Therapy of cancer based on targeting adaptive, innate and/or regulatory component of the immune response

Abstract

The invention relates to a kit of parts, suitable for use in a therapy of cancer, wherein said kit comprises: (i) a recombinant protein comprising one or several polypeptides bearing one or several epitopes of one or several tumor-associated antigens, said polypeptides being inserted in the same or different permissive sites of an adenylate cyclase (CyaA) protein or of a fragment thereof, wherein said CyaA fragment retains the property of said adenylate cyclase protein to target Antigen Presenting Cells or a mixture of such recombinant proteins wherein at least one of said epitopes, or tumor associated antigens, or insertion sites of CyaA protein, or fragment of said CyaA protein is different between the various recombinant proteins in the mixture; and said kit of parts further comprises at least one of the following compounds; (ii) an agent, suitable for modulating a regulatory immune response in a patient ad optionally; (iii) an adjuvant component suitable for activating the innate immune response in a patient.

Claims

1. A method of reducing the growth of a tumor in a patient, the method comprising administering (i) a recombinant adenylate cyclase (CyaA) protein or fragment thereof, and (ii) a toll-like receptor (TLR) agonist to a patient having a tumor; wherein the CyaA protein or fragment thereof comprises at least one inserted polypeptide bearing at least one epitope of an antigen associated with the tumor; wherein the CyaA protein or fragment thereof retains the ability to target the CD11b/CD18 receptor on Antigen Presenting Cells; wherein the TLR agonist is selected from a TLR-9 agonist, a TLR-3 agonist, and a TLR-7 agonist; and wherein the recombinant adenylate cyclase (CyaA) protein or fragment thereof, and the TLR agonist are administered in amounts sufficient to reduce the growth of the tumor in the patient.

2. The method of claim 1, wherein the tumor is an infiltrating tumor.

3. The method of claim 1, wherein the tumor is a vascularized tumor.

4. The method of claim 1, wherein the tumor is a metastatic tumor.

5. The method of claim 1, wherein the tumor is a cervical tumor.

6. The method of claim 1, wherein the tumor is a tumor induced by HPV infection and the tumor associated antigen is a tumor associated antigen of an oncogenic HPV selected from HPV16, HPV18, HPV31, HPV33, HPV35, HPV45, HPV52 and HPV58.

Description

FIGURES

(1) FIG. 1. FIG. 1.1 (A to H): Abrogation of CyaA-E7 therapeutic effects in large tumor-bearing mice. C57BL/6 mice were inoculated on day 0 with 5?10.sup.5 TC-1 cells and then received PBS at day 4 (A) or were treated i.v. with 50 ?g of CyaA-E7 at day 4 (B), 7 (C), 11 (D), 18 (E), 25 (F) or 30 (G). Each curve represents the mean diameter of the tumor in a single mouse. The number of tumor free mice on day 100 versus the total number of animals included, the percentage of survival on day 100 and the p-value of the Likelihood Ratio test to compare the tumor growth versus the tumor growth in the PBS-treated group are shown. H) Kaplan-Meier plot of mice survival. Mice were sacrificed when tumor diameter reached 20 mm or whenever the sanitary status of the animals commanded. Pooled data of two independent experiments are shown;

(2) FIG. 1.2. CTX effect on tumor growth in RAG.sup.?/? mice. A, C57BL/6-RAG-1-.sup.?/? mice were injected on day 0 with 5?10.sup.5 TC-1 cells. On day 24, they received 2.5 mg CTX i.p. or 200 ?l PBS. The number of tumor-free mice on day 60 relating to the total number of animals included, the percent surviving on day 60 and the p-value determined by likelihood ratio test comparing tumor growth in the CTX-treated groups with tumor growth in the PBS-treated group are indicated for each set of experiments. B, Kaplan-Meier plot of mouse survival. The log-rank test was used to compare the group receiving CTX with the group treated with PBS (p=0.22). Mice were killed when tumor diameter reached 20 nm or when necessary due to the sanitary status of the animals. Pooled data of two independent experiments are shown.

(3) FIG. 2. FIG. 2.1 Combined tumor treatment with CyaA-E7 and different TLR-ligands. C57BL/6 mice were inoculated on day 0 with 5?10.sup.5 TC-1 cells. On day 25, they received either PBS (A), 50 ?g of CyaA-E7 alone (B), 30 ?g of CpG-B complexed with 60 ?g of DOTAP (C), or 25 ?g of PIC (D) or they were injected i.v with 50 ?g of CyaA-E7 combined with 50 ?g of R848 (E), or with 30 ?g of pU/DOTAP (F), of CpG-A/DOTAP (0) or of CpG-B/DOTAP (H), or with 25 ?g of PIC (I). Each curve represents the mean diameter of tumor in a single mouse. The number of tumor free mice on day 100 versus the total number of animals included, the percentage of survival on day 100 and the p-value of the Likelihood Ratio test to compare the tumor growth versus the tumor growth in the CyaA-E7-treated group or versus the tumor growth in the PBS-treated group are shown. J) Kaplan-Meier plot of mice survival. Mice were sacrificed when tumor diameter reached 20 mm or whenever the sanitary status of the animals commanded. Pooled data of two independent experiments are shown.

(4) FIG. 2.2. Evaluation of CTX doses. A, C57BL/6 mice were injected on day 0 with 5?10.sup.5 TC-1 cells. On day 24, they were left untreated or received 100 mg/kg or 300 mg/kg of CTX i.p. On day 25, mice were injected with PBS or 50 ?g CyaA-E7. The number of tumor-free mice on day 75 relative to the total number of animals included, the percent surviving on day 75 and the p-value determined by likelihood ratio test comparing tumor growth in the group treated with 300 mg/kg CTX with tumor growth in the group treated with 100 mg/kg CTX are indicated for each set of experiments. B, Kaplan-Meier plot of mouse survival. The log-rank test was used to compare the group receiving 300 mgkg CTX with the group receiving 100 mg/mg CTX (p=0.95). Mice were killed when tumor diameter reached 20 mm or when necessary due to the sanitary status of the animals. Pooled data of two independent experiments are shown.

(5) FIG. 3. Combined tumor treatment with CyaA-E7 and chemotherapy agents. C57BL/6 mice were inoculated on day 0 with 5?10.sup.5 TC-1 cells. On day 24, mice were left untreated (A, B) or received either 2.5 mg cyclophosphamide (CTX) i.p (C, D) or 125 ?g doxorubicin (DOX) i.t. (E). On day 25, mice received i.v either PBS (A, C) or 50 ?g of CyaA-E7 (B, D, E). The number of tumor free mice on day 100 versus the total number of animals included, the percentage of survival on day 100 and the p-value of the Likelihood Ratio test to compare the tumor growth versus the tumor growth in the CyaA-E7-treated group or versus the tumor growth in the PBS-treated group are shown. F) Kaplan-Meier plot of mice survival. Mice were sacrificed when tumor diameter reached 20 mm or whenever the sanitary status of the animals commanded. Pooled data of two independent experiments are shown.

(6) FIG. 4. Combined tumor treatment with CyaA-E7, CpG-B/DOTAP and CTX (tritherapy). C57BL/6 mice were inoculated on day 0 with 5?10.sup.5 TC-1 cells and on day 24, they were left untreated (A, B) or received 2.5 mg CTX i.p (C, D). On day 25, they were. injected i.v either with PBS (A), 50 ?g of CyaA-E7 (B), CpG-B/DOTAP (C) or with 50 ?g of CyaA-E7 and 30 ?g of CpG-B/DOTAP (D). The number of tumor free mice on day 100 versus the total number of animals included, the percentage of survival on day 100 and the p-value of the Likelihood Ratio test to compare the tumor growth versus the tumor growth in the CyaA-E7-treated group or versus the tumor growth in the PBS-treated group are shown. E) Kaplan-Meier plot of mice survival. Mice were sacrificed when tumor diameter reached 20 mm or whenever the sanitary status of the animals commanded. Pooled data of two independent experiments are shown.

(7) FIG. 5. FIG. 5.1 The therapeutic efficacy of tritherapy decreases in advanced tumors. C57BL/6 mice were inoculated on day 0 with 5?10.sup.5 TC-1 cells and were left untreated (A, B) or received 2.5 mg CTX i.p on day 24 (C), 29 (D) or 39 (E). On day 25, control mice were injected either with PBS (A) or with 50 ?g of CyaA-E7 alone (B). On day 25 (C), 30 (D) or 40 (E), groups of mice were treated i.v with 50 ?g of CyaA-E7 and 30 ?g CpG-B/DOTAP. The number of tumor free mice on day 100 versus the total number of animals included, the percentage of survival on day 100 and the p-value of the Likelihood Ratio test to compare the tumor growth versus the tumor growth in the tritherapy group are shown. F) Kaplan-Meier plot of mice survival. Mice were sacrificed when tumor diameter reached 25 mm or whenever the sanitary status of the animals commanded. Pooled data of two independent experiments are shown.

(8) FIG. 5.2. Antitumoral efficacy of a second administration of tritherapy. A, C57BL/6 mice were injected on day 0 with 5?10.sup.5 TC-1 cells. On day 39, they were left untreated or received 2.5 mg CTX i.p. On day 40, control mice were injected with PBS and mice treated with tritherapy were injected i.v with 50 ?g CyaA-E7 and 30 ?g CpG-B/DOTAP. A group of mice were also treated with CTX on day 54 and with CyaA-E7 and CpG-B/DOTAP on day 55. The number of tumor-free mice on day 90 after treatment relative to the total number of animals included, the percent surviving on day 90 after treatment and the p-value determined by likelihood ratio test comparing tumor growth in the groupe treated with tritherapy on day 40 with tumor growth in the PBS-treated group or the group treated with tritherapy on days 40 and 55 versus the group treated with tritherapy on day 40 are indicated for each set of experiments. B, Kaplan-Mier plot of mouse survival. The log-rank test was used to compare the group receiving tritherapy on day 40 with the group treated with PBS (p<0.0001) and the group treated on days 40 and 55 with the group treated on day 40 (p=0.0012). Mice were killed when tumor diameter reached 20 nm or when necessary due to the sanitary status of the animals. Pooled data of two independent experiments are shown.

(9) FIG. 6. Evaluation of the tritherapy in the EL4-E7 model. C57BL/6 mice were inoculated on day 0 with 4?10.sup.6 EL4-E7 cells and were left untreated (A) or received 2.5 mg CTX i.p on day 6 (B), 13 (C) or 20 (D). Mice were injected i.v either with PBS on day 25 (A) or 50 ?g of CyaA-E7 and 30 ?g CpG-B/DOTAP on day 7 (B), 14 (C) or 21 (D). The number of tumor free mice on day 100 versus the total number of animals included, the percentage of survival on day 100 are shown. E) Kaplan-Meier plot of mice survival. Mice were sacrificed when tumor diameter reached 25 mm or whenever the sanitary status of the animals commanded. Pooled data of two independent experiments are shown.

(10) FIG. 7. Changes in immune cells after tumor inoculation. C57BL/6 mice were inoculated on day 0 with 5?10.sup.5 TC-1 cells and three mice were sacrificed on days 0, 10, 25 and 40. Spleen, tumor draining lymph nodes and tumor were excised, ground to prepare the cell suspensions, stained and analyzed by flow cytometry. Pooled data of two experiments are shown (n=6-9 mice). Bars represent mean values and the error bars correspond to standard error of the mean. *<0.05 **<0.01. A) Percentage of CD4.sup.+CD25.sup.+FoxP3.sup.+ cells. B) Percentage of CD4.sup.+ cells that express CD25 and FoxP3. C) Percentage of CD11 c cells. D) Percentage of CD11b.sup.+ cells. E) Percentage of GR1.sup.+ cells. F) Percentage of CD11b+GR1.sup.+ positive cells.

(11) FIG. 8. Analysis of tumor specific immune response in control and tumor-bearing mice after different treatments. C57BL/6 mice were left untreated (A, C) or were inoculated on day 0 with 5?10.sup.5 TC-1 cells (B, D). Twenty-five days later, control and tumor-bearing mice were injected with either 50 ?g of control CyaA or of CyaA-E7, with PBS or with 30 ?g of CpG-B or pU/DOTAP, 25 ?g of PIC, 50 ?g of R848, 2.5 mg of CTX (24 h before) or with CTX and CpG-B/DOTAP. Percentage of CD8.sup.+ tetramer.sup.+ cells in control (A) and in tumor-bearing mice (B). At day 7 after treatment, mice were sacrificed and the percentage of tetramer positive cells was analyzed by flow cytometry. In vivo CTL in control (C) and in tumor-bearing mice (D). At day 7 after treatment, mice were i.v. injected with 5?10.sup.6 splenocytes loaded with CFSE.sup.high and E7.sub.49-57 peptide and with 5?10.sup.6 splenocytes loaded with CFSE.sup.low. Twenty-four hours later, spleens were removed and single-cell suspensions were analyzed by flow cytometry to determine the ratio of CFSE.sup.high to CFSE.sup.low cells. Pooled data of two experiments are shown (n=6). Bars represent mean values and the error bars correspond to standard error of the mean. Data was compared by ANOVA followed by Dunnett post test.

(12) FIG. 9. Changes in immune cells after tritherapy on day 25. C57BL/6 mice were inoculated on day 0 with 5?10.sup.5 TC-1 cells. On day 24, they received 2.5 mg CTX i.p and 24 h after, 50 ?g of CyaA-E7 and 30 ?g CpG-B/DOTAP. Groups of mice were sacrificed before treatment (day 0), 24 hour after CTX alone (day 1) or on days 4, 7 or 11 after tritherapy administration. Spleen, tumor draining lymph nodes and tumor were excised, ground to prepare the cell suspensions, stained and analyzed by flow cytometry. Pooled data of two experiments are shown (n=4-6 mice). Bars represent mean values and the error bars correspond to standard error of the mean. *<0.05 **<0.01. A) Percentage of CD4.sup.+ cells that express CD25 and FoxP3. B) Percentage of CD4.sup.+ cells that are CD25.sup.? and FoxP3.sup.+. C) Percentage of CD4.sup.+ cells that express FoxP3. D) Percentage of CD8.sup.+ cells that are tetramer.sup.+ and CD44+. E) Percentage of CD11c.sup.+ cells. F) Percentage of CD11 b.sup.+ cells. G) Percentage of GR1.sup.+ cells. H) Percentage of CD11b.sup.+GR1.sup.+ positive cells.

(13) FIG. 10. Immunostaining of CD3. C57BL/6 mice were injected on day 0 with 5?10.sup.5 TC-1 cells. On day 24 or 39, they received 2.5 mg CTX i.p. On day 25 or 40, mice were treated i.v with 50 ?g CyaA-E7 and 30 ?g CpG-B/DOTAP. Eleven days after treatment, mice were sacrificed. Non-treated tumor-bearing mice were sacrificed on days 25 and 40. Immunostaining of tumors for CD3 was done as described in Materials and Methods.

(14) FIG. 11. Phenotype of myeloid cells induced by tritherapy and by tumor growth. C57BL/6 mice were injected on day 0 with 5?10.sup.5 TC-1 cells. Tritherapy treated mice received on day 24, 2.5 mg CTX i.p and 24 h later, 50 ?g CyaA-E7 and 30 ?g CpG-B/DOTAP. PBS treated mice received 200 ?l of PBS on day 25. Control mice without tumors received similar treatments. All mice were sacrificed 11 days after treatment (36 days after tumor inoculation). Spleens were excised, ground to prepare the cell suspensions, stained and analyzed by flow cytometry. A representative experiment of two is shown (n=6 mice). Bars represent mean values and the error bars correspond to standard error of the mean. The percentages of CD11b.sup.+CD124.sup.+ and CD11b.sup.+Ly6G.sup.+ cells are presented in panels A and B, respectively.

EXAMPLES

(15) A tritherapy based on the simultaneous targeting of innate (by a TLR9 ligand), adaptive (by the CyaA-E7) and regulatory (by low dose of cyclophosphamide) component of the immune system was shown to induce full eradication of large-tumor in around 90% of treated animals.

Materials and Methods

(16) Mice and Tumors

(17) Specific pathogen-free 5-week-old female C57BL/6 mice were purchased from Charles River (L'Arbresle, France) and were kept in the Pasteur Institute animal facilities under pathogen-free conditions with water and food ad libitum. C57BL/6-RAG1.sup.?/? mice were obtained from the Jackson Laboratory, USA. Experiments involving animals were conducted according to the institutional guidelines for animal care.

(18) TC-1 cells expressing HPV16-E6 and HPV16-E7 proteins derived from primary mouse lung epithelial cells were obtained from the American Type Culture Collection (LGC Promochem, Molsheim, France) (14). EL4-E7 cells, a mouse lymphoma expressing HPV16-E7 (15). Cells were maintained in RPMI 1640 with GlutaMAX supplemented with 10% heat-inactivated fetal calf serum, 100 units/ml penicillin, 100 ?g/ml streptomycin, 0.4 mg/ml geneticin and 5?10.sup.?5 mol/l 2-mercaptoethanol (Life Technologies, Cergy-Pontoise, France).

(19) Tumor cells lines (5?10.sup.5 TC-1 cells or 4?10.sup.6 EL4-E7 cells) were inoculated into the shaved left back of C57BL/6 mice in 200 ?l volume of PBS. Tumor size, presented as the average of two perpendicular diameters (millimeters), was measured at regular intervals.

(20) Reagents

(21) The synthetic peptide E7.sub.49-57 (RAHYNIVTF, one-letter code for amino acid) corresponding to the HPV16-E7 H2-D.sup.b-restricted epitope (16) was purchased from NeoMPS (Strasbourg, France).

(22) The detoxified form of the adenylate cyclase of Bordetella pertussis carrying a truncated form of the E7 protein (CyaA-E7) and the control adenylate cyclase without any insert (CyaA) were purified as described in (5) and as summarized hereafter.

(23) CpG ODNs (Type A, CpG 2216: 5-GGGGGACGATCGTCGGGGGG-3; Type B, CpG 1826: 5-TCCATGACGTTCCTGACGTT-3) were synthesized by Proligo (Paris, France). Boldface nucleotides correspond to phosphorothioate backbone. Polyuridine (pU) was purchased from Sigma (Steinheim, Germany), R848 from PharmaTech (Shanghai, China) and polyinosinic-polycytidylic acid (PIC) from Invivogen (San Diego, Calif., USA). Thirty ?g of CpG ODNs or pU were diluted in 50 ?l of Optimen medium (Gibco, Grand Island, N.Y., USA) and mixed with 60 ?g of DOTAP (Roche, Mannheim, Germany) diluted in 100 ?l of Optimen. The rest of the reagents were diluted in PBS before injection. Cyclophosphamide (CTX) (Sigma, Steinheim, Germany) and doxorubicin (DOX) (Calbiochem, La Jolla, Calif., USA) were diluted respectively in PBS and in sterile water before injection. The different antigenic formulations and adjuvants were injected simultaneously except chemotherapeutic agents that were given 24 h before vaccine. Intravenous administrations were performed by retroorbital injection in a volume of 200 ?l, intratumoral administrations were done by injection in a volume of 50 ?l and subcutaneous administration were performed in 200 ?l.

(24) Construction and Purification of Recombinant B. pertussis Adenylate Cyclase Carrying HPV16-E7 Epitopes.

(25) Recombinant adenylate cyclase used were expressed in E. coli by using derivatives of plasmid pTRACE5 (5) which codes for an enzymatically inactive CyaA (Dadaglio G et al Int Immunol, 15: 1423-1430, 2003) (Gmira S. et al Res Microbiol, 152: 889-900, 2001). Plasmid pTRACE5 is an expression vector for an enzymatically inactive, and therefore non-cytotoxic, variant of B. pertussis CyaA. It also expresses B. pertussis cyaC gene that is required for the postranslational acylation of CyaA. This plasmid is a derivative of the previously described pTRACG plasmid (Gmira et al., 2001, Res. Mic. 152:889). It was obtained by insertion of the hexanucleotide CTGCAG in the EcoRV site located within the 5 part of the cyaA DNA sequence. This results in an in-frame insertion of the dipeptide Leu-Gln between Asp188 and Ile189 of CyaA within an essential part of the catalytic site (Guermonprez et al. 2000, Meth. Enzymol. 326:527). Plasmid pTRACE5 harbors a ColE1 origin of replication and an Ampcillin resistant marker. In this plasmid, the cyaC and the modified cyaA genes are placed in the same transcriptional unit under the control of the ? phage Pr promoter. The pTRCAG plasmid also encodes the thermosensitive ? repressor cl.sup.857 that strongly represses gene transcription at the ? Pr promoter at temperatures below 32? C.

(26) The E. coli strain XL1-Blue (Stratagene, La Jolla, Calif.) was used for all DNA manipulations that were performed according to standard protocols (Maniatis et al.).

(27) CyaA-E7.sub.49-57 contains a 9-amino acid long polypeptide sequence (RAHYNIVTF) inserted between codons 224 and 235 of CyaA. The expression plasmid for CyaA-E7.sub.49-57 was constructed as follows. Two synthetic oligonucleotides (MWG, Courtabceuf, France), BTP1 (5-CTA GCC GTG CCC ATT ACA ATA TTG TAA CCT TTG GTA C-3 coding strand) and BTP2 (5-CAA AGG TTA CAA TAT TGT AAT GGG CAC GG-3 non coding strand) were annealed and ligated into the pTRACE5 digested with Nhel and Kpnl. CyaA-E7.sub.Full contains the entire sequence of the HPV16-E7 protein, i.e., 98 amino acids, inserted at the same 224 position of the enzymatically inactive CyaA deposited at the CNCM (Paris, France) under n? CNCM I-3191 on Mar. 18, 2004. The DNA sequence encoding the E7 protein was amplified from HPV16 DNA (Seedorf K et al Virology 145: 181-185, 1985) using specific primers BTP3, (5-GGG CGC TAG CAT GCA TGG AGA TAC ACC TAC-3), and BTP4 (5-GGG CGG TAC CTG GTT TCT GAG AAC AGA TGG G-3). The resulting PCR product was digested by Nhel and Kpnl and ligated into pTRACE5 cleaved by Nhel and Kpnl. The Sspl site present in the annealed oligonucleotide as well as in the full sequence of HPV16-E7 allowed rapid identification of insertion mutants. CyaA-E7.sub.?30-42 contains the first 29 amino acid residues of HPV16-E7 inserted between codons 319 and 320 of CyaA as well as residues 43 to 98 of HPV16-E7 inserted between codons 224 and 235 of CyaA. The expression plasmid for CyaA-E7.sub.?30-42 was constructed in two steps deposited at the CNCM under n? CNCM I-3190 on Mar. 18, 2004. A first DNA fragment encoding (amino acid residues 1 to 29) of HPV16-E7 was PCR amplified using as a target DNA a synthetic HPV16-E7 gene (optimized for production in E. coli, designed by GTP Technology, Lab?ge, France), and primers BTP5 (5-GGG CAC CGG TAA ACG TAT GCA CGG CGA TAC TCC G-3), and BTP6 (5-CGT GAG CAT CTG GCT TTC ACT AGT ACG TTT GTT CAG CTG CTC GTA GCA-3). A second, DNA fragment encoding codons 320 to 372 of CyaA was PCR amplified using pTRACE5 as target DNA and primers BTP7 (5-GGG CAC TAG TGA AAG CCA GAT OCT CAC GCG CGG G-3), and BTP8 (5-AGT ACA TCC GGC GAG AAC-3). These two DNA fragments (that partly overlap) were purified and combined with primers BTP5 and BTP8 in a third PCR to amplify a 294 bp long DNA fragment. This fragment was digested by Agel and BstBl and inserted between the corresponding sites of pTRACE5 to yield plasmid pTRACE5-E7.sub.1-29. Then, a DNA fragment encoding the amino acid residues 43 to 98 of HPV16-E7 was PCR amplified using the synthetic HPV16-E7 gene as target DNA and primers BTP9 (5-GGG CGC TAG CGG TCA AGC AGA ACC GGA C-3) and BTP10 (5-GGG CGG TAC CAG GTT TTT GAG AGC AAA TCG GAC AAA CAA TCC CCA GAG TAC CCA TC-3). The purified PCR fragment was digested by Nhel and Kpnl and ligated into plasmid pTRACE5-E7.sub.1-29 digested by the same restriction enzymes.

(28) All recombinant adenylate cyclase were produced in the Escherichia coli strain BLR (Novagen, Madison, Wis.) as described previously (26). The recombinant proteins were purified close to homogeneity (FIG. 1B) from inclusion bodies by a two-step procedure that includes DEAE-Sepharose and phenyl-Sepharose chromatography, as described previously (26). An additional washing step with 60% isopropanol in 20 mM Hepes-Na, pH7.5, was added to the phenyl-Sepharose chromatography in order to eliminate most of the contaminating LPS. LPS contents were determined using the kit QCL-1000 (Biowhittaker, Walkersville, Md.). Purified recombinant proteins were analyzed by SDS-gel analysis. Protein concentrations were determined spectrophotometrically from the absorption at 280 nm using a molecular extinction coefficient of 142,000 M.sup.?1.Math.cm.sup.?1.

(29) Flow Cytometric Analysis

(30) To analysis cells changes during tumor growth or after therapy, solid tumors, spleens and inguinal lymph nodes were excised, ground to prepare cell suspensions, and subsequently stained.

(31) Monoclonal antibodies used for staining were FITC-conjugated anti-CD11b, anti-CD4, anti-CD69, anti-CD8; PE-conjugated anti-GR1+, anti-CD124 anti-NK1.1; APC-conjugated anti-CD25, anti-CD44, anti-CD69, anti-CD8, anti-CD11c (all from PharMingen, Erembodegem, Belgium). PE-conjugated H-2D.sup.b/E7.sub.49-57 tetramers were obtained from Beckman Coulter (Fullerton, Calif., USA). T regulatory cells staining was done using Mouse Regulatory Staining kit (E-biosciences, San Diego, Calif., USA).

(32) FACScalibur (Becton Dickinson, Franklin Lakes, N.J., USA) was used for flow cytometry and events were analyzed with CELLQuest software (Becton Dickinson, Franklin Lakes, N.J., USA).

(33) In Vivo Killing Assay

(34) Na?ve spleen cells were pulsed for 30 min with 9 ?M E7.sub.49-57 peptide at 37? C. After extensive washing, cells were labelled with 2.5 ?M CFSE (CFSE.sup.high) (Molecular Probes). Control non-peptide-treated splenocytes were labelled with 0.25 ?M CFSE (CFSE.sup.low). CFSE.sup.high and CFSE.sup.low cells were mixed in a 1:1 ratio and 10.sup.7 cells were injected i.v. into na?ve or immunized animals. Twenty-four hours later, spleens were removed and single-cells suspensions were analyzed by flow cytometry to determine the ratio of CFSE.sup.high to CFSE.sup.low cells. The percentage of specific lysis was calculated as follow: percent-specific lysis=100?(100?(% CFSE.sup.high immunized/% CFSE.sup.low immunized)/(% CFSE.sup.high control/CFSE.sup.low control)).

(35) Anti-CD3 Immunohistochemistry

(36) Tumor samples were fixed for formalin and embedded in paraffin. Three-micrometer sections were microwaved for 10 minutes in Tris-EDTA (0.001 M pH 9) for antigen retrieval and endogenous peroxidase was quenched with Peroxidase Blocking Reagent (Dako, Carpintero, Calif.). Tissue sections were incubated overnight at 4? C. with affinity purified anti-human CD3 (Lab Vision Corporation, Fremont, Calif.) diluted 1:300 in tris-buffered saline. The peroxidase activity was revealed using anti-rabbit EnVision System (Dako) and DAB+ Substrate Chromogen System (Dako). Finally, sections were counterstained with methyl green.

(37) Statistical Analysis

(38) Tumor growth data were analyzed by non-linear mixed effect models using Monolix software (http://www.math.u-psud.fr/?lavielle/monolix/). Mean diameters of tumors over time were fitted using the model described in (17) and treatments were compared using the Likelihood Ratio test. Kaplan-Meier plots were used to analyzed survival, and a log rank test was used to examine statistical significance of differences in the survival curves using Prism software (GraphPad Software, Inc. San Diego, USA). Data from in vivo CTL and tetramer staining were compared by ANOVA followed by Dunnett posttest. p values less than 0.05 were considered to be statistically significant.

Results

(39) CyaA-E7 Therapeutic Effect is Abrogated in Large Tumor-Bearing Mice

(40) We have previously shown that a single injection of the CyaA recombinant protein carrying the HPV E7 antigen (CyaA-E7), 10 days after the graft of 5?10.sup.4 TC-1 tumor cells, induces a full regression of tumor growth and leads to the survival of all treated mice (5). To determine if such therapeutic vaccination is still efficient at later stage of tumor growth, mice were injected with 5?10.sup.5 TC-1 cells and then treated with a single i.v. injection of 50 ?g of CyaA-E7 at various time points. In mice treated 4 days after the injection of TC-1 cells, after an initial phase of growth, tumors were rejected in all animals. Tumor relapse at day 60 was however observed in one mouse (FIG. 1.1 B). A progressive decrease of the CyaA-E7 antitumor efficacy was observed in mice, which received a delayed treatment. Thus, only 20% of mice treated by CyaA-E7 25 days after injection of TC-1 cells were protected from tumor growth. However, the delayed in tumor growth and the increased survival were still significant as compared to PBS treated mice (FIG. 1.1 F, H). In mice treated 30 days after tumor inoculation, we did not observed any significant difference in the tumor growth or the survival as compared to the PBS-treated group (FIG. 1.1 G, H). Thus, when injected one month after the tumor graft, CyaA-E7 lost its capacity to induce therapeutic antitumor responses.

(41) TLR-Ligands Increase the Therapeutic Efficacy of CyaA-E7 on Advanced Tumors

(42) We then tested whether adjuvants can restore the anti-tumor therapeutic responses induced by CyaA-E7 administration in mice suffering from advanced tumors. For these experiments, mice were treated 25 days after tumor cells injection since it was the latter time point where we could detect the CyaA-E7 therapeutic activity.

(43) Toll-like receptor (TLR) ligands have recently received great attention due to their ability of trigger DC maturation in vivo. We selected for these experiments five synthetic TLR ligands: a TLR-3 ligand, polyinosinic-polycytidylic acid (PIC), two TLR-7 ligands, polyuridine (pU) and R848 and two TLR-9 ligands, CpG-A and CpG-B. pU and CpGs were complexed in DOTAP to protect them from degradation and facilitate their uptake.

(44) A significant increase in tumor regression was observed in mice treated 25 days after TC-1 cells injection with CyaA-E7 and with pU or CpGs or PIC but not with the R848. The survival percentage ranged from 41% with pU and PIC to 50% with CpG-B and 58% with CpG-A (FIGS. 2.1 F, G, H, I and J). After injection of CyaA-E7 with these TLR agonists at day 25, the tumors continued to grow for five to seven days, reaching diameters up to 14 mm. Then, the size of most of the tumors started to decrease, reflecting a successful induction of a therapeutic immune response. This phase could last for 25 days, ultimately leading to the total cure of the tumor. However, in some mice, a tumor relapse was observed before its total eradication.

(45) The therapeutic effect of two of these TLR ligands (PIC and CpG-B) administered alone without the CyaA-E7 vaccine was tested to study whether these TLR ligands could enhance the immune response primed by the tumor. However, none of these TLR ligands administered alone had any effect on the tumor growth (FIGS. 2.1 C, D and J). Therefore, E7 delivery by the CyaA is required to achieve regression of these large tumors.

(46) We then analyzed if chemotherapeutic agents could also restore the therapeutic activity of CyaA-E7 when administered to large-tumor bearing mice. We selected two drugs for this purpose: cyclophosphamide (CTX), an alkylating agent of the nitrogen mustard type that have been shown for a long time to enhance the efficacy of antitumor vaccines (20) (21); and doxorubicin (DOX), an intercalating agent that inhibit the action of the enzyme topoisomerase II and induce an immunogenic cell death that can control the growth of tumors after intratumoral injection (22). In our model of large tumors, DOX did not enhance the effect of CyaA-E7 when given either intra-tumorally (FIG. 3 E, F), or intravenously. In contrast, low dose of CTX injected 24 hours before the CyaA-E7 vaccine led to a delayed growth of all tumors and 58% of mice totally eradicated their tumors. Unlike the TLR ligands, the administration of CTX alone significantly delayed tumor growth but this effect was transient and was followed by tumor relapse (FIG. 3 C). This tumor growth delay was not observed in RAG1.sup.?/? mice (FIG. 1.2), strongly suggesting that this effect was mediated by T cells. Finally, the antitumor efficacy of the combined administration of CTX and CyaA-E7 was not improved by a three-fold CTX dose increase (FIG. 2.2). CTX at 100 mg/kg displayed maximal antitumor efficacy, reducing the possible side-effects associated to this combined therapy.

(47) Eradication of Large Tumors by a Tritherapeutic Treatment Combining Chemotherapy, a TLR-9 Ligand and an Antitumor Vaccine

(48) Next, we hypothesized that since CyaA-E7, CTX and CpG have different mechanisms of action, they could have a synergic effect. Thus, we administered CTX to 24 days tumor-bearing mice and 24 hour later, mice were injected with CyaA-E7 and CpG-B in DOTAP. This tritherapy showed a strong antitumor efficacy with a survival rate of 87.5%. Only two out of the sixteen mice receiving the tritherapy did not eradicate the tumor (FIG. 4 D, E). The CyaA-E7 vaccine was essential for this therapeutic effect since the administration of CTX and CpG-B/DOTAP was unable to induce the regression of the tumors, although this treatment induced a significant delay in the tumor growth as previously observed after the administration of CTX alone (FIG. 4 C, E).

(49) To determine the potential of the tritherapy to cure very large tumors, we administered this treatment to tumor-bearing mice 30 or 40 days after tumor inoculation. When the tritherapy was administered on day 30, there was a decrease in the efficacy but still 41.7% of the animals were able to eradicate the tumor (FIG. 5.1 D, F). Even on day 40, when the tumor diameter ranged between 15 and 20 mm, two mice definitively eradicated the tumor. Remarkably, in all treated mice, tumors started to regress reflecting the induction of an effective immune response that last for around 20 days and was then followed by tumor relapse (FIG. 5.1 E, F). However, the regression phase could be prolonged by a second administration of the tritherapy at day 55, leading to tumor eradication in 43% of treated mice (FIG. 5.2).

(50) To expand these results to another model of HPV-induced tumor, we injected mice subcutaneously with EL4 cells transfected with the E7 protein (EL4-E7 cells) (15). The growth of these cells was faster than the TC-1 growth and at day 14, EL4-E7 tumors reached a similar size to TC-1 tumors at day 30 (FIG. 6 A, E). Seven, 14 or 21 days after tumor cells injection, mice were treated by the tritherapy. As shown in FIG. 6, the tritherapy was effective to treat tumor until 14 days after inoculation but failed to eradicate the tumor 21 days after the inoculation. Similarly to the TC-1 model, all tumors started to regress but finally most of them relapsed.

(51) Changes in Immune Cells During Tumor Development

(52) We then investigated the mechanisms underlying the loss of therapeutic activity of CyaA-E7 in large-tumor bearing mice. In particular, we investigated whether expansion of regulatory T cells (Treg) and/or myeloid suppressor cells (MDSCs) could be responsible for such a decrease of CyaA-E7 activity. The percentage of these cell populations in the spleen, inguinal draining lymph nodes (DLN) and tumor was thus analyzed at different time points after the injection of TC-1 cells (FIG. 7).

(53) A high percentage of CD4.sup.+CD25.sup.+FoxP3.sup.+ cells was found infiltrating the tumor at all time points analyzed, indicating a specific recruitment and/or expansion of these cells. This population also increased progressively in spleens and DLNs of tumor-bearing mice. This increase was faster in the DLN and by day 10, the difference with the inguinal lymph node before tumor inoculation was significant (10.8 vs 8.7%; p<0.05) and this difference further increased on day 25 (12.6 vs 8.7% p<0.01) and on day 40 (16.1 vs 8.7%; p<0.01). In the spleen, a significant difference was only detectable on day 40 (20.7 vs 11.0%; p<0.01).

(54) A myeloid derived CD11b.sup.+GR1.sup.+ cell population increased progressively in the spleen of tumor-bearing animals and on day 40, a significant higher percentage of these cells was found in the spleens of tumor-bearing mice as compared to control mice (11.6 vs 1.0%; p<0.01). This population expressed the IL-4 receptor alpha chain (CD124), recently associated with immunosuppressive MDSCs (FIG. 11A) (36).

(55) In conclusion, a marked expansion of Treg was observed inside the tumor and also systematically, although less marked in spleen and lymph nodes. The expansion of myeloid-derived suppressor cells CD11b.sup.+GR1.sup.+ cells was only detectable in spleens of late stage tumor-bearing mice.

(56) Analysis of Anti-Tumor Immune Responses Induced by the Tritherapy in Control and Tumor-Bearing Mice

(57) To study the immune responses induced by the different treatments, we analyzed the CD8.sup.+ T cell response specific for the E7.sub.49-57 CD8.sup.+ T cell epitope by determining the percentage of tetramer.sup.+ cells and the in vivo cytolytic activity generated in control and tumor-bearing mice. In control mice, the only treatment that significantly enhanced the number of tetramer.sup.+ cells in comparison with the CyaA-E7 alone was the immunization with CpG-B/DOTAP and CyaA-E7 independent of the administration of CTX. Surprisingly, the tritherapy was less effective despite its stronger antitumor activity (FIG. 8 A). In contrast, the in vivo CTL assay revealed a significant increase of cytolytic activity following immunization with both CyaA-E7+CpG-B/DOTAP or with the tritherapy.

(58) Mice grafted with TC-1 tumor cells 25 days before immunisation showed a lower increase in tetramer.sup.+ cells than control immunized mice and none of the tested combinations was significantly different from immunization with CyaA-E7 alone (FIG. 8 B). In contrast, a significantly stronger in vivo CTL activity was observed in mice immunized with CyaA-E7+CpG-B/DOTAP in the absence or presence of CTX (p<0.05) than with CyaA-E7 alone (FIG. 8 D). The administration of CTX with CyaA-E7 did not enhance the immune response induced by CyaA-E7, despite its strong antitumor activity.

(59) No tetramer.sup.+ cells were detected in tumor-bearing mice 40 days after TC-1 injection. The in vivo cytolytic activity was low but still detected (FIG. 8E-F). Thus, the expansion of myeloid-derived suppressor cells and Treg associated with TC-1 growth gradually impairs the antitumor immune response induced by tritherapy. Moreover, in agreement with the antitumor data, in tumor-bearing mice, the tritherapy induced the highest in vivo cytotoxic activity of all combinations tested.

(60) Changes in Immune Cells after Tritherapy on Day 25.

(61) Next we evaluated the changes in the immune system induced by the tritherapy while the tumor is rejected. Specifically, we analyzed the evolution of the T regulatory cells, myeloid suppressor cells, tumor-specific CD8.sup.+ T cells and NK cells.

(62) As expected, the administration of cyclophosphamide induced a transient lymphodepletion in spleen and lymph node that reverted by day 7. The CD25.sup.+FoxP3.sup.+CD4.sup.+ population showed a slightly more pronounced decrease than total CD4.sup.+ cells, that did not reach statistical significance in the spleen and draining lymph node (FIG. 9). Interestingly enough, tumor-associated regulatory T cells experienced a significant decrease. The percentage of CD25.sup.+FoxP3.sup.+/CD4.sup.+ cells dropped from 50% in non-treated mice to 30% on day 4 and 7 after treatment (FIG. 9 A). Surprisingly, 11 days after the tritherapy, an increase of CD25.sup.?FoxP3.sup.+ cells was observed in all organs analyzed, leading to a significant increase in the percentage of FoxP3.sup.+ CD4.sup.+ T cells in the spleen and draining lymph node and to a rapid recovery of regulatory T cells percentage in the tumors (FIG. 9 B, C). In parallel to these expansion of FoxP3.sup.+ cells, a significant increase of tumor specific CD8.sup.+ T cells was detected in the spleen, DLN and tumors, where the percentage of tetramer positive cells were especially high (FIG. 9 D). Other evidences of the immune response onset were detected as, for instance, a transient activation of NK cells in spleen and DLN, further indicating the onset of the immune response. Anti-CD3 immunohistochemistry revealed a low T cell infiltration in non-treated tumors that increased dramatically 11 days after tritherapy. Tumors treated on day 40 presented a reduced T cell infiltration as compared to tumor treated on day 25 (FIG. 10).

(63) We found a much higher percentage of CD11b.sup.+GR1.sup.+ cells (up to 30% of the cells) in the spleen of mice receiving tritherapy than in control mice. Though not as pronounced, there was also a higher percentage of CD11b.sup.+GR1.sup.+ cells in the DLN in mice receiving tritherapy than in controls (FIG. 9F, G, H). This population expressed the specific granulocyte marker Ly6G but not the IL-4 receptor alpha chain. These markers identified this population as nonsuppressive granulocytes/neutrophils (FIG. 11). Finally, a strong increase in CD11b+GR1+ myeloid suppressor cells was observed in the spleen and more moderately in the DLN (FIG. 9 F, G, H).

DISCUSSION

(64) The aim of this work was to test combination treatments that could enhance the antitumor activity displayed by the recently developed CyaA-E7 therapeutic vaccine. This vaccine has the remarkable property to induce antitumor immunity in absence of any adjuvant but its effectiveness is abolished as the tumor progresses (FIG. 1). Our results point out that in order to display maximal activity of this vaccine, the innate immune system has to be activated and, simultaneously, the regulatory component of the immune system has to be downregulated.

(65) The activation of the innate immune system can be readily performed by synthetic TLR ligands. Nevertheless, not all the ligands performed well and we could detect differences even among ligands of the same TLR as in the case of the TLR-7 ligands, R848 and pU complexed with DOTAP. An explanation to this observation could be the different pharmacokinetic profile of a small molecule, R848, versus a liposomal formulation of an oligonucleotide, underscoring a fundamental dimension for the development of new adjuvants based of the TLR triggering. The best results were obtained with phosphorothioate CpG oligonuclotides complexed with DOTAP, reaching 50 to 60% of complete eradication of tumors treated 25 days after inoculation. In the rest of the tumors, a delay in the growth could be achieved but finally regrew. In this advance tumor model, the administration of the TLR ligands alone did not have any effect in the tumor growth. Therefore, the simultaneous administration of the CyaA-E7 was required to obtain a therapeutic effect.

(66) A different approach tested was the synergy of immunotherapy and chemotherapy. We chose two widely used drugs, named cyclophosphamide and doxorubicin, that have been shown to enhance antitumor vaccine efficacy (21). Moreover, intratumoral injections of doxorubicin have been recently shown to induce an immunogenic cells death that lead to tumor immune rejection (22). However, in our model only cyclophosphamide had an adjuvant activity when combined with CyaA-E7, inducing the tumor regression in around 60% of treated mice. In contrast, doxorubicin had no effect when used either by intratumoral route or intravenously. A single injection of low dose of cyclophosphamide could transiently control tumor growth but was not able to induce the complete eradication. As in the case of the TLR ligands, the administration of CyaA-E7 was indispensable. Low dose of cyclophosphamide has been recently shown to decrease the number and inactivate Treg (13,23,24). In our model, a high percentage of CD25+FoxP3+ cells versus CD4+ cells was detected in tumor-infiltrated lymphocytes, and this percentage increased in the draining lymph node and spleen as tumor progress. Four days after the administration of cyclophosphamide, a slight decrease of these cells was detected in the spleen and draining lymph node, but the strongest decrease was found in the tumor-infiltrated lymphocyte, where the percentage of CD25+FoxP3+/CD4+ decreased from around 50% to below 30%, reflecting a higher susceptibility to the cyclophosphamide of the tumor-associated T regulatory cells (TA-Treg). The alteration of the TA-Treg homeostasis may be a key step in the disturbance of the tumor stroma cell interactions following CTX treatment (25). CTX treatment was indeed reported to modify the functional profile of tumor-infiltrating T cells, as shown by their inability to produce IFN-? and to change IL-10-producing tumor-infiltrating macrophages into IFN-? producers. This disturbance, although not enough to induce complete regression in our model, pave the way for the effector cells induced by the CyaA-E7. Therefore, it could be anticipated that treatments that enhance the effector cells induced by CyaA-E7, would synergyze with the low dose cyclophosphamide. Indeed, the maximal response was achieved by the combination of low dose cyclophosphamide, CpG-B/DOTAP and the vaccination with CyaA-E7. The combination of CpG, CTX and a vaccine in the form of DC-derived exosomes, have been tested previously by Taieb et al. (23) for the treatment of bulky B16A2/gp100 tumors. In this model however, CpG did not enhance antitumor efficacy of DC-derived exosomes given in combination with CTX. In contrast to these results, we observed strong synergy between the three components, CyaA-E7, CpG-B/DOTAP and CTX, of tritherapy. This tritherapy was able to eradicate large, established tumor in 87% of animals treated 25 days after inoculation with tumors of a mean diameter of 8 mm. The efficacy was reduced when treating larger tumors but even in mice treated 40 days after inoculation, when the tumor mean diameter reached around 2 cm, all the tumor exhibit a transient size reduction, reflecting the induction of an effective immune response. Worthy of note, 2 out of 12 mice bearing these huge tumors completely eradicated them. Moreover, a second administration of the tritherapy 15 days after the first treatment lead to tumor eradication in 43% of treated mice. These results were further corroborated using a different tumor model, based on the inoculation of EL4 cells transfected with the E7 protein.

(67) The analysis of the immune responses induced by the tritherapy showed that the CpG/DOTAP treatment is the only adjuvant tested that can increase significantly the E7-specific CD8.sup.+ T cell response as compared to the CyaA-E7 alone. In tumor bearing animals this difference was reduced and, although that treatment still increased the percentage of tetramer.sup.+ cells, the difference was not statistically significant. The administration of CTX with the CpG/DOTAP and the vaccine reduced the E7-specific CD8.sup.+ T cell response, probably as a consequence of the systemic lymphodepletion. In contrast, the in vivo CTL activity remained at similar level that those of CpG/DOTAP or was even higher in tumor-bearing mice. Thus, the CTLs induced by the tritherapy displayed a higher killing activity in a per cell basis. The administration of CTX slightly increased the immune response in tumor-bearing animals but not in control mice. This increase was lower that that induced by CpG/DOTAP. Therefore, both treatments can enhance the antitumor activity of CyaA-E7 but clearly by different mechanism of action.

(68) An expansion of Treg after vaccination of tumor bearing host has been reported both in mice (26,27) and in human (28), underscoring the potential that therapeutic cancer vaccines could deepen tumor-specific T-cell tolerance. In our tumor model, in parallel to the strong antitumor immunity induced by the tritherapy that lead to the eradication tumor in around 90% of treated mice, an expansion of FoxP3+CD4+ was detected in the spleen and draining lymph node. In addition, the composition of the FoxP3+CD4+ compartment regarding the CD25 marker in the spleen, draining lymph node and tumor is altered after tritherapy, showing an increase in the CD25? cells. This may reflect the expansion of tumor induced regulatory CD25? CD4+ as previously reported in (27). It has been demonstrated that, on a cell per cell basis, CD4.sup.+CD25.sup.?FoxP3.sup.+ cells are as regulatory as CD4.sup.+CD25.sup.+FoxP3.sup.+ cells (31). We observed substantial expansion of neutrophils in the spleen and draining lymph node following tritherapy. As key component of the inflammatory response, neutrophils made important contributions to the recruitment, activation and programming of antigen presenting cells and contribute to the quality of the secondary immune response (33, 34). However, neutrophils expansion may detrimentally affect the on-going antitumor response by the secretion of reactive oxygen species (35).

(69) Moreover, in spleen and draining lymph node, there were a huge expansion of CD11b+GR1+ myeloid suppressor cells, that may exert a detrimental impact on the on-going antitumor response directly by the secretion of NO (29) or indirectly by the simulation of T regulatory cells (30). Interestingly, these cells increased also in the spleen at the late stage of TC-1 growth where the tritherapy were not able to induce complete tumor regression.

(70) Thus, the cyclophosphamide only controls the regulatory component of immune system during the priming phase of the vaccine and the expansion of regulatory systems during the effector phase may be responsible, in part, of the abrogation of the effector immune response in mice immunized 40 days after tumor inoculation. Thus, new strategies to block the induction or to promote the inactivation of the regulatory component of the immune systems during the effector phase of the immune response should be evaluated, tipping the balance in favor of the effector function.

REFERENCES

(71) (1) Guermonprez P, Ladant D, Karimova G, Ullmann A, Leclerc C. Direct delivery of the Bordetella pertussis adenylate cyclase toxin to the MHC class I antigen presentation pathway. J Immunol 1999; 162:1910-6. (2) Ladant D, Ullmann A. Bordatella pertussis adenylate cyclase: a toxin with multiple talents. Trends Microbiol 1999; 7:172-6. (3) Schlecht G, Loucka J, Najar H, Sebo P, Leclerc C. Antigen targeting to CD11 b allows efficient presentation of CD4+ and CD8+ T cell epitopes and in vivo Th1-polarized T cell priming. J Immunol 2004; 173:6089-97. (4) Fayolle C, Ladant D, Karimova G, Ullmann A, Leclerc C. Therapy of murine tumors with recombinant Bordetella pertussis adenylate cyclase carrying a cytotoxic T cell epitope. J Immunol 1999; 162:4157-62. (5) Preville X, Ladant D, Timmerman B, Leclerc C. Eradication of established tumors by vaccination with recombinant Bordetella pertussis adenylate cyclase carrying the human papillomavirus 16 E7 oncoprotein. Cancer Res 2005; 65:641-9. (6) Lowndes C M. Vaccines for cervical cancer. Epidemiol Infect 2006; 134:1-12. (7) Bosch F X, Lorincz A, Munoz N, Meijer C J, Shah K V. The causal relation between human papillomavirus and cervical cancer. J Clin Pathol 2002; 55:244-65. (8) Munger K, Phelps W C, Bubb V, Howley P M, Schlegel R. The E6 and E7 genes of the human papillomavirus type 16 together are necessary and sufficient for transformation of primary human keratinocytes. J Virol 1989; 63:4417-21. (9) von Knebel Doeberitz M, Rittmuller C, zur Hausen H, Durst M. Inhibition of tumorigenicity of cervical cancer cells in nude mice by HPV E6-E7 anti-sense RNA. Int J Cancer 1992; 51:831-4. (10) Hopfl R, Heim K, Christensen N, Zumbach K, Wieland U, Volgger B, et al. Spontaneous regression of CIN and delayed-type hypersensitivity to HPV-16 oncoprotein E7. Lancet 2000; 356:1985-6. (11) Akira S, Takeda K. Toll-like receptor signalling. Nat Rev Immunol 2004; 4:499-511. (12) Yang S, Haluska F G. Treatment of melanoma with 5-fluorouracil or dacarbazine in vitro sensitizes cells to antigen-specific CTL lysis through perforin/granzyme- and Fas-mediated pathways. J Immunol 2004; 172:4599-608. (13) Lutsiak M E, Semnani R T, De Pascalis R, Kashmiri S V, Schlom J, Sabzevari H. Inhibition of CD4(+)25+ T regulatory cell function implicated in enhanced immune response by low-dose cyclophosphamide. Blood 2005; 105:2862-8. (14) Lin K Y, Guarnieri F G, Staveley-O'Carroll K F, Levitsky H I, August J T, Pardoll D M, et al. Treatment of established tumors with a novel vaccine that enhances major histocompatibility class I I presentation of tumor antigen. Cancer Res 1996; 56:21-6. (15) Tindle R W, Croft S, Herd K, Malcolm K, Geczy A F, Stewart T, et al. A vaccine conjugate of ISCAR immunocarrier and peptide epitopes of the E7 cervical cancer-associated protein of human papillomavirus type 16 elicits specific Th1- and Th2-type responses in immunized mice in the absence of oil-based adjuvants. Clin Exp Immunol 1995; 101:265-71. (16) Feltkamp M C, Smits H L, Vierboom M P, Minnaar R P, de Jongh B M, Drijfhout J W, et al. Vaccination with cytotoxic T lymphocyte epitope-containing peptide protects against a tumor induced by human papillomavirus type 16-transformed cells. Eur J Immunol 1993; 23:2242-9. (17) El Halimi R, Oca?a J, Ruiz de Villa M, Escrich E, Solanas M. Modelling tumor growth data using a non-linear mixed-effects model. InterStat 2003; http://interstat.statjournals.net/. (18) Li Q, Normolle D P, Sayre D M, Zeng X, Sun R, Jiang G, et al. Immunological effects of BCG as an adjuvant in autologous tumor vaccines. Clin Immunol 2000; 94:64-72. (19) Facciabene A, Aurisicchio L, La Monica N. Baculovirus vectors elicit antigen-specific immune responses in mice. J Virol 2004; 78:8663-72. (20) Berd D, Maguire H C, Jr., Mastrangelo M J. Induction of cell-mediated immunity to autologous melanoma cells and regression of metastases after treatment with a melanoma cell vaccine preceded by cyclophosphamide. Cancer Res 1986; 46:2572-7. (21) Machiels J P, Reilly R T, Emens L A, Ercolini A M, Lei R Y, Weintraub D, et al. Cyclophosphamide, doxorubicin, and paclitaxel enhance the antitumor immune response of granulocyte/macrophage-colony stimulating factor-secreting whole-cell vaccines in HER-2/neu tolerized mice. Cancer Res 2001; 61:3689-97. (22) Casares N, Pequignot M O, Tesniere A, Ghiringhelli F, Roux S, Chaput N, et al. Caspase-dependent immunogenicity of doxorubicin-induced tumor cell death. J Exp Med 2005; 202:1691-701. (23) Taieb J, Chaput N, Schartz N, Roux S, Novault S, Menard C, et al. Chemoimmunotherapy of tumors: cyclophosphamide synergizes with exosome based vaccines. J Immunol 2006; 176:2722-9. (24) Ercolini A M, Ladle B H, Manning E A, Pfannenstiel L W, Armstrong T D, Machiels J P, et al. Recruitment of latent pools of high-avidity CD8(+) T cells to the antitumor immune response. J Exp Med 2005; 201:1591-602. (25) Ibe S, Qin Z, Schuler T, Preiss S, Blankenstein T. Tumor rejection by disturbing tumor stroma cell interactions. J Exp Med 2001; 194:1549-59. (26) Maksimow M, Miiluniemi M, Marttila-Ichihara F, Jalkanen S, Hanninen A. Antigen targeting to endosomal pathway in dendritic cell vaccination activates regulatory T cells and attenuates tumor-immunity. Blood 2006. (27) Zhou G, Drake C G, Levitsky H I. Amplification of tumor-specific regulatory T cells following therapeutic cancer vaccines. Blood 2006; 107:628-36. (28) Banerjee D, Dhodapkar M V, Matayeva E, Steinman R M, Dhodapkar K. Expansion of FOXP3high regulatory T cells by human dendritic cells (DCs) in vitro and after D C injection of cytokine matured DCs in myeloma patients. Blood 2006. (29) Angulo I, de las Heras F G, Garcia-Bustos J F, Gargallo D, Munoz-Fernandez M A, Fresno M. Nitric oxide-producing CD11b(+)Ly-6G(Gr-1)(+)CD31(E R-M P12)(+) cells in the spleen of cyclophosphamide-treated mice: implications for T-cell responses in immunosuppressed mice. Blood 2000; 95:212-20. (30) Yang R, Cai Z, Zhang Y, Yutzy I V W, Roby K, Roden R. CD80 in Immune Suppression by Mouse Ovarian Carcinoma-Associated Gr-1+CD11b+ Myeloid Cells. Cancer Res 2006; 66:6807-15. (31) Nava-Parada, P. Forni G, Knutson K L, Pease L R, Cells E. Peptide vaccine given with a Toll-like receptor agonist is effective for the treatment and prevention of spontaneous breast tumors. Cancer Res 2007; 67(3):1326-34. (32) Banerjee D, Dhodapkar M V, Matayeva E, Steinman R M, Dhodapkar K. Expansion of FOXP3high regulatory T cells by human dendritic cells (DCs) in vitro and after D C injection of cytokine matured DCs in myeloma patients. Blood 2006; 108: 1298-305. (33) Bennouna S, Bliss S K, Curiel T J, Denkers E Y. Cross-talk in the innate immune system: neutrophils instruct recruitment and activation of dendritic cells during microbial infection. J. Immunol 2003; 171(11): 6052-8. (34) Maletto B A, Ropolo A S, Alignani D O, et al. Presence of neutrophil-bearing antigen in lymphoid organs of immune mice. Blood 2006; 108(9): 3094-102. (35) Schmielau J, Finn O J. Activated granulocytes and granulocyte-derived hydrogen peroxide are the underlying mechanism of suppression of t-cell function in advanced cancer patients. Cancer Res 2001; 61(12): 4756-60. (36) Gallina G, Dolcetti L, Serafini P, et al. Tumors induce a subset of inflammatory monocytes with immunosuppressive activity on CD8+ T cells. The Journal of clinical investigation 2006; 116(10):2777-90.