WO2007030451A2 - Anticorps utilises comme mimetiques de recepteurs de lymphocytes t, leurs methodes de production et leurs applications - Google Patents

Anticorps utilises comme mimetiques de recepteurs de lymphocytes t, leurs methodes de production et leurs applications Download PDF

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WO2007030451A2
WO2007030451A2 PCT/US2006/034547 US2006034547W WO2007030451A2 WO 2007030451 A2 WO2007030451 A2 WO 2007030451A2 US 2006034547 W US2006034547 W US 2006034547W WO 2007030451 A2 WO2007030451 A2 WO 2007030451A2
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peptide
mhc
cells
specific
hla
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PCT/US2006/034547
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WO2007030451A3 (fr
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Jon A. Weidanz
Vaughan Wittman
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Receptor Logic, Ltd.
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Priority to AU2006289683A priority Critical patent/AU2006289683A1/en
Priority to CA002662798A priority patent/CA2662798A1/fr
Priority to EP06814164A priority patent/EP1933864A4/fr
Publication of WO2007030451A2 publication Critical patent/WO2007030451A2/fr
Priority to IL190014A priority patent/IL190014A0/en
Publication of WO2007030451A3 publication Critical patent/WO2007030451A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2833—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against MHC-molecules, e.g. HLA-molecules
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/32—Immunoglobulins specific features characterized by aspects of specificity or valency specific for a neo-epitope on a complex, e.g. antibody-antigen or ligand-receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates generally to a methodology of producing antibodies that recognize peptides associated with a tumorigenic or disease state, wherein the peptides are displayed in the context of HLA molecules. These antibodies will mimic the specificity of a T cell receptor (TCR) such that the molecules may be used as therapeutic, diagnostic and research reagents.
  • TCR T cell receptor
  • Class I major histocompatibility complex (MHC) molecules designated HLA class I in humans, bind and display peptide antigen ligands upon the cell surface.
  • the peptide antigen ligands presented by the class I MHC molecule are derived from either normal endogenous proteins ("self) or foreign proteins ("nonself ) introduced into the cell. Nonself proteins may be products of malignant transformation or intracellular pathogens such as viruses.
  • class I MHC molecules convey information regarding the internal milieu of a cell to immune effector cells including but not limited to, CD8 + cytotoxic T lymphocytes (CTLs), which are activated upon interaction with "nonself peptides, thereby lysing or killing the cell presenting such "nonself peptides.
  • CTLs cytotoxic T lymphocytes
  • Class Il MHC molecules designated HLA class Il in humans, also bind and display peptide antigen ligands upon the cell surface. Unlike class I MHC molecules which are expressed on virtually all nucleated cells, class Il MHC molecules are normally confined to specialized cells, such as B lymphocytes, macrophages, dendritic cells, and other antigen presenting cells which take up foreign antigens from the extracellular fluid via an endocytic pathway.
  • the peptides they bind and present are derived from extracellular foreign antigens, such as products of bacteria that multiply outside of cells, wherein such products include protein toxins secreted by the bacteria that often have deleterious and even lethal effects on the host (e.g., human).
  • class II molecules convey information regarding the fitness of the extracellular space in the vicinity of the cell displaying the class Il molecule to immune effector cells, including but not limited to, CD4 + helper T cells, thereby helping to eliminate such pathogens.
  • the extermination of such pathogens is accomplished by both helping B cells make antibodies against microbes, as well as toxins produced by such microbes, and by activating macrophages to destroy ingested microbes.
  • Class I and class Il HLA molecules exhibit extensive polymorphism generated by systematic recombinatorial and point mutation events during cell differentiation and maturation resulting from allelic diversity of the parents; as such, hundreds of different HLA types exist throughout the world's population, resulting in a large immunological diversity. Such extensive HLA diversity throughout the population is the root cause of tissue or organ transplant rejection between individuals as well as of differing individual susceptibility and/or resistance to infectious diseases. HLA molecules also contribute significantly to autoimmunity and cancer. [0006] Class I MHC molecules alert the immune response to disorders within host cells.
  • Peptides which are derived from viral- and tumor-specific proteins within the cell are loaded into the class I molecule's antigen binding groove in the endoplasmic reticulum of the cell and subsequently carried to the cell surface.
  • the class I MHC molecule and its loaded peptide ligand are on the cell surface, the class I molecule and its peptide ligand are accessible to cytotoxic T lymphocytes (CTL).
  • CTLs survey the peptides presented by the class I molecule and destroy those cells harboring ligands derived from infectious or neoplastic agents within that cell.
  • Discerning virus- and tumor-specific ligands for CTL recognition is an important component of vaccine design.
  • Ligands unique to tumorigenic or infected cells can be tested and incorporated into vaccines designed to evoke a protective CTL response.
  • Several methodologies are currently employed to identify potentially protective peptide ligands.
  • One approach uses T cell lines or clones to screen for biologically active ligands among chromatographic fractions of eluted peptides (Cox et al., 1994). This approach has been employed to identify peptide ligands specific to cancerous cells.
  • a second technique utilizes predictive algorithms to identify peptides capable of binding to a particular class I molecule based upon previously determined motif and/or individual ligand sequences (De Groot et al., 2001); however, there have been reports describing discrepancies between these algorithms and empirical data. Peptides having high predicted probability of binding from a pathogen of interest can then be synthesized and tested for T cell reactivity in various assays, such as but not limited to, precursor, tetramer and ELISpot assays.
  • TAAs tumor associated antigens
  • the tumor suppressor protein p53 is a good example. p53 and similar intracellular tumor associated proteins are normally processed within the cell into peptides which are then presented in the context of either HLA class I or class Il molecules on the surface of the tumor cell. Native antibodies are not generated against peptide-HLA complexes. Third, many of the antigens recognized by antibodies are heterogenic by nature, which limits the effectiveness of an antibody to a single tumor histology. For these reasons it is apparent that antibodies generated against surface expressed tumor antigens may not be optimal therapeutic targets for cancer immunotherapy. [0010] The majority of proteins produced by a cell reside within intracellular compartments, thus preventing their direct recognition by antibody molecules.
  • T cell epitopes are common to a broad range of tumors which have originated from several distinct tissues.
  • the primary goal of epitope discovery has been to identify peptide (tumor antigens) for use in the construction of vaccines that activate a clinically relevant cellular immune response against the tumor cells.
  • the goal of vaccination in cancer immunotherapy is to elicit a cytotoxic T lymphocyte (CTL) response and activate T helper responses to eliminate the tumor.
  • CTL cytotoxic T lymphocyte
  • the immunogen employed in the prior art methods uses MHC which has been "enriched" for one particular peptide, and therefore such immunogen contains a pool of peptide-MHC complexes and is not loaded solely with the peptide of interest.
  • MHC which has been "enriched" for one particular peptide
  • immunization protocols presented in these prior art references had to be carried out over long periods of time (i.e., approximately 5 months or longer).
  • Antibodies with T cell receptor-like specificity of the present invention enable the measurement of antigen presentation on tumors or diseased/infected cells by direct visualization.
  • Previous studies attempting to visualize peptide-HLA complexes using a soluble TCR found that the poor affinity of the TCR made it difficult to consistently detect low levels of target on tumor cells (Weidanz, 2000). Therefore, in addition to being used as targeting agents, TCRm of the present invention serve as valuable tools to obtain information regarding the presence, expression pattern, and distribution of the target peptide-HLA complex antigens on the tumor surface and in tumor metastasis.
  • the present invention relates to a methodology of producing antibodies that recognize peptides associated with a tumorigenic or disease state, wherein the peptides are displayed in the context of HLA molecules.
  • These antibodies will mimic the specificity of a T cell receptor (TCR) such that the molecules may be used as therapeutic, diagnostic and research reagents.
  • TCR T cell receptor
  • the T cell receptor mimics will have higher binding affinity than a T cell receptor.
  • the T cell receptor mimic has a binding affinity of about 10 nanomolar or greater.
  • the present invention is directed to a method of producing a T-cell receptor mimic.
  • the method of the presently disclosed and claimed invention includes identifying a peptide of interest, wherein the peptide of interest is capable of being presented by an MHC molecule. Then, an immunogen comprising at least one peptide/MHC complex is formed, wherein the peptide of the peptide/MHC complex is the peptide of interest. An effective amount of the immunogen is then administered to a host for eliciting an immune response, and the immunogen retains a three-dimensional form thereof for a period of time sufficient to elicit an immune response against the three-dimensional presentation of the peptide in the binding groove of the MHC molecule.
  • Serum collected from the host is assayed to determine if desired antibodies that recognize a three-dimensional presentation of the peptide in the binding groove of the MHC molecule are being produced.
  • the desired antibodies can differentiate the peptide/MHC complex from the MHC molecule alone, the peptide alone, and a complex of MHC and irrelevant peptide. Finally, the desired antibodies are isolated.
  • the peptide of interest may be associated with at least one of a tumorigenic state, an infectious state and a disease state, or the peptide of interest may be specific to a particular organ or tissue.
  • the presentation of the peptide in context of an MHC molecule may be novel to cancer cells, or it may be greatly increased in cancer cells when compared to normal cells.
  • the step of forming an immunogen in the method of the presently disclosed and claimed invention may include recombinantly expressing the peptide/MHC complex in the form of a single chain trimer.
  • the step of forming an immunogen in the method of the presently disclosed and claimed invention may include recombinantly expressing the peptide/MHC complex and chemically cross-linking the peptide/MHC complex to aid in stabilization of the immunogen.
  • the step of forming the immunogen of the present invention includes recombinantly expressing the MHC heavy chain and the MHC light chain separately in E. coli, and then refolding the MHC heavy and light chains with peptide in vitro.
  • the immunogen may be formed by multimerizing two or more peptide/MHC complexes, such as but not limited to, a dimer, a trimer, a tetramer, a pentamer, or a hexamer.
  • the two or more peptide/MHC complexes may be covalently attached, and they may be modified to enable covalent attachment of the peptide/MHC complexes to one another.
  • the two or more peptide/MHC complexes may be non-covalently attached.
  • the two or more peptide/MHC complexes may be attached to a substrate.
  • a tail may be attached to the two or more peptide/MHC complexes to aid in multimerization, wherein the tail may be selected from the group including but not limited to, a biotinylation signal peptide tail, an immunoglobulin heavy chain tail, a TNF tail, an IgM tail, a Fos/Jun tail, and combinations thereof.
  • the peptide/MHC complexes may be multimerized through liposome encapsulation, through the use of an artificial antigen presenting cell, or through the use of polymerized streptavidin.
  • the immunogen may be further modified to aid in stabilization thereof.
  • the modification may be selected from the group consisting of modifying an anchor in the peptide/MHC complex, modifying amino acids in the peptide/MHC complex, PEGalation, chemical cross-linking, changes in pH or salt, addition of at least one chaperone protein, addition of at least one adjuvant, and combinations thereof.
  • the host immunized for eliciting an immune response in the presently disclosed and claimed method may be, for example but not by way of limitation, a rabbit, a rat, or a mouse, such as but not limited to, a Balb/c mouse or a transgenic mouse.
  • the transgenic mouse may be transgenic for the MHC molecule of the immunogen, or the transgenic mouse may be capable of producing human antibodies.
  • the assaying step of the presently disclosed and claimed invention may further include preabsorbing the serum to remove antibodies that are not peptide specific.
  • the step of isolating the desired antibodies of the presently disclosed and claimed invention may further include a method for isolating at least one of B cells expressing surface immunoglobulin, B memory cells, hybridoma cells and plasma cells producing the desired antibodies.
  • the step of isolating the B memory cells may include sorting the B memory cells using at least one of FACS sorting, beads coated with peptide/MHC complex, magnetic beads, and intracellular staining.
  • the method may further include the step of differentiating and expanding the B memory cells into plasma cells.
  • the method of the presently disclosed and claimed invention may further include the step of assaying the isolated desired antibodies to confirm their specificity and to determine if the isolated desired antibodies cross-react with other MHC molecules.
  • the present invention is also directed to a T cell receptor mimic that includes an antibody or antibody fragment reactive against a specific peptide/MHC complex, wherein the antibody or antibody fragment can differentiate the specific peptide/MHC complex from the MHC molecule alone, the peptide alone, and a complex of MHC and an irrelevant peptide.
  • the T cell receptor mimic is produced by immunizing a host with an effective amount of an immunogen comprising a multimer of two or more specific peptide/MHC complexes.
  • the immunogen may be in the form of a tetramer.
  • the peptide of the specific peptide/MHC complex may be associated with at least one of a tumorigenic state, an infectious state and a disease state, or the peptide of the specific peptide/MHC complex may be specific to a particular organ or tissue.
  • the presentation of the peptide of the specific peptide/MHC complex in the context of an MHC molecule may be novel to cancer cells, or may be greatly increased in cancer cells when compared to normal cells.
  • the peptide of the specific peptide/MHC complex may comprise any of SEQ ID NOS: 1-3 and 6.
  • the T cell receptor mimic may have at least one functional moiety, such as but not limited to, a detectable moiety or a therapeutic moiety, bound thereto.
  • the detectable moiety may be selected from the group consisting of a fluorophore, an enzyme, a radioisotope and combinations thereof
  • the therapeutic moiety may be selected from the group consisting of a cytotoxic moiety, a toxic moiety, a cytokine moiety, a bi-specific antibody moiety, and combinations thereof.
  • the present invention is also directed to a hybridoma cell or a B cell producing a T cell receptor mimic comprising an antibody or antibody fragment reactive against a specific peptide/MHC complex, wherein the antibody or antibody fragment can differentiate the specific peptide/MHC complex from the MHC molecule alone, the peptide alone, and a complex of MHC and an irrelevant peptide.
  • the peptide of the specific peptide/MHC complex may be associated with at least one of a tumorigenic state, an infectious state and a disease state, or the peptide of the specific peptide/MHC complex may be specific to a particular organ or tissue.
  • the presentation of the peptide of the specific peptide/MHC complex in the context of an MHC molecule may be novel to cancer cells, or may be greatly increased in cancer cells when compared to normal cells.
  • the peptide of the specific peptide/MHC complex may comprise any of SEQ ID NOS: 1-3 and 6.
  • the present invention is further directed to an isolated nucleic acid segment encoding a T cell receptor mimic comprising an antibody or antibody fragment reactive against a specific peptide/MHC complex, wherein the antibody or antibody fragment can differentiate the specific peptide/MHC complex from the MHC molecule alone, the peptide alone, and a complex of MHC and an irrelevant peptide.
  • the peptide of the specific peptide/MHC complex may be associated with at least one of a tumorigenic state, an infectious state and a disease state, or the peptide of the specific peptide/MHC complex may be specific to a particular organ or tissue.
  • the presentation of the peptide of the specific peptide/MHC complex in the context of an MHC molecule may be novel to cancer cells, or may be greatly increased in cancer cells when compared to normal cells.
  • the peptide of the specific peptide/MHC complex may comprise any of SEQ ID NOS: 1-3 and 6.
  • the present invention is also related to an immunogen used in production of a T cell receptor mimic.
  • the immunogen includes a multimer of two or more identical peptide/MHC complexes, such as a tetramer, wherein the peptide/MHC complexes are capable of retaining their 3-dimensional form for a period of time sufficient to elicit an immune response in a host such that antibodies that recognize a three-dimensional presentation of the peptide in the binding groove of the MHC molecule are produced.
  • the antibodies so produced are capable of differentiating the peptide/MHC complex from the MHC molecule alone, the peptide alone, and a complex of MHC and irrelevant peptide.
  • the peptide of the specific peptide/MHC complex may be associated with at least one of a tumorigenic state, an infectious state and a disease state, or the peptide of the specific peptide/MHC complex may be specific to a particular organ or tissue.
  • the presentation of the peptide of the specific peptide/MHC complex in the context of an MHC molecule may be novel to cancer cells, or may be greatly increased in cancer cells when compared to normal cells.
  • the peptide of the specific peptide/MHC complex may comprise any of SEQ ID NOS:1-3 and 6.
  • Fig. 1 illustrates size exclusion chromatography on a Sephadex S-75 column of a mixture of refolded heavy and light ( ⁇ 2m) chains of HLA-A2 with synthetic peptide (LLGRNSFEV; SEQ ID NO:1).
  • Peptide-HLA-A2 folded monomers were prepared and purified using S-75 size exclusion chromatography.
  • Monomers consisting of peptide-HLA-A2 were prepared by mixing heavy chain (1 ⁇ M) together with beta-2 microglobulin (2 ⁇ M) and 10 mg of the desired peptide in buffer (1 L) optimized to facilitate folding of conformational ⁇ correct peptide loaded HLA complexes.
  • Fig. 1 shows the typical chromatogram profile for the purification of refolded peptide-HLA-A2 monomer. In this Fig., 5 peaks are seen, which are marked as aggregates, refolded monomer, HLA-A2 heavy chain, beta2- microglobulin, and peptide alone.
  • a typical purification will yield 8 to 12 mg of peptide-HLA-A2 monomer.
  • the sample is concentrated to approximately 5 mL using an Amicon concentrator and biotinylated with biotin ligase following standard procedures (Avidity, CO).
  • the biotin labeled monomer was isolated using the same approach as described above (data not shown). The biotin labeled material may then be used for making tetramers as described in Fig. 2.
  • Fig. 2 illustrates preparation and purification of peptide-HLA tetramer using size exclusion chromatography on a Sephadex S-200 column of the multimerized refolded monomer peak of Fig. 1.
  • biotin labeled monomer was mixed with streptavidin at either 4:1 or 8:1 molar ratios. The precise ratio was determined for each peptide-HLA preparation and was based on the ratio of the two proteins which generates the largest amount of tetramer band as determined by gel shift assays by SDS-PAGE.
  • tetramer 8 mg was used, and after mixing with the appropriate amount of streptavidin, the sample (usually in 5 to 10 mL) was applied to the S-200 column for purification by FPLC.
  • Fig. 2 shows the chromatogram profile for a typical tetramer purification run on an S-200 column, and as shown, 4 peaks are present which represent tetramer, trimer, dimerand monomer forms of the peptide-HLA-A2 complex. 3 and 4 mg of purified tetramer was routinely produced.
  • Fig. 3 illustrates the stability of the 264 peptide-HLA-A2 tetramers. Tetramer stability was assessed in mouse serum at 4 0 C and 37 °C. 25 ⁇ g of 264 peptide-tetramer complex was added to 5 mL of 100% mouse serum and incubated at4°C and 37°C for 75 hr. At designated times, 50 ⁇ L aliquots of sample were removed and stored at -20 0 C and remained frozen until completion of the experiment. To determine the integrity of the peptide-HLA tetramer, samples were evaluated using a sandwich ELISA and two antibodies, BB7.2 and W6/32 that bind only conformationally intact peptide-HLA tetramers.
  • An ELISA protocol was developed using 96-well plates (Nunc maxisorb plates) that were coated overnight (OYN) at 4 0 C with 0.5 ⁇ g of BB7.2,washed with buffer (PBS/0.05% Tween-20) and then blocked with 200 ⁇ l of 5% milk for 1 hr at room temperature. Sample (50 ⁇ L) from each time point was assayed in duplicate wells, incubated for 1 hr at room temperature and washed; then, 50 ⁇ l_ of a 1 :1000 dilution of biotin conjugated W6/32 antibody was added to each well and incubated for 1 hr at room temperature.
  • streptavidin-HRP horseradish peroxidase conjugate
  • ABTS substrate ABTS substrate
  • All sample signals were plotted as % of control.
  • Control tetramer was added to serum, mixed, and immediately removed for assaying by ELISA.
  • the stability half-life for the 264-peptide-HLA-A2 tetramerat4°C was greater than 72 hrs, while at 37 °C the stability half-life was approximately 10 hrs.
  • Fig. 4 illustrates the complete structure of the peptide-HLA-A2 tetramer immunogen, as obtained from the tetramer peak of Fig. 2, and recognition of the peptide-HLA epitope by a TCR mimic.
  • FIG. 5 illustrates the development of an ELISA assay to screen mouse bleeds to determine if there are antibodies specific to the peptide-of-interest-HLA-molecule complex present.
  • the schematic illustrates two newly developed screening assays for detection of anti-peptide-HLA specific antibodies from immunized mouse serum.
  • Assay #2 evolved from Assay #1.
  • Fig. 6 illustrates the results from an ELISA of 6 individual bleeds from Balb/c mice immunized with tetramers of 264 peptide-HLA-A2, using assay format #2 as described in Fig. 5.
  • Mice male and female Balb/c; 13 and 12 groups, respectively) were immunized 4 times every 2 weeks by subcutaneous injection in the region behind the head or in the side flanks with 100 ⁇ l containing 50 ⁇ g of 264 peptide-HLA-A2 tetramer and 25 ⁇ g of QuilA (adjuvant). Bleeds were taken at 3 weeks, 5 weeks and just prior to sacrificing the mice.
  • Fig. 6 shows screening results from mice sera after 3 immunizations (week 5).
  • Detection of polyclonal antibodies reactive for 264 peptide-HLA-A2 tetramer was carried out by ELISA (assay #2 described in Fig. 5).
  • the ELISA results demonstrate that a 264 peptide-HLA-A2 antibody response can be elicited in both male (I3M1-M3) and female (I2M1-M3) mice using the immunization protocol and screening assay of the presently disclosed and claimed invention.
  • Fig. 7 illustrates development of cell-based direct and competitive binding assays for screening mouse bleeds for antibodies specific to the peptide-of-interest-HLA-molecule complex.
  • the schematic illustrates two newly developed cell-based screening assays for detection of anti-peptide-HLA specific antibodies from immunized mouse serum.
  • Two cell based assays were developed: Assay #3 is a Cell-based direct binding approach and Assay #4 is a Cell-based competitive binding approach which uses soluble monomer or tetramer peptide-HLA-A2 complexes as competitors and non-competitors. The sensitivity of Assay # 4 is much greater than Assay #3.
  • Fig. 8 illustrates peptide loading of T2 cells.
  • T2 cells HLA-A2 + , TAP deficient
  • BB7 antibody specific for properly folded HLA-A2, ATCC #HB-82
  • 5x10 5 T2 cells were incubated in 100 ⁇ l of buffer containing 100 ⁇ g of either 264 or elF4G peptide for 6 hours at 37 0 C, washed and stained with 0.5 ⁇ g BB7.2 for 20 min. Negative control cells were not pulsed with peptide.
  • Fig. 9 illustrates an example of the cell-based direct binding assay of Fig. 7, and contains the results of staining of 264 peptide-loaded T2 cells with the I3M2 mouse bleed.
  • T2 cells HLA-A2 + , TAP deficient
  • I3M2 immunoglobulin-associated mammase
  • Fig. 9 illustrates an example of the cell-based direct binding assay of Fig. 7, and contains the results of staining of 264 peptide-loaded T2 cells with the I3M2 mouse bleed.
  • T2 cells HLA-A2 + , TAP deficient
  • 5x10 5 T2 cells were incubated in 100 ⁇ l of buffer containing 100 ⁇ g of either 264 or elF4G peptide for 6 hours at 37°C, washed and stained with 100 ⁇ l of a 1 :200 dilution of preabsorbed sera for 20 min. After staining, the reaction was washed once with 3-4 ml wash buffer and resuspended in approximately 100 ⁇ l of wash buffer containing 0.5 ⁇ g of FITC-conjugated goat anti-mouse IgG (Caltag, Burlingame, CA). Cells were washed as above and resuspended in 0.5 ml wash buffer for analysis.
  • Fig. 10 illustrates that pre-bleed samples (mice bleeds taken prior to immunization) show no sign of reactivity to T2 cells pulsed with either the 264- or elF4G peptides.
  • T2 cells HLA-A2 + , TAP deficient
  • 5x10 5 T2 cells were incubated in 100 ⁇ l of buffer containing 100 ⁇ g of either 264 or elF4G peptide for 6 hours at 37 0 C, washed and stained with 100 ⁇ l of a 1 :200 dilution of sera for 20 min. After staining the reaction was washed once with 3-4 ml wash buffer and resuspended in approximately 100 ⁇ l of wash buffer containing 0.5 ⁇ g of FITC-conjugated goat anti-mouse IgG (Caltag, Burlingame, CA). Cells were washed as above and resuspended in 0.5 ml wash buffer for analysis.
  • FIG. 11 depicts development of assays to screen hybridomas to determine if they are producing anti-HLA-peptide specific antibodies.
  • the schematic illustrates two ELISA-based screening assays for detection of anti-peptide-HLA specific monoclonal antibodies from culture supernatant.
  • Assay #1 is an ELISA-based direct binding approach that coats wells of a 96-well plate with 0.5 ⁇ g of either specific or irrelevant tetramer.
  • Hybridoma cell culture supernatant (50 ⁇ l_) was assayed in duplicate by addition to an antibody coated plate blocked with 5% milk for 1hr at room temperature.
  • Assay #2 is an ELISA that uses a competitive binding approach in which cell culture supernatant is incubated in the presence of either 300 ng of competitor or non-competitor (soluble monomer or tetramer peptide-HLA-A2 complexes) in wells on 96-well plates that have been coated with 100 ng of specific peptide-HLA-A2 tetramer and blocked with 5% milk.
  • Fig. 12 illustrates a competitive ELISA assay for evaluation of individual hybridomas (I3M1) reactive against 264p-HLA-A2 complexes.
  • Light grey bar addition of 264p-HLA-A2 tetramer (competitor, 0.3 ⁇ g);
  • Dark grey bar addition of elF4Gp-HLA-A2 tetramer (non-competitor, 0.3 ⁇ g).
  • Hybridoma cell culture supernatant 50 ⁇ L was incubated in the presence of 300 ng of competitor (264 peptide-HLA-A2 tetramer) or non-competitor (elF4G peptide-HLA-A2 tetramer) in wells on a 96-well plate coated previously with 100 ng of 264 peptide-HLA-A2 tetramer. After 1 hr incubation, the plate was washed, probed with goat anti-mouse HRP, developed using TMB or ABTS and read at 450 or 405 nm, respectively.
  • competitor 264 peptide-HLA-A2 tetramer
  • elF4G peptide-HLA-A2 tetramer non-competitor
  • Results were calculated by dividing the absorbance read in the presence of non-competitor by the absorbance read in the presence of competitor [elF4G/264]. Ratios of 2 or greater were considered to be positive, and hybridoma clones with this desired ratio were selected for further analysis.
  • Fig. 12 shows 4 different hybridoma supematants (M1/3-A5, M1/3-F11 , M1/4-G3, and M1/6-A12) with a specific binding ratio [elF4G/264] of 2 or greater. [0043] Fig.
  • Hybridoma cell culture supernatant 50 ⁇ l_ was incubated without any tetramer addition or in the presence of 300 ng of competitor (264 peptide-HLA-A2 tetramer) or non-competitor (elF4G peptide-HLA-A2 tetramer) in wells on a 96-well plate coated previously with 100ng of 264 peptide-HLA-A2 tetramer.
  • Fig. 13 illustrates three different hybridoma supematants with favorable elF4G/264 ratios. These include M1-1 F8, M1-2G5, M1-6C7 and M3-2A6, which were selected for further analysis.
  • Fig. 14 illustrates the characterization of monoclonal antibody I3.M3-2A6 by the cell- based competitive binding assay.
  • T2 cells HLA-A2 + , TAP deficient
  • I3.M3-2A6 immunoglobulin-like cells
  • immunogen 264 tetramers
  • tetramer complex that would compete with specific binding to 264p-HLA-A2
  • tetramer complex that would not compete with specific binding (elF4Gp)
  • no tetramer to demonstrate that the antibody specifically recognizes the 264p-HLA-A2 complex on the cell surface.
  • Cell supernatant was pre-absorbed against 20 ⁇ g of soluble Her2/neu-peptide-HLA-A2 complexes, diluted 1 :200 and added (100 ⁇ l) to a tube containing 1 ⁇ g of either 264p-HLA-A2 tetramer (competitor) or elF4Gp-HLA-A2 tetramer (non competitor) for 15 minutes at room temperature.
  • 5x10 5 T2 cells were incubated in 100 ⁇ l of buffer containing 100 ⁇ g of 264 peptide for 6 hours at 37 0 C, washed, resuspended in 100 ⁇ l, and added to the preabsorbed/tetramer treated supernatant for 20 minutes at room temperature.
  • reaction was washed once with 3-4 ml wash buffer and resuspended in approximately 100 ⁇ l of wash buffer containing 0.5 ⁇ g of FlTC-conjugated goat anti-mouse IgG (Caltag, Burlingame, CA). Cells were washed as above and resuspended in 0.5 ml wash buffer for analysis. Samples were collected on a FACScan (BD biosciences, San Diego, California) and analyzed using Cell Quest software (version 3.3, BD Biosciences).
  • Fig. 15 illustrates a broad outline of the epitope discovery technology described in detail in Hildebrand et al. (US Patent Application Publication No. US 2002/0197672A1 , published December 26, 2002, previously incorporated herein by reference).
  • Soluble HLA- secreting transfectants are created in a cancerous or diseased cell line of interest.
  • a normal (i.e., noncancerous or non-diseased) cell line also transfected with a construct encoding the soluble HLA is grown and cultured.
  • Soluble HLA molecules are collected from both cell lines, and the peptides are eluted.
  • Mass spectrometric maps are generated comparing cancerous (or diseased) peptides to normal peptides. Differences in the maps are sequenced to identify their precise amino acid sequence, and such sequence is utilized to determine the protein from which the peptide was derived (i.e., its "source protein").
  • Fig. 16 illustrates the stability of the elF4Gp-HLA-A2 tetramers. Tetramer stability was assessed in mouse serum at 37°C (•) and at 4°C ( ⁇ ) using the conformational antibodies BB7.2 and W6/32.
  • elF4G peptide-tetramer complex 25 ⁇ g of elF4G peptide-tetramer complex was added to 5 mL of 100% mouse serum and incubated at 4°C and 37°C for 75 hr. At designated times, 50 ⁇ L aliquots of sample were removed and stored at -20 0 C and remained frozen until completion of the experiment. To determine the integrity of the peptide-HLA tetramer, samples were evaluated using a sandwich ELISA and two antibodies, BB7.2 and W6/32, that bind only conformationally intact peptide-HLA tetramers.
  • An ELISA protocol was developed using 96-well plates (Nunc maxisorb plates) that were coated O/N at 4 0 C with 0.5 ⁇ g of BB7.2, washed with buffer (PBS/0.05% Tween-20) and then blocked with 200 ⁇ l of 5% milk for 1hr at room temperature. Sample (50 ⁇ L) from each time point was added in duplicate wells, incubated for 1 hr at room temperature, washed, and then 50 ⁇ L of at 1 :1000 dilution of biotin conjugated W6/32 antibody was added to each well and incubated for 1 hr at room temperature.
  • streptavidin-HRP horseradish peroxidase conjugate
  • ABTS substrate ABTS substrate
  • All sample signals were plotted as % of control.
  • Control tetramer was added to serum, mixed, and immediately removed for assaying by ELISA.
  • the half-life of stability for the elF4G-peptide-HLA-A2 tetramer at 4°C was greater than 72 hrs while at 37°C the half-life was approximately 40 hrs. [0047] Fig.
  • FIG. 17 illustrates the results from an ELISA of bleeds from 6 individual Balb/c mice immunized with tetramers of elF4Gp-HLA-A2.
  • Mouse samples from left to right are I8.M1 , I8.M2, I8.M3, 18.M4, I8.M5, I8.M6.
  • P53-264 264p-HLA-A2 monomer (0.5 ⁇ g/well)
  • Eif4G elF4Gp-HLA-A2 monomer (0.5 ⁇ g/well)
  • Her2/neu Her2/neu peptide-HLA-A2 monomer (0.5 ⁇ g/well).
  • mice female Balb/c
  • mice were immunized 4 times every 2 weeks by subcutaneous injection in the region behind the head or in the side flanks with 100 ⁇ l containing 50 ⁇ g of elF4G peptide-HLA-A2 tetramer and 25 ⁇ g of QuilA (adjuvant).
  • Bleeds were taken at 3 weeks, 5 weeks and just prior to sacrificing mice.
  • Fig. 17 shows results from mice sera after 3 immunizations (week 5).
  • ELISA Detection of polyclonal antibodies reactive for el F4G peptide-HLA-A2 tetramer was carried out by ELISA (assay #2 described in Fig. 5). The ELISA results demonstrate that a 264 peptide-HLA-A2 antibody response can be elicited in female Balb/c (I8.M1-M6) mice using the immunization protocol and screening assay of the presently disclosed and claimed invention.
  • Fig. 18 illustrates T2 cell direct binding assay performed according to the method of Fig. 7.
  • T2 cells HLA-A2 + , TAP deficient
  • BB7.2 antibody specific for HLA-A2
  • T2 cells were incubated in 100 ⁇ l of buffer containing 100 ⁇ g of either 264 or elF4G peptide for 6 hours at 37 C C, washed and stained with 0.5 ⁇ g BB7.2 for 20 min. Negative control cells were not pulsed with peptide.
  • Fig. 19 illustrates the results of staining of elF4Gp-loaded T2 cells with a bleed from an elF4Gp-HLA-A2 immunized mouse.
  • T2 cells HLA-A2 + , TAP deficient
  • I8M2 immunoglobulinous mesenchymal cells
  • Fig. 19 illustrates the results of staining of elF4Gp-loaded T2 cells with a bleed from an elF4Gp-HLA-A2 immunized mouse.
  • 5x10 5 T2 cells were incubated in 100 ⁇ l of buffer containing 100 ⁇ g of either elF4G or 264 peptide for 6 hours at 37 0 C, washed and stained with 100 ⁇ l of a 1:200 dilution of preabsorbed sera for 20 min. After staining, the reaction was washed once with 3-4 ml wash buffer and resuspended in approximately 100 ⁇ l of wash buffer containing 0.5 ⁇ g of FITC-conjugated goat anti-mouse IgG (Caltag, Burlingame, CA). Samples were collected on a FACScan (BD biosciences, San Diego, California) and analyzed using Cell Quest software (version 3.3, BD Biosciences).
  • elF4G peptide-pulsed T2 cells (open trace) shifted significantly to the right of the 264 peptide pulsed T2s when stained, indicating the presence of elF4Gp-HLA-A2 specific antibodies from immunized mice.
  • Fig. 20 illustrates the results of a T2 cell-competitive binding assay, the method of which is outlined in Fig. 7.
  • T2 cells HLA-A2 + , TAP deficient
  • I8M2 immunoglobulin-associated antigen
  • elF4Gp tetramers monomer complex that would compete with specific binding to elF4Gp-HLA-A2; (2) monomer complex that would not compete with specific binding (264p); or (3) no monomer, to demonstrate that the antibody specifically recognizes the elF4Gp-HLA-A2 complex on the cell surface.
  • Cell supernatant was pre-absorbed against 20 ⁇ g of soluble Her2/neu-peptide-HLA-A2 complexes, diluted 1 :200 and added (100 ⁇ l) to tube containing 1 ⁇ g of either elF4Gp-HLA-A2 monomer (competitor) or 264p-HLA-A2 monomer (non competitor) for 15 minutes at room temperature.
  • 5x10 5 T2 cells were incubated in 100 ⁇ l of buffer containing 100 ⁇ g of eIF4G peptide for 6 hours at 37°C, washed, resuspended in 100 ⁇ l, and added to the preabsorbed/monomer treated supernatant for 20 minutes at room temperature.
  • reaction was washed once with 3-4 ml wash buffer and resuspended in approximately 100 ⁇ l of wash buffer containing 0.5 ⁇ g of FITC-conjugated goat anti-mouse IgG (Caltag, Buriingame, CA). Cells were washed as above and resuspended in 0.5 ml wash buffer for analysis. Samples were collected on a FACScan (BD biosciences, San Diego, California) and analyzed using Cell Quest software (version 3.3, BD Biosciences).
  • Fig. 21 illustrates the results of another T2 cell-competitive binding assay similar to the one described in Fig. 20, except that the competitor mixed with the mouse bleed prior to reacting with the T2 cells was in the form of a tetramer rather than a monomer.
  • T2 cells HLA-A2 + , TAP deficient
  • T2 cells were stained with pre-absorbed, diluted serum from mouse I8M2 (immunized with elF4Gp tetramers) in the presence of (1 ) tetramer complex that would compete with specific binding to eIF4Gp-HLA-A2; (2) tetramer complex that would not compete with specific binding (264p); or (3) no tetramer, to demonstrate that the antibody specifically recognizes the eIF4Gp-HLA-A2 complex on the cell surface.
  • Cell supernatant was pre-absorbed against 20 ⁇ g of soluble Her2/neu-peptide-HLA-A2 complexes, diluted 1 :200 and added (100 ⁇ l) to tube containing 1 ⁇ g of either elF4Gp-HLA-A2 tetramer (competitor) or 264p-HLA-A2 tetramer (non competitor) for 15 minutes at room temperature.
  • 5x10 5 T2 cells were incubated in 100 ⁇ l of buffer containing 100 ⁇ g of elF4G peptide for 6 hours at 37°C, washed, resuspended in 100 ⁇ l, and added to the preabsorbed/tetramer treated supernatant for 20 minutes at room temperature.
  • reaction was washed once with 3-4 ml wash buffer and resuspended in approximately 100 ⁇ l of wash buffer containing 0.5 ⁇ g of FITC-conjugated goat anti-mouse IgG (Caltag, Burlingame, CA). Cells were washed as above and resuspended in 0.5 ml wash bufferfor analysis. Samples were collected on a FACScan (BD biosciences, San Diego, California) and analyzed using Cell Quest software (version 3.3, BD Biosciences).
  • elF4G peptide-competition resulted in a significant shift of the T2 cell trace (thick line, open trace) to the left (towards the origin), while the 264 peptide competition (thin line, open trace) resulted in a much smaller shift away from T2s stained in the absence of tetramer, indicating the presence of polyclonal antibodies with a high degree of specificity for the elF4Gp-HLA-A2 complex.
  • Fig. 22 illustrates the binding specificity of mAb 4F7, as determined by ELISA.
  • a 96-well plate was coated with 0.5 ⁇ g of specific monomer (elF4G-peptide-HLA-A2) and non-specific monomers (264, VLQ and TMT peptide-HLA-A2 monomers).
  • the VLQ and TMT peptides are derived from the human beta-chorionic gonadotropin protein, as described in detail herein after. After blocking wells with 5% milk, 100 ng of 4F7 antibody was added to each well and incubated for 1 hr at room temperature.
  • Fig. 23 illustrates 4F7 TCR mimic binding affinity and specificity evaluated by surface plasmon resonance (BIACore).
  • SPR BIACore
  • Various concentrations of soluble monomer peptide-HLA-A2 (10, 20, 50, and 100 nM) were run over a 4F7 coated chip (4F7 coupled to a biosensor chip via amine chemistry), and then BIACore software was used to best fit the binding curves generated.
  • the affinity constant of 4F7 mAb for its specific ligand was determined at 2 x10 "9 M.
  • T2 cells HLA-A2 + , TAP deficient
  • 4F7 immunoglobulin-A2 + , TAP deficient
  • 5x10 5 T2 cells were incubated in 100 ⁇ l of buffer containing 100 ⁇ g of elF4G, 264, or TMT peptide for 6 hours at 37 0 C, washed and stained with 100 ⁇ l of 4F7 culture supernatant for 20 min.
  • Fig. 25 illustrates that 4F7 TCRm detects endogenous elF4G (720) peptide-HLA-A2 complexes on an HLA-A2 positive tumor cell line but not on a normal mammary epithelial cell line.
  • a human mammary epithelial cell line (NHMEC) and (B) a human breast carcinoma cell line (MDA-MB-231 ) were grown in medium specified by the ATCC and were detached using 1Xtrypsin/EDTA (0.25% trypsin/2.21 mM EDTA in HBSS without sodium bicarbonate, calcium and magnesium) (Mediatech, Herndon, VA). Cells were washed and then stained with 5 ⁇ g/ml of isotype control mAb or 4F7 TCRm-FITC in PBS/0.5% FBS/2mM EDTA (staining/wash buffer). FACS analysis was performed on a FACScan (BD Biosciences, San Diego, CA). The results from flow cytometric studies are expressed as mean fluorescence intensity (MFI) in histogram plots.
  • MFI mean fluorescence intensity
  • Fig. 26 illustrates that purified 4F7 mAb binds elF4Gp-HLA-A2 complexes on human breast carcinoma cell line MCF-7.
  • 5x10 5 MCF-7 cells were incubated in 100 ⁇ l of buffer containing 100 ⁇ l of 4F7 culture supernatant plus 1 ⁇ g of either el F4Gp-HLA-A2tetramer (competitor) or264p-HLA-A2tetramer(non competitor) or no addition for 15 minutes at room temperature. After staining, the reactions were washed once with 3-4 ml wash buffer and resuspended in approximately 100 ⁇ l of wash buffer containing 0.5 ⁇ g of PE-conjugated goat anti-mouse IgG (Caltag, Burlingame, CA). Cells were washed as above and resuspended in 0.5 ml wash buffer for analysis.
  • Fig.26-A demonstrate 4F7 binding specificity for endogenous peptide elF4Gp-HLA-A2 complexes on MCF-7 tumor cells.
  • panel B it is shown that 4F7 and BB7.2 do not bind to HLA-A2 negative BT-20 breast cancer cells, further supporting the claim for 4F7 monoclonal antibody binding specificity for elF4G peptide presented in the context of HLA-A2.
  • Fig. 27 illustrates staining of MDA-MB-231 cells with 4F7 mAb (50 ng) in the absence or presence of soluble peptide-HLA-A2 monomers including elF4Gp (competitor; 25 nM), 264p (non-competitor; 25 nM) or Her2/neu peptide (non-competitor; 25 nM).
  • soluble peptide-HLA-A2 monomers including elF4Gp (competitor; 25 nM), 264p (non-competitor; 25 nM) or Her2/neu peptide (non-competitor; 25 nM).
  • 5x10 5 MDA-MB-231 cells were incubated in 100 ⁇ l of buffer containing 100 ⁇ l of 4F7 culture supernatant plus 25 nM of elF4Gp-HLA-A2 tetramer (competitor), 264p-HLA-A2 tetramer or Her-2/neu-HLA-A2 (non competitors) or no addition for 15 minutes at room temperature. After staining, the reactions were washed once with 3-4 ml wash buffer and resuspended in approximately 100 ⁇ l of wash buffer containing 0.5 ⁇ g of PE-conjugated goat anti-mouse IgG (Caltag, Burlingame, CA).
  • Fig.27-A demonstrates 4F7 binding specificity for endogenous elF4Gp-HLA-A2 complexes on MDA-231 tumor cells. Binding of the 4F7 TCR mimic to MDA-MB-231 cells was significantly reduced (see leftward shift with peak) in the presence of 25nM of competitor (elF4Gp-HLA-A2 monomer).
  • Fig. 28 illustrates endogenous elF4G peptide presented by HLA-A2 molecules on the surface of HIV-1 infected and non-infected human CD4+ T cells.
  • HIV-1 exposed CD4+ T cells were gated based on p24 expression and analyzed separately as (D-F) infected-p24 positive (middle panels) or (G-I) non-infected-p24 negative (bottom panels).
  • Fig. 29 illustrates time-dependent expression of elF4G( 720 ) peptide-HLA-A2 complexes on HIV-infected cells.
  • Human CD4+ T cells were infected with HIV-1 (strain Ba-L) at an MOl of 1.0 and stained with (A) 4F7 TCRm or (B) isotype control on days 3 thru 9 post-infection.
  • Non-infected cells p24 negative
  • Non-infected cells are represented by gray bars.
  • HIV-1 infected cells p24 positive
  • black bars are represented by black bars.
  • Fig. 30 illustrates HLA-peptide tetramer inhibition of 4F7 staining of HIV-1 infected cells.
  • Human CD4+ T cells were infected with HIV-1 (strain Ba-L) at an MOI of 1.0 and stained with mAb 4F7 TCRm on (A) day 4 Pl and (B) day 5 Pl in the presence of elF4G( 720 )-HLA-A*0201 -tetramer (competitor), p53( 264 )-HLA-A*0201 -tetramer (non-competitor) or VLQ( 44 )-HLA-A*0201 tetramer (non-competitor) or without tetramer addition.
  • Results are from staining p24 positive CD4+ T cells and are presented as % elF4G( 720 ) expression.
  • Fig. 31 illustrates the characterization of 1 B8 TCRm binding specificity.
  • HLA-A2 tetramer complexes were loaded with 0.1 ⁇ g of each of the following peptides: Her2 (369-377; KIFGSLAFL (SEQ ID NO:3)), VLQ (44-52; VLQGVLPAL (SEQ ID NO:5)), eIF4G (720-748; VLMTEDIKL (SEQ ID NO:2)) and TMT (40-48; TMTRVLQGC (SEQ ID NO:4)).
  • Recombinant proteins were detected by staining with 1 B8 TCR mAb specific for Her-2 369 -A2 complex (A), 3F9 TCRm mAb specific for TMT 40 -A2 complex (B) and BB7.2 mAb specific for HLA-A2.1 (C) followed by ELISA as described herein. Data are representative of three independent experiments.
  • Fig. 32 illustrates the characterization of 1 B8 TCRm binding detection sensitivity.
  • T2 cells (5 x 10 5 ) were incubated in AIM-V medium (Invitrogen, Carlsbad, CA) and loaded with 10 mM Her2 369 , elF4G 720 , TMT 40 peptide or no peptide. After 4 hr, the cells were washed to remove excess peptide and stained with 0.5 ⁇ g/ml of 1 B8 TCRm mAb antibody. Bound mAb was detected using the PE-conjugated goat anti-mouse IgG heavy chain specific polyclonal Ab. Filled area represents T2 cells stained with IgG 1 isotype control.
  • T2 cells were treated with acid to remove endogenous peptide bound to HLA-A2, pulsed with 20 irrelevant peptides or 20 irrelevant peptides plus the Her2 (369) peptide and then stained with 1 B8 TCRm mAb.
  • T2 cells (5 x 10 6 /mL) were acid stripped (0.131 M citric acid, 0.067M Na 2 HPO 4 , pH 3.3) for 45 seconds, washed twice with 50 ml of RPMI supplemented with 2mM Hepes and resuspended at 3.3 x 10 6 /ml in 30 ⁇ g/mL of ⁇ 2-microglobulin (Fitzgerald Industries, Concord, MA) (23, 24).
  • Fig. 33 illustrates that 1 B8 detects endogenous Her2/neu peptide-HLA-A2 complexes on HLA-A2 positive tumor cells.
  • All adherent tumor cell lines were grown in medium specified by the ATCC and were detached using 1X trypsin/EDTA (0.25% trypsin/2.21 mM EDTA in HBSS without sodium bicarbonate, calcium and magnesium (Mediatech, Herndon, VA). Cells were washed and then stained with 5mg/ml of 1 B8 TCRm in PBS/0.5% FBS/2mM EDTA (staining/wash buffer), and the bound TCRm was detected by subsequent incubation with PE-labeled goat anti-mouse IgG.
  • FACS analysis was performed on a FACScan (BD Biosciences, San Diego, CA).
  • the results from flow cytometric studies are expressed either as mean fluorescence intensity (MFI) in histogram plots or as the mean fluorescence intensity ratio (MFIR), the ratio between the MFI of cells stained with the selected mAb and the MFI of cells stained with the isotype-matched mouse Ig.
  • MFI mean fluorescence intensity
  • MFIR mean fluorescence intensity ratio
  • Generation of MFRI values normalizes background staining between the cell lines.
  • Human tumor cell lines were stained with 0.5 ⁇ g of isotype control mAb (thin dark gray line), 3F9 TCRm mAb (thick black line) and 1 B8 TCRm mAb (thick gray line).
  • Fig. 34 illustrates HLA-peptide specific inhibition of human tumor cell staining and CTL killing.
  • MDA-MB-231 cells (5 x 10 5 ) were incubated for 1 h with 0.5 ⁇ g/ml of 1B8 TCRm mAb in the presence of 0.1 or 1.0 ⁇ g/ml of Her2/neu peptide-HLA-A2 tetramer, 1.0 ⁇ g/ml TMT peptide-HLA-A2 tetramer or no tetramer. After staining, the reactions were washed once and resuspended in 100 ⁇ l of wash buffer containing 0.5 ⁇ g of PE-conjugated goat anti-mouse IgG.
  • CTLs (1x10 6 ) were restimulated in 2 ml cultures with 0.2 x 10 6 irradiated Jurkat-A2.1 cells (20,000 rad) that were preincubated with Her-2/neu peptide (15 ⁇ M). Irradiated (3000 rad) C57BL/6 spleen cells (5 x 10 5 ) were added as fillers. Restimulation medium was complete RPMI containing 2% (v/v) supernatant from concanavalin-A stimulated rat spleen cells. T2 cells pulsed with Her2 (369) peptide or not pulsed were incubated with CTL in a 6h 51 Cr release assay at an E:T ratio of 10:1.
  • C MDA-231 cells were either not treated (white bars) or pre-treated for 24 h with rIFN- ⁇ (20 ng/ml) and TNF- ⁇ (3 ng/ml) (black bars).
  • Anti-Her2 (36g) -A2 CTL activity was then evaluated in the absence or presence of 0.5 ⁇ g of 1B8 TCRm or BB7.2 mAbs in a 6 h 51 Cr release assay at an E:T ratio of 10:1. All CTL assays were done in triplicate from 3 independent experiments.
  • T2 cells pulsed with peptides and tumor cells (MDA-MB-231 , Saos-2, MCF-7, SW620 and COLO205) were incubated with 150 ⁇ Ci of 51 Cr-sodium chromate for 1 hour at 37°C. Cells were washed three times and resuspended in complete RPMI medium.
  • 51 Cr-labeled target cells (10 4 ) were incubated at a 10:1 CTL:target ratio in a final volume of 200 ⁇ l in U-bottomed 96-well microtiter plates. Previous studies have shown optimal killing at a 10:1 CTL:tumor cell ratio (Lustgarten et al., 1997).
  • 5x10 5 MDA-MB-231 cells were incubated in 100 ⁇ l of buffer containing 100 ⁇ l of 1 B8 culture supernatant for 15 minutes at room temperature.
  • Fig. 35 demonstrates that 1 B8 TCR mimic has dual specificity and does not bind to Her-2/neu peptide alone.
  • Fig. 36 illustrates the expression of Her2/neu protein in human tumor cell lines.
  • Tumor cell lines were evaluated for the expression of Her2/neu protein by ELISA and flow cytometry.
  • Cellular levels of Her2/neu were determined by preparing tumor cell lysates and quantifying Her2/neu with the c-erbB2/c-neu Rapid Format ELISA (CalBiochem) according to the manufacturer's instructions.
  • Her2/neu protein was detected in a sandwich ELISA using two mouse monoclonal antibodies. The detector antibody was bound to horseradish peroxidase-conjugated streptavidin and color was developed by incubation with TMT substrate (Pierce). The concentration of Her2/neu in the samples was quantified by generating a standard curve using known concentrations of Her2/neu provided in the kit.
  • (A) Tumor cell lysate was prepared from each line and analyzed for Her2/neu levels (ng/10 6 cells) by ELISA.
  • Fig. 37 illustrates expression of HLA-A*0201 and HLA-Her2 (36g) peptide complexes on human tumor cell lines and CTL lysis of human tumor cell lines.
  • Tumor cell lines were evaluated forthe expression of HLA-A2 and Her2 (36g) -A2 complex expression.
  • Tumor cells were stained with (A) anti-HLA-A2.1 mAb (BB7.2) and (B) 1 B8 TCRm. Results are plotted as mean fluorescence intensity ratio (MFIR) with standard deviation from three different experiments.
  • MFIR mean fluorescence intensity ratio
  • C The specificity of the Her2 (369) -A2 reactive CTL line was evaluated against human tumor cell lines not treated. CTL cytotoxic activity was evaluated in a 6 h 51 Cr release assay at an E:T ratio of 10: 1 as described herein above. Regression analysis was determined from flow cytometric and cytotoxic data for MDA-MB-231 , Saos-2, MCF-7, SW620 and Colo205 tumor cell lines. The analyses did not reach significance for peptide-A2 vs. total Her2, tumor lysis vs. total Her2, peptide-A2 vs. HLA-A2, tumor lysis vs. HLA-A2 and peptide-A2 vs. tumor lysis.
  • Fig. 38 illustrates expression of HLA-A*0201 molecules and HLA-Her2 (369) peptide complexes after cytokine treatment of human tumor cell lines.
  • Human tumor cell lines were pre-treated for 24 h with rIFN- ⁇ (20 ng/ml) and TNF- ⁇ (3 ng/ml) and stained with (A) anti-A2.1 BB7.2 or (B) 1 B8 TCRm mAbs. Results are plotted as mean fluorescence intensity ratio (MFIR) with standard deviation from three different experiments.
  • MFIR mean fluorescence intensity ratio
  • C The specificity of the Her2 (369) -A2 reactive CTL line was evaluated against human tumor cell lines pre-treated for 24 h with rlFN- ⁇ (20 ng/ml) and TNF- ⁇ (3 ng/ml). CTL cytotoxic activity was evaluated in a 6 h 51 Cr release assay at an E:T ratio of 10:1 as described herein above.
  • Fig. 39 illustrates the characterization of binding specificity for 3.2G1 TCRm.
  • A Supernatant from hybridoma 3.2G1 was used to probe wells coated with HLA-A2 tetramer refolded with the different peptides indicated. Bound antibody was detected with a goat anti-mouse peroxidase conjugate and developed using ABTS.
  • B Hybridoma supernatant was used to stain 5 x 10 5 T2 cells pulsed with the peptides indicated or no peptide. After washing, cells were probed with a goat anti-mouse secondary antibody, washed and analyzed by flow cytometry.
  • T2 cells were pulsed with 20 ⁇ g/ml GVL peptide and then stained with a preincubated mixture of 1 ⁇ g/100 ⁇ l 3.2G1 TCRm and either GVL tetramer or VLQ tetramer. The tetramer and antibody were preincubated for 40 min before addition to the pulsed cells. Tetramer concentrations ( ⁇ g/stain) ranged from 1 to 0.01 for GVL and 1 to 0.1 for VLQ.
  • Fig. 40 illustrates CDC of peptide-pulsed T2 cells.
  • T2 cells were pulsed with the various peptide mixes for 4 hours, washed and dispensed into wells in 96 well plates at 3 x 10 5 cells/well. Antibody and rabbit complement were added and the reactions allowed to proceed for 4 hours, and then cytotoxicity was analyzed using the LDH assay from Promega.
  • A T2 cells were pulsed with mixes of GVLTMT peptide at the concentrations in mg/ml shown in the legend at the top of the figure for 4 hours before incubating with 2.5 ⁇ g/ml 3.2G1 TCRm or BB7.2 antibody and rabbit complement.
  • T2 cells were pulsed with varying levels of peptide diluted 1 :2 from 50 ⁇ g/ml to 0.1 ⁇ g/ml before incubating with 10 ⁇ g/ml 3.2G1 TCRm or BB7.2 antibody.
  • C T2 cells were pulsed with 20 ⁇ g/ml peptide before addition of a mix containing varying amounts of antibody and either GVL or VLQ tetramer at a final concentration of 2 ⁇ g/ml tetramer. Final antibody concentration was varied from 9 to 0.1 ⁇ g/ml and corresponds to color coding shown in the legend for (C). Bars representing standard error are shown for (A), (B) and (C).
  • Fig.41 illustrates that 3.2G1 detects endogenous GVL-HLA-A2 complexes on human tumor lines, lmmunofluorescent staining was carried out using 3.2G1 , BB7.2, and isotype control antibodies on four human tumor lines. 3.2G1 detects various levels of GVL/A2 on the cells' surface and does not stain the HLA-A2 negative cell line BT20.
  • Fig. 42 illustrates CDC and ADCC of MDA-MB-231 cells by 3.2G1 TCRm.
  • A Complement-dependent cytolysis was carried out using 2 x 10 5 MDA-MB-231 cells well in a 96 well plate.
  • ADCC reactions included 2 x 10 5 MDA-MB-231 cells/well and IL-2 stimulated human PBMC preparations at an E:T ratio of 30:1 with 10 ⁇ g/ml 3.2G1. Lysis was determined using the LDH assay.
  • FIG. 43 illustrates that the 3.2GITCRm prevents tumor growth in athymic nude mice.
  • Female athymic mice were subcutaneously injected between the shoulders with 5x10 6 MDA-MB-231 cells in 0.2 ml containing 1 :1 mixture of medium and Matrigel. Mice were given tumor cells and treated i.p.
  • mice received one injection a week (50 ⁇ g/injection) for three weeks. Tumor growth was initially seen in mice treated with IgG 23 control antibody at week 6 and by week 10 the tumor volume had increased >30-fold ( ⁇ ). In contrast, no tumor growth was seen in mice treated with the 3.2G1 antibody ( ⁇ ). Tumors were monitored and final scoring was tabulated at 69 days after implant at which time all tumors were at least 6 mm in diameter and no new tumors had appeared for 21 days.
  • Fig. 44 illustrates that the 3.2G1 TCRm can be used therapeutically to treat athymic nude mice with established tumors.
  • Female athymic mice were subcutaneously injected in the right flank with 1x10 7 MDA-MB-231 breast cancer cells containing 1 :1 mixture of medium and Matrigel. After 10 days of growth, tumors were measured using calipers with the mean tumor volume (mm 3 ) ranging between 62 and 105 mm 3 .
  • Fig. 45 illustrates a protocol for the generation of peptide-MHC Class I specific TCR mimics of the present invention.
  • Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein.
  • the foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2 nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (1989) and Coligan et al. Current Protocols in Immunology (Current Protocols, Wiley lnterscience (1994)), which are incorporated herein by reference.
  • isolated polynucleotide and isolated nucleic acid segment shall mean a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the "isolated polynucleotide” or “isolated nucleic acid segment” (1) is not associated with all or a portion of a polynucleotide in which the "isolated polynucleotide” or “isolated nucleic acid segment” is found in nature, (2) is operably linked to a polynucleotide which it is not linked to in nature, or (3) does not occur in nature as part of a larger sequence.
  • isolated protein means a protein of cDNA, recombinant RNA, or synthetic origin or some combination thereof, which by virtue of its origin, or source of derivation, the "isolated protein” (1) is not associated with proteins found in nature, (2) is free of other proteins from the same source, e.g., free of murine proteins, (3) is expressed by a cell from a different species, or, (4) does not occur in nature.
  • polypeptide as used herein is a generic term to refer to native protein, fragments, or analogs of a polypeptide sequence. Hence, native protein, fragments, and analogs are species of the polypeptide genus.
  • naturally-occurring refers to the fact that an object can be found in nature.
  • a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory or otherwise is naturally-occurring.
  • operably linked refers to positions of components so described are in a relationship permitting them to function in their intended manner.
  • a control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
  • control sequence refers to polynucleotide sequences which are necessary to effect the expression and processing of coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, generally, such control sequences include promoters and transcription termination sequence.
  • control sequences is intended to include, at a minimum, all components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
  • polynucleotide as referred to herein means a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide.
  • the term includes single and double stranded forms of DNA.
  • oligonucleotide includes naturally occurring, and modified nucleotides linked together by naturally occurring, and non-naturally occurring oligonucleotide linkages.
  • Oligonucleotides are a polynucleotide subset generally comprising a length of 200 bases or fewer. In one embodiment, oligonucleotides are 10 to 60 bases in length, such as but not limited to, 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in length. Oligonucleotides are usually single stranded, e.g., for probes; although oligonucleotides may be double stranded, e.g., for use in the construction of a gene mutant. Oligonucleotides of the invention can be either sense or antisense oligonucleotides.
  • nucleotides include deoxyribonucleotides and ribonucleotides.
  • modified nucleotides includes nucleotides with modified or substituted sugar groups and the like.
  • oligonucleotide linkages includes oligonucleotides linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate, phosphoroamidate, and the like. See e.g., LaPlanche et al. Nucl. Acids Res.
  • An oligonucleotide can include a label for detection, if desired.
  • the term "selectively hybridize” referred to herein means to detectably and specifically bind.
  • Polynucleotides, oligonucleotides and fragments thereof in accordance with the invention selectively hybridize to nucleic acid strands under hybridization and wash conditions that minimize appreciable amounts of detectable binding to nonspecific nucleic acids. High stringency conditions can be used to achieve selective hybridization conditions as known in the art and discussed herein.
  • nucleic acid sequence homology between the polynucleotides, oligonucleotides, and fragments of the invention and a nucleic acid sequence of interest will be at least 80%, and more typically with increasing homologies of at least 85%, 90%, 95%, 99%, and 100%.
  • Two amino acid sequences are homologous if there is a partial or complete identity between their sequences. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum matching. Gaps (in either of the two sequences being matched) are allowed in maximizing matching; gap lengths of 5 or less are preferred with 2 or less being more preferred.
  • two protein sequences are homologous, as this term is used herein, if they have an alignment score of at more than 5 (in standard deviation units) using the program ALIGN with the mutation data matrix and a gap penalty of 6 or greater. See Dayhoff, M. O., in Atlas of Protein Sequence and Structure, pp. 101-110 (Volume 5, National Biomedical Research Foundation (1972)) and Supplement 2 to this volume, pp. 1-10.
  • the two sequences or parts thereof are more preferably homologous if their amino acids are greater than or equal to 50% identical when optimally aligned using the ALIGN program.
  • a polynucleotide sequence is homologous (i.e., is identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to a reference polypeptide sequence.
  • the term “complementary to” is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence.
  • the nucleotide sequence "TATAC” corresponds to a reference sequence "TATAC” and is complementary to a reference sequence "GTATA".
  • reference sequence is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA or gene sequence given in a sequence listing or may comprise a complete cDNA or gene sequence.
  • a reference sequence is at least 18 nucleotides or 6 amino acids in length, frequently at least 24 nucleotides or 8 amino acids in length, and often at least 48 nucleotides or 16 amino acids in length.
  • two polynucleotides or amino acid sequences may each (1) comprise a sequence (i.e., a portion of the complete polynucleotide or amino acid sequence) that is similar between the two molecules, and (2) may further comprise a sequence that is divergent between the two polynucleotides or amino acid sequences, sequence comparisons between two (or more) molecules are typically performed by comparing sequences of the two molecules over a "comparison window" to identify and compare local regions of sequence similarity.
  • a “comparison window”, as used herein, refers to a conceptual segment of at least 18 contiguous nucleotide positions or 6 amino acids wherein a polynucleotide sequence or amino acid sequence may be compared to a reference sequence of at least 18 contiguous nucleotides or 6 amino acid sequences and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions, deletions, substitutions, and the like (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math.
  • sequence identity means that two polynucleotide or amino acid sequences are identical (i.e., on a nucleotide-by-nucleotide or residue-by-residue basis) over the comparison window.
  • percentage of sequence identity is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) or residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • substantially identical denotes a characteristic of a polynucleotide or amino acid sequence, wherein the polynucleotide or amino acid comprises a sequence that has at least 85 percent sequence identity, such as at least 90 to 95 percent sequence identity, or at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 18 nucleotide (6 amino acid) positions, frequently over a window of at least 24-48 nucleotide (8-16 amino acid) positions, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the comparison window.
  • the reference sequence may be a subset of a larger sequence.
  • Examples of unconventional amino acids include: 4-hydroxyproline, Y-carboxyglutamate, ⁇ -N,N,N-trimethyllysine, ⁇ -N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, ⁇ -N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline).
  • the lefthand direction is the amino terminal direction and the righthand direction is the carboxy-terminal direction, in accordance with standard usage and convention.
  • the lefthand end of single-stranded polynucleotide sequences is the 5' end; the lefthand direction of double-stranded polynucleotide sequences is referred to as the 5' direction.
  • the direction of 5' to 3' addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA and which are 5' to the 5' end of the RNA transcript are referred to as "upstream sequences"; sequence regions on the DNA strand having the same sequence as the RNA and which are 3' to the 3' end of the RNA transcript are referred to as "downstream sequences".
  • the term "substantial identity” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, such as at least 90 percent sequence identity, or at least 95 percent sequence identity, or at least 99 percent sequence identity.
  • residue positions which are not identical differ by conservative amino acid substitutions. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine.
  • Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic-aspartic, and asparagine-glutamine.
  • amino acid sequences of antibodies or immunoglobulin molecules are contemplated as being encompassed by the present invention, providing that the variations in the amino acid sequence maintain at least 75%, such as at least 80%, 90%, 95%, and 99%.
  • conservative amino acid replacements are contemplated. Conservative replacements are those that take place within a family of amino acids that are related in their side chains.
  • More preferred families are: serine and threonine are aliphatic-hydroxy family; asparagine and glutamine are an amide-containing family; alanine, yaline, leucine and isoleucine are an aliphatic family; and phenylalanine, tryptophan, and tyrosine are an aromatic family.
  • Structural and functional domains can be identified by comparison of the nucleotide and/or amino acid sequence data to public or proprietary sequence databases.
  • computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and/or function. Methods to identify protein sequences that fold into a known three-dimensional structure are known. Bowie et al. Science 253:164 (1991).
  • Bowie et al. Science 253:164 (1991) demonstrate that those of skill in the art can recognize sequence motifs and structural conformations that may be used to define structural and functional domains in accordance with the invention.
  • Preferred amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and (5) confer or modify other physicochemical or functional properties of such analogs.
  • Analogs can include various mutations of a sequence otherthan the naturally-occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the naturally-occurring sequence (preferably in the portion of the polypeptide outside the domain(s) forming intermolecular contacts.
  • a conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence).
  • Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure ⁇ . Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et at. Nature 354:105 (1991), which are each incorporated herein by reference.
  • polypeptide fragment refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the naturally-occurring sequence deduced, for example, from a full-length cDNA sequence. Fragments typically are at least 5, 6, 8 or 10 amino acids long, such as at least 14 amino acids long or at least 20 amino acids long, usually at least 50 amino acids, long or at least 70 amino acids long.
  • Antibody or "antibody peptide(s)” refer to an intact antibody, or a binding fragment thereof that competes with the intact antibody for specific binding. Binding fragments are produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Binding fragments include Fab, Fab', F(ab') 2 , Fv, and single-chain antibodies. An antibody other than a "bispecific” or “bifunctional” antibody is understood to have each of its binding sites identical.
  • An antibody substantially inhibits adhesion of a receptor to a counterreceptor when an excess of antibody reduces the quantity of receptor bound to counterreceptor by at least about 20%, 40%, 60% or 80%, and more usually greater than about 85% (as measured in an in vitro competitive binding assay).
  • MHC Major Histocompabiiity Complex
  • HLA Human Leukocyte Antigens
  • HLA Human Leukocyte Antigens
  • MHC light chain and “MHC heavy chain” as used herein will be understood to refer to portions of the MHC molecule.
  • class I molecules are heterodimers comprised of two noncovalently bound polypeptide chains, a larger "heavy” chain ( ⁇ ) and a smaller “light” chain ( ⁇ -2-microglobulin or ⁇ 2m).
  • the polymorphic, polygenic heavy chain (45 kDa), encoded within the MHC on chromosome six, is subdivided into three extracellular domains (designated 1 , 2, and 3), one intracellular domain, and one transmembrane domain. The two outermost extracellular domains, l and 2, together form the groove that binds antigenic peptide.
  • the 3 domain of the molecule contains the recognition site for the CD8 protein on the CTL; this interaction serves to stabilize the contact between the T cell and the APC.
  • the invariant light chain (12 kDa), encoded outside the MHC on chromosome 15, consists of a single, extracellular polypeptide.
  • MHC light chain ⁇ -2-microglobulin
  • ⁇ 2m may be used interchangeably herein.
  • epitopic determinants includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor.
  • Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • An antibody is said to specifically bind an antigen when the dissociation constant is ⁇ 1 ⁇ M, or ⁇ 100 nM, or ⁇ 10 nM.
  • antibody is used in the broadest sense, and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., Fab, F(ab') 2 and Fv) so long as they exhibit the desired biological activity.
  • Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules which lack antigen specificity. Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas.
  • Native antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond. While the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end.
  • VH variable domain
  • VL variable domain at one end
  • the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.
  • Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains (Clothia et al., J. MoI. Biol. 186, 651-66, 1985); Novotny and Haber, Proc. Natl. Acad. Sci. USA 82 4592-4596 (1985).
  • An "isolated" antibody is one which has been identified and separated and/or recovered from a component of the environment in which is was produced. Contaminant components of its production environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
  • the antibody will be purified as measurable by at least three different methods: 1) to greater than 50% by weight of antibody as determined by the Lowry method, such as more than 75% by weight, or more than 85% by weight, or more than 95% by weight, or more than 99% by weight; 2) to a degree sufficient to obtain at least 10 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequentator, such as at least 15 residues of sequence; or 3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomasie blue or, preferably, silver stain.
  • Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present.
  • antibody mutant refers to an amino acid sequence variant of an antibody wherein one or more of the amino acid residues have been modified. Such mutants necessarily have less than 100% sequence identity or similarity with the amino acid sequence having at least 75% amino acid sequence identity or similarity with the amino acid sequence of either the heavy or light chain variable domain of the antibody, such as at least 80%, or at least 85%, or at least 90%, or at least 95%.
  • variable domain in the context of variable domain of antibodies, refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen.
  • CDRs complementarity determining regions
  • the more highly conserved portions of variable domains are called the framework (FR).
  • variable domains of native heavy and light chains each comprise four FR regions, largely adopting a ⁇ -sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the ⁇ -sheet structure.
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat et al.)
  • the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector function, such as participation of the antibody in antibody-dependent cellular toxicity.
  • antibody fragment refers to a portion of a full-length antibody, generally the antigen binding or variable region.
  • antibody fragments include Fab, Fab', F(ab') 2 and Fvfragments.
  • Papain digestion of antibodies produces two identical antigen binding fragments, called the Fab fragment, each with a single antigen binding site, and a residual "Fc" fragment, so-called for its ability to crystallize readily.
  • Pepsin treatment yields an F(ab') 2 fragment that has two antigen binding fragments which are capable of cross-linking antigen, and a residual other fragment (which is termed pFc').
  • Fv fragment refers to Fv, F(ab) and F(ab') 2 fragments.
  • An "Fv” fragment is the minimum antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in a tight, non-covalent association (V H -V L dimer). It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the V H -V L dimer. Collectively, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • the Fab fragment [also designated as F(ab)] also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain.
  • Fab' fragments differ from Fab fragments by the addition. of a few residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region.
  • Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains have a free thiol group.
  • F(ab') fragments are produced by cleavage of the disulfide bond at the hinge cysteines of the F(ab') 2 pepsin digestion product. Additional chemical couplings of antibody fragments are known to those of ordinary skill in the art.
  • the light chains of antibodies (immunoglobulin) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (.kappa.) and lambda (.lambda.), based on the amino sequences of their constant domain.
  • immunoglobulins can be assigned to different classes. There are at least five (5) major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., lgG-1 , lgG-2, lgG-3 and lgG4; lgA-1 and lgA-2.
  • the heavy chains constant domains that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , v and ⁇ , respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.
  • the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler and Milstein, Nature 256, 495 (1975), or may be made by recombinant methods, e.g. , as described in U.S. Pat. No. 4,816,567.
  • the monoclonal antibodies for use with the present invention may also be isolated from phage antibody libraries using the techniques described in Clackson et al. Nature 352: 624-628 (1991), as well as in Marks et al., J. MoI. Biol. 222: 581-597 (1991).
  • the monoclonal antibodies of the present invention may require administration of such or similar monoclonal antibody to a subject, such as a human.
  • a subject such as a human
  • administration of such antibodies to a human patient will normally elicit an immune response, wherein the immune response is directed towards the antibodies themselves.
  • Such reactions limit the duration and effectiveness of such a therapy.
  • the monoclonal antibodies of the present invention can be "humanized", that is, the antibodies are engineered such that antigenic portions thereof are removed and like portions of a human antibody are substituted therefor, while the antibodies' affinity for specific peptide/MHC complexes is retained.
  • Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin. Humanization can be performed following the method of Winter and co-workers (Jones et al., 1986; Riechmann et al., 1988; Verhoeyen et al., 1988), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. (See also U.S.
  • F v framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibodies can also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta, 1992).
  • Fc immunoglobulin constant region
  • a treatment protocol that can be utilized in such a method includes a single dose, generally administered intravenously, of 10-20 mg of humanized mAb per kg (Sandborn, et al. 2001).
  • alternative dosing patterns may be appropriate, such as the use of three infusions, administered once every two weeks, of 800 to 1600 mg or even higher amounts of humanized mAb (Richards et al., 1999).
  • the presently disclosed and claimed invention further includes fully human monoclonal antibodies against specific peptide/MHC complexes.
  • Fully human antibodies essentially relate to antibody molecules in which the entire sequence of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed "human antibodies", or “fully human antibodies” herein.
  • Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., Hybridoma, 2:7 (1983)) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., PNAS 82:859 (1985)).
  • Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote, et al., PNAS 80:2026 (1983)) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985).
  • human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom et al., Nucleic Acids Res. 19:4133 (1991); Marks et al., J MoI Biol. 222:581 (1991)).
  • human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos.
  • Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen.
  • transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen.
  • the endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome.
  • the human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments. An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications.
  • nonhuman animal is a mouse, and is termed the XENOMOUSETM as disclosed in PCT publications WO 96/33735 and WO 96/34096.
  • This animal produces B cells which secrete fully human immunoglobulins.
  • the antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies.
  • the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.
  • a method for producing an antibody of interest is disclosed in U.S. Pat. No. 5,916,771, issued to Hori et al. on June 29, 1999, and incorporated herein by reference. It includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing the heavy chain and the light chain.
  • label refers to incorporation of a detectable marker, e.g., by incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods). In certain situations, the label or marker can also be therapeutic.
  • a detectable marker e.g., by incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods).
  • avidin e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods.
  • the label or marker can also be therapeutic.
  • labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, ⁇ -galactosidase, luciferase, alkaline phosphatase), chemiluminescent, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags).
  • labels are attached by spacer arms of various lengths to reduce potential steric hindrance.
  • pharmaceutical agent or drug refers to a chemical compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.
  • Other chemistry terms herein are used according to conventional usage in the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (Parker, S., Ed., McGraw-Hill, San
  • anti-plastic agent is used herein to refer to agents that have the functional property of inhibiting a development or progression of a neoplasm in a human, particularly a malignant (cancerous) lesion, such as a carcinoma, sarcoma, lymphoma, or leukemia. Inhibition of metastasis is frequently a property of antineoplastic agents.
  • substantially pure means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition), and preferably a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80 percent of all macromolecular species present in the composition, such as more than about 85%, 90%, 95%, and 99%. In one embodiment, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.
  • patient includes human and veterinary subjects.
  • a “liposome” is a small vesicle composed of various types of lipids, phospholipids and/or surfactant. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes.
  • Treatment refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.
  • a "disorder” is any condition that would benefit from treatment with the polypeptide. This includes chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question.
  • cancer refers to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hopatoma, breast cancer, colon cancer, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer.
  • mammal for purposes of treatment refers to any animal classified as a mammal, including human, domestic and farm animals, nonhuman primates, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc.
  • the T cell receptor mimic of the presently disclosed and claimed invention may be attached to any of various functional moieties.
  • a T cell receptor mimic of the present invention attached to a functional moiety may be referred to herein as an "immunoconjugate".
  • the functional moiety is a detectable moiety or a therapeutic moiety.
  • a detectable moiety or a therapeutic moiety may be particularly employed in applications of the present invention involving use of the T cell receptor mimic to detect the specific peptide/MHC complex, or to kill target cells and/or damage target tissues.
  • the present invention include the T cell receptor mimics described herein attached to any of numerous types of detectable moieties, depending on the application and purpose.
  • the detectable moiety attached to the T cell receptor mimic may be a reporter moiety that enables specific detection of the specific peptide/MHC complex bound by the T cell receptor mimic of the presently disclosed and claimed invention.
  • reporter moieties may be utilized to detect the specific peptide/MHC complex, depending on the application and purpose, the reporter moiety may be a fluorophore, an enzyme or a radioisotope.
  • Specific reporter moieties that may utilized in accordance with the present invention include, but are not limited to, green fluorescent protein
  • GFP alkaline phosphatase
  • AP alkaline phosphatase
  • OFP orange fluorescent protein
  • FITC fluorescein isothiocyanate
  • Cy-chrome Cy-chrome
  • rhodamine blue fluorescent protein
  • BFP horseradish peroxidase
  • HPR horseradish peroxidase
  • a fluorophore may be employed as a detection moiety enabling detection of the specific peptide/MHC complex via any of numerous fluorescence detection methods.
  • fluorescence detection methods include, but are not limited to, fluorescence activated flow cytometry (FACS), immunofluorescence confocal microscopy, fluorescence in-situ hybridization (FISH), fluorescence resonance energy transfer (FRET), and the like.
  • fluorophores may be employed to detect the specific peptide/MHC complex.
  • suitable fluorophores include, but are not limited to, phycoerythrin, fluorescein isothiocyanate (FITC), Cy-chrome, rhodamine, green fluorescent protein (GFP), blue fluorescent protein (BFP), Texas red, and the like.
  • an enzyme may be utilized as the detectable moiety to enable detection of the specific peptide/MHC complex via any of various enzyme-based detection methods.
  • enzyme-based detection methods include, but are not limited to, enzyme linked immunosorbent assay (ELISA; for example, to detect the specific peptide/MHC complex in a solution), enzyme-linked chemiluminescence assay (for example, to detect the complex on solubilized cells), and enzyme-linked immunohistochemical assay (for example, to detect the complex in a fixed tissue).
  • ELISA enzyme linked immunosorbent assay
  • enzyme-linked chemiluminescence assay for example, to detect the complex on solubilized cells
  • enzyme-linked immunohistochemical assay for example, to detect the complex in a fixed tissue.
  • Numerous types of enzymes may be employed to detect the specific peptide/MHC complex, depending on the application and purpose.
  • suitable enzymes include, but are not limited to, horseradish peroxidase (HPR), ⁇ -galactosidase, and alkaline phosphatase (AP).
  • HPR horseradish peroxidase
  • AP alkaline phosphatase
  • Ample guidance for practicing such enzyme-based detection methods is provided in the literature of the art (for example, referto: Khatkhatay M I. and Desai M., 1999. J Immunoassay 20:151-83; wisdom G B., 1994. Methods MoI Biol. 32:433-40; Ishikawa E. et al., 1983. J Immunoassay 4:209-327; Oellerich M., 1980. J Clin Chem Clin Biochem. 18:197-208; Schuurs A H. and van Weemen B K., 1980. J Immunoassay 1:229-49).
  • HPR horseradish peroxidase
  • AP alkaline phosphatase
  • the present invention include the T cell receptor mimics described herein attached to any of numerous types of therapeutic moieties, depending on the application and purpose.
  • Various types of therapeutic moieties that may be utilized in accordance with the present invention include, but are not limited to, a cytotoxic moiety, a toxic moiety, a cytokine moiety, a bi-specific antibody moiety, and the like.
  • Specific examples of therapeutic moieties that may be utilized in accordance with the present invention include, but are not limited to, Pseudomonas exotoxin, Diptheria toxin, interleukin 2, CD3, CD16, interleukin 4, interleukin 10, Ricin A toxin, and the like.
  • the functional moiety may be attached to the T cell receptor mimic of the present invention in various ways, depending on the context, application and purpose.
  • a polypeptidic functional moiety in particular a polypeptidic toxin, may be attached to the antibody or antibody fragment via standard recombinant techniques broadly practiced in the art (for Example, refer to Sambrook et al., infra, and associated references, listed in the Examples section which follows).
  • a functional moiety may also be attached to the T cell receptor mimic of the presently disclosed and claimed invention using standard chemical synthesis techniques widely practiced in the art [for example, refer to the extensive guidelines provided by The American Chemical Society (for example at: http://www.chemistry.org/portal/Chemistry)].
  • One of ordinary skill in the art, such as a chemist will possess the required expertise for suitably practicing such such chemical synthesis techniques.
  • a functional moiety may be attached to the T cell receptor mimic by attaching an affinity tag-coupled T cell receptor mimic of the present invention to the functional moiety conjugated to a specific ligand of the affinity tag.
  • affinity tags may be employed to attach the T cell receptor mimic to the functional moiety.
  • the affinity tag is a biotin molecule or a streptavidin molecule.
  • a biotin or streptavidin affinity tag can be used to optimally enable attachment of a streptavidin-conjugated or a biotin-conjugated functional moiety, respectively, to the T cell receptor mimic due to the capability of streptavidin and biotin to bind to each other with the highest non covalent binding affinity known to man (i.e., with a Kd of about 10 "14 to 10 '15 ).
  • a pharmaceutical composition of the present invention includes a T cell receptor mimic of the present invention and a therapeutic moiety conjugated thereto.
  • the pharmaceutical composition of the present invention may be an antineoplastic agent.
  • a diagnostic composition of the present invention includes a T cell receptor mimic of the present invention and a detectable moiety conjugated thereto.
  • the present invention relates to methodologies for producing antibodies that function as T-cell receptor mimics (TCR m s) and recognize peptides displayed in the context of HLA molecules, wherein the peptide is associated with a tumorigenic, infectious or disease state.
  • TCR T cell receptor
  • the T cell receptor mimics of the presently disclosed and claimed invention will have a higher binding affinity than a T cell receptor.
  • the T cell receptor mimic produced by the method of the presently disclosed and claimed invention has a binding affinity of about 10 nanomolar or greater.
  • the immunogen comprises a peptide/MHC complex, wherein the 3-dimensional presentation of the peptide in the binding groove is the epitope recognized with high specificity by the antibody.
  • the immunogen may be any form of a stable peptide/MHC complex that may be utilized for immunization of a host capable of producing antibodies to the immunogen, and the immunogen may be produced by any methods known to those skilled in the art.
  • the immunogen is used in the construction of an agent that will activate a clinically relevant cellular immune response against the tumor cell which displays the particular peptide/MHC complex.
  • the peptide epitopes of the peptide/MHC complex of the immunogen are antigens that have been discovered as being novel to cancer cells, and such peptide epitopes are present on the surface of cells associated with a tumorigenic, infectious or disease state, such as but not limited to cancer cells, and displayed in the context of MHC molecules.
  • the peptide may be a known tumor antigen, or a peptide identified in U.S. Patent Application Publication No. US 2002/0197672 A1, filed by Hildebrand et al. on October 10, 2001 and published on December 26, 2002; or U.S. Patent Application Publication No. US 2005/0003483 A1, filed by Hildebrand etal.
  • the immunogen may be produced in a manner so that it is stable, or it may be modified by various means to make it more stable. Two different methods of producing a stable form of an immunogen of the present invention will be described in more detail hereinbelow. However, it is to be understood that other methods, or variations of the below described methods, are within the ordinary skill of a person in the art and therefore fall within the scope of the present invention.
  • the immunogen is produced by a cell-based approach through genetic engineering and recombinant expression, thus significantly increasing the half-life of the complex.
  • the genetically-engineered and recombinantly expressed peptide/MHC complex may be chemically cross-linked to aid in stabilization of the complex.
  • the peptide/MHC complex may be genetically engineered such that the complex is produced in the form of a single-chain trimer.
  • the MHC heavy chain, ⁇ -2 microglobulin and peptide are all produced as a single-chain trimer that is linked together.
  • Methods of producing single-chain trimers are known in the art and are disclosed particularly in Yu et al. (2002).
  • Other methods involve forming a single-chain dimer in which the peptide- ⁇ 2m molecules are linked together, and in the single-chain dimer, the ⁇ 2m molecule may or may not be membrane bound.
  • the immunogen of the presently claimed and disclosed invention is produced by multimerizing two or more peptide/MHC complexes.
  • the term "multimer” as used herein will be understood to include two or more copies of the peptide/MHC complex which are covalently or non-covalently attached together, either directly or indirectly.
  • the MHC molecules of the complexes may be produced by any methods known in the art. Examples of MHC production include but are not limited to endogenous production and purification, or recombinant production and expression in host cells. In one embodiment, the MHC heavy chain and ⁇ 2m molecules are expressed in E. coli and folded together with a synthesized peptide.
  • the peptide/MHC complex may be produced as the genetically-engineered single- chain trimer (or the single-chain dimer plus MHC heavy chain) described hereinabove.
  • each of the peptide/MHC complexes may be modified in some manner known in the art to enable attachment of the peptide/MHC complexes to each other, or the multimer may be formed around a substrate to which each copy of the peptide/MHC complex is attached.
  • the multimer can contain any desired number of peptide/MHC complexes and thus form any multimer desired, such as but not limited to, a dimer, a trimer, a tetramer, a pentamer, a hexamer, and the like.
  • a dimer a trimer
  • a tetramer a pentamer
  • a hexamer a compound having a chiral amide
  • Specific examples of multimers which may be utilized in accordance with the present invention are described hereinbelow; however, these examples are not to be regarded as limiting, and other methods of multimerization known to those of skill in the art are also within the scope of the present invention.
  • Streptavidin has four binding sites for biotin, so a BSP (biotinylation signal peptide) tail may be attached to the MHC molecule during production thereof, and a tetramer of the desired peptide/MHC complex could be formed by combining the peptide/MHC complexes with the BSP tails with biotin added enzymatically in vitro.
  • An immunoglobulin heavy chain tail may be utilized as a substrate for forming a dimer, while a TNF tail may be utilized as a substrate for forming a trimer.
  • An IgM tail could be utilized as a substrate for forming various combinations, such as tetramers, hexamers and pentamers.
  • the peptide/MHC complexes may be multimerized through liposome encapsulation or artificial antigen presenting cell technology (see U.S. Serial No. 10/050,231 , filed by Hildebrand et al. on January 16, 2002, the contents of which are hereby expressly incorporated herein by reference). Further, the peptide/MHC complexes may be multimerized through the use of polymerized streptavidin and would produce what is termed a "streptamer" (see http://www.streptamer.com/streptamer/, which is hereby expressly incorporated herein by reference in its entirety).
  • the immunogen of the present invention may further be modified for providing better performance or for aiding in stabilization of the immunogen.
  • modifications which may be utilized in accordance with the present invention include but are not limited to, modifying anchor/tail or modifying amino acids in peptide/MHC complex, PEGalation, chemical cross-linking, changes in pH or salt depending on the specific peptide of the peptide/MHC complex, addition of one or more chaperone proteins that stabilize certain peptide/MHC complexes, addition of one or more adjuvants that enhance immunogenicity (such as but not limited to the addition of a T cell epitope on a multimer), and the like.
  • the immunogen is produced and stabilized, it is delivered to a host for eliciting an immune response.
  • the host may be any animal known in the art that is useful in biotechnological screening assays and is capable of producing recoverable antibodies when administered an immunogen, such as but not limited to, rabbits, mice and rats.
  • the host is a mouse, such as a Balb/c mouse or a transgenic mouse.
  • the mouse is transgenic for the particular MHC molecule of the immunogen so as to minimize the antigenicity of the immunoge ⁇ , thereby ensuring that the 3-dimensional domain of the peptide sitting in the binding pocket of the MHC molecule is the focus of the antibodies generated thereto and thus is preferentially recognized with high specificity.
  • the mouse is transgenic and produces human antibodies, thereby greatly easing the development work for creating a human therapeutic.
  • a screening assay is performed to determine if the desired antibodies are being produced.
  • the assay requires four components plus the sera of the mouse to be screened.
  • the four components include: (A) a binding/capture material (such as but not limited to, streptavidin, avidin, biotin, etc.) coated on wells of a solid support, such as a microtiter plate; (B) properly folded HLA trimer (HLA heavy chain plus ⁇ 2m plus peptide) molecule containing an irrelevant peptide; (C) properly folded HLA tetramer or trimer containing the peptide of interest; and (D) at least one antibody which recognizes mouse IgG and IgA constant regions and is covalently linked to a disclosing agent, such as but not limited to, peroxidase or alkaline phosphatase.
  • a binding/capture material such as but not limited to, streptavidin, avidin, biotin, etc.
  • the solid support of (A) must be able to bind the HLA molecule of interest in such a way as to present the peptide and the HLA to an antibody without stearic or other hindrance.
  • One configuration of the properly folded HLA trimers in (B) and (C) above is a single-site biotinylation. If single-site biotinylation cannot be achieved, then other methods of capture, such as antibody may be used. If antibody is used to capture the HLA molecule onto the solid support, it cannot cross- react with the anti-mouse IgG and IgA in (D) above.
  • the bleeds from the immunized mice Prior to assaying the serum from immunized mice, it is preferred that the bleeds from the immunized mice be preabsorbed to remove antibodies that are not peptide specific.
  • the preabsorption step should remove antibodies that are reactive to epitopes present on any component of the immunogen other than the peptide, including but not limited to, ⁇ 2m, HLA heavy chain, a substrate utilized for multimerization, an immunogen stabilizer, and the like.
  • One embodiment of methods of assaying serum from immunized mice is described in the attached figures (see for example Fig. 5), as well as in the Examples provided hereinafter.
  • a standard hybridoma fusion protocol can be employed to generate cells producing monoclonal antibodies. These cells are plated such that individual clones can be identified, selected as individuals, and grown up in individual wells in plates. The supernatants from these cells can then be screened for production of antibodies of the desired specificity. These hybridoma cells can also be grown as individual clones and mixed and sorted or grown in bulk and sorted as described below for cells expressing surface immunoglobulin of the desired reactivity.
  • cell sorting is utilized to isolate desired B cells, such as B memory cells, prior to hybridoma formation.
  • One method of sorting which may be utilized in accordance with the present invention is FACS sorting, as B memory cells have immunoglobulin on their surface, and this specificity may be utilized to identify and capture these cells.
  • FACS sorting is a preferred method as it involves two color staining.
  • beads can be coated with peptide/HLA complex (with FITC or PE) and attached to a column, and B cells with immunoglobulin on their surface can be identified by FACS as well as by binding to the complex.
  • a sorting method using magnetic beads such as those produced by Dynal or Miltenyi, may be utilized.
  • the sorted B cells may further be differentiated and expanded into plasma cells, which secrete antibodies, screened for specificity and then used to create hybridomas or have their antibody genes cloned for expression in recombinant form.
  • the antibodies are sorted, they are assayed to confirm that they are specific for one peptide/MHC complex and to determine if they exhibit any cross reactivity with other HLA molecules.
  • One method of conducting such assays is a sera screen assay as described in U.S. Patent Application Publication No. US 2004/0126829 A1 , filed by Hildebrand et al. on September 24, 2003 and published on July 1 , 2004, the contents of which are hereby expressly incorporated herein by reference.
  • other methods of assaying for quality control are within the skill of a person of ordinary skill in the art and therefore are also within the scope of the present invention.
  • the present invention also includes a predictive screen to determine if a particular peptide can be utilized in an immunogen of the present invention for producing the desired antibodies which act as T-cell receptor mimics.
  • These screens include but are not limited to, stability, refolding, IC 50 , K n , and the like.
  • the present invention may provide a threshold of binding affinity of peptide so that a predictive threshold can be created for examining entire proteins of interest for potential peptides. This threshold can also be used as a predictor of yield that can be obtained in the refolding process of producing the peptide/MHC complex.
  • the TCR mimics of the present invention have a variety of uses.
  • the TCR mimic reagents could be utilized in a variety of vaccine-related uses.
  • the TCR mimics could be utilized as direct therapeutic agents, either as an antibody or bispecific molecule.
  • the TCR mimics of the present invention could be utilized for carcinogenic profiling, to provide an individualized approach to cancer detection and treatment.
  • cancer profiling refers to the screening of cancer cells with TCRm's of various specificities to define a set of peptide/MHC complexes on the tumor.
  • TCR mimics of the present invention could be utilized for vaccine validation, as a useful tool to determine whether desired T cell epitopes are displayed on cells such as but not limited to, tumor cells, viral infected cells, parasite infected cells, and the like.
  • the TCR mimics of the present invention could also be used as research reagents to understand the fate of antigen processing and presentation in vivo and in vitro, and these processes could be evaluated between solid tumor cells, metastatic tumor cells, cells exposed to chemo-agents, tumor cells after exposure to a vaccine, and the like.
  • the TCR mimics of the present invention could also be utilized as vehicles for drug transport to transport payloads of toxic substances to tumor cells or viral infected cells.
  • the TCR mimics of the present invention could also be utilized as diagnostic reagents for identifying tumor cells, viral infected cells, and the like.
  • the TCR mimic reagents of the present invention could also be utilized in metabolic typing, such as but not limited to, to identify disease-induced modifications to antigen processing and presentation as well as peptide-HLA presentation and tumor sensitivity to drugs.
  • the human p53 protein is an intracellular tumor suppressor protein. Point mutations in the p53 gene inactivate or reduce the effectiveness of the p53 protein and leave cells vulnerable to transformation during progression towards malignancy. As cells attempt to compensate for a lack of active p53, over production of the p53 protein is common to many human cancers including breast cancer, resulting in cytoplasmic increases in p53 peptide fragments such as the peptide 264-272. There are many reports demonstrating that surface HLA-A2 presents the 264-peptide epitope from wild-type p53 (Theobald et al., 1995; and Theobald et al., 1998).
  • Cytotoxic T lymphocytes have been generated against the 264-peptide-HLA-A2 complexes (referred to herein as 264p-HLA-A2) on breast cancer cells from peripheral blood monolayer cells (PBMC) of healthy donors and individuals with breast cancer (Nikitina et al., 2001; Barfoed et al., 2000; and Gnjatic et al., 1998). Further, several studies have reported successful immunization with the 264 peptide in HLA-A2 transgenic mice (Yu et al., 1997; and Hoffmann et al., 2005).
  • PBMC peripheral blood monolayer cells
  • the filtrate of this mix was concentrated, and the buffer was exchanged with 10 mM Tris pH 8.0.
  • the mix was biotinylated using a recombinant birA ligase for two hours at room temperature and then subjected to size exclusion chromatography on a Sephadex S-75 column (Superdex S-75, Amersham GE Health Sciences) (Fig. 1).
  • a monomer HLA-A2-peptide can be purified from a Sephadex S-75 column, concentrated and then biotinylated using birA ligase for 2 hours at room temperature.
  • the refolded biotinylated monomer peak was reisolated on the S-75 column and then multimerized with streptavidin (SA) at a 4:1 molar ratio.
  • SA streptavidin
  • the multimerized sample was subjected to size exclusion chromatography on a Sephadex S-200 column (Fig. 2).
  • a pre-absorption step was incorporated into an ELISA assay format. This step was designed to remove antibodies against HLA and ⁇ 2-microglobulin from the reaction.
  • biotinylated non-relevant monomers were used to pre-absorb and then remove the formed complexes from the reaction on a sold surface-bound SA.
  • ELISA format sera from immunized mice are first reacted with HLA-A2 monomers containing another irrelevant peptide before reacting them with HLA-A2 complexes of the relevant peptide. The specifics of these assays are described in more detail herein below.
  • Pre-Absorption assay Serum from the immunized mice was used in an ELISA format to identify "peptide-specific" antibody responses.
  • TCR mimics are antibodies having dual specificity for both peptide and HLA.
  • the immunized mice will produce antibody specificities against HLA epitopes. It is these antibodies that the pre-absorption protocol substantially removes from the serum samples. In order to substantially remove antibodies that were not peptide specific, a pre-absorption step was included in the protocol. It was assumed that 12 ⁇ g of IgG is present in 1 ml of mouse serum, and that 10% of the IgG in immunized mouse serum is specific for an epitope on the peptide-HLA-A2 immunogen.
  • 1.2 ⁇ g of IgG in 1 ml of serum from an immunized mouse is potentially specific for some position on the peptide-A2 molecules and is not "peptide specific".
  • 20 ⁇ g of biotinylated Her2/neu-peptide-HLA-A2 (which differs from 264p-HLA-A2 only in the peptide) was added to 1 ml of a 1 :200 dilution of each mouse bleed. Samples were incubated overnight at 4°C with agitation.
  • Fig. 5 demonstrates the development of an ELISA assay for screening mouse bleeds to determine if there are antibodies specific to the peptide-of-interest-HLA-molecule complex present.
  • Pre-absorbed serum samples from six Balb/c mice were individually tested in the ELISA screening assay of Fig. 5 (see Fig. 6). Briefly, 96 well plates (maxisorb; Nunc) were coated the night before with 0.5 ⁇ g of either biotinylated 264p-HLA-A2 monomer or biotinylated elF4Gp-HLA-A2 monomer at4°C.
  • TMB tetramethylbenzidine
  • BB7.2 mAb was used at 50 to 200 ng/well. This mAb recognizes only conformationally correct forms of the refolded peptide-HLA-A2 molecule.
  • a negative control in the assay a peptide-HLA-A2 complex containing an irrelevant peptide was coated on the plate. In this particular assay, the negative control was elF4G peptide-loaded HLA-A2 monomer.
  • the mice used for the production of the antibodies were pre-bled in order to ensure that Balb/c mice do not harbor antibodies specific for the desired antigens before immunization. Assay background was determined using pre-bleed samples at 1:200 and 1 :400 dilution. The highest absorbance reading recorded for pre-bleeds was less than OD 0.06 at 450 nm.
  • Fig. 6 shows the results from an ELISA of six individual bleeds from Balb/c mice immunized with tetramers of 264p-HLA-A2.
  • the data shown in Fig. 6 demonstrates that both male and female mice immunized with 264p-HLA-A2 tetramers make specific antibody to 264p-HLA-A2 monomers.
  • Bleeds incubated in wells containing elF4Gp-HLA-A2 monomers (irrelevant peptide) were used to evaluate non-specific reactivity of bleeds.
  • mice with peptide-A2 tetramers demonstrate minimal reactivity to elF4Gp/A2 with signal to noise ratios ranging from 3 to 6 fold, indicating that immunization of mice with peptide-A2 tetramers leads to the generation of specific antibody responses to the immunogen.
  • T2 binding assay To confirm the ELISA findings, the binding of the different mouse bleed samples to T2 cells pulsed with either the 264 peptide (peptide of interest) or the elF4G peptide (irrelevant peptide) was investigated, as shown in Fig. 7.
  • T2 cells are a human B lymphoblastoid cell line (ATCC CRL-1999) that has been well characterized by Peter Creswell (Wei et al., 1992). T2 cells are useful for studying recognition of HLA-A2 antigens because they are deficient in peptide loading.
  • TAP1/2 proteins which are necessary proteins for transporting peptides from the cytosol into the endoplasmic reticulum for loading HLA class I molecules. Because of the TAP1/2 deficiency, these cells express a low level of empty HLA-A2 molecules on the surface. Thus, these cells can be primed (loaded) with peptides of choice, and the cells will display them appropriately in the context of HLA-A2 molecules on their surface. Addition of peptide to these cells leads to peptide binding to the HLA-A2 molecules which are constantly cycling to the surface and stabilization of the HLA-A2 structure. The more stable structure increases the density of surface displayed HLA-A2 molecules that are loaded with the particular peptide of interest.
  • T2 cells can be loaded with relevant or irrelevant peptide, and the reactivity of immune sera from immunized mice against them can be measured. The larger the difference in the response between T2 cells loaded with relevant or irrelevant peptide, the higher the titer for specific antibodies in the sera.
  • T2 cells were loaded with either the 264 or the elF4G peptide, and then the cells were stained with the BB7.2 antibody to detect the level of HLA-A2 molecules present on the surface of T2 cells.
  • Fig. 8 shows that both 264 and elF4G peptides have been successfully loaded by comparing the BB7.2 staining profile of cells that received peptide versus the cells that did not receive peptide (negative controls). These findings demonstrate that elF4G peptide may be more efficient at loading and stabilizing HLA-A2 on T2 cells than the 264 peptide.
  • Fig. 9 illustrates the results of staining of 264 peptide-loaded T2 cells with the I3M2 mouse bleed.
  • the pre-absorbed mouse sample preferentially binds cells pulsed with 264 peptide.
  • Fig. 10 demonstrates that the pre-bleed samples (mice bleeds taken prior to immunization) show no sign of reactivity to T2 cells pulsed with either the 264- or elF4G peptide.
  • these results clearly demonstrate that a polyclonal peptide-HLA specific antibody response can be generated to the specific three-dimensional, and that these antibodies are specific for the immunogen that was used. They confirm that the antibodies produced also recognize a "native" or natural form of the peptide-HLA-A2 complex and are not restricted in reactivity to the refolded form used to prepare the immunogen.
  • Hybridomas were generated by submitting 12 mice immunized with 264p-HLA-A2 to the Hybridoma Center, Oklahoma State University, Stillwater, Oklahoma, for hybridoma generation using standard technology. In total, the center returned 1440 supernatants from p53-264 hybridoma isolates for screening.
  • Fig. 11 depicts development of assays to screen hybridomas to determine if they are producing anti-peptide-HLA specific antibodies. In a primary ELISA screen, 40 positives were identified, and in a secondary screen, 7 positives against 264p-HLA-A2 were identified. The results from screening hybridoma supernatants by a competitive binding ELISA are shown in Fig. 12.
  • Hybridomas determined positive after a first screening were expanded, and the supernatant was diluted and rescreened by competitive ELISA two weeks after cell growth.
  • Fig. 13 represents data obtained from a competitive ELISA of these positive hybridoma clones.
  • TCRm's specific for 264p-HLA-A2 were determined by showing a reduction in absorbance (read at 450 nm) after addition of competitor (no tetramer versus 264p tetramer), while no change in absorbance was observed after addition of non-competitor (no tetramer versus elF4Gp tetramer).
  • Example 1 clearly demonstrate that the immunogen of the present invention is capable of eliciting an immune response in a host that is specific for an epitope formed by a desired peptide presented in the context of an HLA molecule.
  • results also indicate there is a significant component of the antibody reactivity in most of the immunized mice that recognizes epitopes that are not specific to the peptide in the context of the HLA binding groove. Rather, these antibodies probably recognize other epitopes common to properly folded HLA-A2 molecules (independent of the peptide region) or epitopes which form as the immunogen is processed, unfolded and denatured in the body.
  • the eukaryotic translation initiation factor 4 gamma is a protein which is part of a complex of molecules that are critical in regulating translation.
  • elF4G eukaryotic translation initiation factor 4 gamma
  • MCF-7 and MDA-MB-231 breast carcinoma cell lines
  • a peptide of elF4G has been identified as being presented by HLA molecules on HIV infected cells at a higher frequency than in uninfected cells by the epitope discovery method of Hildebrand et al.
  • Fig. 15 An expression construct encoding a secreted HLA molecule is transfected into a normal cell line and an infected, diseased or cancerous cell line (in this case, an HIV infected cell line), and the cell lines are cultured at high density in hollow-fiber bioreactors. Then, the secreted HLA molecules are harvested and affinity purified, and the peptides bound therein are eluted.
  • the peptides from the uninfected cell line and the HIV infected cell line are then comparatively mapped using mass spectroscopy to identify peptides that are presented by HLA at a higher frequency in the HIV infected than in the uninfected cells.
  • the peptide VLMTEDIKL (SEQ ID NO:2), was identified, and determined to be a peptide fragment of eukaryotic translation initiation factor 4 gamma (elF4G).
  • the peptide of SEQ ID NO:2 is referred to herein as the "elF4G peptide", or "elF4Gp".
  • Tetramer stability was assessed as described in Example 1 for the 264p-HLA-A2 tetramers. In contrast to the 264p-HLA-A2 tetramers, which have a half life of 10 hours at 37°C, elF4Gp-HLA-A2 tetramers have a half life of 20 hours, and 40% of tetramers remain stable after 40 hours of incubation.
  • elF4Gp-HLA-A2 tetramers were utilized to immunize Balb/c mice as described in Example 1, and the mice were bled and sera assayed using the ELISA method described above in Example 1 and in Fig. 5.
  • Sera from a mouse immunized with elF4Gp-HLA-A2 tetramers was pre-absorbed with biotinylated 264p-HLA-A2 monomers.
  • the serum was reacted with SA on a solid surface and then used in an ELISA format.
  • Serum was reacted with solid surface bound (1) 264p-HLA-A2 monomers; (2) elF4Gp-HLA-A2 monomers; or (3) Her2/neu-peptide-HLA-A2 monomers, and the bound antibody was detected with a goat anti mouse (GAM)-HRP conjugate antibody.
  • GAM goat anti mouse
  • the ELISA reactions were then developed with TMB (an HRP chromogenic substrate), and the absorbance read at 450 nm. The results shown in Fig.
  • Fig. 18 demonstrates the detection of HLA-A2 levels on peptide-pulsed T2 cells using BB7.2 mAb. This figure demonstrates the successful and relatively equivalent loading of both the 264 and elF4G peptides on the surface of HLA-A2 T2 cells.
  • Fig. 19 demonstrates the results of staining elF4G and 264 peptide-loaded T2 cells with a bleed from a mouse immunized with elF4Gp-HLA-A2. 264 peptide loaded cells are shown in the solid peak.
  • the pre-absorbed serum sample was used at a dilution of 1 :400 for staining and preferentially binds cells pulsed with the elF4G peptide (as shown by the rightward shift).
  • the pre- bleed sample shows no sign of reactivity to T2 cells pulsed with either peptide (not shown).
  • T2 cell-based competitive assays as described in Example 1 and in Fig.
  • Figs. 20 and 21 were used to further evaluate the specificity of the polyclonal antibody to elF4Gp-HLA-A2, and the results are shown in Figs. 20 and 21.
  • sera from mice immunized with elF4Gp-HLA-A2 tetramers were diluted 1 :200 in PBS and pre-absorbed against Her2/neu-peptide-HLA-A2.
  • the sera was then mixed with elF4Gp-HLA-A2 or with 264p-HLA-A2, either in the form of monomers (Fig. 20) or tetramers (Fig. 21) and before being reacted with T2 cells loaded with elF4G peptide (100 ⁇ g/ml).
  • the maximum staining signal (filled peak) is shown for the anti-serum.
  • a competitor elF4Gp-HLA-A2
  • a non-competitor 264p-HLA-A2
  • the results shown in Figs. 20 and 21 reveal that the addition of the 264p-HLA-A2 monomer ortetramer had little inhibitory activity on anti-serum binding to elF4G peptide-loaded T2 cells.
  • Mouse hybridomas were generated as described in Example 1 using standard technology, and immunogen specific monoclonals were identified using a competitive binding ELISA (as described herein before). From over 800 clones, 27 mAb candidates were identified, and 4F7 mAb (IgGI isotype) was selected for further characterization. After expanding the 4F7 hybridoma cell line by known methods in the art, the mAb was purified from 300 ml of culture supernatant on a Protein-A column that yielded 30 mg of 4F7 mAb.
  • the specificity of antibody binding to relevant peptide-HLA-A2 tetramers and 3 irrelevant peptide-HLA-A2 tetramers was determined by ELISA, as shown in Fig.22.
  • the 4F7 mAb showed specific binding only to elF4Gp- HLA-A2 tetramers; no signal was detected using irrelevant peptide-A2 controls, which included peptide VLQ and TMT, both derived from the human beta chorionic gonadotropin protein, and 264 peptide derived from the human p53 tumor suppressor protein.
  • the binding affinity and specificity of the 4F7 mAb was determined by plasmon surface resonance (BIACore).
  • 4F7 mAb was coupled to a biosensor chip via amine chemistry, and soluble monomers of HLA-A2 loaded with 264 or elF4G peptide were passed over the antibody coated chip.
  • Fig. 23 specific binding of soluble elF4Gp-HLA-A2 monomer to 4F7 mAb was observed, while no binding to 264p-HLA-A2 complexes containing the irrelevant peptide p53-264 was observed.
  • the affinity constant of 4F7 mAb for its specific ligand was determined at 2 x10 "9 M.
  • Figs. 22 and 23 4F7 binding to recombinant elF4Gp-HLA-A2 molecules was demonstrated.
  • Fig. 24 4F7 binding to elF4Gp-HLA-A2 complexes on the surface of T2 cells was demonstrated.
  • cells were pulsed at 10 ⁇ g/ml with the following peptides: elF4G, 264, and TMT. Unpulsed T2 cells were also used as a control.
  • Fig. 24A T2 cells pulsed with irrelevant peptides or no peptide and stained with 4F7 (50 ng) displayed minimal signal.
  • the 4F7 TCRm mAb was used to stain a normal human mammary epithelial cell line and a human breast carcinoma cell line (MDA-MB-231). Although both cell lines expressed similar levels of HLA-A2 on their surface, the 4F7 TCRm mAb stained only the breast carcinoma cell line (Fig. 25), indicating that cancer cells express this peptide-HLA-A2 epitope. In addition, these results support the binding specificity of 4F7 TCRm mAb for the elF4G( 720 ) peptide-HLA-A2 complex.
  • Fig. 26A MCF-7 cells were stained with 100 ng of 4F7 mAb and showed a significant rightward shift compared to the isotype control.
  • soluble tetramers competitor and non-competitor
  • elF4Gp-HLA-A2 tetramer completely blocked 4F7 staining, while the non-competitor, 264p-HI_A-A2, failed to block 4F7 mAb from binding to cells.
  • the HLA-A2 negative breast carcinoma cell line BT-20 was not stained with 4F7 mAb.
  • FIG. 27 three panels are shown in which MDA-231 cells were stained with 4F7 mAb (50 ng) in the absence or presence of soluble peptide-HLA-A2 monomers.
  • the three peptide-HLA- A2 monomers selected were elF4Gp (competitor) and 264p and Her2/neu peptide (non-competitors).
  • 4F7 binds to MDA-231 cells, and its binding is significantly inhibited using competitor.
  • no reduction in binding signal strength was seen with either non-competitor, indicating that 4F7 binds to tumor cells in a specific manner.
  • These data confirm the isolation of a novel TCRm monoclonal antibody with specificity for a peptide derived from the elF4G protein that is presented by HLA-A2 on the surface of breast cancer cells.
  • the staining profiles for4F7 TCRm, 1 B8 TCRm, and IgG 1 isotype control using mock infected HLA-A*0201 positive PBMCs are shown in Fig. 28A.
  • the 4F7 TCRm mAb showed modest staining of mock infected PBMCs, thus validating our Sup-T1 cell findings in which elF4G( 720 ) peptide is constitutively expressed at low levels. In contrast, no cell staining was observed with the two control mAbs.
  • elF4G( 720 ) expression was examined in HLA-A*0201 positive CD4+ T cells infected with the HIV-1 strain IHb and stained with the 4F7 TCRm five days post-infection (Pl).
  • HIV-1 infected CD4+ T cells were identified by HIV-1 p24 expression (Figs. 28D-F and 28G-I) by staining with the anti-p24-PE conjugate. On day 5 Pl , 30.1 % of the cells were p24 positive. At this time the population of cells was stained with the 4F7 TCRm, 1 B8 TCRm or IgGI isotype control mAbs. As shown in Figs.
  • the 4F7 TCRm mAb was used to directly examine the kinetics of elF4G( 720 ) peptide-HLA-A*0201 complex presentation on HIV-1 infected CD4+ T cells for9 days post-infection (Pl).
  • the p24 positive CD4+ T cells had a two-fold increase in 4F7 TCRm staining signal compared to the p24 negative cells by the third day Pl.
  • days 7 and 8 Pl the 4F7 TCRm staining differential had increased by almost 4-fold between the p24 negative and positive groups (Fig. 29A).
  • there were no significant changes in cell staining using the isotype control Ab Fig. 29B. This finding directly validates the expression of the elF4G( 720 )-HLA-A*0201 epitope and reveals the dynamic nature of host-peptide epitope presentation on HIV infected cells.
  • the infected CD4+ T cells were stained with 0.5 ⁇ g of 4F7 TCRm in the presence of either (1) elF4G( 720 )-HLA-A*0201 tetramer complex that would compete with specific binding to elF4G( 720 )-HLA-A*0201 ; (2) p53( 264 )-HLA-A*0201 tetramer complexes; or (3) VLQ( 44 )-HLA-A*0201 tetramer complexes, wherein (2) and (3) would not compete with specific binding to elF4G( 720 )-HLA-A*0201.
  • the 1 B8 TCRm mAb did not stain the infected or non-infected CD4+ T cells (data not shown), further supporting the claim that the 4F7 TCRm specifically recognizes the elF4G( 720 )-HLA-A*Q201 complex.
  • these findings indicate that HIV-1 infection of primary cells leads to the enhancement of host peptide elF4G( 720 ) through which immune receptors (TCRm here) can distinguish the virally infected from non-infected cells.
  • Her-2(9 369 ) represents a common epitope expressed by various tumor types including breast carcinomas (Brossart et al., 1999). Approximately 20-30% of primary breast cancers express Her-2.
  • the Her-2/neu receptor protein is a member of the tyrosine kinase family of growth factor receptors (Coussens et al., 1985) that is frequently amplified and overexpressed in breast cancer (Slamon et al., 2001).
  • the Her-2/neu protein is generally displayed on the surface of cells and, during malignancy, is detected at high levels on tumor cells.
  • Herceptin an anti-Her-2/neu antibody
  • Herceptin oncoprotein contains several HLA-A2-restricted epitopes that are recognized by CTL on autologous tumors.
  • Her-2 epitope spans amino acids 369-377 (Her-2(9 369 )) (KIFGSLAFL; SEQ ID NO:3) (Fisk et al., 1995) and is recognized by tumor associated lymphocytes as well as reactive T cell clones as an immunodominant HLA-A2-restricted epitope.
  • the peptide has been shown to bind with high affinity to HLA-A2 alleles (Fisk et al., 1995; and Seliger et al., 2000).
  • Her-2(9 369 ) epitope binds to HLA-A2 with intermediate affinity (IC 50 ⁇ 50 nM) (Rongcun et al., 1999), and because it is grossly overexpressed on malignant cells, it has been used as a vaccine candidate in several clinical trials. For instance, Knutson et al. (2002) demonstrated that patients immunized with Her-2(9 369 ) could develop interferon-gamma (IFN- ⁇ ) responses to the peptide and exhibited increased Her-2(9 369 )-specific precursor frequencies.
  • IFN- ⁇ interferon-gamma
  • Her2/neu-peptide-HLA-A2 monomers and tetramers were generated as described above in Example 1.
  • Her2/neu-peptide-HLA-A2 tetramers were generated at a lower efficiency than for either 264p-HLA-A2 tetramers (Example 1) or elF4Gp-HLA-A2 tetramers (Example 2), as shown in Table I.
  • the relatively low tetramer yields with Her2/neu peptide do not correlate well with the high affinity of this peptide to HLA-A2.
  • the IC 50 of Her2/neu peptide is lower than p53-264, yet tetramer yield with Her2/neu peptide is two to three fold less than tetramer yield with p53-264.
  • the Her2/neu-peptide-HLA-A2 tetramers were utilized for immunization of Balb/c mice and generation of monoclonal antibodies as described in detail in Examples 1 and 2. Briefly, the 1 B8 TCRm mAb was generated by immunizing mice with soluble recombinant HLA-A*0201 loaded with the Her2/neu 369 peptide epitope.
  • the soluble heavy chains of HLA-A*0201 hereafter designated A2+
  • ⁇ 2-microgiobulin ⁇ 2m
  • the conformation of the refolded protein was assessed using anti-HLA Class I antibody (W6/32) and the anti-HLA-A2 specific mAb BB7.2 (data not shown).
  • the refolded protein served as the immunogen and as the positive control in screening assays of hybridoma supernatants.
  • the elF4G 720 , TMT 40 and VLQ 44 peptide loaded A2+ molecules served as negative controls.
  • Over 2000 hybridomas were screened and the 1 B8 TCRm hybridoma was selected because it specifically recognized the recombinant HLA-A2 protein loaded with the p369 peptide but did not bind recombinant HLA-A2 proteins loaded with irrelevant peptides (Fig.31A).
  • the 3F9 TCRm mAb was used, which is specific for the TMT 40 peptide-HLA-A2 complex. As shown in Fig. 31 B, the 3F9 TCRm mAb binds specifically to the TMT( 40 )-A2 complex without binding to the Her2( 369 )-A2 complex. To demonstrate that recombinant HLA-A2 proteins were properly folded after being loaded with the peptide, they were stained with the BB7.2 anti-A2.1 mAb (Fig. 31C). These data demonstrate that the TCRm antibodies recognize a specific MHC-peptide complex and they do not have detectable cross-reactivity with either A2+ molecules or HLA complexes loaded with irrelevant peptides.
  • 1 B8 TCRm only stains T2 cells pulsed with the Her2/neu peptide but does not bind T2 cells not pulsed or pulsed with irrelevant peptides. These results confirm the fine and unique specificity of the 1 B8 TCRm for the Her2/neu 369 peptide present in the binding pocket of the HLA-A2 complex. [0208] The specificity and sensitivity of the 1 B8 TCRm mAb for the Her2( 369 )-A2 complex was further evaluated using three different methods. In the first series of experiments, T2 cells were pulsed with a cocktail consisting of 20 different irrelevant peptides in the presence or absence of the p369 peptide.
  • HLA-A2 tetramer complexes were loaded with either (1) Her-2/neu peptide that would compete with specific binding to Her2 ( 36g )-A2; or (2) irrelevant TMT peptide that would not compete for binding sites, and then added to the staining reactions.
  • MDA-MB-231 tumor cells were stained with 0.5 ⁇ g of 1 B8 in the presence of Her2( 369 )-A2 tetramer or TMT( 40 )-A2 tetramer complex. The results, shown in Fig.
  • Fig. 35 illustrates that 1 B8 mAb does not bind to soluble Her2/neu peptide.
  • MDA-MB-231 cells were stained with 1B8 in the presence or absence of exogenously added Her-2/neu peptide.
  • Fig. 35 demonstrates that 1B8 TCR mimic has dual specificity and does not bind to Her-2/neu peptide alone.
  • the COLO205 cell line revealed noticeably higher levels of Her2/neu protein than MDA-MB-231, Saos-2, MCF-7 and SW620 tumor cell lines.
  • Her2( 369 )-A2 expression levels (MFIR) of COLO205 were similar to those of Saos-2, SW620 and MCF-7 cell lines and roughly 3-fold lower than MDA-MB-231 cells, even though COLO205 demonstrated significantly higher expression of the Her2/neu antigen (Fig. 36).
  • MFIR Her2( 369 )-A2 expression levels
  • Human chorionic gonadotropin is a member of the glycoprotein hormone family that shares homology with luteinizing hormone, follicle stimulating hormone and thyroid stimulating hormone. Each of these is a heterodimer with a variable ⁇ chain and a common ⁇ chain. hCG is most commonly associated with pregnancy assessment but is also a marker for tumors resulting from tissues associated with placenta or germ cells. In a comprehensive review of hCG in cancer, Stenman et al.
  • ⁇ chain (hCG ⁇ ) is found in the serum of 45-60% of patients with biliary and pancreatic cancers, and 10-30% of other cancers, lmmunohistochemical analysis and urinalysis have been used to demonstrate the presence of hCG ⁇ in lung, gynecological and head and neck cancers.
  • the aggressiveness and resistance to therapy of bladder cell carcinoma expressing hCG ⁇ has been associated with an autocrine anti-apoptotic effect elicited by the free ⁇ chain (Butler et al., 2000).
  • a first step in evaluating the efficacy of therapeutic antibodies is in vitro assessment of their specificity and ability to induce tumor cell lysis via the activation of complement and ADCC.
  • the therapeutic successes of the monoclonal antibodies trastuzumab and rituxamab are thought to be due, at least in part, to their ability to promote ADCC and CDC (Clynes et al., 2000; Spiridon et al., 2004; Harjunpaa et al., 2000; and Golay et al., 2000).
  • the antigen binding specificity, in vitro lytic abilities and in vivo tumor growth inhibition of a TCRm mAb, 3.2G1 which is specific for the GVL peptide (residues 47-55 from hCG ⁇ ) presented in the context of HLA-A2, are demonstrated.
  • Cell culture medium included IMDM and RPMI from Cambrex (Walkerville, MD), L-15 from Mediatech (Herndon, VA), and Hybridoma SFM and AIM-V from Invitrogen (Carlsbad, CA).
  • Media supplements included heat-inactivated fetal bovine serum (FBS) and penicillin/streptomycin from Sigma (St. Louis, MO) and L-glutamine from HyClone (Logan, LJT).
  • Recombinant human IL-2 was obtained from Peprotech (Rockyhill, NJ).
  • All tumor lines were maintained in culture medium containing glutamine, pen/strep and 10% FBS. Cell cultures were maintained at 37°C in 5% CO 2 atmosphere with the exception of MDA and SW620 which were cultured without CO 2 . MDA and SW620 cells were cultured in L-15, SKOV3.A2 and T2 in IMDM, and BT20 in RPMI. When necessary, attached cells were released from flasks using TrypLE Express (Invitr.ogen, Carlsbad, CA ).
  • PBMC Human peripheral blood mononuclear cells
  • Murine hybridoma cells were initially grown in RPMI supplemented with 10% FBS, glutamine and pen/strep (RPMI/10) as described below. After selection for binding specificity, clones were grown in RPMI/10 to provide supernatant containing the antibodies of interest or in SFM to provide supernatant for isolation of purified antibodies from protein G columns (GE Healthcare BioSciences, Piscataway, NJ ).
  • Peptides and HLA-A2 complexes The following peptides were synthesized at the Molecular Biology Resource Facility, University of Oklahoma (Oklahoma City, OK.): KIFGSLAFL (residues 369-377, designated Her-2; SEQ ID NO:3), eukaryotic initiation translation factor 4 gamma VLMTEDIKL (residues 720-728, designated elF4G; SEQ ID NO:2), human chorionic gonadotropin- ⁇ TMTRVLQGV (residues 40-48, designated TMT; SEQ ID NO:4), VLQGVLPAL (residues 44-53, designated VLQ; SEQ ID NO:5), and GVLPALPQV (residues 47-55, designated GVL; SEQ ID NO:6).
  • HLA-A2 extracelluar domain and ⁇ 2 microglobulin were produced as inclusion bodies in E. coli and refolded essentially as described previously. After refolding, the peptide-HLA-A2 mixture was concentrated, and properly folded complex was isolated from contaminants on a Superdex 75 sizing column (GE Healthcare Bio-Sciences AB). This complex, designated the monomer, was biotinylated using the BirA biotin ligase enzyme (Avidity, Denver, CO) and purified on the S75 column. Purified, biotinylated monomer was mixed with streptavidin at an empirically determined ratio to yield higher order complexes.
  • Tetramer concentration was determined by BCA protein assay (Pierce, Rockford, IL).
  • ELISA assays were performed using Maxisorb 96-well plates (Nunc, Rochester, N.Y.). Assays to evaluate binding specificity of the TCRm antibodies were done on plates coated with either 500 ng/well HLA monomer or 100 ng/well HLA tetramer. Bound antibodies were detected with peroxidase-labeled goat anti-mouse IgG (Jackson ImmunoResearch) followed by ABTS (Pierce). Reactions were quenched with 1% SDS. Absorbance was measured at 405 nm on a Victor 11 plate reader (PerkinElmer, Wellesley, MA).
  • the SBA Clonotyping System/HRP and mouse immunoglobulin panel from Southern Biotech were used to estimate the concentration of 3.2G1 (isotype IgG 23 ) in the supernatant of FBS-containing medium.
  • the assay was run according to manufacturer's directions, and 3.2G1 signal was compared with that of an IgG 23 standard supplied by the manufacturer. Development, quenching and analysis of the plate were performed as described above for the other TCRms.
  • T2 is a mutant cell line that lacks transporter-associated proteins (TAP) 1 and 2 which allows for efficient loading of exogenous peptides (Wei et al., 1992).
  • T2 cells were pulsed with the peptides at 20 ⁇ g/ml for 4 hours in growth medium, with the exception of the peptide-titration experiments, in which the peptide concentration was varied as indicated.
  • Cells were washed and resuspended in staining buffer (SB; PBS+0.5% BSA+2 mM EDTA) and then stained with 1 ⁇ g of 3.2G1 , BB7.2 or isotype control antibody for 15 to 30 minutes in 100 ⁇ l SB.
  • SB staining buffer
  • Fig.41 tumor cell lines were stained and evaluated in the same manner as the T2 cells, after being released from plates and washed in SB. Tetramer competition stains were carried out in the same order described above except that tetramer at the appropriate concentration was mixed with the antibody and allowed to stand for 40 minutes before the mix was added to the cells.
  • Cytotoxicity Analysis Specific cell lysis in the complement dependent cytotoxicity (CDC), natural killer cell (NK) and antibody dependent cellular cytotoxicity (ADCC) assays was evaluated using the CytoTox 96 non-radioactive cytotoxicity Lactate Dehydrogenase Assay (LDH assay) from Promega (Madison, Wl), following the instructions provided by the manufacturer.
  • CDC complement dependent cytotoxicity
  • NK natural killer cell
  • ADCC antibody dependent cellular cytotoxicity
  • This assay measures the release of cellular LDH into the culture supernatant after cell lysis. All cells were grown or pulsed with peptide in their appropriate growth medium, but final incubations of cells in the presence of complement (CDC) or human PBMCs (NK and ADCC) was carried out in AIM-V medium for 4 hours at 37°C. CDC analysis of T2 cells took place under three different conditions: (1) the antibody concentration was varied and competing or non-competing tetramer added, (2) peptide mixes were used to pulse cells, or (3) GVL peptide was titrated for use in cell pulsing. CDC analysis of MDA-MB-231 cells using antibody dilutions and tetramer competition was carried out on adherent cells.
  • ADCC reactions using human PBMC effector cells (E) were carried out on MDA-MB-231 target cells (T) using 3.2G1 or W6/32 antibodies at a final concentration of 10 ⁇ g/ml. Effecto ⁇ target ratios (E:T) were varied as indicated in the figures.
  • mice Six week-old female athymic nude mice (CByJ. Cg-Foxn1 ⁇ nu ⁇ /j) were obtained from Jackson Laboratories and housed under sterile conditions in barrier cages. Each of nineteen mice was implanted with 5x10 6 freshly harvested (97% viable) MDA-MB-231 cells in 0.2 ml containing 1 :1 mixture of medium and Matrigel (Sigma, St. Louis, MO) (Ferguson et al., 2005; and Hermann et al., 2005). Mice received an i.p.
  • TCRm antibody 3.2G1 Characterization of the TCRm antibody 3.2G1 : To establish that the 3.2G1 TCRm mAb isolated in the initial screening was HLA-A2 restricted and peptide-specific, a series of assays to characterize its binding specificity were performed. The first assessment utilized refolded peptide/HLA-A2 molecules as targets for testing the 3.2G1 TCRm in an ELISA.
  • Fig. 39A shows the results of ELISA analysis of supernatant from hybridoma 3.2G1 versus HLA-A2/ ⁇ 2 m complex refolded with its cognate peptide GVL or with one of three other irrelevant peptides.
  • the cells were pulsed with the specific peptide GVL, with irrelevant peptides VLQ or TMT, or with no peptide, and then stained with 3.2G1 (Fig. 39B).
  • the concentration of 3.2G1 in supernatant was determined by isotype-specific ELISA, and the antibody was used at 1 ⁇ g per stain. Binding to the surface of the cells was detected with goat anti-mouse FITC labeled secondary antibody and the cells were analyzed by flow cytometry.
  • the GVL pulsed cells shifted significantly (mean fluorescence intensity [MFI] of 141) compared to cells pulsed with the irrelevant peptides containing closely related sequences VLQ and TMT or no peptide (MFI of 7.3, 7.5 and 9.0 respectively).
  • T2 cells were next pulsed with varying levels of GVL peptide.
  • the peptide was serially diluted and added to cells at concentrations ranging from 50 ⁇ g/ml to 0.1 ⁇ g/ml.
  • the net MFI was determined by subtracting the VLQ peptide pulsed T2 cell MFI value from the MFI of GVL pulsed cells.
  • Fig.40A illustrates the CDC results of cells pulsed with various ratios of peptide (GVCTMT) for both the HLA-A2 specific BB7.2 antibody and 3.2G1.
  • BB7.2 is a murine lgG2b antibody, and this isotype also efficiently fixes complement.
  • BB7.2-driven lysis demonstrates that there is little difference between cells pulsed with peptides at the various concentrations.
  • 3.2G1 detects endogenous GVL peptide-HLA-A2 presented on human tumor cell lines: The ability of the 3.2G1 antibody to detect endogenously processed peptide in the context of the HLA-A2 molecule was evaluated by immunofluorescent staining of a series of tumor cell lines (Fig. 41). BB7.2 mAb indicated the level of HLA-A2 expression on cells. SKOV3.A2 and SW620 are ovarian and colon cancer cell lines, respectively, while MDA-MB-231 and BT20 are breast cancer cell lines. Additional analysis of the SKOV3.A2, SW620 and MDA-MB-231 cell lines by ELISA indicated that hCG ⁇ was present in these lines (data not shown).
  • BT20 cells were not evaluated for the presence of hCG ⁇ but were included as an HLA-A2 negative control.
  • the three HLA-A2 positive tumor cell lines displayed different levels of GVL/A2 when stained with the 3.2G1 TCRm and, as might be anticipated, the staining intensity varied in accordance with the level of HLA-A2 on the surface.
  • the HLA-A2 negative cell line, BT-20 was not stained with either 3.2G1 or BB7.2. Because of its consistently high level of expression of GVL/A2 and in order to maximize the target density, the MDA-MB-231 cell line was selected as the target for the following in vitro and in vivo assays.
  • the 3.2G1 TCRm mAb directs killing of a human tumor cell line in vitro:
  • the breast cancer cell line MDA-MB-231 was subjected to competition analysis via tetramer blockade of CDC in the same manner in which the T2 cells were evaluated (described above). Cells were plated and allowed to adhere overnight before antibody or antibody plus tetramer was applied. Antibody concentration was varied from 25 to 1 ⁇ g/ml, and tetramer concentration was held constant at 6 ⁇ g/ml. CDC of cells incubated with antibody in the absence of tetramer showed an antibody concentration-dependent lysis which was paralleled by cells incubated with antibody in the presence of VLQ tetramer.
  • a second mechanism which plays an important role in the ability of a therapeutic antibody to control or eliminate tumors is antibody-dependent cell-mediated cytotoxicity (ADCC) (Liu et al., 2004; Prang et al., 2005; and Clynes et al., 2000).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • peripheral blood mononuclear cells were isolated from the platelet chambers of aphaeresis collection devices from anonymous donors. The cells were held in serum-free medium (AIM-V) containing 200 units/ml rhlL-2 for 2 to 7 days with media changes every 2 to 3 days in order to maintain and activate the NK population (Liu et al., 2002).
  • each preparation was evaluated using the NK-sensitive cell line K562 at the same time the ADCC assays were carried out. All PBMC isolates were shown to exhibit lysis levels of 60% or more with one exception (35%) (data not shown).
  • MDA-MB-231 cells were first evaluated for sensitivity to ADCC as adherent cultures using five different human PBMC preparations to control for variation among the individual donors.
  • Fig. 42B shows the results of these assays, which contained 10 ⁇ g/ml of 3.2G1 TCRm and were run at an E:T ratio of 30:1.
  • the PBMC preparations varied in their ability to lyse MDA cells as might be anticipated due to differences in receptor expression by NK cells.
  • the overall ADCC ranged from 6.8 to 9.6% with an average value of 8.7%.
  • 3.2G1 TCRm orthe pan-HLA antibody W6/32 which is also a murine isotype IgG 23 , were used as targeting agents.
  • Fig. 42C shows the results from an ADCC analysis of MDA-231 cells using two different human donor preparations at an E:T ratio of 20:1 with 3.2G1 and W6/32.
  • the lysis values achieved for W6/32 (14.6-22.6%) were greater than those of 3.2G1 (6.4-13.4%) suggesting that lysis was at least in part dependent on epitope density.
  • these results show a modest but consistent level of tumor-specific ADCC mediated by the 3.2G1 TCRm.
  • Fig. 44 illustrates that the 3.2G1 TCRm can be used therapeutically to treat athymic nude mice with established tumors. Female athymic mice were subcutaneously injected with MDA-MB-231 breast cancer cells and after 10 days of growth, the mice were injected with either the 3.2G1 TCRm antibody or an IgG 23 isotype control antibody.
  • mice then received 3 more injections at weekly intervals. 24 days after initial injection, tumor growth was measured and plotted as tumor volume. Tumor growth in the IgG 23 isotype control group increased almost three-fold from an initial pre-treatment mean of 105 mm 3 to a mean of 295 mm 3 . In contrast, the 3.2G1 treated group had a mean tumor volume of 62 mm 3 that was reduced to a tumor volume of 8 mm 3 after treatment. Even more impressive was that 3 out of 4 mice in the 3.2G1 treated group had no tumors.
  • TCRm mAbs can be used therapeutically to eradicate established tumors in mice, thus demonstrating the therapeutic effectiveness of using TCRm to kill tumors via binding to a specific peptide-MHC complex on the surface of cancer cells.
  • the current study characterizes the functional properties of an antibody with the type of HLA-restricted peptide specificity associated with T cell receptors. The similarity in epitope recognition to a TCR has led us to designate this antibody a TCR mimic (TCRm) and to investigate its potential as a therapeutic agent.
  • TCRm TCR mimic
  • the 3.2G1 TCRm is a murine IgG 23 monoclonal antibody that (1) binds to and mediates both CDC and ADCC lysis of cells bearing the GVL peptide-HLA complex on their surface and (2) inhibits the growth of a human breast cancer cell line when it is implanted into mice.
  • 3.2G1 TCRm immunofluorescent staining intensity was proportional to the antibody concentration and to the amount of peptide present on the surface of the T2 cells. Staining was also blocked in a dose-dependent manner by GVLJA2 tetramers added to the staining buffer.
  • the results obtained with our novel TCRm indicate that (1) the peptide/MHC complex is a legitimate target for cancer therapy by a naked antibody, (2) the level of expression of specific complex is high enough on at least one tumor line to lead to efficient lysis, and (3) there appears to be a threshold level of expression of the complex above which the antibody is effective.
  • a large number of peptide antigens from tumors that are recognized by T cells have been previously characterized (Novellino et al., 2005) and now offer new targets available on the tumor surface for antibody therapy. These antibodies open access to a new range of targets available on the cell surface which are independent of the ultimate location of the original protein to which they are directed.
  • the ability to create effective TCRm recognizing such peptides in the context of MHC antigens presents the opportunity to significantly expand the current repertoire of therapeutic antibodies.
  • FIG. 45 Shown in Fig. 45 is a timeline of the protocol of generating peptide-MHC specific monoclonal antibodies of the presently disclosed and claimed invention. As evidenced by the figure and the examples provided herein above, a rapid method of generating peptide-MHC specific monoclonal antibodies has been demonstrated, wherein the peptide-MHC specific monoclonal antibodies can be generated in 8-12 weeks.
  • the method of the presently disclosed and claimed invention results in hybridoma cells producing high affinity, full-length antibodies to specific peptide-HLA complexes.
  • An example of the affinity range achieved is shown by the 4F7 monoclonal antibody (see for example, Fig. 23 and Example 2), which has a K D of approximately 1 nM. Affinity measurements for the 1B8 monoclonal antibody indicate that it is in the same affinity range.
  • the product produced by the method of the presently disclosed and claimed invention is "ready to use”; it is a whole antibody which is easy to purify and characterize, and does not require any further manipulation to achieve expression of significant quantities of material.
  • the method of the presently disclosed and claimed invention requires significantly less time to product when compared to the prior art methods.
  • the method of the presently disclosed and claimed invention can complete the cycle from immunization to identification of candidate hybridomas in as few as eight weeks, as shown in Fig. 45 and as achieved as described herein for monoclonal antibody 1 B8.
  • the method of the presently disclosed and claimed invention is both rapid and reproducible.
  • the immunogen employed in the method of the presently disclosed and claimed invention is novel.
  • the immunogen consists of peptide-HLA complexes that are loaded solely with the peptide of interest.
  • the immunogens are made in a form which allows production and characterization of milligram quantities of highly purified material which correctly presents the three dimensional structure of the peptide-HLA complex. This complex can be easily manipulated to form higher order multimers. Preliminary data indicates that the use of tetrameric forms of the peptide-HLA immunogen is more efficient at generating a specific response than are monomeric or mixed multimeric forms of the immunogen.
  • the screening processes described in the presently claimed and disclosed invention are unique and completely describe methods to discern the presence of anti-peptide/HLA antibodies in the serum of immunized mice, even in the presence of antibodies which react with other epitopes present on the complex.
  • the screening processes also produce methods to identify and characterize monoclonal antibodies produced after hybridoma fusion.
  • the presently disclosed and claimed invention overcomes obstacles encountered in prior art methods, which reported low yields of specific monoclonal responses (Eastman et al., 1996; Dadaglio et al., 1997; and Andersen et al., 1996).
  • the antibodies generated by the method of the presently disclosed and claimed invention are also clearly distinct from those reported from phage libraries.
  • a phage-derived Fab which recognized hTERT-HLA-A2 complex would stain hTERT-peptide pulsed HLA-A2 positive cells (Levet al., 2002), but would not stain tumor cells (Parkhurst et al., 2004), indicating that this prior art antibody had either low specificity, or low affinity, or both.
  • Such an antibody would not be useful in applications described herein for the presently disclosed and claimed invention, such as but not limited to, epitope validation in vaccine development and other clinical applications.
  • Nikitina E. Y., et al. Dendritic cells transduced with full-length wild-type p53 generate antitumor cytotoxic T lymphocytes from peripheral blood of cancer patients. Clin Cancer Res, 7(1): 127-35 (2001).
  • HLA-A2 molecules in an antigen-processing mutant cell contain signal sequence-derived peptides. Nature, 356(6368): 443-6 (1992).

Abstract

La présente invention concerne une méthode permettant de produire des anticorps qui reconnaissent des peptides associés à un état oncogène ou pathologique, les peptides se présentant dans le contexte de molécules HLA. Ces anticorps produisent un effet semblable à la spécificité d'un récepteur des lymphocytes T (TCR), mais possèdent une affinité de liaison supérieure, ce qui signifie que les molécules peuvent être utilisées comme réactifs dans des applications thérapeutiques et diagnostiques et dans la recherche. La méthode de la présente invention destinée à la production d'un mimétique de récepteur de lymphocytes T consiste à identifier un peptide d'intérêt capable d'être présenté par une molécule CMH, à former un immunogène comprenant au moins un complexe peptide/CMH dont le peptide est le peptide d'intérêt, puis à administrer une dose utile de l'immunogène à un hôte pour déclencher une réaction immunitaire et à analyser le sérum prélevé chez l'hôte pour déterminer si les anticorps désirés qui reconnaissent une présentation tridimensionnelle du peptide dans le sillon de fixation de la molécule CMH sont produits. Ces anticorps désirés peuvent différencier le complexe peptide/CMH par rapport à une molécule CMH seule, le peptide seul, et un complexe formé par un CMH et un peptide sans intérêt. La méthode de l'invention consiste enfin à isoler les anticorps désirés.
PCT/US2006/034547 2005-09-07 2006-09-07 Anticorps utilises comme mimetiques de recepteurs de lymphocytes t, leurs methodes de production et leurs applications WO2007030451A2 (fr)

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AU2006289683A AU2006289683A1 (en) 2005-09-07 2006-09-07 Antibodies as T cell receptor mimics, methods of production and uses thereof
CA002662798A CA2662798A1 (fr) 2005-09-07 2006-09-07 Anticorps utilises comme mimetiques de recepteurs de lymphocytes t, leurs methodes de production et leurs applications
EP06814164A EP1933864A4 (fr) 2005-09-07 2006-09-07 Anticorps utilisés comme mimétiques de récepteurs de lymphocytes t, leurs méthodes de production et leurs applications
IL190014A IL190014A0 (en) 2005-09-07 2008-03-06 Antibodies as t cell receptor mimics, methods of production and uses thereof

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EP2026837A2 (fr) * 2006-06-01 2009-02-25 Receptor Logic, Ltd. Anticorps utiles en tant qu'analogues de récepteur des lymphocytes t, leurs procédés de production et leurs utilisations
EP2262834A2 (fr) * 2008-02-27 2010-12-22 Receptor Logic Inc. Anticorps imitant les récepteurs des lymphocytes t, leurs procédés de production et d utilisation
US9926380B2 (en) 2012-07-10 2018-03-27 Board Of Regents, The University Of Texas System Monoclonal antibodies for use in diagnosis and therapy of cancers and autoimmune disease

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EP2026837A2 (fr) * 2006-06-01 2009-02-25 Receptor Logic, Ltd. Anticorps utiles en tant qu'analogues de récepteur des lymphocytes t, leurs procédés de production et leurs utilisations
EP2026837A4 (fr) * 2006-06-01 2010-06-02 Receptor Logic Inc Anticorps utiles en tant qu'analogues de récepteur des lymphocytes t, leurs procédés de production et leurs utilisations
EP2262834A2 (fr) * 2008-02-27 2010-12-22 Receptor Logic Inc. Anticorps imitant les récepteurs des lymphocytes t, leurs procédés de production et d utilisation
EP2262834A4 (fr) * 2008-02-27 2011-08-17 Receptor Logic Inc Anticorps imitant les récepteurs des lymphocytes t, leurs procédés de production et d utilisation
US9926380B2 (en) 2012-07-10 2018-03-27 Board Of Regents, The University Of Texas System Monoclonal antibodies for use in diagnosis and therapy of cancers and autoimmune disease
US11192958B2 (en) 2012-07-10 2021-12-07 Board Of Regents, The University Of Texas System Monoclonal antibodies for use in diagnosis and therapy of cancers and autoimmune disease

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IL190014A0 (en) 2008-08-07
EP1933864A4 (fr) 2009-12-16

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