Patent Grant
8242239
The present invention relates to tumor-associated peptides that are capable of binding to a molecule of the human class I major histocompatibility complex (MHC). Such peptides are, for example, used in the immunotherapy of tumor diseases.
The recognition of tumor-associated antigens (TAA) by immune system components plays a major role in the elimination of tumor cells by the immune system. This mechanism is based on the prerequisite that there are qualitative or quantitative differences between tumor cells and normal cells. In order to achieve an anti-tumor response, the tumor cells have to express antigens that are targets of an immune response sufficient for elimination of the tumor.
Particularly CD8-expressing cytotoxic T lymphocytes (in the following termed CTL) participate in tumor rejection. In order to elicit such an immune reaction by cytotoxic T cells, foreign proteins/peptides must be presented to the T cells. T cells recognize antigens as peptide fragments only if those are presented on cell surfaces by MHC molecules. These MHC (“major histocompatibility complex”) molecules are peptide receptors that normally bind peptides within the cell in order to transport them to the cell surface. This peptide/MHC molecule complex can be recognized by T cells. Human MHC molecules are termed human leukocyte antigens (HLA).
There are two classes of MHC molecules: MHC class I molecules, present on most nucleated cells, present peptides that are generated by proteolytic degradation of endogenous proteins. MHC class II molecules are only found on professional antigen presenting cells (APC), and present peptides of exogenous proteins that are engulfed and processed by APC during endocytosis. Complexes formed between peptide and MHC class I are recognized by CD8-positive cytotoxic T cells, complexes formed between peptide and MHC class II are recognized by CD4 T helper cells.
In order for a peptide to elicit a cellular immune response, it needs to bind to an MHC molecule. This process depends on the allele of the MHC molecule and on the amino acid sequence of the peptide. Usually, MHC class I-binding peptides have a length of 8-10 residues, and their sequences contain two conserved residues (“anchors”) interacting with the corresponding binding groove of the MHC molecule.
In order for the immune system to be able to elicit an effective CTL response against tumor-derived peptides, such peptides not only have to be able to bind to certain MHC class I molecules being expressed by tumor cells, they also have to be recognized by T cells bearing specific T cell receptors (TCR).
The main purpose for developing a tumor vaccine is the identification and characterization of tumor-associated antigens being recognized by CD8+CTL.
The antigens, or their epitopes, that are recognized by the tumor-specific cytotoxic T lymphocytes can be molecules from all protein classes, such as enzymes, receptors, transcription factors, etc. Another important class of tumor-associated antigens are tissue-specific structures such as CT (“cancer testis”) antigens that are expressed in various types of tumors and in healthy testicular tissue. For the proteins to be recognized as tumor-specific antigens by cytotoxic T lymphocytes, and to be therefore used in a therapy, certain conditions must be met: The antigen should mainly be expressed by tumor cells and not or only in smaller amounts by normal cells in contrast to tumors. Furthermore, it is desirable that the respective antigen is present in high concentration not only in one type of tumor but in other tumor types as well. The presence of epitopes in the amino acid sequence of the antigen is also absolutely mandatory since such peptides that are derived from a tumor-associated antigen (“immunogenic peptides”), should lead to a T cell response, either in vitro or in vivo.
Therefore, TAAs are a starting point for the development of a tumor vaccine. The methods for identifying and characterizing the TAAs are based on the use of CTL that have already been induced in the patients, or they are based on the generation of differential transcription profiles between tumors and normal tissues.
However, the identification of genes overexpressed in tumor tissues or human tumor cell lines, or selectively expressed in such tissues or cell lines, does not provide precise information as to the use of the antigens being transcribed from these genes in the immune therapy. This is because only individual epitopes of these antigens are suitable for such an application since only the antigen epitopes—not the entire antigen—elicit a T cell response through MHC presentation. It is therefore important to select only those peptides from overexpressed or selectively expressed proteins that are presented in connection with MHC molecules, so targets for the specific recognition of primary cells or of tumor cell lines established from primary tumor tissue cells, by cytotoxic T lymphocytes could be obtained.
Based on this background, it is an objective of the present invention to provide at least one novel amino acid sequence for such a peptide that is capable to bind to a molecule of the human major histocompatibility complex (MHC) class I.
According to the invention this objective is addressed by providing a tumor-associated peptide with an amino acid sequence selected from the group consisting of SEQ ID NO. 1 to SEQ ID NO. 577 in the attached sequence protocol, whereby the peptide being capable of binding to a molecule of the human major histocompatibility complex (MHC) class I. In this way, the objective forming the basis of the invention is addressed completely.
It is to be understood that the identified tumor peptides may be synthesized to obtain larger quantities or for the use for the below described purposes, or may be expressed in cells.
The inventors were able to isolate and identify the above mentioned peptides from tumor tissue as specific ligands from MHC class I molecules. The term “tumor-associated” peptides herein refers to peptides that are isolated and identified from tumor material. Thus, these peptides that are presented on true (primary) tumors are subjected to antigen processing in a tumor cell.
The specific ligands could be used in cancer therapy, e.g. to induce an immune response against tumor cells expressing the respective antigens from which the peptides are derived.
Such an immune response in form of an induction of CTL may be obtained in vivo. In order to obtain such an immune response the peptide is administered to a patient suffering from a TAA-associated tumor disease, for example in form of a pharmaceutical composition.
On the other hand, a CTL response against a tumor expressing the antigens from which the peptides are derived may also be elicited ex vivo. In order to do so, the CTL precursor cells are incubated together with antigen presenting cells and the peptides. Then, the thus stimulated CTL are cultivated, and these activated CTL are administered to the patient.
Furthermore, it is possible to load APC with the peptides ex vivo, and to administer these loaded APC to the patient expressing in the tumor tissue the antigens from which the peptide is derived. Then, the APC themselves may present the peptide to the CTL in vivo, and thereby activate them.
However, the peptides according to the invention may also be used as diagnostic reagents.
Thus, using the peptides it could be found out if CTL are present in a CTL population or have been induced by a therapy that are specifically directed against a peptide.
The peptides may also be used to test for the increase of precursor T cells with reactivity against the defined peptide.
Furthermore, the peptide may be used as a marker to track the disease course of a tumor expressing the antigen from which the peptide is derived.
The attached Table 1 lists the identified peptides. The table also contains the proteins from which the peptides are derived, and the respective positions of the peptides in the respective proteins which are named or abbreviated by accepted gene symbols according to the “HUGO Gene Nomenclature Committee” (gene.ucl.ac.uk/nomenclature/ or ncbi.nlm.nih.gov/LocusLink/). The English names of the proteins have been maintained to avoid erroneous translations. Furthermore, the Acc numbers are listed that are used in the gene bank of the “National Center for Bio-technology Information” of the National Institute of Health (see ncbi.nlm.nih.gov).
The inventors were able to isolate the peptides (or ligands) from 8 renal cell tumors and 2 glioblastomas from altogether 10 patients, RCC75, RCC98, RCC100, RCC103, RCC112, RCC115, RCC116, RCC130 and NCH359, as well as NCH361, and from a tumor cell line (J-Y).
From the patients' tumors and the cell line J-Y 577 ligands could be identified that were bound to the HLA subtypes A*03, B*07, B*40 (RCC75), A*01, A*03, B*07, B*18 (RCC98), A*02, A*03, B*07, B*18 (RCC100), A*11, A*25, B*15, B*44 (RCC103), A*01, A*31, B*08, B*27 (RCC112), A*02, A*03, B*15, B*18 (RCC115), A*01, A*02, B*27, B*37 (RCC116), A*02, A*24, B*07, B*44 (RCC130), A*03, A*32, B*07, B*35 (NCH359), A*26, B*38 (NCH361) and A*02, B*07 (J-Y).
Some of the ligands are derived from highly expressed so called “housekeeping” genes that are uniformly expressed in most tissues, however, many are characterized by tissue-specific and tumor-specific expression.
Thus, several peptides could be identified that are derived from proteins which are especially overexpressed in tumor tissue. For example, fragments of tenascin-C (GLAPSIRTK, SEQ ID NO. 2) could be identified (Herold-Mende et al., Clinical impact and Functional Aspects of Tenascin-C Expression during Glioma Progression, 2002, Int. J. Cancer, 98: 362-369).
Also, the inventors were able to identify ligands, among others, that are derived from SOX9 (YPHLHNAEL, SEQ ID NO. 7) and RGS5 (LAALPHSCL, SEQ ID NO. 448).
As primary tumor cells are not suited for in vitro culture, the inventors have chosen a human tumor cell line as an example in order to additionally demonstrate that peptides according to the invention identified from this cell line, are suited to activate cytotoxic T lymphocytes in vitro. Particularly, the inventors could show that using a peptide from the established tumor cell line JY as an example, it was possible to generate cytotoxic T lymphocytes (CTL) in vitro that are specific for the selected peptide with the sequence FPSLREAAL (MAGEA1, position 294-302) and SEQ ID NO. 114 and the HLA allele B*0702. Using these CTL, KM22 target cells loaded with the SEQ ID NO. 114 peptide could be selectively killed, whereas KM22 control target cells not loaded with the SEQ ID NO. 114 peptide were not recognized by the cytotoxic T cells. Thus, it could be shown exemplified that human T cells could be activated in vitro using the peptides according to the invention as epitopes. Furthermore, it could be demonstrated that the cytotoxic T lymphocytes lysing the T2 cells loaded with the SEQ ID NO. 114 peptide also express interferon gamma which has been described as a reliable marker for the activation of T cells.
In a preferred embodiment, it is also possible to use peptides for stimulating an immune response that comprise sequence ID NO. 1 to 577, and in which at least one amino acid has been replaced by another amino acid with similar chemical properties.
Referring to the corresponding MHC subtypes, these are, for example, the anchor amino acids that may be replaced by amino acids with similar chemical properties. Thus, for example, in peptides associated with the MHC subtype HLA-A *2, leucine on position 2 may be replaced by isoleucine, valine or methionine and vice versa, and at the C-terminus leucine by valine, isoleucine and alanine, each containing non-polar side chains.
Furthermore it is possible to use peptides with sequence ID No. 1 to 577 comprising at least one additional amino acid N- or/and C-terminally, or in which at least one amino acid is deleted.
Furthermore, peptides with sequence ID No. 1 to 577 may be used in which at least one amino acid is chemically modified.
The varying amino acid(s) is (are) selected in such way that the variation does not affect the immunogenicity of the peptide, i.e. demonstrates a similar binding affinity to the MHC molecule and the capability for T cell stimulation.
According to the invention, the peptide may be used for treatment of tumor diseases and/or adenomatous diseases.
The tumor diseases to be treated comprise, for example, kidney, brain, mammary, pancreas, gastric, testicular and/or skin cancers and tumor diseases of the nerve system. This list of tumor diseases is only exemplary, and is not intended to limit the area of application. The inventors were able to show in independent studies that the peptides according to the invention are suitable for such use. In these studies it was shown that specifically generated CTL that are specific for certain peptides were able to kill tumor cells effectively and selectively.
Basically, for the use of tumor-associated antigens in a tumor vaccine, several application forms are possible. For example, Tighe et al. 1998, Gene vaccination: plasmid DNA is more than just a blueprint, Immunol. Today 19(2):89-97, described that the antigen may be administered either as recombinant protein together with suitable adjuvants or carrier systems, or in form of the cDNA encoding the antigen in plasmid vectors. For this, the antigen needs to be processed and presented by antigen presenting cells (APC) in the patient's body in order for an immune response to be induced.
Melief et al., 1996, Peptide-based cancer vaccines, Curr. Opin. Immunol. 8:651-657, demonstrated another option, namely the use of synthetic peptides as a vaccine.
For this purpose, the peptide may be used in a preferred embodiment together with added adjuvants, or alone.
As an adjuvant, for example, the granulocyte macrophage colony stimulating factor (GMCSF) may be used. Further examples for such adjuvants are aluminum hydroxide, mineral oil emulsions such as, for example, Freund's adjuvant, saponins or silicon compounds.
The use of adjuvants provides the advantage that the immune response induced by the peptide may be enhanced, and/or the peptide may be stabilized.
In another preferred embodiment, the peptide is used bound onto an antigen presenting cell. This measure provides the advantage that the peptides can be presented to the immune system, particularly cytotoxic T lymphocytes (CTL). By this, the CTL are able to recognize the tumor cells and specifically kill them. Suitable as antigen presenting cells for such an application are, for example, dendritic cells, monocytes or B lymphocytes.
For this purpose, the cells are, for example, loaded with peptides ex vivo. On the other hand, it is also possible to transfect the cells with DNA encoding the peptides or with the corresponding RNA to express the peptide on the cells.
The inventors were able to show in independent studies that it is possible to load dendritic cells (DC) with specific peptides, and that these loaded dendritic cells activate peptide-specific CTL. This means that the immune system can be stimulated to raise CTL against the tumors expressing the respective peptides.
The antigen presenting cells carrying the peptide may be used either directly or may be activated prior to their use, for example with the heat shock protein gp96. This heat shock protein induces the expression of MHC class I molecules and co-stimulatory molecules such as B7, and also stimulates the production of cytokines. Together, this supports the induction of immune responses.
In another preferred embodiment, the peptides are used to label leukocytes, especially T lymphocytes.
This application is advantageous if it is intended to use the peptides to find out if CTL are present in a CTL population that are specifically directed against a peptide.
Furthermore, the peptide may be used as a marker to evaluate the progress of a therapy for a tumor disease.
The peptide may be used in other immunizations or therapies for monitoring the therapy as well. Therefore, the peptide may not only be used therapeutically but also diagnostically.
In another embodiment, the peptides are used to generate an antibody.
Polyclonal antibodies may be obtained conventionally by immunizing animals by injection of the peptides and subsequent purification of the immunoglobulin.
Monoclonal antibodies may be generated according to standard protocols, such as, for example, described in Methods Enzymol. (1986), 121, Hybridoma technology and monoclonal antibodies.
In another aspect the invention also relates to a pharmaceutical composition comprising one or more of the peptides.
This composition is used, for example, for parenteral administration, such as subcutaneous, intradermal or intramuscular, or for oral application. For this, the peptides are solved or suspended in a pharmaceutically acceptable, preferably aqueous carrier. Furthermore, the composition may contain excipients such as buffers, binders, diluents, etc.
The peptides may also be given together with immunostimulatory substances such as cytokines. A comprehensive description of excipients that may be used in such compositions is given, for example in A. Kibbe, Handbook of Pharmaceutical Excipients, 3. Ed., 2000, American Pharmaceutical Association and pharmaceutical press.
The preparation may be used for prevention, prophylaxis and/or therapy of tumor diseases and/or adenomatous diseases.
The pharmaceutical preparation containing at least one of the peptides with sequence ID NO. 1 to 577 will be administered to a patient suffering from a tumor disease with which the respective peptide or antigen is associated. Thus, a tumor-specific immune response based on tumor-specific CTL can be elicited.
The amount of the peptide or peptides in the pharmaceutical composition is present in a therapeutically effective amount. The peptides that are present in the composition may also bind to at least two different HLA types.
In a further aspect, the present invention relates to nucleic acid molecules encoding the peptides with sequence ID NO. 1 to 577.
The nucleic acid molecules may be DNA or RNA molecules, and may also be used for immune therapy of cancer diseases. The peptide which is expressed from the nucleic acid molecule induces an immune response against tumor cells expressing the peptide.
According to the invention, the nucleic acid molecules may also be present in a vector.
Furthermore, the invention relates to cells which have been genetically altered using a nucleic acid molecule encoding the peptides so that they produce a peptide with the sequence ID NO. 1 to 577.
For this purpose, the cells are transfected with the DNA encoding the peptides, or with the respective RNA, whereby the peptides are made to be expressed on the cells. As antigen presenting cells for such an approach are suited, for example, dendritic cells, monocytes or B lymphocytes.
It is to be understood that the features that have been mentioned above and that are yet to be discussed below, are suitable not only in the respectively described combination but also alone, without leaving the scope of the present invention.
Embodiments of the invention are presented and discussed in the following examples and in the attached figures.
a shows the negative control regarding CTL-specific lysis of KM22 target cells (without peptide);
b shows the CTL-specific lysis of KM22 target cells pulsed with peptide and treated with interferon gamma.
Eight samples were obtained from the Department of Urology at the University of Tubingen, Tubingen, Germany, that were derived from patients with histologically confirmed renal cell carcinoma. The patient termed RCC75 had the following HLA haplotype: A*03, B*07, B*40. The patient termed RCC98 had the following HLA haplotype: A*01, A*03, B*07, B*18, The patient termed RCC100 had the following HLA haplotype: A*02, A*03 B*07, B*18. The patient termed RCC103 had the following HLA haplotype: A*11, A*25, B*15, B*44. The patient termed RCC112 had the following HLA haplotype: A*01, A*31, B*08, B*27. The patient termed RCC115 had following the HLA haplotype: A*02, A*03, B*15, B*18, The patient termed RCC116 had the following HLA haplotype: A*01, A*02, B*27, B*37, and the patient termed RCC130 had following HLA haplotype: A*02, A*24, B*07, B*44.
Two samples were obtained from the Department of Neurosurgery, University of Heidelberg, Heidelberg, Germany, that were derived from patients with histologically confirmed glioblastomas. The patient termed NCH359 had the following HLA haplotype: A*03, A*32, B*07, B*35. The patient termed NCH361 had following HLA haplotype: A*26 and B*38.
Shock frozen tumor samples were processed as previously described in Schirle, M. et al., Identification of tumor-associated MHC class I ligands by a novel T cell-independent approach, 2000, European Journal of Immunology, 30:2216-2225. The peptides were isolated according to standard protocols, using the monoclonal antibody W6/32 which is specific for HLA class I molecules, or using the monoclonal antibody BB7.2 which is specific for HLA-A2. Barnstable, C. J. et al., Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens-new tools for genetic analysis, 1978, Cell, 14:9-20, and Parham, P. & Brodsky, F. M., Partial purification and some properties of BB7.2. A cytotoxic monoclonal antibody with specificity for HLA-A2 and a variant of HLA-A28, 1981, Hum. Immunol., 3:277-299, have described generation and use of these antibodies.
The peptides were separated by “reversed phase HPLC” (SMART system, μRPC C2/C18 SC 2.1/19, Amersham Pharmacia Biotech), and the obtained fractions were analyzed by nanoESI MS. The approach corresponded to the approach described by Schirle, M. et al., Identification of tumor-associated MHC class I ligands by a novel T cell-independent approach, 2000, European Journal of Immunology, 30:2216-2225.
The peptides obtained from tumor tissue were identified by capillary LC MS, as just described, however with minor modifications: 100 μL of each sample were loaded, desalted and pre-concentrated on a 300 μm*5 mm C18μ guard column (LC Packings). Solvent and sample were added using a syringe pump (PHD 2000, Harvard Apparatur, Inc.) with a sealed 100 μL syringe (1710 RNR, Hamilton) at a speed of 2 μL/min. For separation of the peptides, the pre-concentration column was installed before a 75 μm*250 mm C18 column (LC Packings). Then, a binary gradient with 25-60% B was run for 70 min. during which time the flow rate was reduced from 12 μL/min to approximately 300 nL/min. using a TEE connector (ZTIC, Valco) and a 300 μm*150 mm C18 column.
To ensure that the system was free from peptide residues, a blank was measured respectively. Online-fragmentation was performed as described, and the spectra of the fragments were analyzed manually. Data base searches (NCBInr, EST) were performed using MASCOT (matrixscience.com).
In the attached sequence protocol and in the attached table 1 the ligands are listed that were bound to the HLA molecules of patients RCC75, RCC98, RCC100, RCC103, RCC112, RCC115, RCC116, RCC130, NCH359 and NCH361. The peptides associated with HLA-A*02 had an allele-specific peptide motif: on position 2, leucine, valine, isoleucine, alanine, or methionine were found, and at the C-terminus leucine, valine, isoleucine, or alanine. Most of the ligands were derived from so-called “housekeeping” proteins, however, ligands from tumor-associated proteins could be identified as well. For example, fragments from tenascin-C could be identified (GLAPSIRTK, SEQ ID NO. 2; GVLKKVIRH, SEQ ID NO. 20). Herold-Mende et al., Clinical Impact and Functional Aspects of Tenascin-C Expression during Glioma Progression, 2002, Int. J. Cancer, 9S: 362-369, show that in general the strength of the expression of the extracellular matrix protein tenascin-C correlates with the severity of the disease and the immigration of tumor cells in healthy tissue.
For detection of peptide-specific T cells, for example specific for the peptide with the sequence FPSLREAAL (SEQ ID NO. 114), mononuclear cells from peripheral blood of healthy subjects were stained with the corresponding HLA-A* subtype tetramers constituted with the respective peptides: To generate the tetramers, recombinant HLA-B* subtype molecules were constituted in vitro with the peptides, purified by gel filtration, biotinylated, and mixed with streptavidin for linking the monomers, as described by Walter S. et al., 2003, Cutting Edge: Predetermined Avidity of Human CDS T Cells Expanded on Calibrated MHC/Anti-CD2S-Coated Microspheres, J. Immunol. 171: 4974-4978.
Basically, the results of the double staining are measured by FACS analysis, and the specific binding of the peptide tetramers is detected (Walter S. et al., 2003, Cutting Edge: Predetermined Avidity of Human CDS T Cells Expanded on Calibrated MHC/Anti-CD2S-Coated Microspheres, J. Immunol. 171: 4974-4978).
To analyze the presentation of the selected peptides by tumor cells and the recognition of the peptides by CTL, CTL were induced in vitro that are specific for the chosen peptides. For this purpose, KM22 and JY target cell lines were used.
2.1. Recovery of the Specific T Lymphocytes
Specific T lymphocytes were isolated from the blood of healthy subjects as described in 1.5 and concentrated by FACS sorting.
2.2. Peptide Synthesis
The peptides chosen as an example were synthesized using F-moc (9-f luorenylmethyloxycarbonyl) protection groups on a peptide synthesizer (432A, Applied Biosystems, Weiterstadt, Germany) and analyzed by “reversed phase” HPLC and mass spectroscopy. Thereby, sufficient amounts of the identified peptides could be produced.
2.3. Induction of an Antigen-Specific CT1 Response Using Restricted Synthetic Peptides
For CTL induction, the T lymphocytes obtained in step 2.1 (5×106 T lymphocytes per well) were co-incubated by in vitro restimulation in 24-well plates with 1×106 irradiated target cells per well in 1.5 mL T cell medium [consisting of RPMI 1640, 25 mM HEPES (Life Technologies/Invitrogen, Karlsruhe, Germany)] with 10% heat-inactivated human AB serum (CC Pro, Neustadt/Weinstraβe, Germany), 2 mM L-glutamine, 50 U/mL penicillin, 50 mg/mL streptomycin, and 20 μg/mL gentamicine (all from Bio Whittaker/Cambrex, Verviers, Belgium). Additionally, 5 ng/mL human IL-12 p70 (R&D Systems) were added. After approximately 4 days co-incubation at 37° C., fresh medium was added with 20 U/mL human IL-2 (R&D Systems), and the cells were incubated for another 3 to 4 days. This stimulation cycle was repeated twice.
2.4. CTL Assay
For the CTL assays, KM22 and JY tumor cell lines were used as target cells. Peptide-pulsed cells were pulsed with 50 μg/mL peptide for 2 hours. All target cells were labeled in RP10 medium (RPMI 1640, supplemented with 10% heat-inactivated fetal calf serum and antibiotics) for 1 hour at 37° C. with [51Cr] sodium chromate (51Cr). Then, 104 cells/well were added to a 96-well round bottom plate. Various numbers of CTL were added to obtain an end volume of 200 μL, with subsequent incubation for 4 hrs at 37° C. Then, supernatants (500 μL/well) were harvested and counted in a beta-plate counter. Specific lysis was calculated in percent as follows: 100×(experimental release−spontaneous release/maximum release−spontaneous release). Each, spontaneous and maximum release were determined in presence of either medium or 2% Triton X-100.
2.5. Results of the CTL Induction
a) CTL Cytotoxic Activity Compared to Peptide-Pulsed Target Cells
In 51Cr release assays (see 2.4.) the cytotoxic activity of induced CTL (see 2.3.) was tested and compared to KM22 and JY cells. Cell lines KM22 and JY are HLA-B*07 positive.
The results of these release assays are shown in
The results show that an antigen-specific kill of the cells can be obtained with CTL cell lines that have been obtained after restimulation for 2 weeks.
In
b shows that KM22 target cells, treated with interferon gamma, positive for HLA allele B*0702 presenting the peptide with the sequence FPSLREAAL (SEQ ID NO: 114) from the tumor antigen MAGE-1 are recognized and lysed by specific cytotoxic T lymphocytes. Thus, only those cells were killed by an increasing number of CTL that presented the respective selected peptides; the control cells loaded with irrelevant peptides were not killed. By this, the specificity of the cytolytic activity could be demonstrated.
b) Production of Interferon Gamma by Peptide-Stimulated T Lymphocytes
In another experiment it was shown that the cytotoxic T lymphocytes lysing the T2 cells loaded with peptide FPSLREAAL (SEQ-ID NO. 114) also expressed interferon gamma which has been described as a reliable marker for activation of T cells.
In
To detect IFN gamma, 1×105 effector cells and stimulator cells that have been pulsed with peptide were grown in T cell medium on 96-well plates (T cell medium: RPMI 1640 with 25 mM HEPES (Gibco/Invitrogen, Karlsruhe, Germany; supplemented with 10% heat-inactivated human AB serum (CC pro, Neustadt/W., Germany; 2 mM L-glutamine, 50 U/mL penicillin, 50 μg/mL streptomycin and 20 μg/mL gentamycine (all from BioWhittaker). The loading with peptides was conducted in X-Vivo 15 medium with the respective peptides for approx. 2 hrs at 37° C.
After 1 to 2 hrs GolgiStop (Becton Dickinson) was added and incubated for another 4 to 5 hrs. Then, the cells were permeabilized and stained by using the Cytofix/Cytoperm Plus kit as well as anti-CD4-FITC, anti-IFN-•-PE and anti-CD8-PerCP according to the manufacturer's recommendations (Becton Dickinson). The cytometric analysis was performed using a FACSCalibur cytometer.
As can be seen from
Furthermore,
c) Peptide-Specific Stimulation of CD8-Positive T Cells
For further determination of the peptide-specific stimulation of CD8-positive T cells the peptide with sequence LAALPHSCL (SEQ ID NO. 448) from protein RGS-5 and the control peptide with sequence ELAGIGILTV (SEQ ID NO. 578) from melanoma antigen MELAN-A (position 26-35, modified by an amino acid exchange of the alanine at position 27 by leucine) were synthesized using standard Fmoc chemistry, with the control peptide also binding to the HLA allele A*02. Biotinylated recombinant A*02 molecules and fluorescent MHC tetramers were generated as described by Altman et al. (“Phenotypic analysis of antigen-specific T lymphocytes”, Science 274:94, 1996). To generate artificial antigen presenting cells (“APCs”), streptavidin-coated polystyrene particles (5.6 μm diameter) with a binding capacity of 0.064 μg biotin-FITC/mg microspheres (Bangs Laboratories, Fishers, Ill., USA) were resuspended with 2×106 particles/mL in a buffer containing the biotinylated MHC and the antibodies, and incubated for 30 min. at room temperature.
For the antigen-specific in vitro stimulation of the human CD8 T cells, PBMC from fresh buffy coat were isolated by standard gradient separation. Untreated CD8 T cells were concentrated by negative depletion using MACS (Miltenyi Biotec, Bergisch Gladbach, Germany). The in vitro stimulations were performed on 24-well plates with 5×106 responder cells and 1×106 beads or 1×106 irradiated APCs per well in 1.5 mL T cell medium (compare supra). 5 ng/mL human IL-12 p70 (R&D Systems, USA) were added together with microspheres. After 3 to 4 days co-incubation at 37° C., fresh medium and 20 U/mL human IL-2 (R&D Systems, USA) were added, and the cells were incubated for another 3 to 4 days. This stimulation cycle was repeated twice.
For the cell surface and intracellular cytometric analysis, tetramer analyses with fluorescent MHC tetramers plus anti-CD8 antibodies (hybridoma UKT8) were conducted on a four-color FACSCalibur (Becton Dickinson).
The results of these analyses are shown in
In the top left of
In the top right of
In
In the bottom row of
In further experiments, peptide-specific T cells from blood from renal cell carcinoma patients previously immunized with peptide-loaded autologous dendritic cells could be detected.
For this detection, a quantitative real-time polymerase chain reaction (RT-PCR) was performed. This RT-PCR was conducted as described by Kammula et al. (Kammula et al., Journal of Immunology 163:6867, 2000). For this, PBMCs were thawed in T cell medium, plated out with 1×106 cells in 500 μL medium and incubated over night with 5% CO2 at 37° C. Then, the synthetic peptides were added with 5 μg/mL for 3 hours, and then an RNA extraction with Trizole (Invitrogen, Karlsruhe, Germany) was performed. The cDNA was transcribed using random hexamer primers (Amersham Biosciences, Freiburg, Germany) and M-MLV reverse transcriptase (Promega GmbH, Mannheim, Germany).
The quantitative RT-PCR was performed on an “ABIPrism 7000 Sequence Detection System” (Applied Biosystems, Darmstadt, Germany) in duplicate in respect to IFN gamma mRNA and CD8 mRNA, using the Taqman PCR master mix (Applied Biosystems), specific primers and fluorescent probes.
The results represent the copy number of IFN gamma mRNA, with each sample being normalized regarding the CD8 mRNA copy number (used as reference gene product) (stimulation index). The gene expression in presence of the tested peptides is relative to the gene expression obtained in presence of the controls (HLA-A*02 epitope derived from HIV1 pol with the sequence ILKEPVHGV, SEQ ID NO. 579) (copy number of the control was set to 1).
In
In
Starting after the 3rd immunization already, strong T cell responses were obtained for GLASFKSFLK (RGS-5 74-83, SEQ ID NO. 153) and SLLTSSKGQLQK (ADFP 369-380, SEQ ID NO. 289), whereby for the last peptide the T cell responses increased after the 4th immunization. Such fluctuations of the T cell response during the course of an immunization therapy are known from other patients as well.
For IARNLTQQL (ADFP 313-321; SEQ ID NO. 233), GPALGRSFL (TNFSF7 78-86, SEQ ID NO. 577) the T cell responses were clearly in the background, and were weaker for a known pan-HLA DR-binding (PADRE) peptide. For these three peptides, an increase of the T cell response was also observed during the course of the immunization therapy (dotted lines). This confirmed that these three latter peptides have elicited a positive T cell response.
The patient was immunized as part of a registered clinical study at the University Hospital in Tubingen, Germany. The clinical study was applied and approved in written form by the Ethical Committee of the University of Tubingen, Germany. The study was conducted according to the rules and regulations of the German Medicines Act (Arzneimittelgesetz) and the good clinical practice (GCP).
In summary, the inventors were thereby able to show that the identified peptides represent promising substances as part of an immunotherapy for a variety of (tumor) diseases.
Tetramer analysis of the microsphere-driven proliferation of B*0702/IARNLTQQL (SEQ ID NO. 233)-specific CD8+ lymphocytes from peripheral blood (see
1×106 CD8+ enriched PBMCs per well from six healthy HLA-A*0201+ donors HD155, HD159, HD161, HD167, 00168 and 00177, were stimulated weekly with microspheres coupled to anti-CD28 plus irrelevant antigen (“irrelevant stimulation”), tumor antigen B*0702/IARNLTQQL (SEQ ID NO. 233) with high density (“HD”) or tumor antigen B*0702/IARNLTQQL (SEQ ID NO. 233) with low density (“LD”) as previously shown [Walter, S, et al. Cutting Edge: Predetermined avidity of human CD8 T cells expanded on calibrated MHC/anti-CD28-coated microspheres. J. Immunol. 171 (1 0):4974-8, 2003] with minor modifications. After three stimulations in vitro, individual wells were stained with antibody CD8 plus tetramer B*0702/IARNLTQQL (SEQ ID NO. 233). Each dot represents the percentage of tetramer+ among CD8+ lymphocytes from one well.
Based on the distribution of observed responses after irrelevant stimulation in individual donors, a significant threshold was calculated, and shown by a horizontal red line using the following formula:
Threshold=upper limit of 95% confidence interval for the mean+3×upper limit of 95% confidence interval for the standard deviation
The numbers in the diagrams show the number of significantly positive wells among total wells for the indicated condition.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 2004 026 135 | May 2004 | DE | national |
This is a divisional of U.S. patent application Ser. No. 11/596,802, now U.S. Pat. No. 8,008,431, issued Aug. 30, 2011, filed Nov. 17, 2006, which is the U.S. National Stage of International Application No. PCT/EP2005/005619, filed May 24, 2005, which in turn claims priority to German Patent Application No. 10 2004 026 135.0, filed May 25, 2004. The contents of all applications are incorporated in their entirety by reference.
| Number | Name | Date | Kind |
|---|---|---|---|
| 6410240 | Hodge et al. | Jun 2002 | B1 |
| 20020061543 | Ross | May 2002 | A1 |
| 20020177567 | Cheever et al. | Nov 2002 | A1 |
| 20030113733 | Khan et al. | Jun 2003 | A1 |
| 20050063967 | Young et al. | Mar 2005 | A1 |
| Number | Date | Country |
|---|---|---|
| 10225144 | Dec 2003 | DE |
| 2003000270 | Jan 2003 | JP |
| Number | Date | Country | |
|---|---|---|---|
| 20100021441 A1 | Jan 2010 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11596802 | US | |
| Child | 12544117 | US |
