RhoC-Based Immunotherapy

Abstract

The present invention relates generally to the field of prophylaxis and therapy of metastatic cancer. In particular there is provided a protein; Ras Homology gene family, member C (RhoC) or peptide fragments thereof that are capable of eliciting anti-cancer immune responses. Specifically, the invention relates to use of RhoC or peptides derived thereof or RhoC specific T-cells for treatment of metastatic cancer. Hence, the invention in one aspect relates to RhoC specific T-cells adoptively transferred or induced in vivo by vaccination as a treatment of cancer.

Also the use of RhoC and immunogenic peptide fragments hereof in cancer treatment, diagnosis and prognosis is provided.

Claims

1. A vaccine composition comprising a) RhoC of SEQ ID no 1 or a functional homologue thereof having at least 70% identity to SEQ ID NO 1 or an immunogenically active peptide fragment comprising a consecutive sequence of said RhoC or said functional homologue thereof or a nucleic acid encoding said RhoC or said peptide fragment; and b) an adjuvant for use as a medicament.

2. The vaccine composition according to claim 1, wherein said immunogenically active peptide fragment consists of a consecutive sequence of in the range of 8 to 50, preferably in the range of 9 to 25 amino acids of said RhoC of SEQ ID no 1 or said functional homologue thereof wherein at the most two amino acid have been substituted.

3. The vaccine composition of claim 1, wherein the vaccine composition is capable of eliciting an immune response against a metastatic cancer expressing RhoC of SEQ ID no 1 or a functional homologue thereof wherein at the most two amino acid have been substituted, when administered to an individual suffering from a metastatic cancer expressing RhoC.

4. An isolated immunogenically active peptide fragment from RhoC of SEQ ID no 1 or a functional homologue thereof consisting of 18 to 25 consecutive amino acids wherein at the most two amino acid have been substituted, wherein the peptide fragment contains at least one of amino acid residues I43, Q123, R140, S141, S152, L 157, E165, G178, V181, K183, N184, R186, R187, R188, P191 or I192 of RhoC of SEQ ID no 1 or of 8 to 10 consecutive amino acids from RhoC of SEQ ID no 1 or a functional homologue thereof wherein at the most two amino acid have been substituted, wherein the peptide fragment contains at least one of amino acid residues I43, Q123, R140, S141, S152, L157, E165, G178, V181, K183, N184, R186, R187, R188, P191 or I192 of RhoC of SEQ ID no 1 or of 26 to 60 consecutive amino acids from RhoC of SEQ ID no 1 or a functional homologue thereof wherein at the most two amino acid have been substituted, wherein the peptide fragment contains at least one of amino acid residues I43, Q123, R140, S141, S152, L157, E165, G178, V181, K183, N184, R186, R187, R188, P191 or I192 of RhoC of SEQ ID no 1.

5. The peptide fragment according to claim 4, which comprises the sequence RXGLQVRKNK, wherein X is selected from the group consisting of alanine and leucine and wherein said peptide fragment is at the most 60 amino acids in length.

6. The peptide fragment according to claim 4, which is an MHC Class I-restricted peptide or an MHC class II-restricted peptide having at least one of the following characteristics: (i) capable of eliciting INF-?-producing cells in a PBL population of a cancer patient at a frequency of at least 1 per 10.sup.4 PBLs as determined by an ELISPOT assay, and/or (ii) capable of in situ detection in a tumor tissue of CTLs that are reactive with the epitope peptide. ((iii) capable of inducing the growth of RhoC specific T-cells in vitro.

7. The peptide fragment according to claim 4, wherein the peptide fragment consists of 8 to 10 or 18 to 25 or 26 to 60 consecutive amino acids from RhoC of SEQ ID no 1.

8. The peptide fragment according to claim 4, wherein the peptide fragment consists of 8 to 10 or 18 to 25 or 26 to 60 consecutive amino acids from RhoC of SEQ ID no 1 wherein at least one amino acid of RhoC of SEQ ID no 1 has been substituted, deleted or added at least.

9. The peptide fragment according to claim 4 that is capable of eliciting INF-?-producing cells in a PBL population of a cancer patient at a frequency of at least 10 per 10.sup.4 PBLs.

10. The peptide fragment according to claim 4, which is capable of eliciting INF-?-producing cells in a PBL population of a patient having a cancer disease where RhoC of SEQ ID no 1 or a functional homologue thereof having at least 70% identity to SEQ ID 1 is expressed.

11. The vaccine composition according to claim 1, wherein the peptide fragment consists of a consecutive sequence of RhoC of SEQ ID no 1 of in the range of 8 to 60 amino acids, preferably of in the range of 9 to 25 amino acids, sequence that is different from the sequences of RhoA of SEQ ID NO 2 or RhoB of SEQ ID NO 3 by at least one amino acid.

12. The vaccine composition according to claim 1, wherein the peptide fragment consists of a sequence selected from the 60 most C-terminal amino acids of RhoC of SEQ ID no 1 or a functional homologue thereof having at least 70% identity to SEQ ID NO 1.

13. The vaccine composition according to claim 1 comprising the peptide fragment according to claim 4.

14. A method of treating a metastatic cancer disease, the method comprising administering to a patient suffering from the disease an effective amount of the composition according to claim 1.

15. The method of claim 14 wherein the disease to be treated is a cancer disease where RhoC is expressed.

16. The method of to claim 15, which is combined with a further cancer treatment.

17. Isolated T-cells, wherein said T-cells are capable of specifically interacting with RhoC of SEQ ID No 1 or a functional homologue thereof having at least 70% identity to SEQ ID NO 1 or an immunogenically active peptide fragment comprising a consecutive sequence of said RhoC or a fragment thereof.

18. The T-cells according to claim 17, wherein said T-cells are capable of specifically interacting with any of the RhoC peptides of claim 4.

19. The T-cells according to claim 17, wherein said T-cells are CDS T-cells.

20. The T-cells according to claim 17, wherein said T-cells are CD4 T-cells.

21. A method of treating a metastatic cancer disease, the method comprising administering to a patient suffering from the disease an effective amount of the peptide of claim 4.

22. A method of treating a metastatic cancer disease, the method comprising administering to a patient suffering from the disease an effective amount of the T-cells of claim 17.

Description

DESCRIPTION OF DRAWINGS

[0251] FIG. 1. Alignment of RhoC, RhoA and RhoB

[0252] Sequence alignments of Human RhoC, Human RhoA and Human RhoB.

[0253] FIG. 2. Binding affinity of Rho1 and Rho1L2.

[0254] Stabilization of Rho1 and Rho1L2 was analyzed by assembly assay. Class I MHC heavy chain bands were quantified on a Phosphorimager. The amount of stabilized HLA-A3 heavy chain is directly related to the binding affinity of the added peptide. The binding of the HLA-A3-restricted positive control peptide Influenza NP265-273 was compared with the peptide Rho1L2 and the native peptide Rho1.

[0255] FIG. 3. HLA-A3 restricted T-cell responses against Rho1L2 as measured by IFN-? ELISPOT.

[0256] The average number of peptide specific IFN-? spots formed in response to Rho1L2 among 5?10.sup.5 in vitro stimulated PBMC from five HLA-A3.sup.+ healthy donors (HD), PBL from 10 Renal Cell Carcinoma patients (RCC), and 10 Melanoma patients (MM). Measurements were made in triplicates. The number of antigen-specific spots was calculated by subtracting the mean number of spots of the control wells from the mean number of spots in the positive wells; in order to prevent that replicates with a high standard variation are accepted as positive results, all replicates were analyzed by Student t-test for unpaired samples, results with a p-value <0.05 were considered as positive.

[0257] FIG. 4. T-cell antigen specificity and cross reactivity

[0258] Cytotoxicity by .sup.51Cr-release assay of a bulk culture stimulated with Rho1L2-loaded autologous DC/autologous PBL. Specific lysis of T2-A3 cells with no peptide or pulsed with Rho1L2 or Rho1. E:T ratio=60:1, measurements were made in duplicates. a) Specificity of a T-cell clone (clone 9) assayed by .sup.51Cr-release assay. Lysis of T2-A3 cells with no peptide, pulsed with Rho1L2, Rho1 or with the HLA-class I specific antibody W6/32 or HLA-A3 specific antibody GAP A3 at different E:T ratios (9:1; 3:1; 1:1; 0.3:1).

[0259] FIG. 5. Functional capacity of RhoC specific T cells

[0260] a) Cytotoxicity of a bulk culture stimulated with Rho1L2-loaded autologous DC/autologous PBL. Specific lysis of the HLA-A3.sup.+ melanoma cell line FM3 without and with the addition of the HLA-class I specific antibody W6/32.

[0261] b) Lysis by a Rho1L2 specific clone of the HLA-A3.sup.+ melanoma cell line FM3, cell lysis with addition of unlabeled T2-A3 cells pulsed with Rho1L2 or no peptide (inhibitor to target ratio=20:1), and cell lysis of the HLA-A3.sup.+ melanoma cell line FM9 and the HLA-A3.sup.? melanoma cell line FM82. Measurements were made in duplicates for all E:T ratios.

[0262] c) Lysis by a Rho1L2 specific clone of the HLA-A3.sup.+ breast cancer cell line BT-20, colon cancer cell line HT-29 and head and neck cancer cell line CRL-2095. Measurements were made in duplicates for all E:T ratios.

EXAMPLES

Example 1: Immune Responses Against RhoC

[0263] 1. Patients

[0264] Peripheral blood lymphocytes (PBL) from HLA-A3.sup.+ cancer patients or peripheral blood mononuclear cells (PBMC) from healthy controls were obtained from the University Hospital Herlev, Denmark. Cells were cryopreserved in FCS with 10% DMSO. Tissue typing was conducted at the Department of Clinical Immunology, The State Hospital, Copenhagen, Denmark. Informed consent was obtained from the patients before any of these measures.

[0265] 2. Assembly Assay for Peptide Binding to MHC Class I Molecules

[0266] The binding affinity of the synthetic peptides (Genscript, Scotch Plains, US) to HLA-A3 molecules was measured by means of the assembly assay as described (13). The assay is based on the stabilization of the class I molecule after loading of different concentrations of peptide to the TAP-deficient cell line T2-A3. Briefly, T2-A3 cells were incubated in methionine-free RPMI 1640 (Gibco BRL, Paisley, UK) with 10% dialysed FCS. Subsequently, cells were metabolically labelled with 50 ?Ci .sup.35S-methionine (Amersham, Birkeroed, Denmark). After incubation, cells were lysed in lysis buffer in the presence of protease inhibitors (100 ?g/ml iodoacetamide, 200 ?g/ml PEFA block and 2 ?g pepstatin (Roche diagnostics, Hvidovre, Denmark)) and with peptide in varying concentrations (4?0.04 Cell nuclei were removed by ultracentrifugation. The supernatant of T2-A3 cells was heated for 5 min at 45? C. in order to reduce background signals by preferentially destabilizing empty HLA-A3. The samples were precleared by addition of Pansorbin (Calbiochem, Darmstadt, Germany) and left on rotation overnight. Stably folded HLA molecules were immune-precipitated using the HLA class I-specific, conformation-dependent mAb W6/32. A-Sepharose beads were added to collect the folded MHC complexes and separated by isoelectric focusing gel electropheresis. MHC heavy chains were quantified using the ImageGauge Phosphorimager program (FUJI Photo Film). 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 stabilization of HLA-A3 is directly related to the binding affinity of the added peptide. The C.sub.50-value was calculated for each peptide as the peptide concentration sufficient for half maximal stabilization.

[0267] 3. Ag Stimulation of PBL

[0268] To extend the sensitivity of the ELISPOT assay, PBL were stimulated once before analysis. At day 0, PBL were thawed and plated in 24-well plates (Nunc, Roskilde, Denmark) in X-Vivo medium (Cambrex Bio Science Copenhagen, Vallensbaek Strand, Denmark) with 5% heat-inactivated human serum in the presence of 10 ?M peptide (GenScript, Scotch Plains, US). The following day 20 U/ml IL-2 (PeproTech, London, UK) was added to the cultures. The cultured cells were tested for reactivity in the ELISPOT on day 8.

[0269] 4. Interferon-Gamma (INF-Gamma ELISPOT Assay)

[0270] The ELISPOT assay was used to quantify peptide epitope-specific INF-? releasing effector cells as described previously (WO 2005/049073, Example 1.2). Briefly, nitrocellulose bottomed 96-well plates (MultiScreen MAIP N45, Millipore, Hedehusene, Denmark) were coated with anti-IFN-? antibody (1-D1K, Mabtech, Nacka, Sweden). The wells were washed, blocked with X-vivo medium before adding 10.sup.4 stimulator T2-A3 cells (with or without 10 ?M peptide; Rho1L2: RLGLQVRKNK; Rho1: RAGLQVRKNK (GenScript, Scotch Plains, US)) and effector cells at different concentrations. The plates were incubated overnight. The following day, medium was discarded and the wells were washed prior to addition of biotinylated secondary antibody (7-B6-1-Biotin, Mabtech, Nacka, Sweden). The plates were incubated for 2 hrs, washed and Avidin-enzyme conjugate (AP-Avidin, Calbiochem, Life Technologies, Inc., Roskilde, Denmark) was added to each well. Plates were incubated at RT for 1 hr before the enzyme substrate NBT/BCIP (Gibco Life Technology, Taastrup, Denmark) was added to each well and incubated at RT for 5-10 min. The reaction was terminated 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. The number of antigen-specific spots was calculated by subtracting the mean number of spots of the control wells from the mean number of spots in the positive wells; responses with a p-value <0.05 (Student t-test for unpaired samples) were considered as positive. The definition is in respect with the CIMT Monitoring Panel inter-laboratory testing project (www.c-init.org).

[0271] 5. Dendritic Cells (DC)

[0272] DC were generated from PBMC by adherence on culture dishes at 37? C. for 60 min in RPMI-1640 enriched with 10% human AB serum. Adherent monocytes were cultured in RPMI-1640 supplemented with 10% human AB serum in the presence of IL-4 (1000 U/ml) and GM-CSF (800 U/ml) for 6 days. DC were matured by addition of IL-1? (2 ng/ml), IL-6 (1000 U/ml), TNF-? (10 ng/ml), and PGE2 (1 ?g/ml). Next day the resulting mature DC were pulsed with 10 ?M peptide for 2 hrs at 37? C., irradiated (20 Gy) and 1?10.sup.5 DC/ml were used for stimulation of 1?10.sup.6 PBL/ml in the presence of 40 U/ml IL-2. IL-2 was added every 3-4 days.

[0273] 6. Establishment of Antigen Specific T-Cell Cultures and Clones

[0274] PBL from a melanoma (MM) patient were stimulated with irradiated (20 Gy) autologous Rho1L2-loaded DC (PBL:DC ratio=3?10.sup.6: 3?10.sup.5). The following day 120 U/ml IL-2 (PeproTech, London, UK) was added. Stimulation of the cultures were carried out every 10 days with Rho1L2-loaded irradiated autologous DC (2?) followed by Rho1L2-loaded irradiated autologous PBL (3?). Hundred and twenty U/ml IL-2 (PeproTech, London, UK) was added after each stimulation. After one month growing cultures were tested for specificity for Rho1L2.

[0275] PBL from a specific culture were cloned by limiting dilution in the presence of cloning mix containing 10.sup.6/ml irradiated (20 Gy) lymphocytes from three healthy donors in X-vivo with 5% heat-inactivated human serum, 25 mM HEPES buffer (GibcoBRL, Paisley, UK), and 120 U/ml IL-2 (PeproTech, London, UK). The plates were incubated at 37? C./5% CO.sub.2. Every 3-4 days 50 ?l fresh media were added containing IL-2 to a final concentration of 120 U/ml. Growing clones were expanded using cloning mix cells (5?10.sup.4 cells/well) and IL-2. After expansion the clones were tested for specificity and cytotoxic potential in a standard .sup.51Cr-release assay.

[0276] 7. Cytotoxicity Assay

[0277] Conventional .sup.51Cr-release assays for CTL-mediated cytotoxicity were carried out as described elsewhere (Andersen et al., (1999) J Immunol 163:3812-3818). Target cells were T2-A3 cells, the HLA-A3.sup.+ breast cancer cell line BT-20, the HLA-A3.sup.+ colon cancer cell line HT-29, the HLA-A3.sup.+ head and neck cancer cell line CRL-2095 (all available at the American Type Culture Collection (ATCC)), the HLA-A3.sup.+ melanoma cell line FM9, the HLA-A3.sup.? melanoma cell line FM82, and the HLA-A3.sup.+ melanoma cell line FM3 (Kirkin et al., (1995) Cancer Immunol Immunother 41: 71-81), with or without added HLA specific mAb W6/32 (Barnstable et al., (1978) Cell 14: 9-20) (2 ?g/100 ?l)(Schmidt et al., (2003) Blood 102: 571-576) or the HLA-A3 specific antibody GAPA3 (Sire et al., (1988) 140: 2422-2430).

Results

[0278] HLA A3 Binding Peptides from RhoC

[0279] RhoC mainly differs from RhoA and RhoB in the C-terminal part of the sequence. Hence, this 20 amino acid region was scrutinized for putative HLA epitopes using the main HLA specific anchor residues as described herein above. We identified a possible HLA-A3 restricted peptide Rho1 (RAGLQVRKNK). However, as Alanine is a poor anchor amino acid in position 2 this peptide was only expected to bind to HLA-A3 with low affinity. As many of the established T-cell epitopes presented by cancer cells such as the melanoma antigens gp100 and MART-1 have relatively low binding affinities to the respective HLA class I molecules, it is common practice to generate heteroclitic peptides from such low-affinity epitopes by substitution of amino acids at specific positions, i.e., the anchor positions, which are crucial for the binding of the peptide to the HLA molecule (Pardoll D M (1998) Nat Med 4: 525-531; Scheibenbogen C., et al, (2002) Int J Cancer 98: 409-414). Consequently, we included a modified counterpart Rho1L2 (RLGLQVRKNK) in our studies, in which position 2 was modified from Alanine to Leucine. The two peptides were synthesized and examined for binding to HLA-A3 by comparison with the HLA-A3 high affinity positive control epitope from Influenza NP265-273 (ILRGSVAHK) by the assembly assay (FIG. 2). The peptide concentration required for half maximal recovery of class I MHC molecules (C.sub.50 value) was 0.3 ?M for the Influenza NP265-273 (FIG. 2). The modified Rho1L2 peptide bound with intermediate affinity (C.sub.50=4), whereas the native peptide Rho1 only bound very weakly to HLA-A3 (C.sub.50>40).

[0280] Spontaneous T-Cell Responses Towards RhoC

[0281] We scrutinized PBL from HLA-A3.sup.+ MM and renal cell carcinoma (RCC) patients for the presence of specific T-cell responses against the modified Rho1L2 (RLGLQVRKNK) peptide by means of the ELISPOT IFN-? secretion assay. As depicted in FIG. 3, specific T-cell responses were present among PBL of 3 out of 10 MM patients. No responses were detected in either RCC patients or healthy controls (HD) against either Rho1L2 or Rho1.

[0282] T-Cell Antigen Specificity and Rho1/Rho1L2 Cross Reactivity

[0283] Having identified patients hosting responses against the Rho1L2 peptide, we used PBL from these cancer patients to generate CTL bulk cultures against this peptide in vitro. Subsequently, we in vitro stimulated PBL from such a patient with Rho1L2-pulsed autologous DC. After four in vitro restimulations, the peptide specificity was tested in standard .sup.51Cr release assays using T2-A3 cells without peptide or loaded with Rho1 or Rho1L2 as target cells (FIG. 4a). This assay revealed that the bulk cultures lysed both T2-A3 cells pulsed with Rho1L2 and Rho1 efficiently, whereas no cytotoxicity was observed against unpulsed T2-A3 cells.

[0284] Next, CTL clones were established from these specific T-cell cultures by limiting dilution. After a short expansion step, the specificity of the growing clones was analyzed in standard .sup.51Cr release assays. The data presented describe the results obtained for one growing clone (clone 9(*)). This clone effectively lysed T2-A3 cells pulsed with both the modified Rho1L2 and the native Rho1 peptide underlining that Rho1L2 specific T cells cross react with the native analogue peptide (FIG. 4b). To examine the HLA restriction of clone 9, we tested the effect of blocking HLA-class I by addition of the HLA specific mAb W6/32 and the HLA-A3 specific mAb GAP A3. Lysis could be completely blocked by incubation of the target cells with both antibodies (FIG. 4b).

[0285] Capacity of RhoC Specific T Cells to Kill Tumor Cells

[0286] First we examined the Rho1L2/Rho1 specific bulk cultures capacity to kill melanoma cells. To this end, the HLA-A3.sup.+ FM3 melanoma cells were killed with high efficacy in a HLA-restricted matter as lysis could be completely blocked by incubation of FM3 target cells with W6/32 (FIG. 5a).

[0287] Likewise clone 9 generated from the specific bulk culture was able to kill FM3 melanoma cells (FIG. 5b). The addition of cold (unlabeled) T2-A3 cells pulsed with the Rho1L2 peptide completely abrogated the killing of FM3 melanoma cells (FIG. 5b). Moreover, the RhoC specific CTL clone was able to lyse the HLA-A3.sup.+ melanoma cancer cell line FM9. As an additional control, we used the HLA-A3.sup.? melanoma cell line FM82 as target cells. No cytotoxicity was observed against this cell line.

[0288] As the expression of RhoC in metastatic cancer has been described in cancers of different origin we further examined the RhoC specific CTL clone capacity to kill other cancer cells than melanoma. Subsequently, the HLA-A3.sup.+ breast cancer cell line BT-20, the HLA-A3.sup.+ head and neck cancer cell line CRL-2095 and the HLA-A3.sup.+ colon cell line HT-29 were used as target cells. The RhoC specific CTL clone lysed all HLA-A3.sup.+ cell lines, although the colon cell line HT-29 only to a limited extent.

Example 2: Non-Limiting Example of Preparation of a Vaccine Composition and Non-Limiting Example of Administration of Vaccine

[0289] Peptide Vaccine

[0290] RhoC peptides can e.g. be synthesized e.g. at the UVA Biomolecular Core Facility with a free amide NH.sub.2 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.

[0291] In practical circumstances, patients will receive a vaccine comprising about 100 ?g of a class I HLA-restricted peptide or a class II HLA-restricted peptide or a combination of both. The patients are vaccinated with e.g. about 100 ?g of the class I HLA peptide in adjuvant alone, or are vaccinated with 100 ?g of the class II HLA peptide in adjuvant alone or are vaccinated with e.g. about 100 ?g of the HLA class I-restricted peptide plus 190 ?g of the class II-restricted peptide. The higher dose of the Class II peptide in the combination 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.

[0292] The above peptides, in 1-ml aqueous solution, can be administered either as a solution/suspension with about 100 ?g of QS-21, or as an emulsion with about 1 ml of Montanide ISA-51 adjuvant.

[0293] 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.

REFERENCE LIST

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