EP2262834A2 - Anticorps imitant les récepteurs des lymphocytes t, leurs procédés de production et d utilisation - Google Patents

Anticorps imitant les récepteurs des lymphocytes t, leurs procédés de production et d utilisation

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Publication number
EP2262834A2
EP2262834A2 EP09715242A EP09715242A EP2262834A2 EP 2262834 A2 EP2262834 A2 EP 2262834A2 EP 09715242 A EP09715242 A EP 09715242A EP 09715242 A EP09715242 A EP 09715242A EP 2262834 A2 EP2262834 A2 EP 2262834A2
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EP
European Patent Office
Prior art keywords
specific peptide
peptide
mhc
mhc complex
cell
Prior art date
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EP09715242A
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German (de)
English (en)
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EP2262834A4 (fr
Inventor
Jon A. Weidanz
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Receptor Logic Inc
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Receptor Logic Inc
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Application filed by Receptor Logic Inc filed Critical Receptor Logic Inc
Publication of EP2262834A2 publication Critical patent/EP2262834A2/fr
Publication of EP2262834A4 publication Critical patent/EP2262834A4/fr
Withdrawn legal-status Critical Current

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    • 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
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
    • 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
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the presently disclosed and claimed invention relates generally to a methodology of utilizing 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 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 Il 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.
  • 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. Once 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.
  • CTL cytotoxic T lymphocytes
  • 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.
  • 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).
  • phage-derived antibodies produced by the prior art methods will not fold right in mammalian cells due to their selection for expression in prokaryotic or simple eukaryotic systems; generally, ⁇ 1% of phage-derived antibodies will efficiently fold in mammalian cells, thus greatly increasing the number of candidates that must be screened and virtually assuring that interesting lead candidates with the most desirable binding properties are non-producible in mammalian cells due to the infrequency of success. Supporting this contention is the fact that very few phage-derived antibodies have proceeded into clinical investigation, and no phage-derived antibody has been approved for use as a therapeutic. All approved therapeutic antibodies have their discovery origin from a mammalian species.
  • the prior art phage-derived antibodies are not useful for making anti- MHC/peptide complexes, as they typically exhibit low affinity, low robustness, low capability to grow and fold, and as they are generally laborious to implement and have not been shown to be viable for approved therapeutic use.
  • Fig. 1 illustrates the expression of HLA/peptide complexes on MDA cells, as detected by T cell receptor mimics RL1 B, RL4B and RL6A.
  • Fig. 2 graphically illustrates that TCRms RL4B and RL6A (A) or RL1 B (B) increase tumor cell cytotoxicity.
  • Fig. 3 graphically illustrates that TCRms RL4B and RL6A (A) or RL1 B (B) increase cell death on tumor cell lines.
  • Fig. 4 graphically depicts that TCRms RL4B and RL6A mediate CDC.
  • Fig. 5 graphically depicts that TCRm RL4B mediates ADCC of breast cancer cells.
  • Fig. 6 illustrates the in vivo efficacy of TCRm RL4B for cancer prophylaxis.
  • Fig. 7 depicts that TCRms RL6A (A) and RL4B (B) retard tumor growth in orthotopic breast cancer models.
  • Fig. 8 illustrates that TCRm RL6A debulks large tumors in orthotopic breast model in mice.
  • Fig. 9 depicts the West Nile Virus (WNV) genome map and peptide sequences.
  • Fig. 10 graphically illustrates titration of RL15A TCRm (anti-WNV3 peptide/HLA-A2 antibody).
  • Fig. 11 graphically depicts peptide titration with various concentrations of WNV-3 peptide and RL15A TCRm.
  • Fig. 12 graphically represents the examination of RL15A TCRm cross-reactivity with WNV peptides 1 , 2, 4, 5 and 6.
  • Fig. 13 graphically represents that RL15A TCRm recognizes dengue type-1 peptide (DT1) peptide-HLA-A2 complex.
  • Fig. 14 graphically represents that RL15A TCRm recognizes dengue type-2 peptide (DT2) peptide-HLA-A2 complex.
  • Fig. 15 graphically represents that RL15A TCRm does not recognize dengue type-3 peptide (DT3) peptide-HLA-A2 complex.
  • Fig. 16 graphically represents that RL15A TCRm recognizes dengue type-4 peptide (DT4) peptide-HLA-A2 complex.
  • Fig. 17 graphically represents that RL15A TCRm recognizes Yellow Fever Virus
  • Fig. 18 graphically represents that RL15A TCRm recognizes JEV/SEV peptide-HLA-
  • Fig. 19 graphically represents that RL15A TCRm recognizes Murray Valley
  • MVEV Encephalitis Virus
  • Fig. 20 graphically depicts the examination of RL15A TCRm cross-reactivity for viral peptide-HLA-A2 epitopes.
  • Fig. 21 graphically illustrates the examination of RL15A TCRm cross-reactivity to cancer-associated peptide-HLA-A2 epitopes.
  • Fig. 22 graphically depicts titration of RL14C TCRm (anti-WNV6 peptide/HLA-A2 antibody).
  • Fig. 23 graphically illustrates peptide titration with WNV-6 peptide and RL14C TCRm.
  • Fig. 24 graphically depicts the examination of RL14C TCRm cross-reactivity with
  • Fig. 25 graphically illustrates the examination of RL14C TCRm cross-reactivity for viral peptide-HLA-A2 epitopes.
  • Fig. 26 graphically depicts the examination of RL14C TCRm cross-reactivity to cancer-associated peptide-HLA-A2 epitopes.
  • Fig. 27 illustrates an affinity determination of RL14C TCRm for cognate peptide-HLA-
  • Fig. 28 demonstrates that TCRm RL15A specifically inhibits anti-SVG9/A2 CTL responses.
  • Fig. 29 illustrates that TCRm antibodies to WNV surface epitopes recognize naturally processed and presented peptide-HLA complexes.
  • Fig. 30 graphically illustrates inhibition of peptide-specific CTL lines using TCRm antibodies.
  • Fig. 31 demonstrates that DCs can cross-present HLA class-l restricted hCG ⁇ epitopes to CD8 + T cells.
  • Fig. 32 illustrates that RL4D TCRm inhibits anti-GVL peptide-A2 reaction CTL after incubation with tumor cell lines.
  • 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.
  • 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.
  • the 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. Alternatively and preferably, two protein sequences (or polypeptide sequences derived from them of at least 30 amino acids in length) 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.
  • 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.
  • the term "corresponds to” is used herein to mean that 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 1 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, a-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 1 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 presently disclosed and claimed 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, valine, 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 other than 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') 2l 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 Histocompability 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, 1 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 it 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. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
  • 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 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. However, the variability is not evenly distributed through the variable domains of antibodies. It is concentrated in three segments called complementarity determining regions (CDRs) also known as hypervariable regions both in the light chain and the heavy chain variable domains.
  • CDRs complementarity determining regions
  • 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 Fv fragments.
  • 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 (CH 1) of the heavy chain.
  • Fab 1 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 (K) and 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 IgA- 2.
  • the heavy chains constant domains that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , y 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 presently disclosed and claimed invention may be made by the hybridoma method first described by Kohler and Milstein, Nature 256, 495 (1975).
  • All monoclonal antibodies utilized in accordance with the presently disclosed and claimed invention will be either (1) the result of a deliberate immunization protocol, as described in more detail herein below; or (2) the result of an immune response that results in the production of antibodies naturally in the course of a disease or cancer.
  • These monoclonal antibodies are distinguished from the prior art antibodies which are phage- derived, because said prior art phage-derived antibodies are not useful for making anti- MHC/peptide complexes, as they typically exhibit low affinity, low robustness, low capability to grow and fold, and as they are generally laborious to implement and have not been shown to be viable for approved therapeutic use.
  • the monoclonal antibodies of the presently disclosed and claimed 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 presently disclosed and claimed 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 therefore, 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 1 , 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). In some cases, 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 invention is not limited to the treatment protocols described above, and other treatment protocols which are known to a person of ordinary skill in the art may be utilized in the methods of the presently disclosed and claimed invention.
  • 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 presently disclosed and claimed 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 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 but not by way of limitation, in U.S.
  • 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, such as a human antibody 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. Various methods of labeling polypeptides and glycoproteins are known in the art and may be used.
  • labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3 H, 14 C, 15 N, 35 S 1 90 Y, 99 Tc, 111 In, 125 I, 131 I) 1 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.
  • the term "pharmaceutical agent or drug” as used herein 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 Francisco (1985)), incorporated herein by reference).
  • the term "antineoplastic 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.
  • 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). Generally, a substantially pure composition will comprise more than about 50% percent of all macromolecular species present in the composition, such as more than about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 99%.
  • 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 and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
  • 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, hepatoma, 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.
  • 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 presently disclosed and claimed invention attached to a functional moiety may be referred to herein as an
  • 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 presently disclosed and claimed 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 presently disclosed and claimed 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 presently disclosed and claimed invention include, but are not limited to, green fluorescent protein (GFP), alkaline phosphatase (AP), peroxidase, orange fluorescent protein (OFP), ⁇ -galactosidase, fluorescein isothiocyanate (FITC), phycoerythrin, Cy-chrome, rhodamine, blue fluorescent protein (BFP), Texas red, horseradish peroxidase (HPR), and the like.
  • GFP green fluorescent protein
  • AP alkaline phosphatase
  • OFP orange fluorescent protein
  • FITC fluorescein isothiocyanate
  • BFP blue fluorescent protein
  • 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.
  • 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
  • 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, refer to: 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 presently disclosed and claimed invention includes 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 presently disclosed and claimed 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 presently disclosed and claimed 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 presently disclosed and claimed 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)].
  • a functional moiety may be attached to the T cell receptor mimic by attaching an affinity tag-coupled T cell receptor mimic of the presently disclosed and claimed 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 presently disclosed and claimed invention includes a T cell receptor mimic of the presently disclosed and claimed invention and a therapeutic moiety conjugated thereto.
  • the pharmaceutical composition of the presently disclosed and claimed invention may be an antineoplastic agent.
  • a diagnostic composition of the presently disclosed and claimed invention includes a T cell receptor mimic of the presently disclosed and claimed invention and a detectable moiety conjugated thereto.
  • the presently disclosed and claimed invention relates to methodologies for uitlizing an agent, such as but not limited to antibodies or antibody fragments that function as T-cell receptor mimics (TCRm's), that recognize peptides displayed in the context of HLA molecules, wherein the peptide is associated with a tumorigenic, infectious, disease or immune dysfunction state.
  • TCR T-cell receptor
  • these antibodies will mimic the specificity of a T cell receptor (TCR) such that the molecules may be used as therapeutic reagents.
  • 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 methods utilize a T-cell receptor mimic, as described in detail hereinabove and in US Serial No. 11/809,895, filed June 1 , 2007, and in US published applications US 2006/0034850, filed May 27, 2005, and US 2007/00992530, filed September 7, 2006, which have previously been incorporated herein by reference.
  • the T- cell receptor mimic utilized in the methods of the presently disclosed and claimed invention comprises 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 specific peptide alone, and a complex of MHC and an irrelevant peptide.
  • the T cell receptor mimic may be produced by any of the methods described in detail in the patent applications listed herein above and expressly incorporated herein by reference; for example but not by way of limitation, the T cell receptor mimic may be produced by immunizing a host with an effective amount of an immunogen comprising a multimer of two or more specific peptide/MHC complexes.
  • the T cell receptor mimic utilized in accordance with the presently disclosed and claimed invention may be produced by a method that includes identifying a peptide of interest, wherein the peptide of interest is capable of being presented by an MHC molecule, and wherein the vaccine composition comprises the peptide of interest.
  • An immunogen comprising a multimer of two or more peptide/MHC complexes is then 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, wherein 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 then assayed to determine if desired antibodies that recognize a three-dimensional presentation of the peptide in the binding groove of the MHC molecule is being produced, wherein the desired antibodies can differentiate the peptide/MHC complex from the MHC molecule alone, the peptide of interest alone, and a complex of MHC and irrelevant peptide.
  • the desired antibodies are then isolated.
  • Table I provides a list of some of the peptides that have been utilized to produce TCRm's by the methods described in detail in US Serial No. 11/809,895, filed June 1 , 2007, and in US published applications US 2006/0034850, filed May 27, 2005, and US 2007/00992530, filed September 7, 2006, which have previously been incorporated herein by reference.
  • the use of TCRm's produced using any of the peptides of SEQ ID NOS: 1-97 is specifically contemplated by the presently disclosed and claimed invention.
  • the presently disclosed and claimed invention is not limited to TCRm's produced using said peptides, but rather the scope of the presently disclosed and claimed invention encompasses TCRm's raised against any specific peptide/MHC complex.
  • any agent capable of directly detecting peptide/MHC complexes on the surface of a cell and differentiating the peptide/MHC complex from the MHC molecule alone, the specific peptide alone and a complex of MHC and irrelevant peptide may be utilized in accordance with the presently disclosed and claimed invention.
  • agents that may be utilized include, but are not limited to, soluble T-cell receptors, extracted T-cell receptors, antibodies, antibody fragments and the technologies described in any of the following US patents/publications: US Publication No.
  • US 2006/0270604 A1 published on November 30, 2006 and filed by Lipovsek et al., on July 7, 2006; US Publication No. US 2008/0139791 A1 , published on June 12, 2008 and filed by Lipovsek et al., on June 12, 2008; US Publication No. US 2006/0286603 A1 , published on December 21 , 2006 and filed by Kolkman et al., on March 28, 2006; US Publication No. US 2005/0053973 A1 , published on March 10, 2005 and filed by Kolkman et al, on may 5, 2004;US Publication No.
  • US 2006/0234299 A1 published on October 19, 2006 and filed by Stemmer et al., on November 16, 2005; US Publication No. US 2008/0003611 A1 , published on January 3, 2008 and filed by Silverman et al., on July 12, 2006;US Publication No. US 2006/0286066 A1 , published on December 21 , 2006 and filed by Basran on December 22, 2005; US Publication No. US 2006/0257406 A1 , published on November 16, 2006 and filed by Winter et al., on May 31 , 2005; US Publication No. US 2006/0106203 A1 , published on May 18, 2006 and filed by Winter et al., on December 28, 2004; US Patent No.
  • the presently disclosed and claimed invention relates to a method of mediating lysis of tumorigenic cells expressing at least one specific peptide/MHC complex on a surface thereof, wherein the specific peptide of the at least one specific peptide/MHC complex is associated with a tumorigenic state.
  • tumorigenic cell as used herein will be understood to refer to a cell showing aberrant growth or structural phenotype such that given time, that phenotype will not act cooperatively with normal body processes.
  • tumor associated antigens includes those associated with fully transformed cells as well as those associated with a precancerous state.
  • an agent comprising a composition reactive against a specific peptide/MHC complex; the agent can differentiate the specific peptide/MHC complex from the MHC molecule alone, the specific peptide alone, and a complex of MHC and an irrelevant peptide.
  • the agent is contacted with tumorigenic cells expressing at least one specific peptide/MHC complex on a surface thereof, such that the agent mediates lysis of the tumor cells expressing the at least one specific peptide/MHC complex on a surface thereof.
  • the agent is a T cell receptor mimic comprising an antibody or antibody fragment reactive against a specific peptide/MHC complex, wherein 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 specific peptide may be associated with any cancer, including but not limited to, at least one of breast cancer, ovarian cancer, prostate cancer, lung cancer, multiple myeloma, biliary cancer, and pancreatic cancer.
  • the specific peptide is at least one of SEQ ID NOS:4, 5, 10, 18, 26, 29, 33 and 43.
  • the mechanism of mediation of cell lysis may comprise at least one of activation of complement-dependent cytotoxicity (CDC), activation of antibody- dependent cellular toxicity (ADCC), induction of apoptosis, and activation of an antiproliferative effect.
  • CDC complement-dependent cytotoxicity
  • ADCC antibody- dependent cellular toxicity
  • induction of apoptosis activation of an antiproliferative effect.
  • the presently disclosed and claimed invention is also directed to a method of mediating lysis of infected cells expressing at least one specific peptide/MHC complex on a surface thereof, wherein the specific peptide of the at least one specific peptide/MHC complex is associated with an infectious disease state.
  • an agent is provided, wherein the agent comprises a composition reactive against a specific peptide/MHC complex, wherein the agent can differentiate the specific peptide/MHC complex from the MHC molecule alone, the specific peptide alone, and a complex of MHC and an irrelevant peptide.
  • the agent is then contacted with infected cells expressing at least one specific peptide/MHC complex on a surface thereof, such that the agent mediates lysis of the infected cells expressing the at least one specific peptide/MHC complex on a surface thereof.
  • the agent is a T cell receptor mimic comprising an antibody or antibody fragment reactive against a specific peptide/MHC complex, wherein 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 specific peptide may be associated with a bacterial infection. That is, the peptide may have been identified as being expressed in complex with a MHC molecule on the surface of a bacterially-infected cell and not expressed on a surface of a normal, non-infected cell.
  • the specific peptide may be associated with a viral infection. That is, the peptide may have been identified as being expressed in complex with a MHC molecule on the surface of a virally-infected cell and not expressed on a surface of a normal, non-infected cell.
  • Examples include, but are not limited to, specific peptides associated with an HIV infection, such as but not limited to, SEQ ID NOS:37 and 43; a flavivirus, including but not limited to, West Nile virus (such as but not limited to, SEQ ID NOS:63 and 78-82), and influenza virus (such as but not limited to, SEQ ID NOS:1 , 28, 37, 50, 53, 54 and 83-88); hepatitis B; hepatitis C; human papilloma virus (HPV); herpes virus; cytomegalovirus (CMV) and Epstein-Barr virus (EBV).
  • SEQ ID NOS:37 and 43 a flavivirus, including but not limited to, West Nile virus (such as but not limited to, SEQ ID NOS:63 and 78-82), and influenza virus (such as but not limited to, SEQ ID NOS:1 , 28, 37, 50, 53, 54 and 83-88)
  • hepatitis B hepatit
  • the mechanism of mediation of cell lysis may comprise at least one of activation of complement-dependent cytotoxicity (CDC), activation of antibody- dependent cellular toxicity (ADCC), induction of apoptosis, and activation of an antiproliferative effect.
  • CDC complement-dependent cytotoxicity
  • ADCC antibody- dependent cellular toxicity
  • the presently disclosed and claimed invention is also directed to a method of blocking autoreactive T cells activated by cells expressing at least one specific peptide/MHC complex on a surface thereof, wherein the specific peptide of the at least one specific peptide/MHC complex is associated with an autoimmune state.
  • an agent comprising a composition reactive against a specific peptide/MHC complex, wherein the agent can differentiate the specific peptide/MHC complex from the MHC molecule alone, the specific peptide alone, and a complex of MHC and an irrelevant peptide.
  • the agent is then contacted with cells expressing at least one specific peptide/MHC complex on a surface thereof, such that the agent binds to the surface of the cell and blocks binding and activation of autoreactive T cells.
  • the agent is a T cell receptor mimic comprising an antibody or antibody fragment reactive against a specific peptide/MHC complex, wherein 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 specific peptide may be any peptide associated with an autoimmune state, including but not limited to, at least one of SEQ ID NOS:2 and 4.
  • 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, while 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 presently disclosed and claimed invention is also related to a method of killing or damaging a target cell expressing or displaying an antigen-presenting portion of a complex composed of a human antigen-presenting molecule and an antigen derived from a pathogen.
  • the method involves exposing the target cell to a T cell receptor mimic as defined herein above, thereby killing or damaging a target cell expressing or displaying an antigen-presenting portion of a complex composed of a human antigen-presenting molecule and an antigen derived from a pathogen.
  • EXAMPLE 1 Detection of endogenously processed and presented peptide/HLA-A2 epitopes using RL1 B, RL4B and RL6A TCRms.
  • the inventors have previously observed that the RL1B, RL4B and RL6A TCR mimic monoclonal antibodies (TCRms) recognize recombinant HLA-A2 protein or T2 cells pulsed with the Her2/neu p369 peptide (SEQ ID NO:5), hCGb p47 peptide (SEQ ID NO:4) or the RNA helicase p68 peptide (SEQ ID NO:10), respectively.
  • TCRms monoclonal antibodies
  • RL4B and RL6A TCRm mAbs stained cells with significantly stronger intensity than cells stained with the RL1 B TCRm suggesting that the Her2/neu peptide/HLA-A2 epitope is present on MDA-MB-231 tumor cells at a lower copy number than the epitopes recognized by RL4B and RL6A.
  • Brightest staining was observed using BB7.2 mAb, and no cell staining was seen with any of the isotype control antibodies.
  • TCRm mAbs can be used in the detection and validation of epitopes which are endogenously processed and presented on the surface of tumor cells.
  • TCRm the cytotoxic properties of these agents was investigated in vitro.
  • TCRm antibodies RL4B and RL6A were incubated with MDA-MB-231 tumor cells and assessed for cytotoxic effects using an MTT cell viability assay.
  • the MTT assay measures cell viability through assessing mitochondrial reductase enzyme activity. The results from this study are shown in Figure 2A and reveal that both RL4B and RL6A TCRm reduce tumor cell viability.
  • TCRm antibodies induce tumor cell death.
  • uptake of Annexin-V and propidium iodide (Pl) was assessed.
  • MDA-MB-231 tumor cells were grown for 24h in the absence or presence of isotype control antibody, BB7.2, RL4B TCRm or RL6A TCRm.
  • Cell death was evaluated after 24h incubation using Annexin-V tagged with APC fluorophore and propridium iodide (Pl) by flow cytometric analysis. Shown in Figure 3A, the percent of dead and/or dying cells are plotted after treatment with respected antibody or TCRm.
  • the BB7.2 mAb was found to induce cell death that was approximately 2-fold greater than the isotype control antibody while both RL4B and RL6A TCRms induced far greater cell death at almost 3-fold more than the isotype control.
  • Figure 3B shows the results of RL1 B TCRm mediated tumor cell death. As seen in this figure, BB7.2 induced approximately 3-fold greater cell death than the isotype control while the RL1 B TCRm generated tumor cell death that was >5-fold higher than the isotype control antibody.
  • RL4B and RL6A TCRm mAbs direct complement dependent cytotoxic (CDC) killing of a human tumor cell line in vitro.
  • the breast cancer cell line MDA-MB-231 was subjected to TCRm mediated CDC in the same manner in which the T2 cells were evaluated (data not shown). Tumor cells were plated and allowed to adhere overnight before antibody was applied. Antibody concentration was varied from 10 to 1.25 ⁇ g/ml.
  • Murine lgG2a antibodies have been found to efficiently direct complement dependent cytolysis (CDC) while the IgGI isotype does not.
  • FIG. 4 illustrates the CDC results of MDA-MB-231 tumor cells for the HLA-A2 specific BB7.2 antibody, RL4B and RL6A TCRms.
  • TCRm antibodies are potent activators of complement resulting in tumor cell lysis and offers a novel approach for targeting and killing tumor cells.
  • RL4B TCRm mediates antibody dependent cell-mediated cytotoxicity
  • ADCC antibody-dependent cell- mediated cytotoxicity
  • 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.
  • Figure 5 shows the results of these assays, which contained 10 ⁇ g/ml of RL4B TCRm and were run at an effector cell to target cell ratio (E:T) 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% after subtraction and normalization of lgG2a isotype control.
  • RL4B TCRm or the pan-HLA antibody W6/32 which is also a murine isotype lgG2a, were used as targeting agents.
  • the lysis values achieved for W6/32 (14.6-22.6%) were greater than those of RL4B (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 RL4B TCRm.
  • RL4B TCRm anti-tumor activity in nude mice implanted with MDA-MB-231 tumor cells.
  • nude mice were implanted with MDA-MB-231 tumor cells.
  • Antibody treatment was initiated at the time of implantation with an intra-peritoneal (i.p.) injection of either RL4B TCRm or an isotype control antibody at a dose of 1.5mg/kg. Tumors began to appear in the isotype control-treated mice between 36 and 43 days (week 6) after implantation, while none were evident in any of the mice treated with RL4B.
  • TCRms prevent tumor growth in breast cancer orthotopic model.
  • athymic nude mice were implanted in the right mammary fat pads with a formulation mixture that was comprised of 5 x 10 6 MDA-MB- 231 tumor cells and Matrigel. Matrigel was used to create a nutrient rich environment that led to rapid tumor growth resulting in palpable tumors in 4 to 5 weeks after cell implantation. Tumors were allowed to grow to a mean volume of approximately >35mm 3 prior to initiation of treatment with RL4B or isotype control antibody (Figure 7A) and RL6A or isotype control antibody ( Figure 7B).
  • mice were treated with weekly injections of either TCRm or control antibody at a concentration of 1.5mg/kg.
  • Athymic nude mice implanted with MDA-MB-231 tumor cells in Matrigel were treated with RL6A TCRm (Figure 7B).
  • one group of mice received 5-weekly injections of isotype control antibody at 1.5mg/kg.
  • Tumor volume at week 0 was >35mm 3 and grew to a mean tumor volume of ⁇ 800mm 3 by week 5.
  • the mean tumor volume in athymic nude mice treated with RL6A ( ⁇ 3) at a dose of 1.5mg/kg initially grew but mean tumor size was reduced by the fourth week and not even palpable after the fifth week after RL6A treatment.
  • no palpable tumors were detected in the RL6A treated mice for an additional 4 weeks (data not shown).
  • TCRm are useful reagents for debulking large established tumors.
  • a significant test at demonstrating the anti-tumor properties of an antibody in vivo is for the antibody to shrink or debulk large established tumors in mice.
  • TCRm's have potent anti-tumor activity in vivo and support the uses of TCRm's as agents for tumor shrinkage.
  • MB-231 (breast) was obtained from the American Type Culture Collection (ATCC).
  • ATCC American Type Culture Collection
  • the murine lgG2a isotype control Abs was purchased from Sigma-Aldrich.
  • Fresh blood buffy coats containing peripheral blood mononuclear cells were obtained from anonymous blood donations from Coffee Memorial Blood Bank (Amarillo, TX).
  • IgG heavy chain-phycoerythrin was purchased from Caltag Laboratories (Burlingame, CA).
  • Isotype control antibodies mouse IgGI , lgG2a and lgG2b, were purchased from Southern Biotech (Birmingham, AL).
  • the BB7.2 anti-HLA A2.1 mAb expressing mouse hybridoma cell line was purchased from the ATCC.
  • KIFGSLAFL (residues 369- 377, designated as Her-2369; SEQ ID NO:5)
  • RNA Helicase p68 YLLPAIVHI (residues 720- 728, designated as p68; SEQ ID NO: 10)
  • human chorionic gonadotropin- ⁇ GVLPALPQV (residues 47-55, designated as GVL47; SEQ ID NO:4) were synthesized at the University of Oklahoma Health Science Center, Oklahoma City, OK, using a solid-phase method and purified by HPLC to greater than 90%.
  • Cell culture included IMDM from Cambrex. Medium supplements included heat-inactivated FBS and penicillin/streptomycin from Sigma-Aldrich and L-glutamine from HyClone. All tumor lines were maintained in culture medium containing glutamine, penicillin/streptomycin and 10% FBS. When necessary, attached cells were released from flasks using TrypLE express (Invitrogen Life technologies).
  • Tumor Cell staining Tumor cells (3x10 5 ) were washed and resuspended in
  • Cells were washed with sterile 1x PBS and then resuspended in 100 ⁇ l culture media containing IOOOng TCRmimics (RL4B, RL6A, RL1B), isotype controls (lgG2a, IgGIb) or an anti-HLA-A2 antibody (BB7.2a). The cells were incubated for 24 hrs at 37 0 C with 5% CO 2 . MTT (Promega, Madison, Wl) was added to wells at a concentration of 10 ⁇ l in each well and allowed to develop overnight. Stop solution was added at 100 ⁇ l in each well, and plates were read for absorbance at 560nm. [00159] b) Cell Death assay.
  • MDA-MB231 cells were plated on a 12 well plate at a density of 20,000 cells/well in 2ml of culture media and allowed to adhere for 24 hrs at 37°C incubator with 5% CO 2 . Plates were washed with sterile 1x PBS and resuspended in 2ml of culture media containing IOOOng, 2000ng and IOOOng respectively of TCRm antibodies RL4B, RL6A, and RL1 B; IOOOng of isotype controls (lgG2a, IgGI); IOOOng of the anti-HLA- A2 antibody (BB7.2A); or were left untreated.
  • the cells were then washed resuspended in 500 ⁇ l of 1x binding buffer, followed by addition of 10 ⁇ l Propidium Iodide (Pl) (BD Pharmingen) to each tube, except for blank controls (untreated-unstained cells and untreated-Annexin V stained cells).
  • Pl Propidium Iodide
  • the samples were then immediately read on a FACS Canto followed by analysis on DIVA software (BD Biosciences).
  • PBMCs peripheral blood mononuclear cells
  • AIM-V serum-free medium
  • W6/32 positive control
  • mice The Jackson Laboratory and housed under sterile conditions in barrier cages.
  • mice were implanted with 5x10 6 freshly harvested MDA-MB-231 cells at 97% viability in Matrigel (Sigma-Aldrich).
  • c. and 50 ⁇ g of either 3.2G1 or isotype control Ab weekly for the following 3 wk (total injections 4).
  • Animals were held for at least 1 week after the appearance of the last tumor in the isotype control group (a total of 70 days) before totaling frequency of occurrence. All tumors reached >3 mm in diameter before being scored as positive.
  • FIG. 9 a total of four nonamer peptides having strong binding affinity for HLA-A * 0201 were identified.
  • WNV3 SVGGVFTSV; SEQ ID NO:63
  • WNV6 SEQ ID NO:82
  • RL14C anti-WNV6 peptide/HLA-A2
  • RL15A anti-WNV3 peptide/HLA-A2
  • RL15A TCRm showed optimum staining of WNV3 peptide pulsed T2 cells at a concentration of 120ng/ml and fluorescence intensity decreased with titration of the TCRm concentration. Background staining was established using unpulsed T2 cells stained with either RL15A TCRm (120ng/ml) or with mouse IgG 1 isotype control antibody (120ng/ml). Data are representative of 3 independent experiments.
  • T2 cells were pulsed for four hours with WNV-3 peptide at concentrations ranging from 1 to 2 x10 4 nM and then stained with purified RL15A (120ng/ml). Maximum RL15A TCRm staining was observed with T2 cells pulsed with 1x10 4 nM of WNV3 peptide. Fluorescent signal was weakly detected for T2 cells pulsed with 1OnM of peptide.
  • RL15A TCRm cross-reactivity for five other WNV peptides that bind HLA-A2 complexes with high affinity was then examined, as shown in Figure 12.
  • T2 cells were pulsed with WNV- peptides (1, 2, 3, 4, 5 & 6; SEQ ID NOS: 78, 79, 63, 80, 81 and 82, respectively) at 20 ⁇ M concentration and then stained with purified RL15A (120ng/ml).
  • RL15A TCRm did not stain T2 cells without peptide or pulsed with WNV peptides 1 , 2, 4, 5, & 6. Background signal was determined using unpulsed T2 (UP T2) cells or pulsed with WNV-3 peptide (SEQ ID NO:63) and then stained with mouse isotype control antibody (120ng/ml). Data are representative of 3 independent experiments.
  • T2 cells were pulsed with dengue type-1 peptide (SEQ ID NO:57) at 20 ⁇ M concentration and then stained with purified RL15A at the following concentrations (90, 120, 250 and 500ng/ml).
  • the maximal signal (geometric mean fluorescent intensity fluorescent intensity ⁇ 15) for DT1 peptide pulsed T2 cells was observed using 500ng/ml of RL15A TCRm.
  • Signal strength was greater for DT1 peptide (geometric mean fluorescent intensity fluorescent intensity >14) than WNV-3 peptide (geometric mean fluorescent intensity fluorescent intensity ⁇ 11) pulsed T2 cells when stained with RL15A TCRm at 120 ng/ml.
  • Background signal was determined using unpulsed T2 (UP T2) cells stained with RL15A TCRm or mouse isotype control antibody (120ng/ml). Data are representative of 3 independent experiments.
  • T2 cells were pulsed with yellow fever virus peptide (YFV; SEQ ID NO: 1;
  • T2 cells were pulsed with Murray Valley Encephalitis virus
  • MVEV MV
  • SEQ ID NO:93 purified RL15A at the following concentrations (90, 120, 250 and 500ng/ml).
  • Background signal was established using unpulsed T2 (UP T2) cells stained with RL15A TCRm or mouse isotype control antibody (120ng/ml). Data are representative of 3 independent experiments.
  • T2 cells were pulsed with 20 ⁇ M of the following peptides: WNV-3 (SEQ ID NO:63), CMVpp65 (NLVPMWATV; SEQ ID NO:95), HPV18 E7-1 (TLQDIVLHL; SEQ ID NO:66), Epstein Barr Virus (YLLEMLWRL; SEQ ID NO:76) and Influenza M1 (GILGFVTL; SEQ ID NO:28) and stained with purified RL15A (120ng/ml). RL15A TCRm cross-reactivity to non-related viral peptides was not observed as geometric mean fluorescent intensity values were comparable to background single intensity.
  • T2 cells were pulsed with 20 ⁇ M of the following peptides: WNV-
  • RL15A TCRm cross-reactivity to non-related viral peptides was not observed as geometric mean fluorescent intensity values were comparable to background single intensity.
  • Background signal was determined by staining unpulsed T2 (UP T2) with RL15A TCRm (120ng/ml) or with mouse IgG 1 isotype control antibody (120ng/ml). In addition, only background signal was detected for T2 cells pulsed with 20 ⁇ M of WNV- 3 peptide and stained with mouse isotype control antibody. Data are representative of 3 independent experiments.
  • RL14C monoclonal antibody RL14C was further characterized.
  • purified RL14C was used at concentrations of 100, 200, 300, 400, and 500ng/ml to stain T2 cells pulsed with 20 ⁇ M of WNV-6 peptide (SEQ ID NO:82). Background signal was established using unpulsed T2 (UP T2) cells stained with either RL14C TCRm (500ng/ml) or with mouse IgG 1 isotype control antibody (500ng/ml). Data are representative of 3 independent experiments.
  • RL14C TCRm crossreactivity for five other WNV peptides that bind HLA-A2 coplex with high affinity was examined.
  • T2 cells were pulsed with WNV- peptides (1 , 2, 3, 4, 5, & 6; SEQ ID NOS:78, 79, 63, 80, 81 , and 82, respectively) at 20 ⁇ M concentration and then stained with purified RL14C (200ng/ml).
  • T2 cells were pulsed with 2OmM of the following peptides: WNV- 6 (SEQ ID NO:82), CMVpp65 (NLVPMWATV; SEQ ID NO:95), HPV18 E7-1 (TLQDIVLHL; SEQ ID NO:66), Epstein Barr Virus (YLLEMLWRL; SEQ ID NO:76), HPV16 (YMLDLQPETT; SEQ ID NO:77) and Influenza M1 (GILGFVTL; SEQ ID NO:28) and stained with purified RL14C (200ng/ml).
  • WNV- 6 SEQ ID NO:82
  • CMVpp65 NLVPMWATV
  • HPV18 E7-1 TQDIVLHL
  • SEQ ID NO:66 Epstein Barr Virus
  • YLLEMLWRL SEQ ID NO:76
  • HPV16 YMLDLQPETT
  • Influenza M1 GILGFVTL; SEQ ID NO:28
  • RL14C TCRm cross-reactivity to non-related viral peptides was not observed as geometric mean fluorescent intensity values were comparable to background signal intensity.
  • Background signal was determined by staining unpulsed T2 (UP T2) cells with RL14C TCRm (200ng/ml) or with mouse IgG 1 isotype control antibody (200ng/ml). In addition, only background signal was detected for T2 cells pulsed with 20 ⁇ M of WNV-6 peptide and stained with mouse isotype control antibody. Data are representative of 3 independent experiments.
  • T2 cells were pulsed with 20 ⁇ M of the following peptides: WNV-
  • MART-1 (ALGIGILTV; SEQ ID NO: 17), Her2/neu (KIFGSLAFL; SEQ ID NO:5), CD19 (KLMSPKLYV; SEQ ID NO:46), NY-ESO-1 (SLLMWIQTV; SEQ ID NO:59), gP100 (YLEVGPVTA; SEQ ID NO:97), human chorionic gonadotropin- ⁇ (GVLPALPQV; SEQ ID NO:4), p53 tumor suppressor protein (LLGRNSFEV; SEQ ID NO:8), CD19 (TLAYLIFCL; SEQ ID NO:11), human chorionic gonadotropin- ⁇ (VLQGVLPAL; SEQ ID NO:3), p68 (YLLPAIVHI; SEQ ID NO:10), topoisomerase (FLYDDNQRV; SEQ ID NO:27), hTERT:540 (ILAKFLHWL; SEQ ID NO:14), and ODC-1 (ILDQKINEV; SEQ ID NO:33
  • RL14C TCRm cross-reactivity to non-related cancer-associated peptides was not observed, as geometric mean fluorescent intensity values were comparable to background signal intensity.
  • Background signal was determined by staining unpulsed T2 (UP T2) cells with RL14C TCRm (200ng/ml) or with mouse IgG 1 isotype control antibody (200ng/ml). In addition, only background signal was detected for T2 cells pulsed with 20 ⁇ M of WNV-6 peptide and stained with mouse isotype control antibody. Data are representative of 3 independent experiments.
  • the affinity for RL14C was determined using surface plasmon resonance, as shown in Figure 27.
  • a rat anti-mouse IgG antibody was immobilized to a sensor chip via standard amine coupling chemistry.
  • a 1OnM solution of RL14C TCRm was passed over the sensor chip and captured by the immobilized anti-mouse IgG mAb.
  • WNV-6 peptide/HLA-A2 monomer complexes at 24, 48 and 96nM were run over the chip, and binding to RL14C was observed and reported as response units (RU).
  • the rates of association and dissociation were determined as 2.27x10 5 and 1.58 x 10 "3 , respectively.
  • the data presented in Figure 29 reveal for the first time the presentation of two novel WNV peptide epitopes on infected cells using TCRms.
  • Cells were also stained with the pan-HLA-A2 antibody, BB7.2, which showed maximum HLA-A2 expression.
  • BB7.2 pan-HLA-A2 antibody
  • the TCRm staining profiles were markedly weaker than that of the BB7.2 mAb.
  • the TCRm detected both WNV3 and WNV6 peptide epitopes on viral infected cells.
  • RL14C were used at concentrations ranging from 30-120ng/ml to stain T2 cells pulsed with various concentrations of selected WNV peptides, selected flavivirus peptides, cancer- associated peptides, or irrelevant peptides, as indicated by the figures. Background staining was established using unpulsed T2 cells (UP T2) stained with either TCRm of interest (120ng/ml or as indicated in figure) or with mouse IgG 1 isotype control antibody (120ng/ml or as indicated in figure).
  • TCRm binding was detected using goat-anti-mouse IgG-PE conjugate (250-500ng/ml), and the geometric mean fluorescent intensity (GMFI) was determined by flow cytometric analysis utilizing either a FACS Canto or FACS Scan (BD Biosciences). Data were analyzed by either FACS Diva or CellQuest software (BD Biosciences) and are representative of 3 independent experiments.
  • GMFI geometric mean fluorescent intensity
  • HelaA2 cells were infected with WNV at MOIs of 10, 3 and 1 and then stained with RL14C and RL15A at 48h post-infection. Detection of bound TCRm was carried out using a goat-anti-murine IgGI-PE conjugate and flow cytometric analysis.
  • IgG antibody was immobilized to a sensor chip via standard amine coupling chemistry.
  • a 1OnM solution of RL14C TCRm was passed over the sensor chip and captured by the immobilized anti-mouse IgG mAb.
  • WNV-6 peptide/HLA-A2 monomer complexes at 24, 48 and 96nM were run over the chip, and binding to RL14C was observed and reported as response units (RU), utilizing a SensiQ (ICx Nomadics, OKC). Association, disassociation, and affinity constants (K D ) were determined using manufacturer supplied analysis software and algorithms.
  • TCRm Immune modulation by TCRm represents a novel application for these agents that could be applied to the inhibition of antigen specific T cell responses.
  • TCRm could be used to specifically block activation and expansion of auto-reactive T cells or T cells that are involved in mediating chronic inflammation.
  • the use of TCRm in this manner represents a unique paradigm shifting technology and an approach to immunotherapy not previously conceived.
  • TCRm specific for two different peptides derived from human chorionic gonadotropin beta (hCG ⁇ ) protein that show specific inhibition of antigen reactive CD8 + T cell lines.
  • TCRms generation of TCRms, characterization of binding to specific peptide, and demonstration of target display on tumor cells.
  • TCT or GVL peptide
  • SEQ ID NOS:2 or 4 peptide
  • splenocytes isolated from immunized mice were prepared for fusion with the P3X-63Ag8.653 myeloma cell line and plated in a semi-soft cellulose medium. After about two weeks, colonies were identified, picked to individual wells of a 96 well plate for expansion and the hybridoma supematants were screened for reactive antibodies.
  • Table Il shows the results from hybridoma fusions for each peptide-HLA-A2 immunogen.
  • IgGI , lgG2a and lgG2b antibodies were selected from each immunization group.
  • each TCRm recognizes its cognate peptide-A2 target in coated wells, it was unclear whether these mAbs would recognize the specific peptide when loaded into HLA-A * 0201 complexes expressed on a cell surface.
  • their binding to T2 cells pulsed with 20 ⁇ M of specific, irrelevant peptides or no peptide was analyzed. Both TCRms stain T2 cells pulsed with only specific peptide (data not shown).
  • CTL lines were generated against the TMT and GVL peptide-HLA-A * 0201 epitopes using autologous dendritic cells.
  • CTL peptide specificity was determined using T2 cells alone or T2 cells pulsed with relevant peptide.
  • TMT and GVL peptide-specific CTL lines responded to T2 cells presenting relevant peptide but not to T2 cells alone.
  • granzyme B production by CTL lines specific for TMT and GVL peptide-epitopes was inhibited by the addition of anti-TMT and anti-GVL TCRm, respectively.
  • peptide-epitope specific TCRm were used to confirm CTL recognition specificity for the TMT peptide and GVL peptide epitopes.
  • TMT and GVL peptide-epitope specific TCRm were processed and presented in the context of HLA-A * 0201 in vaccine-treated DCs and that TCRm antibodies are useful agents in validating the recognition specificity of the CTL response.
  • RL4D TCRm specifically blocks anti-GVL peptide/A2 CTL from reacting to tumor cell lines.
  • the ability of a TCRm to specially inhibit a CTL response to an antigen positive tumor cell line was examined.
  • the RL4D TCRm was used to stain the tumor cell lines. Only the Colo205 and MDA-MB-231 tumor cells showed staining with RL4D TCRm, demonstrating that the GVL peptide/A2 epitope was expressed on the surface of these cells (see histogram plots).
  • the left side of the bottom panel shows GVL peptide-specific CTL production of granzyme B measured by ELISpot assay after 6hr incubation with tumor cell lines.
  • Antibodies and synthetic peptides The conjugated polyclonal antibodies goat anti-mouse-lgG (H + L chains)-horseradish peroxidase (HRP) and goat anti-mouse IgG heavy chain-phycoerythrin (PE) were purchased from Caltag Biosciences (Burlingame, CA). The mouse lgd isotype control antibody was purchased from Southern Biotech (Birmingham, AL).
  • TMTRVLQGV human chorionic gonadotropin- ⁇ peptide designated as TMT (40) ; (SEQ ID NO:2)] and GVLPALPQV [residues 47-55, human chorionic gonadotropin- ⁇ peptide, designated as GVLf 47) ; (SEQ ID NO:4)] were synthesized at the University of Oklahoma Health Sciences Center, Oklahoma City, OK, using a solid- phase method and purified by HPLC to greater than 90%.
  • the human lymphoblastoid cell line T2 (HLA-A * 0201) and the
  • P3X-63Ag8.653 murine myeloma cell line used as a fusion partner were purchased from the American Type Culture Collection (ATCC, Manassas, VA).
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • Recombinant human IL-4 and GM-CSF were obtained from Peprotech (Rockyhill, NJ). After 5-6 days, the immature dendritic cells were detached from the flask by incubation at 4°C for 20 - 60 min., centrifuged, counted and either used immediately or frozen at -80°C for later use.
  • T2 is a mutant cell line that lacks transporter-associated proteins (TAP) 1 and 2 which allows for efficient loading of exogenous peptides (Wei, M. L. & Cresswell, P. HLA-A2 molecules in an antigen-processing mutant cell contain signal sequence-derived peptides. Nature 1992, 356(6368), 443-446]).
  • 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.
  • T2 cells were pulsed for 4 hours with decreasing amounts of specific peptide (2,000 - 0.15 nM). T2 cells (5 x 10 5 ) were then washed in SB to remove excess peptide and stained with each TCRm-PE conjugate, 3F9 and 1B10 TCRms at 1mg/ml_ of SB).
  • T cells were stimulated as bulk cultures in vitro on a 8-10 day cycle for 3-4 weeks with autologous immature DCs previously exposed to the vaccine (contains hCG ⁇ antigen) or the vehicle (no hCG ⁇ antigen) at 30 ⁇ g/ml, two wells were untreated, and the plate was incubated for up to 3 days at 37° C, 5% CO 2 and matured with Poly I:C) at a ratio of 10:1 in the presence of cytokines sequentially added (10ng/ml each of IL-7 on day 0 and IL-2 on day 1) every 3 days.
  • cytokines sequentially added (10ng/ml each of IL-7 on day 0 and IL-2 on day 1
  • CD8 + T cells from HLA-A2 * donors were repeatedly stimulated with hCG ⁇ synthetic peptides (TMTRVLQGV (SEQ ID NO:2) and GVLPALPQV (SEQ ID NO:4)) loaded on to matured autologous DCs. Effector T lymphocytes were expanded on anti-CD3 and anti-CD28 Dynal immunomagnetic beads (Invitrogen, Carlsbad, CA) and restimulated with vaccine on day 14, and CD8 + and CD4 + T cells were purified using a commercial T cell enrichment kit (Miltenyi MACS, Auburn, CA).
  • CD8 + CTL activity of vaccine or peptide-stimulated CD8 + T cells was assessed using vaccine-treated DCs or peptide-loaded T2 cells in the presence of 3 ⁇ g/ml ⁇ 2 microglobulin.
  • CD8 + CTL response was measured in a cell-based IFN- ⁇ cytokine or granzyme B production ELISpot assay (MabTech, Sweden and Cell Sciences, Canton, MA for ELISpot kits).
  • the DEAD box protein p68 a novel transcriptional coactivator of the p53 tumor suppressor. The EMBO Journal. 24: 543-553 (2005).
  • Tumor- associated antigen human chorionic gonadotropin beta contains numerous antigenic determinants recognized by in vitro-induced CD8+ and CD4+ T lymphocytes. Cancer Immunol lmmunother 2002;50(12):673 — 81.
  • Gauduin MC Intracellular cytokine staining for the characterization and quantitation of antigen-specific T lymphocyte responses. Methods 2006;38(4):263 — 73.
  • 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).
  • Novellino L 1 Castelli C Purani G. A listing of human tumor antigens recognized by T cells: March 2004 update. Cancer Immunol Immunother 2005;54(3):187 — 207. Oka Y, Tsuboi A 1 Kawakami M, Elisseeva OA, Nakajima H, Udaka K, et al. Development of WT 1 peptide cancer vaccine against hematopoietic malignancies and solid cancers. Curr Med Chem 2006;13(20):2345— 52.
  • HER-2/neu is expressed in human renal cell carcinoma at heterogeneous levels independently of tumor grading and staging and can be recognized by HLA-A2.1- restricted cytotoxic T lymphocytes, lnt J Cancer, 87(3): 349-59 (2000).
  • HLA-A2 molecules in an antigen-processing mutant cell contain signal sequence-derived peptides. Nature 1992;356(6368):443 — 6.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Virology (AREA)
  • Oncology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé permettant de produire et d'utiliser 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 peuvent être utilisés dans le cadre de procédés thérapeutiques destinés à la médiation de lyses cellulaires.
EP09715242A 2008-02-27 2009-02-27 Anticorps imitant les récepteurs des lymphocytes t, leurs procédés de production et d utilisation Withdrawn EP2262834A4 (fr)

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US6732808P 2008-02-27 2008-02-27
US19187108P 2008-09-12 2008-09-12
PCT/US2009/001290 WO2009108372A2 (fr) 2008-02-27 2009-02-27 Anticorps imitant les récepteurs des lymphocytes t, leurs procédés de production et d’utilisation

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AU2012236068A1 (en) 2011-04-01 2013-10-17 Eureka Therapeutics, Inc. T cell receptor-like antibodies specific for a WT1 peptide presented by HLA-A2
GB201501017D0 (en) * 2014-12-23 2015-03-04 Immatics Biotechnologies Gmbh Novel peptides and combination of peptides for use in immunotherapy against hepatocellular carcinoma (HCC) and other cancers
LT3626729T (lt) 2014-12-23 2021-10-25 Immatics Biotechnologies Gmbh Nauji peptidai ir peptidų deriniai, skirti naudoti imunoterapijai prieš hepatoceliulinę karcinomą (hck) ir kitų rūšių vėžį
MX2017012352A (es) 2015-04-03 2018-01-26 Eureka Therapeutics Inc Construccion dirigida a complejos de peptido de alfa-fetoproteina/complejo principal de histocompatibilidad (afp/cph) y usos de los mismos.

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WO2007030451A2 (fr) * 2005-09-07 2007-03-15 Receptor Logic, Ltd. Anticorps utilises comme mimetiques de recepteurs de lymphocytes t, leurs methodes de production et leurs applications
US20070092530A1 (en) * 2004-05-27 2007-04-26 Weidanz Jon A Antibodies as T cell receptor mimics, methods of production and uses thereof
WO2007143104A2 (fr) * 2006-06-01 2007-12-13 Receptor Logic, Ltd. Anticorps utiles en tant qu'analogues de récepteur des lymphocytes t, leurs procédés de production et leurs utilisations

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US20090042285A1 (en) * 2004-05-27 2009-02-12 Weidanz Jon A Antibodies at T cell receptor mimics, methods of production and uses thereof

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US20070092530A1 (en) * 2004-05-27 2007-04-26 Weidanz Jon A Antibodies as T cell receptor mimics, methods of production and uses thereof
WO2007030451A2 (fr) * 2005-09-07 2007-03-15 Receptor Logic, Ltd. Anticorps utilises comme mimetiques de recepteurs de lymphocytes t, leurs methodes de production et leurs applications
WO2007143104A2 (fr) * 2006-06-01 2007-12-13 Receptor Logic, Ltd. Anticorps utiles en tant qu'analogues de récepteur des lymphocytes t, leurs procédés de production et leurs utilisations

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Title
NEETHLING F A ET AL: "Assessing vaccine potency using TCRmimic antibodies", VACCINE, ELSEVIER LTD, GB, vol. 26, no. 25, 25 February 2008 (2008-02-25), pages 3092-3102, XP022710591, ISSN: 0264-410X, DOI: DOI:10.1016/J.VACCINE.2008.02.025 [retrieved on 2008-02-25] *
See also references of WO2009108372A2 *

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EP2262834A4 (fr) 2011-08-17
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