Patent Application
20080050396
The invention will now be illustrated by the following, non-limiting examples and the drawings wherein
Immune responses against Bcl-2 in breast cancer patients
1. Patients
Peripheral blood lymphocytes (PBL) were collected from breast cancer patients. PBL were isolated using Lymphoprep separation, HLA-typed (Department of Clinical Immunology, University Hospital, Copenhagen, Denmark) and frozen in FCS with 10% DMSO. None of the patients received immunotherapy prior to sampling of blood.
2. Assembly Assay for Peptide Binding to MHC Class I Molecules
The binding affinity of the synthetic peptides (Invitrogen, Carlsbad, Calif., USA) to HLA-A2 molecules, metabolically labelled with [35S]-methionine, was measured in the assembly assay, as described previously. The assay is based on peptide-mediated stabilisation of empty HLA molecules released upon cell lysis, from the TAP-deficient cell line T2. Stably folded HLA-molecules were immune-precipitated using the HLA class I-specific, conformation-dependent mAb W6/32, and separated by isoelectric focusing (IEF) gel electrophoresis. MHC heavy chain bands were quantified using the ImageGauge Phosphorimager program (FUJI photo film Co., Carrollton, Tex., USA). The intensity of the band is directly related to the amount of peptide-bound class I MHC complex recovered during the assay. Subsequently, the extent of stabilisation of HLA-A2 is directly related to the binding affinity of the added peptide. The recovery of HLA-A2 was measured in the presence of 50, 5, 0.5, 0.05 μM of the relevant peptide. The C50 value was calculated for each peptide as the peptide concentration sufficient for half maximal stabilisation.
3. Antigen Stimulation of PBL
To extend the sensitivity of the ELISPOT assay, PBL were stimulated once in vitro prior to analysis. At day 0, PBL or crushed lymph nodes were thawed and plated in 2 ml/well at a concentration of 2×106 cells in 24-well plates (Nunc, Denmark) in X-vivo medium (Bio Whittaker, Walkersville, Md.), 5% heat-inactivated human serum, and 2 mM of L-glutamine in the presence of 10 μM of peptide. Two days later 20 IU/ml recombinant interleukin-2 (IL-2) (Chiron, Ratingen, Germany) was added to the cultures. The cultured cells were tested for reactivity in the ELISPOT on day 12.
4. ELISPOT Assay
The ELISPOT assay was used to quantify peptide epitope-specific interferon-γ releasing effector cells as described previously (4). Briefly, nitrocellulose bottomed 96-well plates (MultiScreen MAIP N45, Millipore, Hedehusene, Denmark) were coated with anti-IFN-γ antibody (1-D1K, Mabtech, Nacka, Sweden). The wells were washed, blocked by X-vivo medium, and cells added in duplicates at different cell concentrations. Peptides were then added to each well and the plates were incubated overnight. The following day, media was discarded and the wells were washed prior to addition of biotinylated secondary antibody (7-B6-1-Biotin, Mabtech). The plates were incubated for 2 hours, washed and Avidin-enzyme conjugate (AP-Avidin, Calbiochem, Life Technologies) was added to each well. Plates were incubated at RT for 1 hour and the enzyme substrate NBT/BCIP (Gibco, Life Technologies) was added to each well and incubated at RT for 5-10 min. The reaction was terminated by washing with tap-water upon the emergency of dark purple spots. The spots were counted using the ImmunoSpot® Series 2.0 Analyzer (CTL Analyzers, LLC, Cleveland, US) and the peptide specific CTL frequency could be calculated from the numbers of spot-forming cells. All assays were performed in triplicates for each peptide antigen.
5. Results
The amino acid sequence of the Bcl-2 protein was screened for the most probable HLA-A2 nona- and decamer peptide epitopes, using the main HLA-A2 specific anchor residues (2). Thirteen Bcl-2 derived peptides were synthesised and examined for binding to HLA-A2 by comparison with the HLA-A2 high affinity positive control epitope from HIV-1 pol476-484 (ILKEPVHGV) (SEQ ID NO:39) by the assembly assay. The assembly assay is based on stabilisation of the class I molecule after loading of different concentrations of peptide to the TAP-deficient cell line T2. Subsequently correctly folded stable MHC heavy chains are immunoprecipitated using conformation-dependent antibodies. The extent of stabilisation of class I MHC molecules is directly related to the binding affinity of the added peptide as exemplified in
aThe value range listed in subscript indicates the position of the first amino acid in the sequence
bThe C50 value is the concentration of the peptide required for half maximal binding to HLA-A2
Using the ELISPOT IFN-γ secretion assay, we examined the presence of specific T-cell responses against the Bcl-2 derived peptides in peripheral blood T cells from breast cancer patients. This method has previously been highly effective when identifying tumour specific CTL in cancer patients.
PBL from 15 HLA-A2 positive breast cancer patients were stimulated once in vitro before examination in the ELISPOT. This procedure was chosen to extend the sensitivity of the ELISPOT as described (4). Since many described CTL epitopes are in fact low affinity peptides we included all thirteen Bcl-2 deduced peptides in the first line of experiments. Responses were detected against Bcl172, Bcl180, Bcl208, and Bcl214 and only data from these peptides are given in
Herein, we describe spontaneous T-cell reactivity against Bcl-2 in peripheral blood from patients suffering from unrelated tumor types, i.e., pancreatic cancer, AML and CLL. Additionally, we show that these Bcl-2 reactive T cells are indeed peptide specific, cytotoxic effector cells. Thus, Bcl-2 may serve as an important and widely applicable target for anti-cancer immunotherapeutic strategies, e.g., in the combination with conventional radiation- and chemotherapy.
The Bcl-2 family comprises several key players in the regulation of apoptosis and includes both proapoptotic as well as antiapoptotic molecules. Bcl-2 is a critical cellular factor contributing to the pathogenesis and progression of cancer. In the present study, we examined the natural cellular immunogenicity of Bcl-2 in cancer patients.
Patients
PBL were isolated using Lymphoprep separation, HLA-typed (Department of Clinical Immunology, University Hospital, Copenhagen, Denmark) and frozen in FCS with 10% DMSO. None of the patients received immunotherapy prior to sampling of blood. Informed consent was obtained from the patients prior to any of theses measures. Peripheral blood lymphocytes (PBL) were collected from thirteen HLA-A2 positive breast cancer patients presenting with progressive disease with distant metastases defining stage IV disease; the majority of patients had more than one tumor location (8/13 patients). Prior treatment included chemotherapy, endocrine therapy, and radiation therapy. Eight patients were previously treated with chemotherapy, while five patients had only received endocrine therapy and no chemotherapy prior to study inclusion. Furthermore, twelve HLA-A2 positive patients with localized operable breast cancer were included and blood samples were collected prior to primary surgery and chemotherapy. Additionally, PBL were collected from two HLA-A2 positive pancreatic cancer patients presenting with progressive disease with distant metastases defining stage IV disease. Finally, PBL from ten HLA-A2 newly diagnosed CLL patients and three AML were collected prior to therapy. PBL from twelve HLA-A2 positive healthy individuals served as controls.
The Granzyme B (GrB) ELISPOT assay was used for measuring antigen-specific CTL cytotoxicity as described. Briefly, nitrocellulose bottomed 96-well plates (MultiScreen MAIP N45, Millipore) were coated with GrB Capture Antibody (BD Biosciences, Brondby, Denmark). The wells were washed and blocked by X-vivo medium with 5% human serum. The cells were added at different cell concentrations. T2 cells and peptides were then added to each well and the plates were incubated 4 hours, medium was discarded and the wells were washed prior to addition of GrB Detection Antibody (BD Biosciences). The plates were incubated for 2 hours, washed and Avidin horseradish peroxidase (BD Biosciences) was added to each well. Plates were incubated at RT for 1 hour AEC Substrate Reagent (BD Biosciences) was added to each well and incubated at RT for 5-10 min. The reaction was terminated by washing with tap-water upon the emergency of red spots. The spots were counted and the peptide specific CTL frequency was calculated like for the INF-γ ELISPOT. All assays were performed in duplicate or triplicates for each peptide antigen.
Antigen specific cells were isolated by means of Bcl208/HLA-A2 coated magnetic beads as previously described. Biotinylated monomers (ProImmune, Oxford, UK) were coupled to streptavin coated magnetic beads (Dynabeads M-280, Dynal A/S, Oslo, Norway) by incubating 2.5 μg monomers with 5×106 beads in 40 μl PBS, for 20 min at room temperature. The magnetic complexes were washed three times in PBS in a magnetic field (Dynal A/S, Oslo, Norway) and subsequently mixed with PBLs, at a ratio of 1:10 in PBS with 5% BSA, and rotated very gently for 1 h. Antigen specific CD8+ T cells associating with the magnetic complexes were gently washed three times. Isolated cells were resuspended numerous times in X-vivo with 5% HS, and incubated for 2 h, before the magnetic beads were released and removed from the cell suspension. The isolated cells were cultured in a 48-well plate in X-vivo, 5% HS and 106 anti-CD28, anti-CD3 coated artificial cell-based antigen presenting cells (K32/41 BBL) that expresses 4-1BB ligand (4-1BBL) (kindly provided by Dr. Carl H. June, Department of Pathology and Laboratory Medicine, University of Pennsylvania). One day after isolation 20 units/ml IL-2 was added, and on day 5 the capacity of these cells to kill target cells was tested either in standard 51Cr release assays.
CTL clones were established from the isolated cultures by limiting dilution in 96-well plates using irradiated PBMC as feeder cells in the presence of 40 IU/ml IL-2 and 1 μg/ml HA in X-vivo with 5% HS. Fresh medium and IL-2 were added to the clones every 3-4 day.
Conventional [51Cr]-release assays for CTL-mediated cytotoxicity was carried out as described elsewhere. Target cells were T2-cells with or without the relevant peptide, the HLA-A2-positive breast cancer cell line MDA-MB-231, and the HLA-A2-negative breast cancer cell line ZR75-1. Both breast cancer cell lines expressed Bcl-2 as examined by reverse transcription-PCR (data not shown).
CTL Responses Against Bcl-2 Derived Peptides
To examine whether Bcl-2 specific T cells were also present in PBL from leukemia patients we examined PBL from ten HLA-A2 positive CLL patients and three AML patients for reactivity against the two peptides bcl208 and bcl214. Bcl-2 responses were present in five of the CLL patients and two of the AML patients (
Bcl-2 Specific Granzyme B Release in PBL
Using the GrB ELISPOT we assessed whether the bcl-2 specific T cells detected in PBL exhibit cytotoxic function. Thus, PBL from three of the bcl-2 reactive breast cancer patients (pt. no.: 19, 20 and 22) were analyzed for reactivity against the two epitopes bcl208 and bcl214 (
The Functional Capacity of Bcl-2-reactive CTL
To further characterize the functional capacity of Bcl-2-reactive CTL, these cells were enriched by means of magnetic beads coated with HLA-A2/bcl208-complexes as described. Cells were stimulated once with peptide in vitro prior to isolation. A small fraction of the isolated cells were cloned by limiting dilution. The expanding cultures were examined for recognition of T2 cells either without peptide or pulsed with bcl208 in a GrB ELISPOT. Several of these clones showed specific recognition of bcl208-pulsed T2 cells (data not shown). However, unfortunately we were not able to expand these clones for further analysis.
One day after isolation IL-2 was added to the remaining cells, and on day 5 the capacity of the cells to kill peptide loaded T2 cells was tested in standard 51Cr release assays. To this end, either unloaded T2 cells or T2 cells loaded with bcl208 peptide served as targets. This assay revealed that only T2 cells pulsed with bcl208 were killed (
Immunogenicity of Bcl-X(L) in cancer patients
Here, we demonstrate that Bcl-XL is a target for T-cell recognition in cancer patients. Thus, we describe spontaneous HLA-A2- and HLA-A3-restricted cytotoxic T-cell responses against peptide epitopes derived from Bcl-XL by means of ELISPOT and flow cytometry stainings. Thus, cellular immune responses against apoptosis inhibitors like the Bcl-2 family proteins appear to represent a general phenomenon in cancer, and consequently, this group of proteins represents attractive universal target proteins for anti-cancer immunotherapy. Additionally, since elevated expression of these proteins in cells is correlated with drug resistance, the combination of immunotherapy with cytotoxic chemotherapy is a very appealing way to treat cancer.
The antiapoptotic protein Bcl-XL is produced from the long alternative splice form of the bcl-x gene, while proapoptotic Bcl-XS is derived from the short alternative splice form of the same gene. Bcl-XL plays an important role in cancer as it has been directly linked to resistance to conventional forms of therapies and poor prognosis. The functional inhibition of Bcl-XL restore the apoptotic process and render neoplastic cells sensitive to chemical and radiation therapies, whereas manipulation of cancer cell lines to express high levels of Bcl-XL results in a multi-drug reistance phenotype. Increased expression of Bcl-XL has been reported in a variety of different malignancies including AML and multiple myeloma as well as solid cancers like bladder cancer, breast cancer, pancreatic cancer and melanoma.
Ideal targets for immunotherapy are gene products silenced in normal tissues, overexpressed in cancer cells, and directly involved in tumor cell survival and progression.
Patients
Peripheral blood lymphocytes (PBL) were collected from patients suffering from cancer of different origin and from healthy controls and were isolated using Lymphoprep separation, HLA-typed (Department of Clinical Immunology, University Hospital, Copenhagen, Denmark) and frozen in FCS with 10% DMSO. None of the patients received immunotherapy prior to sampling of blood. Informed consent was obtained from the patients prior to any of theses measures.
Flow Cytometry (FACS)
PBL from a breast cancer patient was stimulated once in vitro with the relevant peptide and at day seven the CD8+ cells were isolated from PBL using the Dynal CD8 negative isolation kit (Dynal Biotech ASA, Oslo, Norway). The resulting CD8 positive T cell culture were stained with PE couplet Pro5™ MHC pentamers (ProImmune, Oxford, UK), followed by antibody staining with the flourochrome coupled mAbs: CD8-APC and CD3-FITC (Becton Dickinson, Immunocytometry Systems, San Jose, Calif.). Both stainings were performed in PBS+2% FCS, for 30 min, 4° C., in the dark. The Pro5™ MHC pentamer complexes used were: HLA-A2/Bcl-XL173-182 (YLNDHLEPWI)(SEQ ID NO:42) and HLA-A2/HIV-1 pol476-484 (ILKEPVHGV)(SEQ ID NO:39). The samples were analysed on BD FACS aria, using DIVA software (BD, San Jose, Calif.).
The bcl-x gene is transcribed into two mRNAs through alternative splicing. The antiapoptotic protein Bcl-XL is produced from the long alternative splice, while proapoptotic Bcl-XS is derived from the short alternative splice form of this gene. The protein product of the larger BCL-XL differs from Bcl-XS protein by an inserted region (amino acids 126-188). Thus, to investigate if Bcl-XL is a natural target for T-cells in cancer patients we scrutinized this inserted region (including nine amino acids at each end) for putative HLA-A2 epitopes using the main HLA-A2 specific anchor residues. Subsequently, we synthesized seven Bcl-XL deduced peptides (Bcl-XL158-166 (EMQVLVSRI)(SEQ ID NO:44), Bcl-XL118-126 (TAYQSFEQV)(SEQ ID NO:43), Bcl-XL173-182 (YLNDHLEPWI)(SEQ ID NO:42), Bcl-XL165-174 (RIAAWMATYL)(SEQ ID NO:45), Bcl-XL169-178 (WMATYLNDHL)(SEQ ID NO:46), Bcl-XL161-170 (VLVSRIAAWM)(SEQ ID NO:48), Bcl-XL141-150 (VAFFSFGGAL)(SEQ ID NO:49)) and scrutinized PBL from HLA-A2+ cancer patients of different origin by means of ELISPOT against these peptides. This method has previously been shown to be highly effective to identify tumor specific CTL in cancer patients. Indeed, strong and frequent CTL responses were detected against four of the examined peptides (Bcl-XL173-182, Bcl-XL141-150, Bcl-XL161-170, and Bcl-XL165-174) in cancer patients of different origin (
Bcl-XL Specific Granzyme B Release in PBL
Using the GrB ELISPOT we assessed whether the Bcl-XL specific T cells detected in PBL exhibit cytotoxic function. Thus, PBL from two of the Bcl-XL reactive breast cancer patients (pt. no.: 35 and 36) were analyzed for reactivity against Bcl-XL173-182 (
FACS Analyses of Bcl-XL Specific T Cells
The spontaneous occurrence of Bcl-XL173-182 specific CTL in PBL from breast cancer patients was further evaluated using FACS analyses and Pro5™ MHC Pentamer staining. PBL from breast cancer patient no. 36 were stimulated once in vitro with peptide and the CD8 positive cells were isolated. This culture was stained with the HLA-A2/BCL-X pentamer complex. FACS analyses revealed an easily detectable population of pentamer positive T cells constituting 0.24% of the CD8+ T cells (
Additional HLA-A2 Restricted Epitopes Against Bcl-X(L)
We scrutinized PBL from HLA-A2+ cancer patients of different origin by means of ELISPOT against Bcl-XL118-126 (TAYQSFEQV)(SEQ ID NO:43) (
HLA-A3-restricted Responses Against Bcl-X(L)
Additionally, we scrutinized the inserted region (including nine amino acids at each end) for putative HLA-A3 epitopes using the main HLA-A3 specific anchor residues. Subsequently, we synthesized two peptides; Bcl-XL165-173 (RIAAWMATY)(SEQ ID NO:50) and the Bcl-XL149-157 (ALCVESVDK)(SEQ ID NO 51). Next, we scrutinized PBL from HLA-A3+ cancer patients of different origin by means of ELISPOT against the Bcl-XL165-173 (RIAAWMATY)(SEQ ID NO: 50) and the Bcl-XL149-157 (ALCVESVDK)(SEQ ID NO:51) peptide. This method has previously been shown to be highly effective to identify tumor specific CTL in cancer patients. Indeed, strong and frequent CTL responses were detected against Bcl-XL165-173 (RIAAWMATY)(SEQ ID NO:50) in cancer patients of different origin, We were able to detect a response against the Bcl-XL165-173 in HLA-A3+ PBL in four out of five examined breast cancer patients (responders are defined as average number of antigen specific cells±½ standard deviation >25 per 105 cells), four out of four examined melanoma patients, two out of two examined pancreatic cancer patients as well as one out of four examined multiple myeloma patients (
Here, we demonstrate that Mcl-1 is a target for T-cell recognition in cancer patients. Thus, we describe spontaneous HLA-A1- and HLA-A3-restricted cytotoxic T-cell responses against peptide epitopes derived from Mcl-1 by means of ELISPOT
Myeloid cell factor-1 (Mcl-1) is a death-inhibiting member of the Bcl-2 family that is expressed in early monocyte differentiation and can promote viability on transfection into immature myeloid cells. Mcl-1 in transgenic mice promotes survival in a spectrum of hematopoietic cell types and immortalization of myeloid cells. Elevated levels of Mcl-1 have been reported for a number of human cancers including prostate cancers, pancreatic cancers, melanoma, breast cancers, ovarian cancer patients, and cervical cancer, as well as B-cell chronic lymphocytic leukemia (B-CLL) and in AML and ALL upon relapse. In B-CLL patients, higher levels of Mcl-1 are strongly correlated with failure to achieve complete remission after single-agent therapy. In multiple myeloma, Mcl-1 plays an important role in the survival of malignant cells. In this regard it has been demonstrated that mice expressing a mcl-1 transgene under control of its own promoter develop B-cell neoplasias with high frequency, ranging from follicular lymphoma to diffuse large cell lymphoma.
HLA-A3-restricted Responses Against Mcl-1
To investigate whether Mcl-1 is a natural target for T-cells in cancer patients we examined the protein sequence for the most probable HLA-A3 nona- and deca-mer peptide epitopes, using the main HLA-A3 specific anchor residues. Subsequently, we synthesized six Mcl-1 deduced peptides (Mcl-1185-194 (YLREQATGAK)(SEQ ID NO:52), Mcl-1293-302 (SITDVLVRTK)(SEQ ID NO:53), Mcl-1267-276 (LISFGAFVAK)(SEQ ID NO:54), Mcl-195-103 (RLLFFAPTR)(SEQ ID NO:55), Mcl-1300-308 (RTKRDWLVK)(SEQ ID NO:56), Mcl-1236-244 (DIKNEDDVK)(SEQ ID NO:57)) and scrutinized PBL from HLA-A3+ cancer patients of different origin for reactivity against these peptides, taking advantage of the ELISPOT assay. This method has previously been shown to be highly efficient for identification of tumor specific CTL in cancer patients. Indeed, strong and frequent CTL responses were detected against two Mcl-1 derived peptides in cancer patients of different origin (Mcl-195-103 and Mcl-1300-308) (
breast cancer patients hosted an immune response against one of these two Mcl-1 peptides. Thus, five breast cancer patients hosted a response against Mcl-195-103 (responders are defined as average number of antigen specific cells±½ standard deviation >25 per 105 cells), and three patients hosted a response against Mcl-1300-308 (
HLA-A1-restricted Responses Against Mcl-1
To investigate whether Mcl-1 is a natural target for T-cells in cancer patients we examined the protein sequence for the most probable HLA-A1 nona- and deca-mer peptide epitopes, using the main HLA-A1 specific anchor residues. Subsequently, we synthesized four Mcl-1 deduced peptides (Mcl-1166-175 (PAEEEEDDLY)(SEQ ID NO:58), Mcl-1121-129 (SPEEELDGY)(SEQ ID NO:59), Mcl-1177-185 (QSLEIISRY)(SEQ ID NO:60), Mcl-1339-347 (AGVGAGLAY)(SEQ ID NO:61)) and scrutinized PBL from HLA-A1+ cancer patients of different origin for reactivity against these peptides, taking advantage of the ELISPOT assay. Indeed, CTL responses were detected against two Mcl-1 derived peptides in cancer patients of different origin (Mcl-1166-175 and Mcl-1177-185) (
Modified Peptide Responses
The immunogenicity of the HLA-A3 restricted peptide Mcl-1300-308 was increased by replacing threonine at position 2 with a better HLA-A3 anchor residue namely Leucine (Mcl-1300-308L2 (RLKRDWLVK)(SEQ ID NO:62)). Spontanous immune responses were detected in two Breast cancer patients against Mcl-1300-308L2 (data not shown). Likewise, to generate more immunogenetic epitope we modified the HLA-A1 restricted peptide Mcl-1177-185 (QSLEIISRY)(SEQ ID NO:60) at position 3 generating the two peptides Mcl-1177-185D3 (QSDEIISRY)(SEQ ID NO:63) and Mcl-1177-185E3 (QSEEIISRY)(SEQ ID NO:64).
Almost all malignancies are characterized by defects in apoptosis signaling. This renders the malignant cells resistant to endogenous apoptotic stimuli, as well as exogenous stimuli such as chemotherapeutic drugs and radiation. The defective apoptosis seen in human cancers are often results from overexpression of antiapoptotic proteins in the Bcl-2 protein family, i.e., Bcl-2, Bcl-XL, and Mcl-1, Bcl-w, Bfl-1A1, Bcl-b, and Bcl2-L-10 Using such inhibitors of apoptosis proteins for vaccination purposes is advantegous because downregulation or loss of expression of these proteins as some form of immune escape would impair sustained tumor growth, since survival of tumor cells requires functionally active members of the Bcl-2 family. For therapeutic strategies, targeting of antigens that plays an insignificant role in relation to tumor cell growth and survival, the selection of antigen deficient tumors is a well-recognized limitation. In addition, since elevated expression of Bcl-2 family proteins in cells is correlated with drug resistance, the combination of a Bcl-2 family-based immunotherapy with cytotoxic chemotherapy is a very exciting new way to treat cancer.
We scanned the Bcl-2, Bcl-X(L) and Mcl-1 proteins for the presence of peptide binding motifs and used these to search for specific T-cell responses in cancer patients. To this end, spontaneous T-cell reactivity was detected against all members of the Bcl-2 family in patients suffering from unrelated tumor types, i.e., pancreatic cancer, breast cancer, melanoma AML and CLL by means of ELISPOT. The presence of Bcl-XL specific CD8+ cells in PBL from cancer patients was confirmed by CD8/pentamer FACS stainings. Taken together, these data shows that CTL defined epitopes from these proteins might be broadly applicable in therapeutic vaccinations against cancer and are therefore of substantial immunotherapeutic value.
In addition, eleven of the breast cancer patients possessed Bcl-2 specific CTLs, eight of these patients were previously treated with at least one type of chemotherapy. In two patients (pt. no: 14 and 17) no CTL responses to the four different Bcl-2 peptides were detectable. Both patients had previously received anti-hormonal therapy but no chemotherapy. Similarly, we were not able to detect any responses in patients with primary localized breast cancer prior to chemotherapy. Thus, in breast cancer patients Bcl-2 responses were only detected in the patients who had received chemotherapy. Although, tumor load may play an important role, this might indicate that the immune responses are introduced or increased as a consequence of the treatment-induced increase of Bcl-2 expression. It points to a scenario in which the combination of a Bcl-2-family based immunotherapy with cytotoxic chemotherapy might in a synergy improve current response rates. The treatment status of the patients examined for Bcl-X(L) and Mcl-1 responses was not available.
In the present study we took advantage of the GrB ELISPOT assay to demonstrate that the Bcl-2 or Bcl-X(L) specific CTL in the patients PBL are indeed cytotoxic effector cells. To further prove this notion, we enriched Bcl-2 reactive T cells from patient PBL, and showed that the resulting T-cell line was able to lyse peptide-pulsed T2-cells in a conventional 51Cr-release assay. Moreover, this Bcl-2 reactive T-cell line was capable of killing a HLA-matched breast cancer cell line, whereas HLA-A2 negative target cells was not killed. These findings shows that cancer cells indeed process and present the Bcl-2 peptide in the context of the HLA-A2 molecule. Finally, we were able to clone these isolated cells and showed that they reacted highly specific against the Bcl-2 peptide epitope.
When peptides derived from melanocyte differentiation antigens were first used to treat patients with stage IV melanoma it was envisioned that this might lead to pronounced destruction of melanocytes, which in turn would manifest clinically, i.e., vitiligo or retinitis. However, clinical experience demonstrated that the incidence of vitiligo in patients receiving vaccinations was not significantly higher than the incidence of melanoma associated hypopigmentation in patients receiving other forms of therapy. Additionally, no serious site-effects have been reported in various vaccination trails against self-antigens. Our data taken together prove that cellular immune responses against the group of Bcl-2 family proteins are a general feature in cancer. In attempt to maximize the impact of immunotherapy, an exciting strategy would be to consider the expression profile and prognostic significance of the chosen target in the particular disease, or disease stage, being treated. Thus, while coexpression of Bcl-2, Mcl-1 and Bcl-XL is seen in some cancers, or a particular stage of disease, other cancers exhibit exclusive expression of one or the other protein. Thus, in some diseases like ovarian cancer, expression of Mcl-1, but not Bcl-2, is associated with advanced stage and poor survival for which reason Mcl-1 might be the prime antigen, whereas in diseases such as CLL, where Bcl-2 and Mcl-1 are co-over expressed, simultaneous targeting of both proteins may represent a more effective strategy than targeting either molecule alone. Similary, Tanaka et al described that the presence of another inhibitor-of-apoptosis protein survivin in breast carcinoma was strongly associated with expression of Bcl-2 and with reduced apoptotic index (AI) and poor overall survival. A similar association between survivin and Bcl-2 has been described in neuroblastoma, gastric cancer, colorectal cancer, and high-grade non-Hodgkin's lymphoma. The safety and potential efficacy of survivin derived peptides in therapeutic vaccinations against cancer is currently being investigated in phase I/II clinical trials (J. Becker, personal communication). Thus, an exciting immunotherapeutic strategy would be to target both Bcl-2 protein family and survivin especially since they execute their anti-apoptotic function though different cellular pathways.
Bcl-2 protein family peptides can e.g. be synthesized e.g. at the UVA Biomolecular Core Facility with a free amide NH2 terminus and free acid COOH terminus. Each is provided as a lyophilized peptide, which is then reconstituted in sterile water and diluted with Lactated Ringer's solution (LR, Baxter Healthcare, Deerfield, Ill.) as a buffer for a final concentration of 67-80% Lactated Ringer's in water. These solutions are then sterile-filtered, placed in borosilicate glass vials, and submitted to a series of quality assurance studies including confirmation of identity, sterility, general safety, and purity, in accordance with FDA guidelines, as defined in IND 6453. Tests of peptide stability demonstrated no decrease in purity or in the peptide concentration, when these peptide solutions were stored at −20° C. for 3 years.
In practical circumstances, patients will receive a vaccine comprising about 100 μg of a class I HLA-restricted peptide with or without a class II HLA-restricted helper peptide. The patients are vaccinated with e.g. about 100 μg of the class I HLA peptide in adjuvant alone, or are vaccinated with e.g. about 100 μg of the HLA class I-restricted peptide plus 190 μg of the class II-restricted helper peptide. The higher dose of the helper peptide is calculated to provide equimolar quantities of the helper and cytotoxic epitopes. Additionally, patients can be vaccinated with a longer peptide comprising the amino acid sequences of both peptides.
The above peptides, in 1-ml aqueous solution, can be administered either as a solution/suspension with about 100 μg of QS-21, or as an emulsion with about 1 ml of Montanide ISA-51 adjuvant.
Patients are immunized e.g. at day 0 and months 1, 2, 3, 6, 9, and 12, with the peptides plus adjuvant, for a total of seven immunizations. With rare exceptions, the vaccinations are administered to the same arm with each vaccine. The peptides are preferably administered s.c.
| Number | Date | Country | Kind |
|---|---|---|---|
| PA 2003 01716 | Nov 2003 | DK | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/DK04/00799 | 11/18/2004 | WO | 00 | 2/28/2007 |
| Number | Date | Country | |
|---|---|---|---|
| 60523119 | Nov 2003 | US |
