence encoding a leader peptide sequence, and can be, for example, about 2 to 10 codons in length, and can be positioned before the coding region for the peptide ligand. A non-limiting example of a restriction site is the AflII site, although other cleavage sites also can be incorporated before the peptide ligand coding region. As discussed supra, use of such a restriction site in combination with a second restriction site, typically positioned at the beginning of the sequence coding for the linker, can allow rapid and straightforward insertion of sequences coding for a wide variety of peptide ligands into a DNA construct encoding an SCT.
A number of strategies can be employed to express an SCT. For example, the SCT can be incorporated into a suitable vector by known methods such as by use of restriction enzymes and ligases (see, e.g., Sambrook et al., supra). A vector can be selected based on factors relating to the cloning protocol. For example, the vector should be compatible with, and have the proper replicon for the host that is being employed. Suitable host cells include eukaryotic and prokaryotic cells, and can be cells that can be easily transformed and exhibit rapid growth in culture medium. Examples of hosts cells include prokaryotes such as E. coli and Bacillus subtilis, and eukaryotes such as animal cells and yeasts, such as, for example, S. cerevisiae. Non-limiting examples of mammalian cells which can be used as hosts to express an SCT include J558, NSO, SP2-O and CHO. Other examples of possible hosts include insect cells such as Sf9, which can be grown using conventional culturing conditions. See Sambrook, et al., supra. In various configurations, cells expressing an SCT can be identified using known methods. For example, expression of an SCT can be determined by an ELISA or Western blot using an antibody probe directed against the MHC heavy chain portion of the SCT.
An expressed SCT can be isolated and purified by known methods. For example, an SCT containing a human HLA-DR1 sequence can be purified by affinity chromatography on a monoclonal antibody L243-SEPHAROSE column by procedures that are generally known and disclosed, e.g., see Harlow, E. et al., Antibodies, A. Laboratory Manual (1988). The L243 monoclonal antibody is specific to a conformational epitope of the properly folded HLA-DR1 molecule (J. Gorga et al. J. Bioi. Chem., 262: 16087-16094), and therefore can be used for purifying a biologically active SCT. In some configurations, an SCT also can also contain a sequence to aid in purification; e.g., a 6×His tag.
In some embodiments, an SCT can be useful in mediating cell immunity as evidenced by an ability to expand a population of cytotoxic T lymphocytes specific for a class I/peptide complex. Furthermore, plasmid DNA that encodes an SCT can be used to produce an SCT which can be used an immunogen for producing an antibody. Such antibody can be, for example, a monoclonal antibody which can recognize and bind a complex comprising an MHC antigen peptide and an MHC class I molecule. A monoclonal antibody can be produced using methods well known to skilled artisans. In some configurations, a monoclonal antibody can be tagged with any tag known to skilled artisans, such as, for example, a fluorophore. An antibody of these configurations can be used in research and/or diagnostically, for example as part of a flow cytometry analysis of a sample from a human patient.
In other embodiments, an SCT or a composition containing an antigen peptide bound to an SCT can be used as immunogen for the preparation of antibodies directed against the antigen or the SCT. A polyclonal or monoclonal antibody against an SCT can be prepared by any of a variety of methods known to skilled artisans (see, e.g., Kohler et al., Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et al., In: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981), and U.S. Pat. No. 2,658,197). In some configurations, an antibody can be labeled. Examples of labels that can be employed include, but are not limited to, enzymes, radioisotopes, fluorophores, chromophores, chemiluminescent compounds, bioluminescent compounds, and metal chelates.
Examples of enzymes include malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, biotin-avidin peroxidase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β.-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholine esterase.
Examples of isotopes are 3H, 125I, 32P, 35S, 14C, 51Cr, 36Cl, 57Co, 58Co, 59Fe, and 75Se. Among the most commonly used fluorescent labeling compounds are fluoroscein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine Examples of typical chemiluminescent labeling compounds are luminal, isoluminol, theromatic acridinium ester, imidazole, acridinium salts, oxalate ester, and dioxetane.
Those of ordinary skill in the art will know of other suitable labels for binding to antibodies, or will be able to ascertain the same by the use of routine experimentation. Furthermore, the binding of these labels to antibodies can be accomplished using standard techniques commonly known to those of ordinary skill in the art. Bioluminescent compounds for purposes of labeling include luciferin, luciferase and aequorin.
In some aspects, antibodies and antigens of the present teachings can be comprised by a kit. A kit can comprise, in various embodiments, a carrier such as a carrier compartmentalized to receive one or more containers, such as vials or tubes.
In various embodiments, an SCT, a composition containing antigen bound to an SCT, and antibodies to these substances can be used in diagnostic applications. For example, an SCT can be used to target lymphocyte receptors, such as CD4+ and CD8+ receptors of T lymphocytes, and the resulting bound determinant can be assayed, for example, by use of an antibody to the bound determinant. In addition, it will be understood that an SCT can be labeled in a manner previously described for antibodies. In this case, the label on the molecule can be detected and quantified. Compositions comprising an antigen bound to an SCT can be used in a similar manner with MHC-restricted receptors recognizing the antigen and a determinant. Typical examples of assays based on the antibodies of the invention are radioimmunoassays (RIA), enzyme immunoassays (ETA), enzyme-linked immunosorbent assays (ELISA), and immunometric or sandwich immunoassays, including simultaneous sandwich, forward sandwich, and reverse sandwich immunoassays.
In another embodiment, an SCT, a composition containing an antigen bound to an SCT, and antibodies to these substances can be administered to a mammal to produce a therapeutic effect. For example, an immune response to self components can cause an autoimmune disease. In some configurations, an SCT, or an antibody against an antigen peptide-MHC complex which is generated against an SCT as immunogen, can be used administered therapeutically to a patient in need thereof. Thus, for example, an SCT can be utilized to treat T cell mediated autoimmune diseases, such as thyroiditis or multiple sclerosis. Other therapeutic uses include therapeutics for bacterial and viral infections, as well as for cancer treatments. In various aspects, these SCTs can be soluble SCTs.
This present teachings also provide an SCT for use in therapeutic or vaccine compositions. Conventional modes of administration can be employed. For example, administration can be carried out by oral, respiratory, or parenteral routes. Intradermal, subcutaneous, and intramuscular routes of administration are preferred when the vaccine is administered parenterally. A therapeutic or vaccine formulation can be prepared by mixing a single chain trimer with an excipient, and dosages and administration routes can be determined according to methods well-known in the art, for example, using methods provided by standard reference texts such as Remington: the Science and Practice of Pharmacy (Alfonso R. Gennaro ed. 19th ed. 1995); Hardman, J. G., et al., Goodman & Gilman's The Pharmacological Basis of Therapeutics, Ninth Edition, McGraw-Hill, 1996; and Rowe, R. C., et al., Handbook of Pharmaceutical Excipients, Fourth Edition, Pharmaceutical Press, 2003.
In some configurations, the ability of an SCT to exhibit a therapeutic or immunizing effect can be enhanced by emulsification with an adjuvant, incorporation in a liposome, coupling to a suitable carrier or even in cells or by combinations of these techniques. For example, the molecules and compositions can be administered with a conventional adjuvant, such as aluminum phosphate and aluminum hydroxide gel, in an amount sufficient to mediate humoral or cellular immune response in the host. Other suitable water soluble adjuvants, such as the Ribi adjuvant system available from Corixa, Seattle, Wash.
In addition, any of the common liquid or solid vehicles can be employed, which are acceptable to the host and do not have any adverse side effects on the host nor any detrimental effects on the reagents of the invention. Conveniently, phosphate buffered saline at a physiological PH can be employed as the carrier. One or more injections may be required, particularly one or two additional booster injections. It will be understood that conventional adjuvants, such as SAF-1, complete Freund's adjuvant and incomplete Freund's adjuvant, or oil-based adjuvants, such as mineral oil, can be administered with an SCT to elicit or enhance an antibody or cell-mediated immune response.
In some aspects, a humanized antibody against an SCT can be prepared using procedures well known in the art (using either chimeric antibody production or CDR grafting technology). U.S. Pat. No. 4,816,567 Cabilly et al., EPA 0120694 Publication No., assigned to Celltech, EPA 0173494 Publication No. assigned to Stanford University, and EPA 0125023 Publication No. assigned to Genentech, describing chimeric antibody procedures and EPA 0194276 Publication No. assigned to Celltech describing CDR grafting procedures.
A humanized antibody can be used therapeutically in humans so as to avoid the problems associated with the use of non-human antibodies in human therapy.
EXAMPLES
The following examples are illustrative, and are not intended to limit the scope of the claims. The description of a composition or a method in an example does not imply that a described composition or article has, or has not, been produced, or that a described method has been performed, irrespective of verb tense used.
Example 1
This example, as illustrated in FIG. 8, provides a representative experiment in which CD8+ T cells from healthy human volunteer (11G38295) were activated with autologous dendritic cells pulsed with P495 peptide (SEQ ID NO: 1) and cultured for 14 days. At the start of culture, 4.6% CD8+ T cells were specific for P495 peptide antigen as assessed by tetramer statining. At day 14, the percentage increased to 61.1%. The CD8+ T cells were then washed and placed in culture with SCT transfectants (called C5-Gle299 cells) in the presence of interleukin-2 (IL-2). Fourteen days later (day 28 of culture), 96.3% of the cells were specific for P495 as assessed by tetramer analysis. The growth curve (11G38295 secondary stimulation) for this experiment is shown: CD8: T cells purified on a cell sorter (MoFlo, diamonds) compared to unpurified CD8+ cells (squares).
Example 2
This example, as illustrated in FIG. 9, demonstrates that large numbers offunctional antigen-specific human CD8+ T cells can be generated ex vivo using K562 cells expressing single chain HLA-A2/CMV peptide/B-microglobulin trimer (K562-SCT). Fresh peripheral blood CD8+ T cells were first stimulated with autologous DC and pp65 peptide (NLVPMVATV (SEQ ID NO: 1)) for 14 days (primary stimulation), harvested and stained using HLA-A2/CMV pp65 tetramer. (A) For the secondary restimulation, CD8+ cells were purified by high speed sorting based on tetramer staining and restimulated for 14 days using the REM method (open circles) or alternatively, unsorted CD8+ T cells were re-stimulated for 14 days in the presence of K562-SCT (closed circles). A representative experiment shows CD8+ T cell growth (total cell number, mean of triplicate determinations) in a secondary 14 days culture period for each group; (B) Flow cytometry analysis of CD8+ T cells during the culture period. Percentages of HLA-A2-CMV pp65 peptide tetramer positive (tet+) CD8+ T cells at initiation of the culture (day 0, 0.49%) after DC stimulation (day 14, 49.9%) and after REM (day 28, 78.7%) or K562-SCT (day 28, 85.9%) expansion. The entire culture period is 28 days.
The results demonstrate a rapid (28 day) and efficient 2-step stimulation method for the expansion of antigen-specific (tet+) CD8+ T cells. These methods do not require the isolation of antigen-specific cells prior to expansion, and can achieve at least about 104-fold increase in antigen-specific CD8+ T cells (see Table 4).
Example 3
This example, as illustrated in FIG. 10, demonstrates retention of CD28 expression and display of a phenotype consistent with central memory, effector memory and effector T cell subsets in CD8+ T cells expanded with K562-SCT. K562-SCT expanded CD8+ T cells retain CD28 expression and display a phenotype consistent with central memory, effector memory and effector T cell subsets. CD8+ T cells were expanded using DC and peptide in primary stimulation for 14 days followed by K5652-SCT in secondary stimulation. On day 18 after the secondary stimulation, cells were harvested and stained using PE-conjugated HLA-A2/CMV-pp65 tetramers (tet−) and FITC-conjugated anti-CD8, -CD45RA, -CD45RO, -CD-28, -CD27, -CD62L, -CCR7 and -CD127 (IL-7Rα).
Adoptive transfer of tumor-specific CD8+ T cells in tumor-bearing mice has shown that terminally differentiated effector T cells are less potent at curing animals with advanced tumors (Gattinoni et al (2005) J. Clin. Invest. 115: 1616). Other published methods on ex vivo expansion of T cells require multiple rounds of in vivo stimulation resulting in terminally differentiated effector T cells (ref). We show that K562-SCT expanded CD8+/tet+ T cells express CD45RO (memory t cells) and retained CD28 expression. A significant percentage of tetramer+ T cells retain expression of CD 27, CD62L, CCR7 and CD127. These results demonstrate that a 2-step method of T cell stimulation using K562-SCT allows for the expansion of multiple T cell subsets including central memory (CD45RO/CCR7+/CD127+), effector memory (CD45RO/CCR7−/CD127+) and effector (CD45RO/CCR7−/CD127−) T cells.
Example 4
This example, as illustrated in FIG. 11, demonstrates the K562-SCT expanded CD8+ T cells show potent lytic activity. In these experiments, CD8+ T cells were expanded using K562-SCT cells as described in Example 3. K562-SCT expanded CD8+ T cells show potent lytic activity. CD8+ T cells were expanded using K562-SCT cells. T cells were tested for their ability to kill CMV pp65 peptide-pulsed (solid bars) T2 target cells in a standard 4 hr51 Cr release assay. Target cells with no peptide (dashed bars) are shown as the control. Data is presented as the mean percent specific lysis of triplicate samples. Results have been repeated in 3 donors.
Example 5
This example, as illustrated in FIG. 12, demonstrates tumor regression following adoptive transfer of K562-SCT expanded CD98+ cytomegalovirus-specific human CD8+ T cells. Adoptive transfer of K562-SCT expanded CMV-specific human CD8+ T cell results in tumor regression. CD8+ T cells were expanded using K562-SCT as described in FIG. 10. On day 0, NOD/Scid β2m (NSB) null mice were injected intra-peritoneal (i.p.) with CMV pp65-expressing tumor cells (5×106 cells) and CD8+ expanded T cells (5×107 cells/dose). Mice received 2 additional injections of T cells (5×107 cells/dose) on days 3 and 6. At the indicated times, mice were injected with an aqueous solution of luciferin (150 mg/kg, i.p.) 5 minutes prior to imaging. Animals were placed in chamber with a CCD camera system (IVIS, Xenogen) and tumor growth was quantitated by measuring light emission (bioluminesence). Mice A1-3 received tumor and CMV-specific T cells, mice B1-2 received tumor but no T cells. Bioluminescence images are shown in (A) and light emission quantification is shown in (B).
Inhibition of tumor growth is observed in mice treated with K562-SCT expanded CD8+ CMV-specific T cells. These results indicate that K562-SCT expanded CD8+ T cells effectively recognize tumor cells expressing CMV antigen in vivo resulting in tumor growth inhibition. On autopsy, no visible tumor was found in any animal given T cells while large tumor deposits were found in both control mice.
Example 6
This example, as illustrated in Tables 3a and 3b, provides an example of the number of T cells at initiation of culture (day 0):







TABLE 3a







Primary stimulation - Dendritic cells + peptide










Donor





ID

Initial #
Final #





1A
Total T cells (% tet+)
1.4 × 10 7 (.49%)
7.4 × 10 6 (49.9%)



tet+ T cells
6.9 × 10.4
3.7 × 10 6


1B
Total T cells (% tet+)
6 × 10 6 (1.5%)
1.6 × 10 7 (34%)



tet+ T cells
9 × 10 4
5.4 × 10 6


2
Total T cells (% tet+)
2.67 × 10 7
2.2 × 10 8 (20.1%)



tet+ T cells
(0.07%)
4.42 × 10 7




1.86 × 10 4
















TABLE 3b







Secondary stimulation - REM vs K562-SCT











1A
REM

1.5 × 10 5
1 × 10 9



K562-CMV SCT pp65
Total T cells (% tet+)
2 × 10 6 (49.9%)
5 × 10 10




tet+ T cells
9.9 × 10 5
(85.9%)






4.2 × 10 10


1B
REM

1.5 × 10 5
6 × 10 9



K562-CMV SCT pp65
Total T cells (% tet+)
2 × 10 6 (34%)
2.8 × 10 9 (55%)




tet+ T cells
6.8 × 10 5
1.5 × 10 9


2
REM

1.5 × 10 5
8.6 × 10 9



K562-CMV SCT pp65
Total T cells (% tet+)
2 × 10 6 (20.3%)
3.5 × 10.9




tet+ T cells
4 × 10 5
(70.4%)






2.5 × 10 9









Shown is the number of T cells at initiation of culture (day 0) and at the end of the primary culture (day 14) and the secondary culture (day 28). ID 1A and 1B represent the same donor in 2 separate experiments. In the parenthesis are the percentages of antigen-specific CD8+ T cells as determined by staining with PE-HLA-A2/CMV pp65 tetramer and anti-CD8 Abs and analyzed by FACS. Absolute CD8+/Tet+ T cell numbers are calculated based on percentages obtained by FACS analysis. Cell sorting was performed on day 14 for REM stimulated cultures only.
Example 7
This example, as illustrated in Table 4, presents a comparison of T cell expansion using rapid expansion methods (REM) vs. K562-SCT mediated expansion methods.










TABLE 4





Stimuli
Expt.
tet+ Fold Increased
% tet + cells


















DC + REM
1
2.8 × 10 5
78.7



2
1.7 × 10 6
70



3
9.7 × 10 7
73


DC + K562 SCT
1
2.3 × 10 6
85.9



2
1.3 × 10 5
55



3
1.4 × 10 7
70.4









Shown are CD8+/tet+ T cell fold-expansion obtained at 28 days in 3 independent experiments. Antigen-specific T cells fold increases are calculated based on percentages of tet+ cells as described in Table 3. Cell sorting was performed on day 14 for REM stimulated cultures only. No cell sorting is needed for K562-SCT stimulated cultures.
Example 8
This example sets forth many materials and methods used in experiments presented herein. In these experiments, single chain trimers, in which all three components of the completely assembled class I molecules are covalently attached to each other via flexible peptide linkers, were produced. Each of the SCTs consisted of the following elements beginning with the amino terminus: a leader sequence of β2-microglobulin, an antigen peptide sequence, a first flexible linker of 10 or 15 amino acid residues, the mature portion of murine or human β2-microglobulin, a second flexible linker of 15 or 20 amino acid residues, and a mature portion of an MHC class I heavy chain, including extracellular, transmembrane, and intracellular domains.
To serve as controls, constructs were also made with only β2-microglobulin covalently attached to a heavy chain. The control constructs consisted of the entire coding region of β2-microglobulin linked via a 15 or 20 amino acid residue linker to the mature portion of the respective heavy chain.
These constructs were stably introduced into mouse or human cell lines and cloned by limiting dilution. Structural integrity of these constructs was then examined by serological as well as functional assays.
Mice
B6 (H-2b), BALB/c (H-2d) and (C3H×B6)F1 (H-2k×b) were purchased from Charles River Laboratory (Wilmington, Mass.) and housed in the barrier animal facility at Washington University School of Medicine (St. Louis, Mo.). OT-1 transgenic mice (Hogquist et al., 1994) were obtained from R. Lorenz, the Washington University School of Medicine.
Cell Lines, Antibodies and Peptides
Cell lines used in this study were RMA, LM1.8, DLD-1, and B6/WT-3. RMA is a Rauscher leukemia virus-induced cell line of C57BL/6 (H-2b) origin. LM1.8 was obtained from INSERM, Institut Pasteur, France and was derived by introducing the mouse ICAM-1 cDNA into the mouse Ltk− fibroblast line DAP-3 under HAT selection (Jaulin et al., 1992). DLD-1 cells which were derived from human colon carcinomas (Dexter et al., 1979) were purchased from ATCC (American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209). The B6/WT-3 cells were derived by SV40 transformation of C57BL/6 embryo fibroblasts as described by Pretell et al. (1979) and were obtained from Louisiana State University Health Sciences Center. Shreveport, La.
MAbs used in this study included the followings: 30-5-7 and 64-3-7 which recognize the folded and open forms of Ld, respectively (Lie et al., 1991 and Smith et al., 1992); mAbs B8-24-3 and 15-5-5 (purchased from ATCC) which recognize folded Kb and Dk, respectively; and mAb 25D-1.16 (obtained from, NIH, Rockville, Md.) which recognizes Kb+SIINFEKL (SEQ ID NO: 18) peptide (Porgador et al., 1997). The OVA-derived peptide SIINFEKL (SEQ ID NO: 18) and SIYR peptide SIYRYYGL (SEQ ID NO: 20) were synthesized on a peptide synthesizer (model 432a; PE Applied Biosystems, Foster City, Calif.). All cells were maintained in complete medium (either DMEM or RPMI 1640) which included 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 2 mM glutamine, 25 μM HEPES, and 100 U/ml penicillin/streptomycin and supplemented with 10% heat inactivated bovine calf serum (HyClone Laboratories, Logan, Utah).
The QL9 peptide QLSPFPFDL (SEQ ID NO: 19), the OVA-derived peptide SIINFEKL (SEQ ID NO: 18) and SIYR peptide SIYRYYGL (SEQ ID NO: 20) were synthesized using Merrifield's solid phase method (1963) on a peptide synthesizer (model 432A: Applied Biosystems, Foster City, Calif.). Peptides were purified by reverse phase HPLC and purity (>95%) was assessed as described by Gorka et al. (1989).
DNA Constructs
Table 5 lists single chain constructs and sequences of some peptide antigens and some linkers. All PCRs were performed using Expandase (Roche Molecular Biochemicals, Indianapolis, Ind.) under standard conditions and the amplified portions of each construct were sequenced for verification.







TABLE 5







Composition of the single chain constructs










Name of Construct
Peptide
First Linker
Second Linker














β2mb.Ld
n.a.
n.a.
(G4S)3






(SEQ ID NO: 13)


 


β2mb(120).Kb
n.a.
n.a.
(G4S)4





(SEQ ID NO: 14)


 


QL9.β2mb.Ld
QLSPFDFDL
(G4S)2
(G4S)3



(SEQ ID NO: 19)
(SEQ ID NO: 11)
(SEQ ID NO: 13)


 


MCMV.β2mb.Ld
YPHGMPTNL
(G4S)2
(G4S)3



(SEQ ID NO: 20)
(SEQ ID NO: 11)
(SEQ ID NO: 13)


 


p29.β2mb.Ld
YPNVNIHNF
G3ASG4S
(G4S)3



(SEQ. ID NO: 21)
(SEQ ID NO: 27)
(SEQ ID NO: 13)


 


OVA.β2mb.Kb (10/15)
SIINFEKL
G3ASG4S
(G4S)3



(SEQ ID NO: 18)
(SEQ ID NO: 27)
(SEQ ID NO: 13)


 


OVA.β2mb.Kb (10/20)
SIINFEKL
G3ASG4S
(G4S)4



(SEQ ID NO: 18)
(SEQ ID NO: 27)
(SEQ ID NO: 14)


 


OVA.β2mb.Kb (15/20)
SIINFEKL
G3AS(G4S)2
(G4S)4



(SEQ ID NO: 18)
(SEQ ID NO: 12)
(SEQ ID NO: 14)









The β2-microglobulin.Ld and β2-microglobulin Kb constructs were made in two steps. First, an XbaI/BamHI cut PCR fragment encoding the β2-microglobulin coding sequence and the first 10 amino acid residues of the linker were cloned into the XbaI/BamHI sites of the mammalian expression vector RSV5.neo (Long, E. O., et al., Human Immunol. 31: 229-235, 1991) to create RSV.5.neo.β2-microglobulin+linker. Second, a BamHI cut PCR fragment encoding the last 7 amino acid residues of the linker and the mature portion of either Ld or Kb cDNA were cloned into the Bam HI site of RSV.5.neo.β2-microglobulin+linker to create RSV.5.neo.β2-microglobulin.Ld/Kb.
Constructs were produced using standard techniques and were confirmed by DNA sequence analysis. The β2mb Kb constructs encode, in amino-to-carboxy terminal order, β2-mb, a linker of 15 or 20 residues consisting of (G4S)3-4, and the mature Kb H chain sequence. The OVA.β2mb.Kb constructs encode, in amino-to-carboxy terminal order, the leader sequence of β2mb, the SIINFEKL sequence, a first linker of 10 or 15 residues (G4S)2-3, the mature β2mb sequence, a second linker of 15-20 residues, (G4S)3-4, then the mature Kb class I heavy chain sequence. Constructs were expressed from the pIRES.neo vector (Clontech Laboratories, Palo Alto, Calif.). The 64-3-7 epitope-tagged Kb mutant (KbR48Q.R50P) was described previously (Myers, N. B. et al., J. Immunol. 165: 5656-5663, 2002.
The QL9.β2-microglobulin.Ld construct was generated by engineering an Avr II site at the junction between the QL9 peptide and the beginning of the linker. Two PCR fragments, one encoding the β2-microglobulin ignal peptide and the QL9 peptide and cut with Xba I/Avr II and the other one encoding the linker+β2-microglobulin residues 1-27 and cut with Avr II/Sna BI cells were cloned into the Xba I and Sna BI sites of RSV.5.neo. β2-microglobulin Ld by 3-piece ligation with the Rapid DNA Ligation Kit (Roche Molecular Biochemicals), to create RSV.5.neo.QL9.β2-microglobulin.Ld. To increase expression efficiency after stable transfection, all these constructs were subcloned into the pIRES.neo vector (Clontech, Palo Alto, Calif.).
The MCMV.β2-microglobulin.Ld, p29.β2-microglobulin.Ld and OVA.β2-microglobulin.Kb constructs were prepared using the same method. The epitope tagged Kb mutant (KbR48Q, R50P) was described previously (Myers, N. B. et al. J. Immunol. 165: 5656-5663, 2000). The different linker variants were made by PCRs using Nhe I and Bsp EI sites engineered into the first and second linkers, respectively. The K3 cDNA was amplified by PCR from a K3 encoding plasmid kindly obtained from Washington University, St. Louis, Mo. and cloned into the Eco RI and Bam HI sites of pIRES.puro2 (Clontech). The various constructs were transfected into LM1.8, DLD-1 or B6/WT-3 cells using transfection reagents LIPOFECTIN (Life Technologies, Gaithersburg, Md.) or FUGENE 6 (Roche Molecular Biochemicals) according to manufacturer's instructions, Neomycin resistance was selected in 0.6 mg/ml genetic-in (Life Technologies) and puromycin resistance was selected in 5 11 g/ml puromycin (Sigma, St. Louis. MO).
Cytotoxic Lymphocyte (CTL) Generation and Maintenance
The Ld-alloreactive CTL clone, 2C, was obtained from MIT, Cambridge, Mass. It was grown in sensitization medium [complete RPMI 1640 supplemented with 10% heat inactivated fetal calf serum (HyClone Laboratories), 50 μM 2-ME, 10 U/ml Ri1-2] and maintained by weekly restimulation with irradiated (2,000R) BALB/c splenocytes (2.5×105 responders and 5×106 stimulators) in 24 well plates at 2 ml per well. The OT-1 T cells were derived by stimulating 2.5×106 OT-1 splenocytes with 5×106 irradiated B6 splenocytes in sensitization medium in the presence of 5×10−6M SIINFEKL (SEQ ID NO: 18) but without Ri1-2 for 5 days. Thereafter, the OT-1 line was restimulated weekly with 10 U/ml Ri1-2 at 5×105 responders per 5×106 stimulators. To test the immunogenicity of the single chain constructs, 7.5×106 responding (C3H×B6) F1 splenocytes were co-cultured with 3.5×105 irradiated (10,000R) LM1.8-β2-microglobulin L20).etKb cells in the presence of 1×10−4M SIINFEKL peptide or LM1.8-OVA.β2mb.etKb (15/20) cells in 24-well Linbro trays containing 2 ml sensitization medium without Ri1-2. After 5 days, they were restimulated in sensitization medium without IL-2 at 2.5×106 responders per 3.5×105 stimulators with 1×10−4M SIINFEKL peptide (for LM1.8-β2m(L20).etKb cells). Thereafter, they were restimulated weekly in the presence of 10 U/ml Ri1-2 at 2.5-5×105 responders per 3.5×105 stimulators with 1×10−5M SIINFEKL peptide (for LM1.8-β2m(L20).etKb cells).
51Cr Release Assay
1×106 target cells were labeled with 150-200 μCi of 51Cr (Na51CrO4, NEN, Boston, Mass.) in 0.2-0.3 ml of complete RPMI 1640 medium+10% bovine calf serum at 37° C. in 5% CO2 for 1-2 hours. Effector cells were plated into round bottom 96-well microtiter plates at various concentrations in the absence or continuous presence of peptide, and 2×103 washed target cells per well were added. The plates were centrifuged at 50×g for 1 minute and incubated for 4 hours at 37° C. in 5% CO2. Radioactivity in 100 μl of supernatant was measured in an Isomedic gamma counter (ICN Biomedicals, Huntsville, Ala.). The mean of triplicate samples was calculated, and percentage 51Cr release was determined according to the following equation: percentage 51Cr release=100×((experimental 51Cr release−control 51Cr release)/(maximum 51Cr release−control 51Cr release)), where experimental 51Cr release represents counts from target cells mixed with effector cells; control 51Cr release represents counts from target cells incubated with medium alone (spontaneous release); and maximum 51Cr release represents counts from target cells lysed by the addition of 5% Triton X-100. Spontaneous release ranged from 5-20%.
Flow Cytometry and Peptide Induction
3-5×105 cells were washed and incubated on ice in FACS medium (PBS containing 1% BSA and 0.1% NaN3) in the presence of a saturating concentration of mAb for 30-60 minutes, washed twice in FACTS medium, and incubated on ice with a saturating concentration of FITC-labeled, Fc-specific goat anti mouse-IgG F(ab′)2 (ICN Biomedicals, Aurora, Ohio) or PE-labeled, goat anti mouse IgG (Pharmingen, San Diego, Calif.) for 20 min. Cells were washed twice and resuspended in FACS medium. Viable cells, gated by forward and side scatter, were analyzed and a FACSCALIBUR (Becton Dickinson, San Jose, Calif.) equipped with an argon ion laser tuned to 488 nm and operating at 150 Mw. The data are expressed as linear fluorescence values obtained from log-amplified data using CELLQUEST Software (Becton Dickinson). Cells incubated with an irrelevant primary mAb followed by secondary antibodies were used as negative controls. For peptide incubation, 1×106 cells were incubated with the indicated concentration of peptide in a final volume of 2 ml complete medium at 37° C. overnight in a 6 well plate.
Immunoprecipitation and Western Blotting
Immunoprecipitations and Western blots. Cells were lysed in 10 mM Tris buffered saline pH 7.4 (TBS) containing 1% digitonin Wako, Richmond, Va.) with 20 mM iodoacetamide (IAA) and 0.2 mM of freshly added PMSF (Sigma). Saturating amounts of the primary antibody were added to the lysis buffer. Post-nuclear lysates were added to protein A-SEPHAROSE CL-4B (Amersham Pharmacia, Uppsala Sweden) for 60 minutes on ice and protein A-bound material was washed in 0.1% digitonin in TBS. Immunoprecipitates were eluted from protein A by boiling for 5 minutes in elution buffer (LDS sample buffer; Invitrogen, Carlsbad, Calif.). Samples were electrophoresed on 7% tris-acetate polyacrylamide gels (Invitrogen) and transferred to IMMOBILON-P PVDF membranes (Millipore, Bedford, Mass.). After overnight blocking in 10% dried milk in PBS-0.05% Tween 20, membranes were incubated with mAb 64-3-7 for 1 hour, washed three times with PBS-0.05% Tween 20, and incubated for 1 hour with biotin-conjugated goat anti-mouse IgG2b (Caltag, San Francisco, Calif.). Following three washes with PBS-0.05% Tween 20, membranes were incubated for hour with streptavidin-conjugated HRP (Zymed, San Francisco, Calif.), washed three times with PBS-0.03% Tween 20, and incubated with ECL chemiluminescent reagents (Amersham Pharmacia Biotech, Piscataway, N.J.) prior to exposure to BIOMAX-MR film (Eastman Kodak, Rochester, N.Y.).
Pulse-chase and immunoprecipitations. After a 45 min pre-incubation in Met/Cys-free medium (DMEM with 5% dialyzed FCS), cells (at 20×106 cells/ml) were pulse labeled with Express 35S-Met/Cys labeling mix (Perkin Elmer Life Sciences, Boston, Mass.) at 300 μCi/ml for 10 min. Cells were then washed extensively, an aliquot removed for the zero time point, and the remaining cells returned to culture at 37 degrees for the indicated times. For immunoprecipitations, labeled cells were lysed in 1% NP-40 (Sigma) dissolved in TBS with 20 mM IAA and 5 mM PMSF. Post-nuclear lysates were pre-cleared over protein A-SEPHAROSE CL-4B for 30 min on ice. Lysates were then transferred to protein A-SEPHAROSE pellets containing the appropriate pre-hound mAbs. After binding for 45 min on ice, protein A pellets were washed 4 times with 0.1% NP-40 in TBS, and bound proteins were eluted by boiling in 10 mM tris-Cl, PH 6.8+0.5% SDS+1% 2-mercaptoethanol. Eluates were mixed with an equal volume of 100 mM sodium acetate, pH 5.4 and digested overnight with 1 Mu endoglycosidase H (ICN, Costa Mesa, Calif.) that was reconstituted in 50 mM sodium acetate, pH 5.4. Following SDS-PAGE, gels were treated with Amplify (Amersham), dried, and exposed to BIOMAX-MR film.
Example 9
This example illustrates correlation of the level and quality of surface expression of SCT molecules with the known affinity of peptide binding to an MHC class I molecule when non-covalently attached. To serologically determine the quality of an SCT and test the role of peptide affinity, an SCT was prepared containing a QL9 antigen peptide, β2-microglobulin and a Ld heavy chain.
Monoclonal antibodies (mAbs) are available which distinguish Ld heavy chain conformation as determined by occupancy with high affinity peptide ligands (Lie et al. 1991; Smith et al., 1992 and 1993; Yu et al., 1999). More specifically, two mAbs, 30-5-7 and 64-3-7, recognize the folded (peptide loaded) and open (peptide empty) conformers of Ld. Evidence for the reciprocal specificity of the two mAbs includes the fact that incubation with high affinity peptide ligands leads to an increase in 30-5-7+ Ld and a decrease in 64-3-7+ Ld affinity, whereas acid stripping leads to a sharp decrease in 30-5-7+ Ld and a proportional increase in 64-3-7+ Ld affinity. Thus these two mAbs can be used in tandem to assess the effect of covalent linkage on the expression of the resultant SCT.
For the antigen peptide portion of an SCT, a sequence encoding the nonomeric peptide termed QL9, QLSPFPFDL, (SEQ ID NO: 19) (Sykulev et al. 1994) was initially used to make the single chain construct QL9. B2-microglobulin.Ld. The QL9 peptide is recognized by a well characterized Ld-restricted alloreactive CTL clone 2C (Udaka et al., 1992). As a peptide minus control construct, β2-microglobulin.Ld, was generated by linking β2-microglobulin and Ld together with a 15 residue flexible linker. These two constructs, QL9.β2-microglobulin.Ld and β2-microglobulin.Ld, were then stably transfected into the human cell line DLD-1, which fails to express endogenous β2-microglobulin Bicknell et al., 1994). Clonal transfectants expressing QL9.β2-microglobulin.Ld or β2-microglobulin.Ld were then examined by flow cytometry with mAbs 30-5-7 and 64-3-7.
As shown in FIG. 1A (panels a and b), both constructs were expressed on the surface of the DLD-1 transfectants indicating that covalent linkage of β2-microglobulin can override the requirement for endogenous β2-microglobulin, in agreement with published observations (Toshitani et al., Proc. Nat'l Acad. Sci. 93: 236-240, 1996). In addition, it was found that the QL9.β2-microglobulin.Ld construct containing all three elements of fully assembled Ld can fold correctly and be expressed on the cell surface as detected by the mAb 30-5-7 that detects an Ld conformation acquired after binding high affinity peptide ligands.
A comparison of the percentage of QL9.β2-microglobulin.Ld vs. β2-microglobulin.Ld in the open vs. folded conformation was also made. Whereas 39% of surface β2-microglobulin.Ld molecules were detected in an open conformation, only 22% of surface QL9.β2-microglobulin.Ld were detected in an open conformation. This difference suggests that covalent attachment of peptide improved the efficiency of peptide loading and reduced, but did not eliminate peptide dissociation. Relative to other class I molecules the Ld molecule is known to be highly susceptible to peptide and β2-microglobulin issociation (Hansen, T., et al., Immunol. Today 21: 83-88, 2000). Indeed this propensity to disassemble results in normal (unattached) Ld having a lower level of surface expression relative to other class I molecules. The propensity to disassemble makes Ld an ideal candidate to test the role of peptide affinity in expression of SCT molecules. For these comparisons, SCT molecules were constructed that included two different Ld ligands, MCMV (Reddehase, M. J., et al. Nature 337: 651-653, 1989) and p29 (Corr, M., et al., J. Exp. Med. 176: 1681-1692, 1992). In previously published data, it was determined that QL9/Ld and MCMV/Ld complexes have a half life of about 2 hours, whereas p29/Ld complexes have a half life of greater that 6 hours (Smith, J. D. et al., J. Exp. Med. 175: 191-202, 1992). Indeed the p29 peptide was the only peptide to fold recombinant Ld heavy chains to a sufficient extent to obtain crystals (Balendiran, G. K., et al., Proc. Nat'l. Acad. Sci. USA 94: 6880-6885, 1997). In agreement with the studies using Ld ligands in solution, the MCMV.β2-microglobulin.Ld construct behaved very similarly to QL9.β2-microglobulin.Ld in that 22% of the surface MCMV.β2-microglobulin.Ld molecules were detected in the open conformation (FIG. 1A, panel c). By contrast, the p29.β2mb.Ld construct exhibited a higher level of expression of the folded conformers and a much lower expression of the open conformers which corresponds to roughly 8% of the surface pool (FIG. 1A, panel d). Identical FACS profiles were obtained when a second independent transfection of DLD-1 cells was performed with these constructs (data not shown). Thus, SCT with peptides known to bind better in solution also make more stable single chain molecules. Therefore, it was found that the level and quality of surface expression of non-covalently bound SCT correlates with the affinity of peptide bind to class I.
Example 10
This example illustrates SCT recognition by T cells and mAb specific for class I/peptide complexes. In these experiments, SCT constructs were tested with the 2C CTL clone to see if they maintained structural integrity as seen by specific T cells. The CTL clone specifically recognizes Ld/QL9 complexes (Sykulev, Y., et al., Proc. Nat'l. Acad. Sci. USA 91: 236-240, 1994). The β2-microglobulin.Ld construct expressed on DLD-1 cells were not recognized by 2C T cells unless exogenous QL9 peptide was added (FIG. 1B). By comparison, DLD-1 cells expressing QL9.β2-microglobulin.Ld molecules were recognized by 2C T cells in a dose dependent manner, similar to 2C T cell recognition of DLD-1 cells expressing the β2-microglobulin.Ld construct when treated with exogenous QL9 peptide. Similar recognition by Ld/MCMV specific T cells was seen with the DLD-1 cells transfected with the MCMV.β2-microglobulin.Ld construct (data not shown). Thus SCTs function as targets for antigen-specific T cells.
SCT constructs were also prepared containing a Kb heavy chain. Kb was chosen because it is a prototypical class I molecule that has been used extensively for structure-function analyses. Furthermore, an mAb (25D-1.16) is available that specifically recognizes Kb+ the ovalbumin derived OVA peptide, SIINFEKL (SEQ ID NO: 18) (Porgador, A., et al. Immunity 6: 715-726, 1997). This reagent allowed the Kb/OVA complexes to be monitored serologically. Thus, a new construct, OVA.β2-microglobulin.Kb, was made by replacing the sequence encoding the p29 peptide and Ld heavy chain from p29.β2-microglobulin.Ld with sequence encoding the OVA peptide and the Kb heavy chain. A corresponding β2-microglobulin.Kb construct (β2-microglobulin+15 residue linker+Kb) was made for comparison. These constructs were transfected into mouse L cells co-expressing ICAM-1 (LM1.8) or DLD-1. The flow cytometry profiles of the LM1.8 transfectants are shown in FIG. 2A. When stained with anti-Kb mAb B8-24-3 that is conformationally sensitive but not peptide specific, both constructs gave high level of expression. In accordance with its specificity, mAb 25D-1.16 was unreactive with the β2-microglobulin.Kb construct unless exogenous OVA peptide was provided (Porgador et al. 1997). By contrast, the OVA.β2-microglobulin.Kb construct was reactive with mAb 25D-1.16. This could explain the relatively low level of 25D-1.16 expression. In parallel, the integrity of the OVA.β2-microglobulin.Kb construct was also tested by T cell recognition. In this case, Kb/OVA specific T cells derived from OT-1 transgenic mice were used (Hogquist et al., 1994). As shown in FIG. 2B, the OVA.β2-microglobulin.Kb transfectants were lysed by these OT-1 derived T cells. Thus, the SCT made with both Ld and Kb are capable recognition by peptide specific T cells. In addition, the Kb/OVAN SCT can be detected by an mAb specifically recognizing this particular class I/peptide combination.
Example 11
This example illustrates accessibility of SCT to loading with exogenous peptide. To assess the stability of the covalent peptide which is anchored in the peptide binding groove, peptide competition assays were performed. In this assay, the relative accessibility of the OV A.β2-microglobulin.Kb construct to a different Kb ligand was monitored. To do this, the 2C CTL clone was again utilized because it also recognizes Kb/SIYR (SEQ ID NO: 42) complex. SIYR (SEQ ID NO: 42) is a synthetic peptide identified from a peptide library (U daka et al., 1996) and has been reported to be as avid a Kb binder as is SIINFEKL (SEQ ID NO: 18) (Tallquist, M.D., et al., J. Immunol. 160: 802-908, 1998). When LM1.8-β2-microglobulin.Kb or LM1.8-OVA.β2-microglobulin.Kb transfectants were compared as targets for 2C T cells after overnight incubation with graded doses of SIYR (SEQ ID NO: 42) peptide (FIG. 3), the OVA.β2-microglobulin.Kb construct was completely resistant to displacement by exogenous SIYR (SEQ ID NO: 42) peptide at a concentration as high as 10−7M. Contrary to this, there was significant lysis of LM1.8-β2-microglobulin.Kb transfectants at a concentration as low as 10−10M. This finding suggests that the OVA.β2-microglobulin.Kb construct is more than 1000-fold less accessible to loading by an exogenous peptide of comparable affinity, when compared with the β2-microglobulin.Kb constructs loaded with endogenous peptides. Thus, the covalent peptide is stably bound in the SCT peptide binding groove.
Example 12
This example illustrates the effects of varying the linker length on the immune recognition of a single chain trimer molecule. In these experiments, another set of OVA.β2-microglobulin.Kb constructs with longer linkers compared to those disclosed above was created. In addition to varying the linker length, a double mutation R48Q, R50P was introduced into the Kb heavy chain to allow the transfer of the epitope detected by the mAb 64-3-7 which recognizes the open conformers (Yu, Y. Y. L., Int. Immunol. 11: 1897-1906, et al., 1999). This epitope tagging (et) has been successfully applied to a number of class Ia and class Ib molecules including Kb, Kd, HLA-B27 and H2-M3, and found to remain specific for open conformers of the epitope tagged molecule without altering peptide binding specificity (Myers, N. B. et al. J. Immunol. 165: 5656-5663, 2000; Yu, Y. Y. L., Int. Immunol. 11: 1897-1906, et al., 1999; Harris, M. R., et al., Int. Immunol. 13: 1275-1282, 2001; Lybarger, L., et al., J. Immunol. 167: 2097-2105, 2001). A total of three constructs which were named OVA.β2-microglobulin.Kb followed by a bracket indicating the length of the two linkers were made. Thus, for example, OVA.β2-microglobulin.Kb (10/15) has a 10 residue linker between the OVA peptide and the β2-microglobulin and a 15 residue linker between β2-microglobulin and the Kb heavy chain. The other two linker combinations were 10/20 and 15/20. These constructs were compared by flow cytometry to unattached Kb or β2-microglobulin (L20).Kb (20 residue linker between β2-microglobulin nd Kb) molecules. As shown in FIG. 4A, all of these constructs gave rise to high levels of expression of folded Kb (B8-24-3) on LM1.8 cells. However, when examined for the presence of open conformers (64-3-7), only the Kb (part a) and β2-microglobulin (L20).Kb (part b) constructs expressed an appreciable amount (>10% when expressed as a fraction of the sum of B8-24-3 and 64-3-7 fluorescence intensity). Furthermore, the open conformers associated with the β2-microglobulin (L20).Kb construct all but disappeared upon exogenous feeding with the OVA peptide (data not shown) thus reaffirming their “peptide-empty” nature. In stark contrast, the other three transfectants, namely, LM1.8-OVA.β2-microglobulin.Kb (10/15), LM1.8-OVA.β2-microglobulin.Kb (10/20) and LM1.8-OVA.β2-microglobulin.Kb (15/20) expressed less than 1% open conformers. Thus, the covalent OVA peptide must be able to occupy the Kb peptide binding groove virtually all the time. When mAb 25D-1.16 reactivity was compared, it was apparent that the linker combination of (15/20) was significantly better than the other two combinations. In parallel with the FACS profiles, the recognition by OT-1 derived T cells was also the best for the transfectants (FIG. 4B). Thus increasing the length of the flexible linkers results in improved recognition of the OVA.β2-microglobulin.Kb construct by both the mAb 25D-1.16 and OT-1 T cells. This improved recognition with longer linkers in SCT could reflect better peptide positioning and/or reduced steric hindrance for TCR and Ab interaction. All subsequent experiments were preformed using OVAβ2-microglobulin b.Kb (15/20) molecules with such optimal linkers.
Example 13
This example illustrates biochemical integrity of the SCT. To examine whether all of the components of the SCT remain intact at the cell surface (FIG. 3), biochemical analyses were performed to compare Kb, β2-microglobulin.Kb, and OVA.β2-microglobulin.Kb. Each of these molecules was immunoprecipitated from respective L cell transfectants and immunoblotted to compare the relative molecular weights of all three Kb constructs. As shown in FIG. 5A, mAb 64-3-7 (specific for open heavy chains) precipitated high levels of Kb, but low to undetectable amounts of β2-microglobulin Kb and OVA.β2-microglobulin Kb. By contrast, B8-24-3 (specific for folded Kb) was able to precipitate significant amounts of all three constructs. The differential reactivity with these two mAbs demonstrate that the covalent attachments to Kb reduced the levels of open conformers existing at steady-state. In addition, this experiment demonstrated that the β2-microglobulin.Kb and OVA.β2-microglobulin.Kb covalent constructs exhibit a slower migration consistent with their expected molecular weights. Indeed, the migration of the OVA.β2-microglobulin,Kb construct was even slower than β2-microglobulin.Kb, indicating that the covalent OVA peptide and linker remain attached. No breakdown products were evident, including fragments that would correspond in size to Kb heavy chains from which the covalent appendages have undergone proteolysis. These results indicate that the preponderance of the single chain molecules, at steady-state, are structurally intact. The doublet bands seen with these constructs represent Endo H-sensitive (ER-resident) vs. Endo H-resistant (post-ER) (FIG. 5B). The β2-microglobulin.Kb molecules were predominantly Endo H-sensitive, whereas the OVA.β2-microglobulin.Kb molecules were predominantly Endo H-resistant. This observation suggests that addition of the covalent peptide facilitates faster ER to Golgi transport.
To demonstrate that the OVA peptide was not undergoing proteolysis from the SCT and then rebinding as an unattached peptide, precipitates were preformed using mAb 25-D1.16. To compare OVA.β2-microglobulin.Kb molecules with Kb molecules loaded with non-covalently attached OVA peptide, 25-D1.16 precipitates were also formed with β2-microglobulin.Kb and Kb constructs subsequent to overnight incubation with exogenous OVA peptide. FIG. 5C demonstrates that mAb 25-D1.16 precipitated OVA.β2-microglobulin.Kb, as well as β2-microglobulin.Kb and Kb molecules after incubation with exogenous OVA peptide. Importantly, the SCT migrated slightly slower than the β2-microglobulin.Kb construct that was precipitated from cells incubated with exogenous peptide. Thus, these precipitates with mAb 25-D1.16 demonstrate that OVA.β2-microglobulin Kb molecules retain covalently attached OVA peptide, rather than rebinding free OVA peptide after proteolysis of the SCT.
Example 14
This example illustrates accelerated folding and maturation of SCTs. To test whether direct covalent attachment of either β2-microglobulin or peptide/β2-microglobulin to the heavy chain increases the efficiency of folding, the maturation kinetics of the various Kb constructs were compared using pulse-chase analysis. The data indicate that newly synthesized single chain molecules do, indeed, mature more quickly than Kb alone. This was apparent both in terms of initial peptide-induced folding (revealed by a loss of 64-3-7 reactivity) and ER to Golgi transport (acquisition of Endo H resistance). Approximately one-half of the Kb molecules were Endo H-resistant after 90 minutes, whereas virtually all of the SCTs were resistant at this time point (see mAb B8-24-3 precipitates). Furthermore, addition of the covalent OVA peptide appeared to enhance folding to a greater extent than addition of β2-microglobulin alone, since the 64-3-7+ conformers of OVA.β2-microglobulin.Kb were lost more rapidly than the 64-3-7+.β2-microglobulin.Kb molecules. Taken together, these data indicate that, by covalently appending all of the subunits required for full assembly, class I molecules can assume a folded conformation and traffic from the ER with high efficiency. These findings indicate that the kinetics of assembly with β2-microglobulin and peptide contribute to the overall rate of class I maturation and ER-to-Golgi transport.
Example 15
This example illustrates immunogenicity of SCTs. To test the ability of the single chain class I molecule to generate a T cell response, the ability of LM1.8 (H2k) cells expressing OVA.β2-microglobulin.Kb and β2-microglobulin.Kb fed exogenous OVA peptide (10−4M) to induce Kb/OVA specific T cell in vitro was compared. For this experiment, responder T cells from [C3H (H2k)×B6 (H2b)] F1 mice were used that potentially should respond to only Kb/OVA complexes presented by either OVA.β2-microglobulin.Kb or peptide fed β2-microglobulin.Kb. Successful generation of antigen-specific CD8+ T cells typically requires in vivo priming, intracellular peptide loading or antigen pulsed, purified dendritic cells (Carbone and Bevan, J. Exp. Med. 169: 603-612, 1989: Mayordomo et al. Nature Med. 1: 1297-302, 1995). However, specific lysis was attainable after just 4 weekly rounds of stimulation splenocytes with cells expressing the OVA. β2-microglobulin.Kb construct (data not shown). High levels Kb/OVA-specific lysis was observed after 5 weekly rounds of stimulation with this same construct (FIG. 2A, panel a). By comparison, after 5 weekly rounds of stimulation with cells expressing the β2-microglobulin.Kb construct and fed 10−4M continuous OVA peptide, little if any Kb/OVA-specific lysis was observed (FIG. 2A, panel b). Thus the single chain class I construct including peptide is superior in stimulating peptide specific T cells. Given that the OVA.β2-microglobulin.Kb construct is more than a 1000 fold less accessible to exogenous peptide than the β2-microglobulin Kb construct (FIG. 3), it is highly unlikely that the OVA.β2-microglobulin.Kb construct is a more potent stimulator due to the covalent OVA peptide being clipped off and rebinding as a free peptide.
To demonstrate that SCTs retain immune recognition as intact structures in vivo, mice were vaccinated with DNA encoding OVA.β2-microglobulin.K and then tested for antibody production. DNA vaccination was preformed using allogeneic BALB/c mice to eliminate the possibility of cross presentation of the OVA peptide on self Kb molecules. After only two injections of DNA, 2/6 BALB/c recipients made significant antibodies (titer 1:16). These antibodies were found to be predominantly Kb/ova specific, since they did not detect Kb loaded with endogenous peptides (FIG. 6B), or an irrelevant peptide. Together these findings demonstrate that the OVA.β2-microglobulin.Kb single chain construct is highly immunogenic due to its capacity to remain covalently attached and to stimulate peptide-specific, class I restricted, CD8 T cells and antibodies.
Example 16
This example illustrates resistance of SCT to down regulation by the K3 protein of γ-HV68. To test the resistance of a single chain construct to down regulation by a virus protein, the K3 protein encoded by murine γ-HV68 was tested. In a recent report γ-HV68 K3 expression was shown to severely reduce Kb and Db expression by inducing a rapid turnover of immature (EndoH-sensitive) class I molecules (Stevenson, P.G., et al., Proc. Nat'l Acad. Sci, USA 97: 8455-8460, 2000). To test whether single chain class I molecules were also susceptible to K3 mediated down regulation, a K3 cDNA was stably introduced into the LM1.8 transfectant expressing the OVA.β2-microglobulin.Kb construct. As can be seen in FIG. 6B (panels a and b), the introduction of K3 almost completely shut down the endogenous Dk expression while the OVA.β2-microglobulin.Kb expression remained largely unscathed. As a control, stable expression of K3 was found to sharply reduce the amount of endogenous Kb (lacking any attachments) expressed on the cell surface of B6/WT-3 cells. Thus, the OVA.β2-microglobulin.Kb single chain class I construct effectively escapes K3-mediated down regulation.
It is to be understood that the present invention has been described in detail by way of illustration and example in order to acquaint others skilled in the art with the invention, its principles, and its practical application. Particular formulations and processes of the present invention are not limited to the descriptions of the specific embodiments presented, but rather the descriptions and examples should be viewed in terms of the claims that follow and their equivalents. While some of the examples and descriptions above include some conclusions about the way the invention may function, the inventors do not intend to be bound by those conclusions and functions, but put them forth only as possible explanations.
It is to be further understood that the specific embodiments of the present invention as set forth are not intended as being exhaustive or limiting of the invention, and that many alternatives, modifications, and variations will be apparent to those of ordinary skill in the art in light of the foregoing examples and detailed description. Accordingly, this invention is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and scope of the following claims.
All publications, patents, patent applications and other references cited in this application are herein incorporated by reference in their entirety as if each individual publication, patent, patent application or other reference were specifically and individually indicated to be incorporated by reference.
Aspects of the Present Teachings
Disclosed herein are various inventive teachings, including, without limitation, the following aspects.
ASPECT 1. A recombinant DNA molecule comprising a DNA sequence encoding a single chain trimer of a mature MHC molecule, the single chain trimer comprising in sequence:

    
    
        (1) a peptide ligand segment;
        (2) a first linker;
        (3) a β2m segment;
        (4) a second linker; and
        (5) a class I heavy chain segment,
    
    


wherein the peptide ligand segment has a carboxy end, the β2m segment has amino and carboxy ends, and the heavy chain segment has an amino end, and wherein the peptide ligand segment is covalently linked via its carboxy end to the amino end of the β2m segment by the first linker, and wherein the β2m segment is covalently linked via its carboxy end to the amino end of the heavy chain segment by the second linker.
ASPECT 2. The recombinant DNA molecule of aspect 1 wherein the class I heavy chain segment is comprised of a HLA-A, HLA-B, HLA-C, 1a, 1b, H-2-K, H-2-Dd or H-2-Ld heavy chain.
ASPECT 3. The recombinant DNA molecule of aspect 1 wherein the class I heavy chain segment contains a mutated conserved residue.
ASPECT 4. The recombinant DNA molecule of aspect 3 wherein the tyrosine at position 84 is mutated.
ASPECT 5. The recombinant DNA molecule of aspect 1 wherein the first linker is comprised of at least 10 amino acid residues.
ASPECT 6. The recombinant DNA molecule of aspect 5 wherein the first linker is comprised of at least 15 amino acid residues.
ASPECT 7. The recombinant DNA molecule of aspect 6 wherein the first and second linkers are comprised of at least about 80 percent glycine, alanine or serine residues.
ASPECT 8. The recombinant DNA molecule of aspect 1 wherein the second linker is comprised of least 15 amino acid residues.
ASPECT 9. The recombinant DNA molecule of aspect 8 wherein the second linker is comprised of at least 20 amino acid residues.
ASPECT 10. The recombinant DNA molecule of aspect 9 wherein the first and second linkers are comprised of at least about 80 percent glycine, alanine or serine residues.
ASPECT 11. The recombinant DNA molecule of aspect 1 wherein the peptide ligand segment comprises an antigenic peptide.
ASPECT 12. The recombinant DNA molecule of aspect 11 wherein the peptide ligand segment contains from about 4 to 30 amino acid residues.
ASPECT 13. The recombinant DNA molecule of aspect 12 wherein the peptide ligand segment contains from about 6 to 20 amino acid residues.
ASPECT 14. The recombinant DNA molecule of aspect 13 wherein the peptide ligand segment contains from about 8 to 12 amino acid residues.
ASPECT 15. The recombinant DNA molecule as claimed in aspect 1, wherein the DNA sequence is contained in a vector.
ASPECT 16. A host transformed with the vector of aspect 15.
ASPECT 17. A recombinant DNA molecule comprising a DNA sequence encoding a single chain trimer of a mature MHC molecule, the single chain trimer comprising in sequence:

    
    
        (1) an antigenic peptide ligand segment containing from about 4 to 30 amino acid residues;
        (2) a first linker comprising at least 10 amino acid residues;
        (3) a β2m segment;
        (4) a second linker comprising at least 15 amino acid residues; and
        (5) a heavy chain segment comprising an HLA-A, HLA-B, HLA-C, 1a, 1b, H-2-K, H-2-Dd or H-2-Ld heavy chain,
    
    


wherein the peptide ligand segment has a carboxy end, the β2m segment has amino and carboxy ends, and the heavy chain segment has an amino end, and wherein the peptide ligand segment is covalently linked via its carboxy end to the amino end of the β2m segment by the first linker, and wherein the β2m segment is covalently linked via its carboxy end to the amino end of the heavy chain segment by the second linker.
ASPECT 18. The recombinant DNA molecule of aspect 17 wherein the class I heavy chain segment contains a mutated conserved residue.
ASPECT 19. The recombinant DNA molecule of aspect 18 wherein a tyrosine at position 84 is mutated.
ASPECT 20. The recombinant DNA molecule of aspect 17 wherein the first linker comprises at least 15 amino acid residues and the second linker comprises at least 20 amino acid residues.
ASPECT 21. The recombinant DNA molecule of aspect 17 wherein the peptide ligand segment contains from about 8 to 12 amino acid residues.
ASPECT 22. A class I heavy chain containing a mutated conserved residue.
ASPECT 23. The class I heavy chain of aspect 22 wherein the tyrosine at position 84 is mutated.
ASPECT 24. The recombinant DNA molecule as aspected in aspect 17, wherein the DNA sequence is contained in a vector.
ASPECT 25. A host transformed with the vector of aspect 24.
ASPECT 26. A single chain trimer of a mature Class I MHC molecule comprising

    
    
        (1) a peptide ligand segment having a carboxy end;
        (2) a first linker;
        (3) a β2m segment having amino and carboxy ends;
        (4) a second linker; and
        (5) a class I heavy chain segment having an amino end, wherein the β2m segment and the heavy chain segment are encoded by a mammalian Class I MHC locus, wherein the carboxy end of the peptide ligand segment is covalently linked to the amino end of the β2m segment via a first flexible peptide linker, and wherein the carboxy end of the β2m segment is covalently linked to the amino end of the class I heavy chain segment via a second flexible peptide linker.
    
    


ASPECT 27. The single chain trimer of aspect 26 wherein the class I heavy chain segment is comprised of an HLA-A, HLA-B, HLA-C, 1a, 1b, H-2-K, H-2-Dd or H-2-Ld heavy chain.
ASPECT 28. The single chain trimer of aspect 26 wherein the class I heavy chain segment contains a mutated conserved residue.
ASPECT 29. The single chain trimer of aspect 28 wherein the tyrosine at position 84 is mutated.
ASPECT 30. The single chain trimer of aspect 26 wherein the first linker comprises at least 10 amino acid residues.
ASPECT 31. The single chain trimer of aspect 30 wherein the first linker comprises at least 15 amino acid residues.
ASPECT 32. The single chain trimer of aspect 31 wherein at least about 80 percent of the linkers comprise glycine, alanine or serine residues.
ASPECT 33. The single chain trimer of aspect 32 wherein the second linker comprises at least 15 amino acid residues.
ASPECT 34. The single chain trimer of aspect 33 wherein the second linker comprises at least 20 amino acid residues.
ASPECT 35. The single chain trimer of aspect 34 wherein at least about 80 percent of the linkers comprise glycine, alanine or serine residues.
ASPECT 36. The single chain trimer of aspect 26 wherein the peptide ligand comprises an antigenic peptide.
ASPECT 37. The single chain trimer of aspect 36 wherein the peptide ligand contains from about 4 to 30 amino acid residues.
ASPECT 38. The single chain trimer of aspect 37 wherein the peptide ligand contains from about 6 to 20 amino acid residues.
ASPECT 39. The single chain trimer of aspect 38 wherein the peptide ligand contains from about 8 to 12 amino acid residues.
ASPECT 40. A single chain trimer of a mature Class I MHC molecule comprising:

    
    
        (1) an antigenic peptide ligand segment containing from about 4 to 30 amino acid residues and having a carboxy end
        (2) a first linker comprising at least 10 amino acid residues;
        (3) a β2m segment having amino and carboxy ends;
        (4) a second linker comprising at least 10 amino acid residues; and
        (5) a heavy chain segment comprising an HLA-A, HLA-B, HLA-C, 1a, 1b, H-2-K, H-2-Dd, and H-2-Ld heavy chain having an amino end, wherein the β2m segment and the heavy chain segment are encoded by a mammalian Class I MHC locus, wherein the carboxy end of the peptide ligand segment is covalently linked to the amino end of the β2m segment via a first flexible peptide linker, and wherein the carboxy end of the β2m segment is covalently linked to the amino end of the class I heavy chain segment via a second flexible peptide linker.
    
    


ASPECT 41. The single chain trimer of aspect 40 wherein the class I heavy chain segment contains a mutated conserved residue.
ASPECT 42. The single chain trimer of aspect 41 wherein a tyrosine at position 84 is mutated.
ASPECT 43. The single chain trimer of aspect 42 wherein the first linker comprises at least 15 amino acids and the second linker comprises at least 15 amino acids.
ASPECT 44. The single chain trimer of aspect 40 wherein the peptide ligand contains from about 8 to 12 amino acid residues.
ASPECT 45. A mutein of a class I heavy chain molecule having an amino acid other than tyrosine substituted at position 84.
ASPECT 46. A composition comprising an antigen bound to a single chain trimer of a mature Class I MHC molecule, the single chain trimer comprising:

    
    
        (1) a peptide ligand segment having a carboxy end;
        (2) a first linker;
        (3) a β2m segment having amino and carboxy ends;
        (4) a second linker; and
        (5) a class I heavy chain segment having an amino end, wherein the β2m segment and the heavy chain segment are encoded by a mammalian Class I MHC locus, wherein the carboxy end of the peptide ligand segment is covalently linked to the amino end of the β2m segment via a first flexible peptide linker, and wherein the carboxy end of the β2m segment is covalently linked to the amino end of the class I heavy chain segment via a second flexible peptide linker.
    
    


ASPECT 47. The composition in accordance with aspect 46 wherein the antigen is a lymphocyte receptor.
ASPECT 48. The composition in accordance with aspect 47 wherein the lymphocyte receptor is a CD4+ or and CD8+ receptor.
ASPECT 49. An immunogenically effective composition comprising a single chain trimer of a mature Class I MHC molecule, the single chain trimer comprising:

    
    
        (1) a peptide ligand segment having a carboxy end;
        (2) a first linker;
        (3) a β2m segment having amino and carboxy ends;
        (4) a second linker; and
        (5) a class I heavy chain segment having an amino end, wherein the β2m segment and the heavy chain segment are encoded by a mammalian Class I MHC locus, wherein the carboxy end of the peptide ligand segment is covalently linked to the amino end of the β2m segment via a first flexible peptide linker, and wherein the carboxy end of the β2m segment is covalently linked to the amino end of the class I heavy chain segment via a second flexible peptide linker and a physiologically acceptable carrier.
    
    


ASPECT 50. An immunogenically effective composition comprising an antigen bound to a single chain trimer of a mature Class I MHC molecule, the single chain trimer comprising:

    
    
        (1) a peptide ligand segment having a carboxy end;
        (2) a first linker;
        (3) a β2m segment having amino and carboxy ends;
        (4) a second linker; and
        (5) a class I heavy chain segment having an amino end, wherein the β2m segment and the heavy chain segment are encoded by a mammalian Class I MHC locus, wherein the carboxy end of the peptide ligand segment is covalently linked to the amino end of the β2m segment via a first flexible peptide linker, and wherein the carboxy end of the β2m segment is covalently linked to the amino end of the class I heavy chain segment via a second flexible peptide linker and a physiologically acceptable carrier.
    
    


ASPECT 51. The immunogenically effective composition in accordance with aspect 50 wherein the antigen is a lymphocyte receptor.
ASPECT 52. The immunogenically effective composition in accordance with aspect 51 wherein the lymphocyte receptor is a CD4+ or and CD8+ receptor.
ASPECT 53. A method of eliciting an immune response in a mammal comprising administering to the mammal an effective amount of a single chain trimer of a mature Class I MHC molecule, the single chain trimer comprising:

    
    
        (1) a peptide ligand segment having a carboxy end;
        (2) a first linker;
        (3) a β2m segment having amino and carboxy ends;
        (4) a second linker; and
        (5) a class I heavy chain segment having an amino end, wherein the β2m segment and the heavy chain segment are encoded by a mammalian Class I MHC locus, wherein the carboxy end of the peptide ligand segment is covalently linked to the amino end of the β2m segment via a first flexible peptide linker, and wherein the carboxy end of the β2m segment is covalently linked to the amino end of the class I heavy chain segment via a second flexible peptide linker.
    
    


ASPECT 54. The method in accordance with aspect 53 wherein the mammal suffers from a T-cell mediated autoimmune disease.
ASPECT 55. The method in accordance with aspect 53 wherein the T-cell mediated autoimmune disease is thyroiditis or multiple sclerosis.
ASPECT 56. The method in accordance with aspect 53 wherein the mammal suffers from a bacterial or a viral infection.
ASPECT 57. The method in accordance with aspect 53 wherein the mammal suffers from cancer.
ASPECT 58. A method of eliciting an immune response in a mammal comprising: administering to the mammal an effective amount of composition comprising an antigen bound to a single chain trimer of a mature Class I MHC molecule, the single chain trimer comprising

    
    
        (1) a peptide ligand segment having a carboxy end;
        (2) a first linker;
        (3) a β2m segment having amino and carboxy ends;
        (4) a second linker; and
        (5) a class I heavy chain segment having an amino end, wherein the β2m segment and the heavy chain segment are encoded by a mammalian Class I MHC locus, wherein the carboxy end of the peptide ligand segment is covalently linked to the amino end of the β2m segment via a first flexible peptide linker, and wherein the carboxy end of the β2m segment is covalently linked to the amino end of the class I heavy chain segment via a second flexible peptide linker.
    
    


ASPECT 59. The method in accordance with aspect 58 wherein the mammal suffers from a T-cell mediated autoimmune disease.
ASPECT 60. The method in accordance with aspect 58 wherein the T-cell mediated autoimmune disease is thyroiditis or multiple sclerosis.
ASPECT 61. The method in accordance with aspect 58 wherein the mammal suffers from a bacterial or a viral infection.
ASPECT 62. The method in accordance with aspect 58 wherein the mammal suffers from cancer.
ASPECT 63. A method of vaccinating a mammal comprising administering to the mammal an effective amount of a single chain trimer of a mature Class I MHC molecule, the single chain trimer comprising:

    
    
        (1) a peptide ligand segment having a carboxy end;
        (2) a first linker;
        (3) a β2m segment having amino and carboxy ends;
        (4) a second linker; and
        (5) a class I heavy chain segment having an amino end, wherein the β2m segment and the heavy chain segment are encoded by a mammalian Class I MHC locus, wherein the carboxy end of the peptide ligand segment is covalently linked to the amino end of the β2m segment via a first flexible peptide linker, and wherein the carboxy end of the β2m segment is covalently linked to the amino end of the class I heavy chain segment via a second flexible peptide linker.
    
    


ASPECT 64. The method in accordance with aspect 63 wherein the single chain trimer is administered by an oral, a respiratory or a parenteral routes.
ASPECT 65. The method in accordance with aspect 63 wherein the single chain trimer is administered by an intradermal, a subcutaneous or an intramuscular route.
ASPECT 66. A method of vaccinating a mammal comprising administering to the mammal an effective amount of a composition comprising an antigen bound to a single chain trimer of a mature Class I MHC molecule, the single chain trimer comprising:

    
    
        (1) a peptide ligand segment having a carboxy end;
        (2) a first linker;
        (3) a β2m segment having amino and carboxy ends;
        (4) a second linker; and
        (5) a class I heavy chain segment having an amino end, wherein the β2m segment and the heavy chain segment are encoded by a mammalian Class I MHC locus, wherein the carboxy end of the peptide ligand segment is covalently linked to the amino end of the β2m segment via a first flexible peptide linker, and wherein the carboxy end of the β2m segment is covalently linked to the amino end of the class I heavy chain segment via a second flexible peptide linker.
    
    


ASPECT 67. The method in accordance with aspect 66 wherein the single chain trimer is administered by an oral, a respiratory or a parenteral routes
ASPECT 68. The method in accordance with aspect 66 wherein the single chain trimer is administered by an intradermal, a subcutaneous or an intramuscular route.
ASPECT 69. An antibody which binds to a single chain trimer of a mature Class I MHC molecule, the single chain trimer comprising:

    
    
        (1) a peptide ligand segment having a carboxy end;
        (2) a first linker;
        (3) a β2m segment having amino and carboxy ends;
        (4) a second linker; and
        (5) a class I heavy chain segment having an amino end, wherein the β2m segment and the heavy chain segment are encoded by a mammalian Class I MHC locus, wherein the carboxy end of the peptide ligand segment is covalently linked to the amino end of the β2m segment via a first flexible peptide linker, and wherein the carboxy end of the β2m segment is covalently linked to the amino end of the class I heavy chain segment via a second flexible peptide linker.
    
    


ASPECT 70. The antibody in accordance with aspect 69, which is a monoclonal antibody.
ASPECT 71. The antibody in accordance with aspect 69, which is a polyclonal antibody.
ASPECT 72. An antibody which binds to a composition comprising an antigen bound to a single chain trimer of a mature Class I MHC molecule, the single chain trimer comprising:

    
    
        (1) a peptide ligand segment having a carboxy end;
        (2) a first linker;
        (3) a β2m segment having amino and carboxy ends;
        (4) a second linker; and
        (5) a class I heavy chain segment having an amino end, wherein the β2m segment and the heavy chain segment are encoded by a mammalian Class I MHC locus, wherein the carboxy end of the peptide ligand segment is covalently linked to the amino end of the β2m segment via a first flexible peptide linker, and wherein the carboxy end of the β2m segment is covalently linked to the amino end of the class I heavy chain segment via a second flexible peptide linker.
    
    


ASPECT 73. The antibody in accordance with aspect 72, which is a monoclonal antibody.
ASPECT 74. The antibody in accordance with aspect 72, which is a polyclonal antibody.
ASPECT 75. A method of detecting MHC gene products in biological preparations comprising:
(a) contacting a biological preparation with the antibody of aspect 69 under conditions allowing the antibody to bind the MHC gene product; and
(b) detecting the presence of the thus bound MHC gene product.
ASPECT 76. A composition comprising the antibody of aspect 69 and a pharmaceutically acceptable carrier.
ASPECT 77. The antibody of aspect 69 which is labeled.
ASPECT 78. The antibody of aspect 77 wherein the label is selected from the group consisting of enzymes, radioisotopes, fluorescent compounds, chemiluminescent compounds, bioluminescent compounds, and metal chelates.
ASPECT 79. A hybridoma that produces the antibody of aspect 69.
ASPECT 80. A method of detecting MHC gene products in biological preparations comprising:
(a) contacting a biological preparation with the antibody of aspect 72 under conditions allowing the antibody to bind the MHC gene product; and
(b) detecting the presence of the thus bound MHC gene product.
ASPECT 81. A composition comprising the antibody of aspect 72 and a pharmaceutically acceptable carrier.
ASPECT 82. The antibody of aspect 72 which is labeled.
ASPECT 83 The antibody of aspect 82 wherein the label is selected from the group consisting of enzymes, radioisotopes, fluorescent compounds, chemiluminescent compounds, bioluminescent compounds, and metal chelates.
ASPECT 84 A hybridoma that produces the antibody or fragment thereof of aspect 72.
ASPECT 85. A method of eliciting an immune response in a mammal comprising vaccinating a mammal with a recombinant DNA molecule encoding a single chain trimer of a mature Class I MHC molecule comprising:

    
    
        (1) an antigenic peptide ligand segment containing from about 4 to 30 amino acid residues and having a carboxy end;
        (2) a first linker comprising at least 10 amino acid residues;
        (3) a β2m segment having amino and carboxy ends;
        (4) a second linker comprising at least 10 amino acid residues; and
        (5) a heavy chain segment encoded by an MHC class I gene or class I like gene, wherein the carboxy end of the peptide ligand segment is covalently linked to the amino end of the β2m segment via a first flexible peptide linker, and wherein the carboxy end of the β2m segment is covalently linked to the amino end of the class I heavy chain segment via a second flexible peptide linker.
    
    


ASPECT 86. A method of aspect 85 wherein the recombinant DNA molecule elicits an immune response against a tumor.
ASPECT 87. A method of aspect 85 wherein the recombinant DNA molecule elicits an immune response against of a virus.
ASPECT 89. The method of aspects 86, 87, or 88 wherein the immune response is selected from the group consisting of T cell mediated and B cell mediated.
ASPECT 90. A method of obtaining antibodies against class I/peptide ligand complexes comprising;
a) vaccinating a mammal with a recombinant DNA molecule encoding a single chain trimer of a mature Class I MHC molecule comprising:

    
    
        (1) an antigenic peptide ligand segment containing from about 4 to 30 amino acid residues and having a carboxy end;
        (2) a first linker comprising at least 10 amino acid residues;
        (3) a β2m segment having amino and carboxy ends;
        (4) a second linker comprising at least 10 amino acid residues; and
        (5) a heavy chain segment from an MHC class I gene or class I like gene, wherein the carboxy end of the peptide ligand segment is covalently linked to the amino end of the β2m segment via a first flexible peptide linker, and wherein the carboxy end of the β2m segment is covalently linked to the amino end of the class I heavy chain segment via a second flexible peptide linker.
    
    


ASPECT 91. A single chain trimer molecule comprising, in amino-to-carboxy terminal order, an antigen peptide sequence, a β2-microglobulin sequence, and a human MHC class I heavy chain sequence.
ASPECT 92.A single chain trimer molecule in accordance with aspect 91, wherein the antigen peptide sequence comprises from about 8 amino acids up to about 13 contiguous amino acid residues.
ASPECT 93. A single chain trimer molecule in accordance with aspect 91, wherein the antigen peptide sequence comprises 9 contiguous amino acids.
ASPECT 94. A single chain trimer molecule in accordance with aspect 91, wherein the antigen peptide sequence comprises a sequence selected from the group consisting of NLVPMVATV (SEQ ID NO: 1), GLCTLVAML (SEQ ID NO: 2), GILGFVFTL (SEQ ID NO: 3); IMDQVPFSV (SEQ ID NO: 4) and YLEPGPVTV (SEQ ID NO: 5).
ASPECT 95.A single chain trimer molecule in accordance with aspect 91, wherein the β2-microglobulin sequence is a human β2-microglobulin sequence.
ASPECT 96.A single chain trimer molecule in accordance with aspect 95, wherein the sequence of the human β2-microglobulin is set forth as SEQ ID NO: 6.
ASPECT 97.A single chain trimer molecule in accordance with aspect 91, wherein the human MHC class I heavy chain sequence is selected from the group consisting of an HLA-A MHC class I heavy chain sequence and an HLA-B MHC class I heavy chain sequence.
ASPECT 98.A single chain trimer molecule in accordance with aspect 97, wherein the HLA-A MHC class I heavy chain sequence is an HLA-A*0201 MHC class I heavy chain sequence.
ASPECT 99.A single chain trimer molecule in accordance with aspect 91, wherein the MHC class I heavy chain sequence is a human MHC class I heavy chain sequence consisting of the sequence set forth as SEQ ID NO: 7.
ASPECT 100. A single chain trimer molecule in accordance with aspect 91, wherein the MHC class I heavy chain sequence is a human MHC class I heavy chain sequence consisting of the sequence set forth as SEQ ID NO: 8, wherein the amino acid at position 84 is a non-aromatic amino acid other than proline.
ASPECT 101. A single chain trimer in accordance with aspect 91, wherein the human MHC class I heavy chain comprises the sequence set forth as SEQ ID NO: 9.
ASPECT 102. A single chain trimer molecule in accordance with aspect 91, further comprising a leader peptide sequence.
ASPECT 103. A single chain trimer molecule in accordance with aspect 102, wherein the leader peptide sequence comprises about 20 contiguous amino acids comprised by the amino terminal sequence of a β2-microglobulin.
ASPECT 104. A single chain trimer molecule in accordance with aspect 103, wherein the leader peptide sequence comprises the sequence set forth as SEQ ID NO: 10.
ASPECT 105. A single chain trimer molecule in accordance with aspect 91, further comprising a first linker sequence extending between the antigen peptide sequence and the β2-microglobulin sequence.
ASPECT 106. A single chain trimer molecule in accordance with aspect 105, wherein the first linker sequence comprises at least about 10 amino acids up to about 15 amino acids.
ASPECT 107. A single chain trimer molecule in accordance with aspect 105, wherein at least about 80 percent of the amino acids comprising the first linker are glycines.
ASPECT 108. A single chain trimer molecule in accordance with aspect 105, wherein the first linker comprises a sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13.
ASPECT 109. A single chain trimer molecule in accordance with aspect 91, further comprising a second linker sequence extending between the β2-microglobulin sequence and the MHC class I heavy chain sequence.
ASPECT 110. A single chain trimer molecule in accordance with aspect 109, wherein the second linker sequence comprises at least about 15 amino acids up to about 20 amino acids.
ASPECT 111. A single chain trimer molecule in accordance with aspect 109, wherein at least about 80 percent of the amino acids comprising the second linker are glycines.
ASPECT 112. A single chain trimer molecule in accordance with aspect 109, wherein the second linker comprises a sequence selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 14.
ASPECT 113. A single chain trimer molecule in accordance with aspect 91, wherein the molecule comprises, in amino-to-carboxy terminal order, an antigen peptide sequence, a first linker sequence, a β2-microglobulin sequence, a second linker sequence, and an MHC class I heavy chain sequence.
ASPECT 114. A single chain trimer molecule in accordance with aspect 91, wherein the molecule has a sequence selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17.
ASPECT 115. A cell comprising the single chain trimer molecule of aspect 91.
ASPECT 116. A cell in accordance with aspect 115, wherein the cell is an antigen presenting cell.
ASPECT 117. A cell in accordance with aspect 116, wherein the antigen presenting cell is selected from the group consisting of a dendritic cell, a B cell and a macrophage.
ASPECT 118. A nucleic acid comprising a sequence encoding a single chain trimer molecule of aspect 91.
ASPECT 119. A nucleic acid in accordance with aspect 118, wherein the antigen peptide sequence comprises a sequence selected from the group consisting of NLVPMVATV (SEQ ID NO: 1), GLCTLVAML (SEQ ID NO: 2), GILGFVFTL (SEQ ID NO: 3); IMDQVPFSV (SEQ ID NO: 4) and YLEPGPVTV (SEQ ID NO: 5).
ASPECT 120. A vector comprising a promoter operably linked to the sequence encoding a single chain trimer molecule of aspect 118.
ASPECT 121. A cell comprising the nucleic acid of aspect 118.
ASPECT 122. A cell in accordance with aspect 121, wherein the cell is an antigen presenting cell.
ASPECT 123. A method of expanding a population of antigen-specific CD8+ T cells ex vivo, the method comprising:
a) co-culturing, in a first culture, a population of antigen-specific CD8+ T cells with a first population of antigen presenting cells comprising an MHC antigen peptide; and
b) co-culturing, in a second culture, the population of antigen-specific CD8+ T cells with a population of cells comprising a single chain trimer of aspect 1, wherein the single chain trimer comprises a segment comprising the sequence of the MHC antigen peptide, a segment comprising a human β2-microglobulin sequence, and a segment comprising a human MHC class I heavy chain sequence.
ASPECT 124. A method in accordance with aspect 123, wherein the first population of antigen presenting cells are selected from the group consisting of dendritic cells, B cells, macrophages and a combination thereof
ASPECT 125. A method in accordance with aspect 123, wherein the first population of antigen presenting cells are dendritic cells.
ASPECT 126. A method in accordance with aspect 123, wherein the population of cells comprising the single chain trimer comprise a second population of antigen presenting cells.
ASPECT 127. A method in accordance with aspect 123, wherein the second population of antigen presenting cells are selected from the group consisting of K562 cells, dendritic cells, B cells, macrophages and a combination thereof
ASPECT 128. A method in accordance with aspect 127, wherein the second population of antigen presenting cells are K562 cells.
ASPECT 129. A method in accordance with aspect 123, wherein the MHC class I heavy chain sequence is an HLA-A*0201 sequence.
ASPECT 130. A method in accordance with aspect 123, wherein the antigen peptide sequence comprises at least about 8 contiguous amino acids up to about 13 contiguous amino acids.
ASPECT 131. A method in accordance with aspect 130, wherein the antigen peptide sequence consists of 9 contiguous amino acids.
ASPECT 132. A method in accordance with aspect 123, wherein the antigen peptide sequence comprises an amino acid sequence identical to a sequence selected from the group consisting of an antigen peptide sequence comprised by a cell infected with a viral pathogen, a bacterial pathogen, or a combination thereof
ASPECT 133. A method in accordance with aspect 132, wherein the MHC antigen peptide sequence is selected from the group consisting of NLVPMVATV (SEQ ID NO: 1), GLCTLVAML (SEQ ID NO: 2) and GILGFVFTL (SEQ ID NO: 3).
ASPECT 134. A method in accordance with aspect 123, wherein the MHC antigen peptide sequence comprises an amino acid sequence identical to an MHC antigen sequence of a protein comprised by a cell of a cancer selected from the group consisting of melanoma, glioma, lung carcinoma, prostate carcinoma, breast carcinoma, colon carcinoma, leukemia, lymphoma, myeloma, and pancreatic carcinoma.
ASPECT 135. A method in accordance with aspect 134, wherein the MHC antigen peptide sequence is selected from the group consisting of IMDQVPFSV (SEQ ID NO: 4) and YLEPGPVTV (SEQ ID NO: 5).
ASPECT 136. A method in accordance with aspect with aspect 123, wherein the antigen-specific CD8+ T cells are autologous to a patient in need of adoptive T cell immunotherapy.
ASPECT 137. A method in accordance with aspect 123, wherein the first population of antigen presenting cells are autologous to a patient in need of adoptive T cell immunotherapy.
ASPECT 138. A method in accordance with aspect 123, wherein the expanding comprises obtaining about 1×109 of the antigen-specific CD8+ T cells in less than about two months after initiating the co-culturing of the antigen-specific CD8+ T cells with the first population of antigen presenting cells.
ASPECT 139. A method in accordance with aspect 123, wherein the expanding comprises obtaining at least about 1×109 of the antigen-specific CD8+ T cells within about 28 days after initiating the co-culturing of the antigen-specific CD8+ T cells with the first population of antigen presenting cells.
ASPECT 140. A method in accordance with aspect 123, wherein expanding the population of antigen-specific CD8+ T cells comprises expanding the population at least about 10,000 fold in less than about 2 months after initiating the co-culturing of the antigen-specific CD8+ T cells with the first population of antigen presenting cells.
ASPECT 141. A method in accordance with aspect 123, wherein expanding the population of antigen-specific CD8+ T cells comprises expanding the population at least about 100,000 fold within about 28 days after initiating the co-culturing of the antigen-specific CD8+ T cells with the first population of antigen presenting cells.
ASPECT 142. A method in accordance with aspect 123, wherein the co-culturing the population of antigen-specific CD8+ T cells with the first population of antigen presenting cells in a first culture comprises co-culturing for about 14 days.
ASPECT 143. A method in accordance with aspect 123, wherein the co-culturing the population of antigen-specific CD8+ T cells with a population of cells comprising a single chain trimer comprising the MHC antigen peptide sequence in a second culture comprises co-culturing for about 14 days.
ASPECT 144. A method in accordance with aspect 123, wherein the population of cells comprising a single chain trimer comprise cells are comprising a nucleic acid encoding the single chain trimer, wherein the cells express the single chain trimer.
ASPECT 145. A method in accordance with aspect 123, wherein the cells comprising the single chain trimer are human hematopoietic cells expressing the single chain trimer.
ASPECT 146. A method in accordance with aspect 144, wherein the human hematopoietic cells expressing the single chain trimer are K-562 cells comprising a nucleic acid encoding the single chain trimer.
ASPECT 147. A method in accordance with aspect 123, wherein the population of antigen-specific CD8+ T cells following the expanding is at least about 55% pure.
ASPECT 148. A method in accordance with aspect 123, wherein the population of antigen-specific CD8+ T cells following the expanding are at least about 95% pure.
ASPECT 149. A method in accordance with aspect 123, wherein the population of antigen-specific CD8+ T cells following the expanding comprises at least about 1.1×105 fold increase in Tetramer-positive cells.
ASPECT 150. A method in accordance with aspect 123, wherein the population of antigen-specific CD8+ T cells following the expanding comprise at least about 4.1×106 fold increase in Tetramer-positive cells.
ASPECT 151. A method of treating infection in a human subject, the method comprising:
expanding a population of antigen-specific CD8+ T cells autologous to a human subject in need of treatment of a pathogen infection in accordance with the method of aspect 123; and
administering the expanded population to the human subject.
ASPECT 152. A method of treating cancer in a human subject, the method comprising:
expanding a population of antigen-specific CD8+ T cells autologous to a human subject in need of treatment of a cancer in accordance with the method of aspect 123; and
administering the expanded population to the human subject.

## Claims

1. A method of expanding a population of antigen-specific CD8+ T cells ex vivo, the method comprising:
establishing a first culture comprising a population of antigen-specific CD8+ T cells, a first population of antigen presenting cells comprising an MHC antigen peptide and a culture medium comprising IL-7;
co-culturing the first culture for no greater than 14 days, wherein IL-2 is added to the first culture every 2-4 days; and
subsequently establishing a second culture comprising the population of antigen-specific CD8+ T cells, a population of cells comprising a single chain trimer and a culture medium comprising IL-2;
co-culturing the second culture for about 14 days, wherein the single chain trimer comprises, in amino-to-carboxy terminal order, a segment comprising the sequence of the MHC antigen peptide, a segment comprising a human β2-microglobulin sequence, and a segment comprising a human MHC class I heavy chain sequence.

2. A method in accordance with claim 1, wherein the first population of antigen presenting cells is selected from the group consisting of dendritic cells, B cells, macrophages and a combination thereof.

3. A method in accordance with claim 1, wherein the population of cells comprising the single chain trimer is a population of antigen presenting cells expressing the single chain trimer.

4. A method in accordance with claim 3, wherein the population of antigen presenting cells expressing the single chain trimer is a population of human hematopoietic cells expressing the single chain trimer.

5. A method in accordance with claim 1, wherein the MHC antigen peptide comprises a sequence selected from the group consisting of NLVPMVATV (SEQ ID NO: 1), GLCTLVAML (SEQ ID NO: 2), GILGFVFTL (SEQ ID NO: 3), IMDQVPFSV (SEQ ID NO: 4) and YLEPGPVTV (SEQ ID NO: 5).

6. A method in accordance with claim 1, wherein expanding the population of antigen-specific CD8+ T cells comprises obtaining about 1×109 of the antigen-specific CD8+ T cells.

7. A method in accordance with claim 1, wherein expanding the population of antigen-specific CD8+ T cells comprises expanding the population at least about 10,000 fold.

8. A method in accordance with claim 1, wherein expanding the population of antigen-specific CD8+ T cells comprises expanding the population at least about 100,000 fold.

9. A method in accordance with claim 1, wherein the population of antigen-specific CD8+ T cells following the expanding is at least about 55% pure.

10. A method in accordance with claim 1, wherein the population of antigen-specific CD8+ T cells following the expanding is at least about 95% pure.

11. A method in accordance with claim 1, wherein the population of antigen-specific CD8+ T cells following the expanding comprises at least about 1.1×105 fold increase in tetramer-positive cells.

12. A method in accordance with claim 1, wherein the population of antigen-specific CD8+ T cells following the expanding comprises at least about 4.1×106 fold increase in tetramer-positive cells.