WO2009151487A1 - Procédés de dosage de la puissance d’un vaccin - Google Patents

Procédés de dosage de la puissance d’un vaccin Download PDF

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Publication number
WO2009151487A1
WO2009151487A1 PCT/US2009/001144 US2009001144W WO2009151487A1 WO 2009151487 A1 WO2009151487 A1 WO 2009151487A1 US 2009001144 W US2009001144 W US 2009001144W WO 2009151487 A1 WO2009151487 A1 WO 2009151487A1
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Prior art keywords
peptide
vaccine
mhc
cell
antigen presenting
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PCT/US2009/001144
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English (en)
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Jon A. Weidanz
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Receptor Logic, Inc.
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Priority claimed from US12/196,885 external-priority patent/US20090075304A1/en
Application filed by Receptor Logic, Inc. filed Critical Receptor Logic, Inc.
Publication of WO2009151487A1 publication Critical patent/WO2009151487A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70539—MHC-molecules, e.g. HLA-molecules

Definitions

  • the present invention relates generally to a methodology of producing antibodies that recognize peptides associated with a tumorigenic or disease state, wherein the peptides are displayed in the context of MHC molecules. These antibodies will mimic the specificity of a T cell receptor (TCR) such that the molecules may be used as therapeutic, diagnostic and research reagents.
  • TCR T cell receptor
  • Class I major histocompatibility complex (MHC) molecules designated HLA class I in humans, bind and display peptide antigen ligands upon the cell surface.
  • the peptide antigen ligands presented by the class I MHC molecule are derived from either normal endogenous proteins ("self) or foreign proteins ("nonself") introduced into the cell. Nonself proteins may be products of malignant transformation or intracellular pathogens such as viruses.
  • class I MHC molecules convey information regarding the internal milieu of a cell to immune effector cells including but not limited to, CD8 * cytotoxic T lymphocytes (CTLs), which are activated upon interaction with "nonself peptides, thereby lysing or killing the cell presenting such "nonself peptides.
  • CTLs cytotoxic T lymphocytes
  • Class Il MHC molecules designated HLA class Il in humans, also bind and display peptide antigen ligands upon the cell surface. Unlike class I MHC molecules which are expressed on virtually all nucleated cells, class Il MHC molecules are normally confined to specialized cells, such as B lymphocytes, macrophages, dendritic cells, and other antigen presenting cells which take up foreign antigens from the extracellular fluid via an endocytic pathway.
  • the peptides they bind and present are derived from extracellular foreign antigens, such as products of bacteria that multiply outside of cells, wherein such products include protein toxins secreted by the bacteria that often have deleterious and even lethal effects on the host (e.g., human).
  • class 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. [0011] Recent years have seen an increase in the development and testing of therapeutic cancer vaccines (Itoh et al., 2006; Markovic et al., 2006; and Hersey et at., 2005).
  • Therapeutic vaccines for cancer and certain types of viral infections are aimed at stimulating cell-mediated immune responses, in particular those mediated by cytotoxic T lymphocytes (CTL) (Oka et al., 2006; Adotevi et al., 2006; and Xia et al., 2006). Therefore, the development of a cytotoxic effector arm of an anti-tumor response to vaccines requires that the epitopes be presented in the context of human leukocyte antigen (HLA) class I molecules on antigen-presenting cells.
  • HLA human leukocyte antigen
  • TAA tumor-associated antigens
  • CTL lines or clones and T cell hybridomas exposed to vaccine-treated cells are often used to assess epitope presentation by measuring cell proliferation, target cell lysis and cytokine production (Keilholz et al., 2006; and Whiteside et al., 2003).
  • These assays suffer from several limitations including but not limited to, inconsistent assay reproducibility and difficulty in producing and maintaining high quality reagents.
  • potency assays are twofold: (1) to ensure that a given vaccine has at least a predefined minimum level of potential biological activity such as stimulation of antigen-specific CTL lines or clones and (2) that lot-to-lot consistency of the manufactured product can be readily monitored.
  • TCRm antibodies have high affinity and avidity for MHC — peptide complexes and are capable of detecting low densities of the specific MHC — peptide complex present on tumor cells.
  • Fig. 1A graphically depicts that HLA class I molecules display peptides processed from intracellular proteins, and present said complex to T-cell receptors. Recognition of non- self peptides stimulates the cellular immune system to eliminate the diseased cell.
  • Fig. 1 B graphically depicts that T-CeII Receptor mimics (TCRm's) exhibit similar binding specificity to cytotoxic T-lymphocyte recognition of particular peptide-HLA complexes and act as a soluble reagent serving as an alternative to cell-based assays.
  • TCRm's T-CeII Receptor mimics
  • Fig. 2 illustrates a flow cytometry assay where T2 cells (lacking antigen presenting functions and presenting exogenously supplied peptides) are separately pulsed with either Peptide 1 (VLQGVLPAL; SEQ ID NO:3) or closely related Peptide 2 (VLQAVLPPL; SEQ ID NO:69) and then stained with a TCRm that was raised against the Peptide 1/HLA-A*0201 complex. A shift is only observed with cells pulsed with the cognate Peptide 1.
  • Fig. 3 graphically illustrates TCRm's show no cross reactivity to different HLA class I alleles.
  • Fig. 4 graphically depicts affinity binding data for TCRm's RL08A and RL09A.
  • Affinity determination for RL08A (left panel) and RL09A (right panel) was carried out on a SensiQ surface plasmon resonance instrument (ICX Nomadics, Oklahoma City, OK, USA).
  • protein A/G was coupled to a sensor chip to capture approximately 6nM of either RL08A or RL09A antibody.
  • Fig. 4A shows the binding affinity data for RL08A.
  • Monomers of Gp100-peptide/HLA-A2 complexes were run over the sensor chip at concentrations of 12, 24, 48, 96, 192, 364 and 786 nM.
  • Binding values were obtained with on- and off-rates of 2.275 x 10 4 (M-1s-1) and 4.97 x 1O -4 (s-1), respectively, resulting in a final KD of 21.8 nM. These values are approximately 3-fold lower than those reported by Denkberg et al. (Eur. J. Immunol, 2004; 34:2919), who found that their Gp100-peptide/HLA-A2 monoclonal antibody had a KD of 60 nM. Monomers of NY-ESO-1-peptide/HLA-A2 complex were then passed over the RL09A coated chip at concentrations of 12, 24 and 48 nM.
  • T2 cells which lack the ability to process antigens, but specifically load exogenous peptides
  • T2 cells which lack the ability to process antigens, but specifically load exogenous peptides
  • T2 cells which lack the ability to process antigens, but specifically load exogenous peptides
  • the appropriate peptide "A” Gp100 peptide-YLEPGPVTV; SEQ ID NO:75
  • R08A cognate TCRm
  • Fig. 6 illustrates a peptide titration study that demonstrates sensitivity of the T cell receptor mimic (TCRm) RL08A.
  • An antigen presenting cell line was pulsed with decreasing amounts of relevant Gp100 peptide-YLEPGPVTV (SEQ ID NO:75) and then stained with a constant amount (250 ng/ml) of RL08A.
  • Bound RL08A was detected using rat anti-mouse mAb-phycoerythrin (PE) conjugate and flow cytometric analysis.
  • PE rat anti-mouse mAb-phycoerythrin
  • Fig. 7 graphically depicts PolyTest peptide competition assays for affinity determination of HLA-A*0201 peptide-epitopes.
  • Two hCG ⁇ peptides (TMT and GVL) were evaluated using a constant concentration of activated soluble HLA-A*0201 in the presence of 2.2 nM standard FITC-labeled peptide. After reaching equilibrium conditions, fluorescence polarization expressed in mP was measured. Values obtained at different peptide dilutions were graphed and inhibitory concentrations expressed as log[IC50]'s determined by fitting the data to a dose — response model. Results show that both epitopes are of high affinity with very similar binding strength.
  • Fig. 8 graphically depicts characterization of anti-hCG ⁇ -HLA-A*0201 TCRm binding specificity.
  • ELISA was performed in a plate coated with 0.1 ⁇ g of peptide-HLA- A*0201-tetramer complexes that included the following: TMT (40 ) (40-48,TMTRVLQGV; SEQ ID NO:2), VLQ (44) (44—52, VLQGVLPAL; SEQ ID NO:3), GVL (47 ) (47-55, GVLPALPQV; SEQ ID NO:4), and Her2/neu (369) (369-377, KIFGSLAFL; SEQ ID NO:5).
  • Binding specificity for TMT (40) and GVL (47) was determined by adding 0.25 ⁇ g of the following antibodies to wells: (A) 3F9 TCRm specific for TMT (40) -HLA-A * 0201 complex and (B) 1 B10 TCRm specific for GVL (47) -HLA-A*0201.
  • Fig. 9 graphically depicts characterization of anti-hCG ⁇ TCRm mAbs for detection of TMT (40) -HLA-A*0201 and GVL (47) -HLA-A*0201 complexes on T2 cells.
  • T2 cells were incubated with 20 ⁇ M of (A and B) TMT (40) , VLQ (44) or GVL (47) peptides. Cells were then stained either with (A) 3F9 TCRm or IgGI isotype control (filled area), or (B) 1B10 or isotype control (filled area). In all experiments bound antibody was detected using goat anti-mouse PE conjugate.
  • HRP horseradish peroxidase
  • Fig. 10 graphically depicts that Vaccine-treated DCs elicit Ag-specific CTL response.
  • Antigen-specific T cells were generated as described in Methods section. Briefly, DCs were either treated with vaccine or vehicle (control) and matured for 24 h with Poly I:C and then added to B11-hCG ⁇ -specific CTL at a 1 :1 ratio. Supernatant was collected at 24 and 48 h post-incubation and tested for interferon- ⁇ production (10 pg/ml) using the BD OptEIA ELISA Kit II.
  • Fig. 11 graphically depicts inhibition of peptide-specific CTL lines using TCRm antibodies.
  • hCG ⁇ peptide-specific T cells were co-cultured with T2 cells as such or loaded with a specific hCG ⁇ peptide (100 ng/ml) in the presence or absence of an HLA-A2.1-hCG ⁇ peptide complex specific TCRm (50 ng/ml). Cytolytic granule granzyme-B production by Ag- specific CTL was measured in a GrB ELISpot assay.
  • Fig. 12 graphically depicts that DCs can cross-present HLA class l-restricted hCG ⁇ epitopes to CD8+ T cells.
  • Cytolytic T cells generated to hCG ⁇ antigen by repeated stimulation with vaccine (20 ⁇ g/ml B11-hCG ⁇ ) + poly IC (50 ng/ml)-activated DCs recognize cross-presented hCG ⁇ epitopes.
  • hCG ⁇ -specific TCRm 50 ng/ml
  • only can effectively block a specific hCG ⁇ -directed response since a TCRm to an unrelated antigen (NY-ESO-1) does not.
  • Fig. 13 graphically depicts that Vaccine-treated DCs reveal time-dependent presentation of CTL epitopes.
  • Immature DCs were treated with vaccine (B11-hCG ⁇ fusion protein) or with control vaccine (B11-CEA fusion protein) for up to 3 days before maturation with Poly I:C reagent (50 ⁇ g/ml).
  • mDCs were then stained with TCRms, anti-TMTpeptide- HLA-A2 (3F9) and anti-GVL peptide-HLA-A2 (1B10). Detection of bound 3F9 and 1B10 was performed using a goat-anti-mouse — FITC conjugate.
  • Fig. 14 graphically depicts the characterization of TCRm binding detection sensitivity.
  • T2 cells were incubated with decreasing concentrations (2000 — 0.150 nM) of (A) TMT peptide and (B) GVL peptide and stained with (A) 3F9 (B) 5E12 or (C) 4A3 TCRm-PE conjugates.
  • the number of specific complexes was determined by plotting the TCRm staining intensity on to a standard curve generated using BD-Calibrite PE-beads. Numbers plotted above bars for peptide concentrations of 0.15 nM and 78 nM indicate the total specific peptide-HLA-A*0201 complexes detected on peptide-pulsed T2 cells.
  • FIG. 15 illustrates a time course analysis using vaccine containing Gp100 antigen: Gp100 peptide-YLEPGPVTV (SEQ ID NO:75) presentation.
  • Antigen presenting cells were treated with vaccine containing Gp100 and subjected to intracellular staining with anti-Gp100 (purple shading - bottom 3 panels) as well as cell surface staining with RL08A (purple shading - top 3 panels) at 24 h, 48 h and 72 h post treatment. Separation from isotype control (green line) is shown by flow cytometry.
  • TCRm-RL08A enables monitoring of de novo processing of Gp100, allowing for direct analysis of Gp100 processing kinetics and presentation of peptide-YLEPGPVTV/HLA-A2 complexes on the surface of vaccine treated antigen presenting cells.
  • TCRm's offer this functionality with a variety of vaccine formats, including but not limited to: virus expression vectors, nucleic acid, microbial vectors, protein/peptide, and the like.
  • Fig. 16 illustrates peptide/HLA epitope presentation visualized by TCRm staining and immunocytochemistry.
  • Antigen presenting cells were treated with vaccine containing Gp100 followed by incubation at 250 ng/ml with RL08A (left panel) and a control TCRm (right panel). Specific binding of RL08A to cells treated with vaccine containing Gp100 (left panel) was detected using a goat anti-mouse-FITC conjugate (green) and fluorescence microscopy. Dapi blue nuclear stain (right panel) was used to indicate the presence of antigen presenting cells attached to the glass slide.
  • Fig. 17 illustrates CTL activity and TCRm specificity for GP100 peptide- YLEPGPVTV (SEQ ID NO:75) and NY-ESO-1 peptide-SLLMWITQV (SEQ ID NO:13). Specificity of RL08A and RL09A was demonstrated in a competition assay where each respective TCRm was able to decrease CTL stimulation by blocking the T-cell receptor's ability to recognize and bind Gp100 peptide-YLEPGPVTV/HLA-A2 and NY-ESO-1 peptide- SLLMWITQV/HLA-A2 complexes. Blue bars represent cells without TCRm added and red bars represent addition of specific TCRm. Interferon-gamma cytokine production is significantly reduced at antigen dose levels of 1.0 x and 0.1x (top & bottom right-side panels).
  • Fig. 18 illustrates that HLA-peptide complex density correlates with the level of CTL stimulation and intensity of TCRm binding.
  • ⁇ MFI Mean Fluorescence Intensity
  • Fig. 19 illustrates the benchmarking of TCRm staining of CTL stimulation.
  • the minimal acceptable CTL stimulation activity was determined (blue bar) and set as acceptance threshold value (blue dashed line) for both Vaccine Antigens A (gp 100) and B (NYESO1).
  • Parallel studies were carried out quantitating the number of specific HLA-peptide from gp100 and NYESO-1 complexes present on antigen presenting cells (purple and green bars, respectively).
  • the complex numbers determined by TCRm staining of each antigen was determined at the threshold dose of each vaccine.
  • the Established CTL threshold was used to derive Correlative TCRm staining thresholds.
  • Fig. 20 illustrates the use of CTL threshold as pass/fail criteria in the TCRm vaccine potency test of the presently disclosed and claimed invention.
  • the potency of nine different Gp 100 Vaccine formulations were compared using the TCRm quantitative potency assays measuring the numbers of HLA-GpIOO peptide complexes.
  • a Gp100 vaccine standard was used to compare the various vaccine formulations and the CTL threshold for the Gp100 TCRm-RL08A, determined previously, was used as the pass/fail benchmark for the formulations. Using this basis, formulations 1 through 8 were deemed acceptable while formulation 9 failed based on the CTL activity threshold benchmark.
  • Fig. 21 graphically depicts three different batches of antigen presenting cells that were exposed to a constant dose of Gp100 antigen (Antigen "A”; SEQ ID NO:75) and assayed using RL08A-TCRm (TCRm #1) or control TCRm using flow cytometry.
  • the ⁇ MFI values were calculated from the individual flow cytometry plots, averaged, and then presented graphically with standard deviation bars.
  • Fig. 22 illustrates TCRm staining adapted to QuantiBRITETM PE bead system from BD Biosciences. Adaptation of TCRm staining readout from qualitative assay results to quantify specific peptide/HLA complexes/cell. Antigen presenting cells were treated with vaccine containing Gp100 and then stained with RL08A to determine the quantity of specific Gp100 peptide-YLEPGPVTV(SEQ ID NO:75)/HLA-A2 complexes present on the cell surface at 72 h post infection. Linear regression was performed using the geometric means of the four QuantiBRITETM PE bead populations (low, medium low, medium high and high) and the mean number of PE molecules per bead (lot #05765) according to the manufacturer's instructions.
  • Fig. 23 illustrates quantitative measurement of peptide/HLA-A2 Gp100 epitope complexes.
  • Bound antibody was detected using rat anti-mouse IgG-PE conjugate.
  • QuantiBRITETM PE beads were run in parallel according to description given in Fig. 17. Linear regression was performed. Anti- isotype control antibody values are subtracted from the RL08A values.
  • Fig. 24 illustrates quantitative measurement of peptide/HLA-A2 NY-ESO-1 epitope complexes.
  • Antigen presenting cells treated with vaccine expressing NY-ESO-1 at doses of 10.0x, 1.0x and 0.1x and then stained with RL09A (red) and RL08A (blue/control) at 24 h, 48 h, 72 h and 96 h post-infection. Both TCRm's were used at [250ng/ml_]. Bound antibody was detected using rat anti-mouse IgG-PE conjugate.
  • QuantiBRITETM PE beads were run in parallel according to description given in Fig. 10. Linear regression was performed. Anti- isotype control antibody values are subtracted from the RL09A values illustrating that detection of peptide-HLA complexes using TCRm's is possible down to the lowest tested multiplicity of infection (MOI) of 0.1 beginning as early as 24 h post-infection (top left panel). Results are plotted as molecules/cell (specific peptide/HLA complexes/cell) versus antigen dose.
  • MOI multiplicity of infection
  • Fig. 25 illustrates quantitative measurement of all HLA A*02 molecules. Antigen presenting cells were treated with two different doses of Gp100 antigen vaccine (Antigen "A"; SEQ ID NO:75) and harvested at 24, 48, 72 or 96 hours post treatment. The number of specific Gp100 antigen-peptide epitope complexes was quantified using the QuantiBRITETM system and RL08A-TCRm. The total number of HLA A*02 molecules were quantified using an anti-HLA A*02 mAb and the QuantiBRITETM system. The percentage of Gp100 antigen occupied HLA molecules were calculated and presented in graphical format. [0042] Fig. 26 illustrates that TCRm's establish a quantitative baseline for ELISpot assays.
  • ELISpot assay was conducted with the contents described below each individual sample result.
  • the inclusion of the specific TCRm antibody reduces the assay background (in red) to virtually zero, whereas non-specific TCRm shows no effect.
  • the significance between the sample with and without the vaccine is greatly enhanced by the inclusion of the TCRm antibody.
  • 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 (2nd 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.
  • naturally occurring nucleotides includes deoxyribonucleotides and ribonucleotides.
  • modified nucleotides referred to herein 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. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984); Stein et al. Nucl. Acids Res. 16:3209 (1988); Zon et al.
  • 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 "TATAC” corresponds to a reference sequence 1 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 1 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, ⁇ -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 1 direction.
  • the direction of 5' to 3' addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA and which are 5' to the 5 1 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-leuci ⁇ e-isoleuci ⁇ e, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic-aspartic, and asparagine-glutamine.
  • amino acid sequences of antibodies or immunoglobulin molecules are contemplated as being encompassed by the present invention, providing that the variations in the amino acid sequence maintain at least 75%, such as at least 80%, 90%, 95%, and 99%.
  • conservative amino acid replacements are contemplated. Conservative replacements are those that take place within a family of amino acids that are related in their side chains.
  • More preferred families are: serine and threonine are aliphatic- hydroxy family; asparagine and glutamine are an amide-containing family; alanine, 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')2, Fv, and single- chain antibodies. An antibody other than a "bispecific” or “bifunctional” antibody is understood to have each of its binding sites identical. An antibody substantially inhibits adhesion of a receptor to a counterreceptor when an excess of antibody reduces the quantity of receptor bound to counterreceptor by at least about 20%, 40%, 60% or 80%, and more usually greater than about 85% (as measured in an in vitro competitive binding assay).
  • MHC Major Histocompability Complex
  • ⁇ LA 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 is was produced.
  • Contaminant components of its production environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or no ⁇ protei ⁇ aceous 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. There are at least two techniques for determining CDRs:
  • 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 (VH -VL 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 VH -VL 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' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH 1 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 present invention may be made by the hybridoma method first described by Kohler and Milstein, Nature 256, 495 (1975), or may be made by recombinant methods, e.g., as described in U.S. Pat. No. 4,816,567.
  • the monoclonal antibodies for use with the present invention may also be isolated from phage antibody libraries using the techniques described in Clackson et al. Nature 352: 624-628 (1991), as well as in Marks et al., J. MoI. Biol. 222: 581-597 (1991).
  • 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).
  • marked avidin e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods.
  • 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, 90 Y, 99 Tc, 111 In, 125 1, 131 I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, ⁇ -galactosidase, luciferase, alkaline phosphatase), chemiluminescent, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags).
  • labels are attached by spacer arms of various lengths to reduce potential steric hindrance.
  • substantially pure means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition), and preferably a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80 percent of all macromolecular species present in the composition, such as more than about 85%, 90%, 95%, and 99%. In one embodiment, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.
  • 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 cancer 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, hopatoma, breast cancer, colon cancer, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer.
  • mammal for purposes of treatment refers to any animal classified as a mammal, including human, domestic and farm animals, nonhuman primates, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc.
  • antigen presenting cell will be understood to include any cell that can present peptides in the context of MHC molecules.
  • the antigen presenting cell must also be capable of processing proteins/polypeptides into peptides that may be presented in the context of MHC molecules.
  • antigen presenting cells that may be utilized in accordance with the presently disclosed and claimed invention include, but are not limited to, dendritic cells (DCs), macrophages and B cells.
  • Active immunotherapy offers exciting prospects to direct the body's own immune responses to resolve localized or systemic disease.
  • Antigen processing is central to active immunotherapy, whether the approach seeks to elicit cytotoxic T-lymphocyte (CTL) responses to treat cancer and intracellular pathogen infection, or if the goal is to induce T- cell anergy, removing T-cell subsets responsible for damaging autoimmune responses.
  • CTL cytotoxic T-lymphocyte
  • Active immunotherapies most often require the intracellular expression of a disease- associated protein or antigen and processing through the Human Leukocyte Antigen (HLA) class I or class Il system (also known as the Major Histocompatibility Complex; MHC).
  • HLA Human Leukocyte Antigen
  • MHC Major Histocompatibility Complex
  • Antigen expression alone is insufficient to predict the activity of a given immunotherapy - appropriate antigen processing and presentation must be measured if the mode of action and associated potency of the immunotherapy can be addressed. Potency is important to measure in an immunotherapeutic product, especially at product release - to compare lot to lot variability and during stability analyses to insure time, transport and storage conditions have not compromised the drug product.
  • HLA class I is expressed on the surface of all nucleated human cells and, via its display of restricted peptide processed from intracellular proteins, presents a regular snapshot of the expressed proteins within a cell - acting like a proteomic biomarker chip for cellular status and antigen processing.
  • T-cell receptor The interaction between the T-cell receptor and the peptide-HLA complex is central to the adaptive immune response - however its complicated nature presents particular challenges for integration into medical diagnosis and therapy.
  • MAb monoclonal antibody
  • TCRm T- CeII Receptor mimic
  • Fig. 1 T- CeII Receptor mimic
  • TCRm antibodies show high affinity to the particular restricted peptide displayed in the context of the cognate HLA molecule used to produce the antibody.
  • Fig. 2 shows an example of the specificity where a TCRm was raised against Peptide 1 and is unable to recognize (as displayed via a flow cytometry staining assay) Peptide 2, which differs from Peptide 1 in only two of the nine amino acid positions.
  • TCRm antibodies have expected properties of monoclonal antibodies. They have high binding specificity to very specific peptide-HLA complexes and as demonstrated in Fig. 3, do not cross react with non-target HLA.
  • TCRm's have binding affinities that are similar to that of the T-cell receptor with Kd values of many TCRm antibodies ⁇ 25 nM as determined by peptide titration and plasmon resonance experiments.
  • TCRm antibodies also show dramatic dynamic range with regards to sensitivity, where T2 cells pulsed with picomolar concentrations of peptides can be readily identified by the appropriate TCRm antibody (Figs. 5 and 6). These data establish that TCRm antibodies have all the desired properties of monoclonal antibodies widely used in various quality control assays for biologic products.
  • the invention is to be understood to not be limited to the use of TCRm's.
  • any agent capable of directly detecting peptide/MHC complexes on the surface of a cell and are capable of quantitatively measuring the number of peptide/MHC complexes present on the surface of a cell through a binding event 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 2005/0089932 A1 , published on April 28, 2005 and filed by Kolkman et al., on June 17, 2004; 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. US 2006/0263768 A1 , published on November 23, 2006 and filed by Tomlinson et al, on April 28, 2006; US Publication No.
  • TCRm monoclonal antibodies are utilized to directly detect a relative density of processed peptide-epitopes presented on the surface of vaccine-treated mDCs.
  • the TCRm antibodies generated recognize specific peptide-HLAA2 epitopes derived from the hCG ⁇ antigen.
  • the vaccine is an antibody — antigen fusion protein developed at Celldex Therapeutics that specifically targets the mannose receptor on DCs and upon binding initiates rapid vaccine internalization (Ramakrishna et al., 2004).
  • the processing and presentation of the antigen in the vaccine was enabled by further treatment with an adjuvant such as Poly I:C and confirmed using peptide-specific T cell lines.
  • the presently disclosed and claimed invention demonstrates that the TCRm antibody was useful in corroborating the observed CTL activity by: (1) specifically inhibiting T cell stimulation, and (2) detection of HLA-A2-TMT and HLA-A2-GVL peptide complexes in vaccine-treated mDCs.
  • the presently disclosed and claimed invention enables the use of agents, such as but not limited to TCRm mAbs, for the detection and quantitation of a relative density of specific peptide-HLA class I complexes on vaccine-treated mDCs and represents an important tool to measure the potency of CTL-inducing vaccines.
  • the presently disclosed and claimed invention is related to methods of assaying vaccine potency.
  • the "potency of a vaccine composition” is defined as a pre-defined minimum level of potential biological activity, such as but not limited to, stimulation of antigen-specific CTL lines or clones. It has been shown that a density of specific peptides displayed by MHC class I complexes directly correlates with the CTL response to virus and cancer, and therefore the present invention is related to the use of antibodies specific for peptide-MHC class I complexes to measure the potency of CTL-inducing vaccines.
  • the measurement of peptide-MHC class I complexes can be quantitatively determined using the methods described using TCRm antibodies. Said quantitative measurement may be related to a relative number of peptide/MHC complexes per cell, or may be related to an actual number of peptide/MHC complexes per cell.
  • 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 present 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 incorporated herein; briefly, 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 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: 1-81 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.
  • the agents such as but not limited to, T cell receptor mimics, described and claimed herein are capable of directly detecting low densities of specific MHC-peptide complexes present on the surface of cells, such as tumor or infected cells.
  • the agents such as but not limited to, T cell receptor mimics, can thereby be utilized to detect the presence of specific peptide/MHC complexes present on the surface of cells treated with a vaccine, wherein the peptide of the specific peptide/MHC complex is a product of the degradation of a vaccine (or, the vaccine itself, when the vaccine is directly delivered in peptide form).
  • T cell receptor mimic When a T cell receptor mimic is utilized as the agent, T cell receptor mimic may have a binding affinity for the specific peptide/MHC complex of about 10 nanomolar or greater.
  • the agent utilized in accordance with the presently disclosed and claimed invention may be provided with a detection moiety bound thereto to aid in measuring the level of specific peptide/MHC complex present on the surface of the antigen presenting cell.
  • Any detection moiety known in the art or otherwise contemplated by a person having ordinary skill in the art for use with the presently disclosed and claimed invention is encompassed by the scope of the presently disclosed and claimed invention.
  • Particular non- limiting examples of detection moieties that may be utilized in accordance with the presently disclosed and claimed invention have been described in detail herein above.
  • the methods of the present invention include the step of providing a vaccine composition and delivering the vaccine composition to at least one antigen presenting cell to provide a vaccine-treated cell.
  • the vaccine composition may be provided in any form known in the art; for example but not by way of limitation, the vaccine composition may be directly provided as at least one protein/polypeptide that may be processed into peptides by the antigen presenting cell.
  • the vaccine composition may be provided in the form of a nucleic acid segment encoding the at least one protein/polypeptide, wherein the antigen presenting cell expresses the nucleic acid segment and produces the protein/polypeptide encoded by the nucleic acid segment.
  • the vaccine composition may be provided in the form of a specific peptide known to be an epitope expressed in the context of MHC molecules.
  • the vaccine composition may be a nucleic acid segment encoding such peptide epitope (wherein the antigen presenting cell expresses said nucleic acid segment and produces said peptide epitope).
  • the antigen presenting cell to which the vaccine composition is delivered may be any cell that is capable of presenting peptides in the context of MHC molecules.
  • the antigen presenting cell When the vaccine composition is presented in the form of a protein/polypeptide (or a nucleic acid segment encoding same), the antigen presenting cell must also be capable of processing proteins/polypeptides into peptides that may be presented in the context of MHC molecules.
  • antigen presenting cells examples include, but are not limited to, dendritic cells, macrophages, B cells and combinations thereof.
  • the agent such as but not limited to the T cell receptor mimic, whereby the agent binds to the cell surface if the specific peptide/MHC complex utilized to produce the agent is present on the cell surface.
  • the number of specific peptide/MHC complexes present on the surface of the vaccine-treated antigen presenting cell are then quantitatively measured; said methods of quantitative measurement may include both relative quantitation based on delta MFI ( ⁇ MFI) values as well as absolute complex number determinations.
  • Methods of quantitatively measuring the number of specific peptide/MHC complexes include, but are not limited to, correlating TCRm binding ⁇ MFI values derived from flow cytometry with appropriate standard, where a known quantity of the staining reagent, such as but not limited to PE, APC or other materials, is present on a number of standards that allow separation via flow cytometry, ⁇ MFI determination and linear regression formula determination.
  • ⁇ MFI values of unknown samples can be measured by flow cytometry, and quantitative differences can be determined based on relative number of peptide-MHC complexes.
  • unknown samples are analyzed, such as by TCRm staining, and the ⁇ MFI values are compared with the linear regression formula to determine the numbers of staining reagent present.
  • the number of staining reagent present on the antibody measured with flow is then used to determine the average number of peptide-MHC molecules present per cell in an assay.
  • Quantitative measurement and “quantitatively measuring” as used herein will be understood to refer to establishing a differential value related to the number of peptide-MHC complexes present on the surface of vaccine treated cells by relative means, such as but not limited to, by using ⁇ MFI values (which directly correlates with the number of peptide-MHC complexes) or a process to convert these relative values into absolute numbers of peptide-MHC complexes as described above.
  • the potency of the vaccine is then determined, based on the quantitative measurement of the number of specific peptide/MHC complex present on the surface of the vaccine-treated antigen presenting cell.
  • Potency is measured by comparing the threshold amount or activity of the vaccine to induce a T-cell response, such as but not limited to a CTL response or T-cell anergy, such that it is meaningful to a biological effect in vivo.
  • a T-cell response such as but not limited to a CTL response or T-cell anergy, such that it is meaningful to a biological effect in vivo.
  • the T cell receptor mimic binding assay determines the correlative density of the HLA-peptide complexes on the antigen presenting cell.
  • the method is quantitative and yields affinity values with a high degree of accuracy for each of the three peptides used in this example.
  • several hCG ⁇ -derived peptides were found to exhibit HLA-A*0201 binding capabilities. Three of them, namely TMTRVLQGV (40-48; SEQ ID NO:2), VLQGVLPAL (44—52; SEQ ID NO:3) and GVLPALPQV (47—55; SEQ ID NO:4) seemed of high affinity able to stabilize HLA complexes on T2 cell surfaces (Table II).
  • TCRm's generation of TCRm's, characterization of binding to specific peptide, and demonstration of target display on tumor cells.
  • TCT or GVL peptide 1 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 supernatants were screened for reactive antibodies.
  • Table III shows the results from hybridoma fusions for each peptide-HLA-A2 immunogen.
  • IgGI , lgG2a and lgG2b antibodies were selected from each immunization group.
  • each of the antibodies selected has no detectable crossreactivity with either the HLA complex or any of a series of HLA complexes loaded with various peptides, which also bind HLA-A2.
  • each TCRms recognize its cognate peptide-A2 target in coated wells, it was unclear whether these mAbs would recognize the specific peptide when loaded into HLAA*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.
  • Fig. 9 shows that both TCRms stain T2 cells pulsed with only specific peptide.
  • Vaccine-treated DCs elicit Ag-specific CTL response.
  • DCs were treated for 3 days with either the B11-hCG ⁇ vaccine or the B11-CEA control vaccine to target DCs for 3 days and then matured for 24 h using Poly I:C.
  • the CTL line was then incubated with vaccine or vehicle-treated DCs at a ratio of 1 :1 for 24 and 48 h.
  • CTL reactivity was measured by sampling culture supernatant for IFN- ⁇ production. As seen in Fig.
  • the IFN- ⁇ response was significantly higher for CTL incubated for 24 h with DC treated with the B11-hCG ⁇ vaccine (50 pg/ml) than with control treated DCs (15 pg/ml).
  • CTL stimulation for 48 h resulted in even a greater difference in IFN- Y levels between vaccine-treated and vehicle-treated DC, indicating an hCG ⁇ -specific CTL response for peptide epitopes presented on 3 day vaccine-treated DCs.
  • Inhibition of CTL stimulation with peptide-epitope specific TCRm 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. As shown in Fig. 11 , TMT and GVL peptide-specific CTL lines responded to T2 cells presenting relevant peptide but not to T2 cells alone. Further, 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. In this example, 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.
  • TCRm antibodies stain vaccine-treated dendritic cells.
  • the use of TCRms to inhibit CTL response indicated indirectly the expression of specific peptide-epitope on the surface of DCs.
  • TCRm mAbs for direct validation of peptide-epitope expression on vaccine-treated DCs has been examined.
  • the hypothesis that hCG ⁇ peptides presented on the surface of vaccine treated DCs via HLA-A*0201 class I molecules are detectable using peptide-epitope specific TCRms was tested.
  • the kinetics of expression and the hierarchy of peptide presentation on the DCs was examined.
  • TCRm detection sensitivity Next, the sensitivity of each antibody as a staining reagent was evaluated. This was done using flow cytometric analysis of T2 cells loaded with peptide ranging from 2000 nM down to 0.15 nM concentrations. Both TCRm clones (3F9 and 1B10) were able to stain T2 cells loaded with as little as 150 pM of peptide (Fig. 14). These findings indicate TCRm mAbs display detection sensitivity limits comparable to the lower detection limits reported for several high avidity CTL lines making TCRm antibodies highly sensitive tools for visualizing and quantitating specific peptide — MHC class I complexes on cells. [00122] Discussion
  • Dendritic cells are potent activators of CD4+ and CD8+ T cells and anti-tumor responses and have been extensively examined as a potentially useful immunotherapeutic approach for cancer treatment. This has led to the direct use of DCs as antigen delivery vehicles in a variety of experimental systems (Steinman, 1996; and Lou et al., 2004). The inventors and others have delivered antigens to DC by way of gene transduction (Chiriva- lnternati et al., 2003) and via receptor-mediated endocytosis of whole proteins using receptor-specific antibodies (Ramakrishna et al., 2004; and He et al., 2004).
  • mDCs have been successfully exploited as vehicles to deliver exogenously loaded synthetic peptides (Nakamura et al., 2005; and Godelaine et al., 2003).
  • Specific targeting of vaccines to antigen-presenting cells such as DCs provides a model system for evaluating whether antigen processing has occurred and which immunogenic peptides have been presented by MHC molecules.
  • current potency assays cannot directly measure specific peptide — MHC complexes.
  • TCRm mAbs generated to two overlapping peptide-epitopes from the TAA hCG ⁇ were used to directly show that presentation of both hCG ⁇ -derived peptide-epitopes readily occurs on the surface of vaccine-treated DCs.
  • MHC — peptide presentation is assessed by indirect means by monitoring a biological response of antigen-specific CTL to proliferate, mediate cell lysis or produce cytokines such as IL-2 and IFN- ⁇ (Whiteside et al., 2003; and Gauduin, 2006). These responses, however, are not instantaneous, are labor and time intensive and are not quantitative (Petricciani et al., 2006).
  • Her2/neu (369)-HLA-A2-specific CTL line mediated lysis of target cells was dependent on the level of expression of Her2/peptide-HLA-A2 complexes on tumor cells (Weidanz et al., 2006). Still others have recently reported that a key variable that may be a determinant of T cell function is the density of epitope presented at the surface of APCs (Bullock et al., 2000; Wherry et al,. 1999; Wherry et al., 2002; and Bullock et al., 2003).
  • TCRm antibodies can be used to directly detect and quantitate specific peptide-HLA class I epitopes on many cells including dendritic cells (Zehn et al., 2006; Zehn et al., 2004; and Kukutsch et al., 2000).
  • the TCRm mAbs used in this example were found to exhibit unique binding specificity and extraordinar detection sensitivity that was demonstrated by staining T2 cells pulsed with a low concentration of specific peptide ( ⁇ 150 pM).
  • High avidity CTL lines reactive to TAA peptide-epitopes have been shown to have a lower detection limit in the 100 pM range (Kageyama et al., 1995; Yee et al., 1999; and Yang et al., 2002).
  • a quantitative method using PE-labeled beads revealed that both the anti-TMT and anti-GVL TCRm mAbs recognized their cognate peptide-epitope at less than 60 peptide- epitope copies per cell.
  • the TCRm mAbs and high avidity CTL lines have comparable detection sensitivity limits.
  • the hCG ⁇ tumor-associated antigen was selected because it is widely expressed by tumors of different histological origins and the B11-hCG ⁇ antibody fusion vaccine has been previously shown to be internalized and capable of inducing CTL responses against the hCG ⁇ peptide-epitopes including TMT peptide-HLA-A2 (He et al., 2004). He et al. reported that CTL generated using DC-treated with the B11-hCG ⁇ vaccine lysed T2 cells pulsed with TMT peptide substantiating the immunogenicity of these two peptide epitopes.
  • the presently disclosed and claimed invention further strengthens this concept using TCRm mAbs in assays not only to measure the potency of a manufactured vaccine lot but to also potentially be able to type tumor sections and DTH punch biopsies.
  • TCRm reagents for anomalies in tumor biomarker expression such as antigen loss variants (HLA, TAA, etc.).
  • An important goal would be to determine whether HLA-A2 TCRms will clearly discriminate between intact HLA from those with structural mutations (polymorphisms) in the binding groove as also ⁇ 2m loss variants.
  • Antibodies and synthetic peptides were purchased from Caltag Biosciences (Burlingame, CA).
  • the mouse IgGI isotype control antibody was purchased from Southern Biotech (Birmingham, AL).
  • TMTRVLQGV human chorionic gonadotropin- ⁇ peptide designated as TMT (40) ; SEQ ID NO:2], VLQGVLPAL [residues 44 — 52, human chorionic gonadotropin- ⁇ peptide, designated as VLQ (44) ; SEQ ID NO:3], GVLPALPQV [residues 47—55, human chorionic gonadotropin- ⁇ peptide, designated as GVL (47) ; SEQ ID NO:4], KIFGSLAFL [residues 369—377, Her2/neu peptide designated as Her2 (369) ; SEQ ID NO:5], EVDPIGHLY [residues 161—169, MAGE-3 cancer testis antigen peptide designated as MAGE-1 (161 )1 SEQ ID NO:6], and GPRTAALGLL [residues 4—13,
  • P3X-63Ag8.653 murine myeloma cell line used as a fusion partner were purchased from the American Type Culture Collection (ATCC, Manassas, VA).
  • mice (Balb/c) were repeatedly immunized with 50 ⁇ g of purified peptide-HLA-A * 0201 complex and Quil-A adjuvant (Sigma, St. Louis, MO).
  • fusions were carried out using the Clonacell-HY Kit (Stem Cell Technologies, Vancouver, BC). Single clones were picked and screened for appropriate mAb production by ELISA (as described below); all three antibodies produced by the resulting hybridomas used in this study were IgGI isotype. Large amounts of antibody- containing supernatant were generated and purified by affinity chromatography as previously described.
  • HLA-A*0201 -peptide MAGE-3 peptide-HLA-A*0101 or Reticulocalbin peptide- HLA-B*0702 in PBS.
  • purified mAb was added to the plate and incubated for 2 h at room temperature (RT). Bound antibody was detected by incubation with a horseradish peroxidase (HRP)-goat anti-mouse IgG and color was developed with ABTS substrate (Pierce, Rockford, IL). OD was measured at 405 nm.
  • HRP horseradish peroxidase
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • T-80 Nalge-Nunc, Rochester, NY
  • T-175 Cornning, Acton, MA
  • Non-adherent cells were removed, the flask was washed twice with PBS, and then 15 — 30 ml supplemented AIM-V (10% heat-inactivated FBS, L-glutamine and Pen/Strep) was added to the flask, as well as IL-4 (50 ng/ml) and GM-CSF (25 ng/ml), stimulating differentiation of monocytes into dendritic cells.
  • AIM-V AIM-V (10% heat-inactivated FBS, L-glutamine and Pen/Strep
  • IL-4 50 ng/ml
  • GM-CSF 25 ng/ml
  • 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 0 C for 20 — 60 min, centrifuged, counted and either used immediately or frozen at -8O 0 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 et al., 1992).
  • T2 cells were pulsed with the peptides at 20 ⁇ g/ml for 4 h 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 h with decreasing amounts of specific peptide (2000 — 0.15 nM). T2 cells (5x10 5 ) were then washed in SB to remove excess peptide and stained with each TCRm-PE conjugate, 3F9 and 1B10 TCRms at 1 ⁇ g/ml of SB).
  • Dendritic cells were harvested and plated into 4 wells of a 24-well tissue culture plate. Either the vaccine (B11-hCG ⁇ ) or the monoclonal antibody alone ("vehicle, B11") were added at 30 ⁇ g/ml, two wells were untreated, and the plate was incubated for up to 3 days at 37 0 C, 5% CO 2 . Cells were matured by addition of Poly I:C (Sigma, St. Louis, MO) at 50 ng/ml to the vaccine- and vehicle-treated well, as well as one of the untreated wells, then incubated for 12 — 18 h. Mature or immature (untreated) DCs were harvested as before, then centrifuged and divided into the appropriate number of aliquots for staining and analysis by flow cytometry.
  • the vaccine B11-hCG ⁇
  • vehicle monoclonal antibody alone
  • 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 (B11-hCG ⁇ ) and matured with Poly I:C) at a ratio of 10:1 in the presence of cytokines sequentially added (10 ng/ml each of IL-7 on day 0 and IL-2 on day 1) every 3 days.
  • 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 cytokine or granzyme-B production ELISpot assay (MabTech, Sweden and Cell Sciences, Canton, MA for ELISpot kits). Spot formation was evaluated by Dr. Sylvia Janetzki (Zellner Consulting, Inc., Fort Lee, N.J.).
  • vaccine or vehicle-treated DCs were added to B11-hCG ⁇ -specific CTL at a 1 :1 ratio unless otherwise noted (see Figs. 11 and 12).
  • the TCRm mAbs were added (10 ⁇ g) to both vaccine- and vehicle-treated DCs + CTLs, and a mouse IgGI isotype was also added as a control.
  • Supernatant 100 ⁇ l/well was collected at 24 and 48 h of incubation. Supernatant samples were frozen at -2O 0 C until testing was performed for lnterferon- ⁇ production using an IFN ⁇ cytokine secretion assay (OptEIA Human IFN- ⁇ ELISA Kit II, BD San Diego, CA).
  • HLA-A*0201 complexes were prepared from inclusion bodies essentially as described by Altma ⁇ et al. (1996).
  • the human HLA-A*0101 and HLAA*0201 heavy chain genes were amplified by PCR and cloned into the pAC4 plasmid containing the birA amino acid sequence (Avidity, Denver, CO).
  • the human beta-2 microglobulin gene was previously cloned into an expression vector for production in an E. coli strain BL-21 (Parker et al., 1989).
  • Refolded monomer was concentrated and purified on an S-75 size exclusion column by FPLC (Pharmacia, Kalamazoo, Ml) and then biotinylated using the biotin ligase enzyme according to the manufacturer's instructions (Avidity). Tetramers were formed by mixing the biotin tagged refolded HLA-A2-peptide complex with streptavidin at a molar ratio of A — 1, respectively. Tetramers were purified on an S-200 Sephadex size exclusion column and the protein concentration was determined by BCA protein assay (Pierce, IL). Soluble intact monomer of HLA-B*0702 protein was produced by LCL-721 B cell transfectants, purified by Protein-G and loaded with reticulocalbin-2 peptide (4 aa.i3aa) for use in ELISA.
  • each individual well of a black 96-well LJL HE PS microplate was loaded with 5 ⁇ l of an 8* ⁇ 2m solution (160 ⁇ g/ml) (Fitzgerald Industries International; Concord, MA), 10 ⁇ l of 4* competitor at various dilutions, 5 ⁇ l of an 8* pFITC preparation (16 nM) and 20 ⁇ l of 2 ⁇ activated sHLA (80 ⁇ g/ml). Soluble HLA was activated by incubating at 53 0 C for 15 min. For all preparations, 1 * BGG/PBS was used as buffer. Specific control groups included: (a) protein only, (b) tracer only and (c) buffer only.
  • milli-polarization 1000(S-GP)/(S + GP), where S and P are background-subtracted intensities of the fluorescence measured in the parallel (S) and perpendicular (P) directions, respectively, and G (grating) is the instrument and assay dependent correction factor.
  • TCRm antibodies can readily detect de novo antigen processing and presentation in cells actively treated with an active immunotherapeutic (e.g. a vaccine composition) or from natural antigen expression (e.g. in virally infected or oncogenic tissues). These events can be tracked using flow cytometry staining as well as immunocytochemistry, with associated quantitation of observed values (Figs. 15 and 16). An example of these studies is presented in Fig. 15, with data from control vaccine or target vaccine (Gp100 antigen) treated antigen presenting cells. There is a strong correlation between TCRm binding of HLA-peptide complexes present on the surface of vaccine treated cells and the presence of intracellular antigen.
  • an active immunotherapeutic e.g. a vaccine composition
  • natural antigen expression e.g. in virally infected or oncogenic tissues.
  • HLA-peptide complexes The temporal relationship between intracellular antigen detection and the appearance of specific HLA-peptide complexes will vary depending on the type of vaccine employed, e.g. peptide, intact protein, nucleic acid, viral vector, etc. Nevertheless, TCRm antibody binding activity correlates with intracellular antigen presence regardless of vaccine-type and properties.
  • induced CTL activities measured by incubating vaccine treated cells with appropriate CTL lines can be effectively correlated with quantitative measurement of peptide-MHC complexes on the surface of the vaccine treated cells as determined by TCRm antibodies.
  • Data presented in Fig. 19 displays both CTL activity and TCRm staining data, thus allowing benchmarking of TCRm staining to CTL stimulation.
  • the minimal acceptable CTL stimulation activity was determined (blue bar) and set as acceptance threshold value (blue dashed line) for both Vaccine Antigens gp100 and NYESO1.
  • Fig. 21 shows data from three separate experiments using TCRm staining of gp100 vaccine treated cells, conducted with different antigen presenting cell populations during different weeks of study. The three studies show very small standard deviations, establishing the reproducibility of the TCRm binding assays. When one compares these standard deviations with those associated with the CTL assays presented in Fig 18, one clearly sees the increased reproducibility and reliability of the data provided. [00148] Using the QuantiBRITETM system (BD Biosciences, Inc.), peak volumes from the flow cytometry plots and ⁇ MFI values can be used to determine the number of HLA- peptide complexes present in a given number of cells (Fig. 22).
  • Standard materials with known quantities of PE molecules are supplied by manufacturer and separated using flow cytometry.
  • the delta MFI values for these know samples are plotted and a linear regession formula is derived allowing unknown samples to be analyzed.
  • the unknowns are reacted with a TCRm antibody and a secondary PE-labeled antibody which binds the TCRm antibody. This interaction will show a measureable delta MFI value.
  • This value is placed in the regression formula and a number of PE molecules correlating with this value is determined. Due to loading efficiency of our secondary antibodies at known amount of PE molecule(s) per antibody, this allow this number to establish the number of peptide-MHC complexes identified by the TCRm antibody.
  • FIGs. 23 and 24 show experiments investigating the temporal kinetics of HLA-peptide presentation.
  • antigen presenting cells were treated with Gp100 and NY-ESO-1 antigens respectively and samples were taken at 24 h, 48 h, 72 h and 96 hours.
  • Cells were incubated with both RL08A-TCRm and RL09A-TCRm and subjected to quantitative analysis as described above.
  • HLA-specific antibody such as BB7.1 which binds HLA A*02
  • TCRm measurement of a specific peptide-HLA complex allows the percentage of HLA molecules occupied by a given antigen-specific peptide to be determined as shown in Fig. 25 using gp100 vaccine to treat cells.
  • TCRm antibody-based assays can be the basis for a quantitative, bio-potency assay for active immunotherapeutic products. Assays can be performed solely using TCRm antibodies. These assays are first benchmarked using CTL-specific activities and then performed in the absence of CTLs to provide reproducible, quantitative data concerning the potency of a given therapy preparation. These assay show the dynamic range required to quantitatively assess differences in therapeutic preparations. Potency differences can be compared with threshold values answering necessary quality questions. Further, TCRm antibodies assist with cell- based assays to remove assay background allowing more significant and comparable data to emerge. TCRm antibodies provide a highly sensitive and selective reagent, in a soluble and stable form, to empower accurate and quantitative measurement of potency of active immunotherapy drugs.
  • the TCRm monoclonal antibody is an ideal biological tool for developing a quantitative bio-potency assay for CTL vaccines.
  • the quantitative methodology using TCRm antibody staining has been developed, and a quantitative dynamic range has been demonstrated for peptide/HLA-A2 epitopes at ⁇ 50 specific complexes on treated cells. Additionally, a quantitative dynamic range has been demonstrated for peptide/HLA-A2 epitopes at ⁇ 2% of total HLA on treated cells.
  • CTL activities have been quantitatively correlated with TCRm's to same vaccine modality and dose. Therefore, a prototype quantitative bio-potency assay has been successfully established.
  • Fig. 26 shows the dramatic difference in assay significance with and without use of the TCRm antibody.
  • TCRm antibodies demonstrate the ability of TCRm antibodies to enhance the quality of established cell-based assays.
  • Background in ELISpot, intracellular cytokine staining and direct CTL assays renders these assays semi-quantitative at best.
  • Inclusion of TCRm antibodies in these cell-based assays can reduce this natural background and enhance the significance of individual assays allowing assay comparability when performed at different times or with different samples. This is illustrated in Fig. 26 where the background present in an assay when dendritic cells are NOT treated with a vaccine are incubated with CD8+ T cells. This background makes the significance of the value seen with vaccine treated cells less impressive.
  • TCRm antibody-based assays can be the basis for a quantitative, biopotency assay for active immunotherapeutic products, eliminating the need for animal-based experimentation. Assays can be performed solely using TCRm antibodies.
  • TCRm antibodies assist with cell- based assays to remove assay background allowing more significant and comparable data to emerge. TCRm antibodies provide a highly sensitive and selective reagent, in a soluble and stable form, to empower accurate and quantitative measurement of potency of active immunotherapy drugs.
  • the normal human male lung fibroblast cell line MRC-5 (ATCC CCL-171TM) was cultured in BioWhittaker ® EMEM (Lonza) supplemented with 2mM HyQ ® l-glutamine (HyClone), HyQ ® penicillin-streptomycin solution (HyClone), and 10% GibcoTM Fetal Bovine Serum (FBS, Invitrogen Corp.). Cells were maintained in T-175 flasks and upon reaching confluence (approximately 8x10 6 cells/flask) were trypsinized, washed, and subcultured at a 1:4 dilution.
  • the ALVAC(2)-TRICOM viral vectors employed in MRC-5 infections consisted of vCP2264 (gp100/Mage1-3mini-hLFA- 3/hlCAM-1/hB7.1-wE3L/wK3L), VCP2292 (NY-ESO-1-hLFA-3/hlCAM-1/hB7.1- wE3L/vvK3L), and vCP2041 (hl_FA-3/hlCAM-1/hB7.1-wE3L/wK3L) provided by sanofi pasteur.
  • PBMC Peripheral blood mononuclear cell preparation.
  • PBMCs were prepared via centrifugation of whole human blood diluted 1:1 in BioWhittaker ® X-VIVO-10TM (Lonza) medium over Ficoll-PaqueTM PLUS (GE Healthcare). Separations were carried out in 50 mL conical tubes containing 35 mL of the blood dilution and 15 mL of Ficoll-PaqueTM PLUS. Cells collected from the interface were counted, washed twice, and then frozen down in 1.5 mL aliquots of 5x10 7 cells in 90% FBS with 10% DMSO (Fisher Scientific) and stored at -80 0 C until use.
  • DC Dendritic cell generation.
  • Non-manipulated monocytes were purified from PBMCs using the human Monocyte Isolation Kit Il (Miltenyi Biotec Inc.) according to the manufacturer's instructions. DCs were then generated as previously described (1).
  • monocytes were cultured in 24-well plates at 5x10 5 cells/well in 1 mL volumes of BioWhittaker ® RPMI (Lonza) supplemented with l-glutamine, penicillin-streptomycin solution, 10% human AB (hAB) serum (Valley Biomedical, Inc.), 100 ⁇ g/mL recombinant human GM- CSF (R&D Systems), and 200 ng/mL recombinant human IL-15 (R&D Systems).
  • Immature DC were activated on day 3 by the addition of LPS (E coli strain 026: B6, Sigma) at a concentration of 10 ng/mL and used as mature DCs on day 4.
  • CD8 + T cells were purified from autologous PBMCs using the human CD8 + T Cell Isolation Kit Il (Miltenyi Biotec Inc.) according to the manufacturer's instructions.
  • Mature DCs were treated with 10 ⁇ g/mL mitomycin C (Sigma) for 45 min at 37°C, washed twice, and loaded in the presence of 3 ⁇ g/mL purified human beta-2-microglobulin ( ⁇ 2 m, Lee Biosolutions, Inc.) with 10 ⁇ g/mL of either YLEPGPVTV peptide (gp100-derived epitope; SEQ ID NO:75) or SLLMWITQV peptide (NY-ESO-1 -derived epitope; SEQ ID NO:13) for 2 h in the 24-well plates at 37°C.
  • mitomycin C Sigma
  • CD8 + T cells were then added at 1x10 6 cells/well in 1 mL volumes of RPMI/10% hAB containing 10 IU/mL recombinant human IL-7 (R&D Systems) and placed at 37°C for 7 days.
  • Adherent APCS were prepared from autologous PBMCs and used to restimulate CTL lines essentially as described (2).
  • 4x10 6 mitomycin C-treated PBMCs were added per well to 24-well plates in 0.5 mL volumes of RPMI/10% hAB and incubated for 2 to 3 hours at 37°C for adherence. The media was then carefully removed and replaced with 0.5 mL fresh media containing 3 ⁇ g/mL ⁇ 2 m and 10 ⁇ g/mL of the relevant peptide for 2 h at 37°C.
  • CTLs harvested from either initial priming or previous restimulation were added at 1x10 6 cells/well in 1 mL volumes of RPMI/10% hAB containing 10 IU/mL recombinant human IL-2 (R&D Systems). The cultures were fed every 3-4 days with 0.5 mL fresh media containing IL-2 and restimulated at 7-10 days.
  • MRC-5 Viral infection of MRC-5.
  • MRC-5 cells were seeded in 6-well plates at 2x10 5 cells/well (2 mL ⁇ /vell) approximately 24 h prior to infection. An extra plate was seeded for the purposes of harvesting and counting prior to infection for MOI calculations.
  • Virus stocks were thawed at room temperature from -80 0 C storage and then kept on ice.
  • MRC-5 Intracellular staining of MRC-5 with gp100 and NY-ESO-1 monoclonal antibodies (MAbs).
  • MRC-5 cells were harvested from 6-well plates by removing all media, adding 1 mL/well Cellgro ® Trysin EDTA (Mediatech Inc.), and incubating at 37°C for 2-3 minutes; 2 ml_/well of RPMI/10% hAB was then added per well and the cells collected. The cells were washed, resuspended in 5 ml. of RPMI/10% hAB, and counted; they were maintained in human serum-containing medium for at least 10 min prior to staining in order to block non-specific binding sites.
  • MRC-5 cells were harvested, counted, and incubated in RPMI/10% hAB as described above. Once the assay layout for staining in 96-well U-bottom plates was established, between 3x10 5 to 5x10 5 cells/well were plated, spun down, and resuspended in 100 ⁇ L/well of FACS buffer. Primary antibodies (TCRms) were added to indicate wells in 100 ⁇ L volumes of FACS buffer at final concentrations of 250 ng/mL The plates were incubated on ice for 30 min, after which they were spun and then washed twice with 200 ⁇ L/well FACS buffer.
  • TCRms Primary antibodies
  • CTL lines were harvested, washed, and resuspended in MRC-5 medium containing 1 ⁇ L/mL of BD GolgiPlug (BD Bioscience) at day 7 before adding 4x10 6 cells/well in 3 mL volumes to 72 h cultures of infected MRC-5 cells in 6-well plates.
  • MRC-5 cells were pulsed with 10 ⁇ g/mL of peptide for 2 h at 37 0 C prior to addition of CTLs.
  • TCRm blockade was accomplished through pre-incubation of MRC-5 cells with 10 ⁇ g/mL of the corresponding TCRm for 30 min at 37°C.
  • CTLs were incubated with MRC-5 for 5 h at 37°C and then harvested.
  • Assay layout for staining in 96-well U-bottom plates was established, between 7x10 5 to 8x10 5 cells/well were plated, spun down, and resuspended in 100 ⁇ L/well of FACS buffer containing 20 ⁇ L/well of APC-labeled anti-human CD8a (RPA-T8, eBioscience). The plates were incubated in the dark on ice for 20 min, after which 100 ⁇ L/well of FACS buffer was added and the plates centrifuged.
  • the cells were then washed once with 200 ⁇ L/well of FACS buffer prior to resuspension in 100 ⁇ L/well of BD Cytofix/CytopermTM Fixation/Permeabilization solution and incubation on ice for 20 min.
  • the cells were then washed in BD Perm/WashTM buffer as described above for intracellular staining of MRC-5.
  • the PE-labeled anti-human IFN- ⁇ antibody (4S.B3, eBioscience) was added to indicated wells in 100 ⁇ L volumes of Perm/Wash buffer at a concentration of 1 ⁇ L per well.
  • the plates were incubated in the dark on ice for 30 min, after which they were washed as before. After washing twice in 200 ⁇ L/well of FACS buffer, the samples were transferred into tubes for data acquisition on a FACSCanto.
  • 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.
  • Novellino L 1 Castelli C 1 Purani G A listing of human tumor antigens recognized by T cells: March 2004 update. Cancer Immunol lmmunother 2005;54(3):187 — 207.
  • HLA-A2 molecules in an antigen-processing mutant cell contain signal sequence-derived peptides. Nature 1992;356(6368):443— 6.

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  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Toxicology (AREA)
  • Food Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

La présente invention concerne des procédés de dosage de la puissance d’une composition vaccinale, la puissance étant un niveau minimum prédéfini d’activité biologique potentielle pour la composition vaccinale. Le procédé comprend la fourniture d’une composition vaccinale et son administration à une cellule présentatrice d’antigènes, la composition vaccinale étant transformée en peptides et les peptides étant présentés par les complexes CMH sur la surface cellulaire. Un agent, tel qu’un mimétique de récepteurs des cellules T, qui réagit contre un complexe spécifique peptide/CMH est fourni et réagi avec la cellule présentatrice d’antigènes traitée avec le vaccin, moyennant quoi l’agent se lie à la surface cellulaire de la cellule présentatrice d’antigènes traitée avec le vaccin si le complexe spécifique peptide/CMH reconnu par l’agent est présent sur la surface cellulaire. Une densité du complexe spécifique peptide/CMH sur la surface de la cellule présentatrice d’antigènes traitée avec le vaccin est mesurée par liaison de l’agent. La puissance du vaccin est alors déterminée en se basant sur la densité mesurée du complexe spécifique peptide/CMH présent sur la surface de la cellule présentatrice d’antigènes traitée avec le vaccin.
PCT/US2009/001144 2008-06-13 2009-02-24 Procédés de dosage de la puissance d’un vaccin WO2009151487A1 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US6153408P 2008-06-13 2008-06-13
US61/061,534 2008-06-13
US12/196,885 US20090075304A1 (en) 2004-05-27 2008-08-22 Methods of assaying vaccine potency
US12/196,885 2008-08-22
US19187108P 2008-09-12 2008-09-12
US61/191,871 2008-09-12

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WO2009151487A1 true WO2009151487A1 (fr) 2009-12-17

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140141455A1 (en) * 2004-05-27 2014-05-22 Receptor Logic, Inc. Methods of assaying vaccine potency
JP2021104033A (ja) * 2014-12-23 2021-07-26 イマティクス バイオテクノロジーズ ゲーエムベーハー 肝細胞がん（ｈｃｃ）およびその他のがんに対する免疫療法で使用するための新規ペプチドおよびペプチド組み合わせ
US11679147B2 (en) 2014-12-23 2023-06-20 Immatics Biotechnologies Gmbh Method of eliciting a CD8+ cytotoxic response in hepatocellular carcinoma patients with a population of activated T cells

Citations (2)

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Publication number Priority date Publication date Assignee Title
WO2002014870A2 (fr) * 2000-08-14 2002-02-21 Akzo Nobel N.V. Utilisation d'anticorps contre des complexes de peptides mhc specifiques
US20070092530A1 (en) * 2004-05-27 2007-04-26 Weidanz Jon A Antibodies as T cell receptor mimics, methods of production and uses thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002014870A2 (fr) * 2000-08-14 2002-02-21 Akzo Nobel N.V. Utilisation d'anticorps contre des complexes de peptides mhc specifiques
US20070092530A1 (en) * 2004-05-27 2007-04-26 Weidanz Jon A Antibodies as T cell receptor mimics, methods of production and uses thereof

Non-Patent Citations (1)

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Title
REAY, P. A ET AL.: "Determination ofthe relationship between T cell responsiveness and the number ofMHC-peptide complexes using specific monoclonal antibodies", JOURNAL OF IMMUNOLOGY, vol. 164, no. 11, 1 June 2000 (2000-06-01), US, pages 5626 - 5634 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140141455A1 (en) * 2004-05-27 2014-05-22 Receptor Logic, Inc. Methods of assaying vaccine potency
JP2021104033A (ja) * 2014-12-23 2021-07-26 イマティクス バイオテクノロジーズ ゲーエムベーハー 肝細胞がん（ｈｃｃ）およびその他のがんに対する免疫療法で使用するための新規ペプチドおよびペプチド組み合わせ
US11679147B2 (en) 2014-12-23 2023-06-20 Immatics Biotechnologies Gmbh Method of eliciting a CD8+ cytotoxic response in hepatocellular carcinoma patients with a population of activated T cells

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