KR101665410B1 - p16INK4a DERIVED PEPTIDES FOR PROPHYLAXIS AND THERAPY OF HPV-ASSOCIATED TUMORS AND OTHER p16INK4a EXPRESSING TUMORS - Google Patents

Abstract

The present invention relates to a specific fragment of the cyclin dependent kinase inhibitor p16 which is capable of increasing the IFN-gamma secretion of T cells or inducing the proliferation of T cells, and a specific fragment of HPV-related or other p16 ^INK4a expressing carcinoma, Lt; RTI ID = 0.0 > immunization < / RTI >

Description

P16INK4a DERIVED PEPTIDES FOR PROPHYLAXIS AND THERAPY OF HPV-ASSOCIATED TUMORS AND OTHER p16INK4a EXPRESSING TUMORS FOR PREVENTION AND TREATMENT OF HPV-TUMOR AND OTHER P16INK4a EXPRESSION TUMORS [

The invention relates to specific fragments of the cyclin dependent kinase inhibitor p16 ^INK4a and to the use of said fragments in immunizing individuals against p16 ^INK4a -expressing tumors.

Millions of people globally are sick or die from cancer each year. Despite intensive therapies, these mortality rates have remained unchanged for many years. Until now, patients suffering from carcinoma have had to undergo nearly cancer-removal surgery or chemotherapy or radiation therapy. However, this approach involves a number of side effects that affect the mortality of patients suffering from carcinoma. It is interesting to note that HPV is associated with more than 5% of all cancers. (Parkin and Bray, 2006). HPV vaccination is already possible but has not been shown to be effective in patients already infected (Hildesheim et al., 2007). Therefore, a new treatment method is required.

Accordingly, the present invention provides a means of preventing and treating HPV-associated tumors and other p16 ^INK4a expressing tumors.

According to the present invention, such prevention and treatment is carried out by the contents of the claims in this specification. The study leading to the present invention is from observations that the cell protein p16 ^INK4a is expressed in HPV infected cells when the cell is transformed and becomes a malignant phenotype due to the collapse of the negative feedback loop by the HPV cancer gene product E7 (Sano et al. 1998; Klaes et al., 2001). Thus, p16 ^INK4a is virtually strongly expressed in all HPV-induced carcinomas and high grade pre-neoplasias including the cervix, vulva, vagina, penis, anus and head and neck tumors (Ishikawa et al., 2006; 2006, Issos et al., 2006; Santos et al., 2006; Roma et al., 2008; Hafkamp et al., 2003). Under physiological conditions, p16 ^INK4a is expressed only in cells undergoing subsequent senescence and is therefore rarely expressed in normal tissues (Beausejour et al., 2007). In addition to HPV- induced cancer gene p16 ^INK4a expression in the HPV- related tumors, p16 ^INK4a is, the melanoma is found in many tumors, but the tumors or HPV viral carcinogenesis did not prove this is not associated with HPV infection, such tumors (Ning et al., 2004; Busch et al., 2010), lung cancer (Leversha et al., 2003; Esposito et al., 2004), esophageal cancer, gastric cancer and colorectal cancer Some of these are cancer, bladder cancer, ovarian cancer, endometrial cancer and breast cancer (Ikuerowo et al., 2007; Buza et al., 2010; Mutations in the retinoblastoma tumor suppressor gene are known to ^{result in} upregulation of p16 ^INK4a expression (Okamoto et al., 1994). However, the underlying mechanism of strong p16 ^INK4a expression in this case is quite heterogeneous and not completely understood.

Overexpression of endogenous gene products in cancer cells has long been recognized as a valuable source of tumor-associated antigens. Immune responses to these antigens have been observed in many cancer patients and their use in immunotherapy approaches has been increasing (Jager et al., 2003; Finn, 2008; Rescigno et al., 2007).

During the course of the experiments leading to the present invention, isolated T ^lymphocytes from peripheral vascular samples of healthy individuals can be specifically stimulated in vitro with peptides derived from p16 ^INK4a , and CD4 + and CD8 + T cells derived from cervical cancer patients It is ^reported that increasing the cytotoxic T cell line which shows spontaneous reactivity to the same p16 ^INK4a peptide and which can attack and kill the HLA-matched p16 ^INK4a- loaded cells and the cervical cancer cell lines I could show it. In other words, the fragments of p16 ^INK4a is that it shows a high immunogenic to induce a very strong immune response against p16 ^INK4a. It has also been shown that the humoral immune response is detectable for p16 ^INK4a .

The discovery of a spontaneous immune response to p16 ^{INK4a that} is not associated with any autoimmune disease and the p16 ^INK4a expression pattern described above makes p16 ^INK4a a promising candidate for immunization of patients with p16 ^INK4a expressing tumors. The ^actively induced strong immune response to p16 ^INK4a can destroy particularly ^HPV -transformed cells and other p16 ^INK4a -expressing cancer cells. Vaccination of donor T cells with these p16 ^INK4a peptides was performed in cell culture experiments. Additional experiments with p16 ^INK4a peptides showed spontaneous T cell responses in cervical cancer patients, ^confirming that p16 ^INK4a peptides are immunogenic in vivo. Therefore, the prevention and treatment of cancer based on vaccination using the peptide has various benefits for the patient. Since p16 ^INK4a is not observed in normal tissues, almost, who did not observe the developing immune from individuals with spontaneous immune responses to p16 ^INK4a serious side effects of p16 ^INK4a immunization it is not expected. Finally, since p16 ^INK4a expression is ^complexly associated with the malignant phenotype of cancer cells, there will be little immune avoidance due to antigen loss.

Accordingly, the present invention relates to specific fragments of inhibiting cyclin-dependent kinases capable of eliciting an immune response against p16 ^INK4a chair p16 ^INK4a. The immune response may be defined as a condition that meets at least one of the following categories: 1. cytotoxicity test or IFN-gamma secretion or perforin expression or granzyme B expression or CD8-positive T cells Induction of measurable CD8-positive T cells as the background by ELISpot or intracellular cytokine staining or cytokine ELISA or equivalent methods as detectable by other cytokines. 2. Induction of CD4-positive T cells detectable by measurable cytokine secretion as the background by ELISpot or intracellular cytokine staining or cytokine ELISA or equivalent method. The cytokines include but are not limited to IFN-alpha, IFN-gamma, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-17, TNF- Or other cytokines that can be produced by CD4-positive T cells. 3. Detection of antibodies by Western blotting, ELISA and homologous or related methods. 4. Induction of any cellular immune response that is not mediated by CD8-positive or CD4-positive T cells such as those described in 1 and 2.

The p16 ^INK4a fragment consists of

(a) any one of the following amino acid sequences:

(a ₁ ) MEPAAGSSMEPSADWLATAAARGRV;

(a ₂ ) TAAARGRVEEVRALLEAGALPNAPNSY;

(a ₃ ) LPNAPNSYGRRPIQVMMMGSARVAELL;

(a ₄ ) VMMMGSARVAELLLLHGAEPNCADPATLTR;

(a ₅ ) ADPATLTRPVHDAAREGFLDTLWLHRAGARL;

(a ₆ ) HRAGARLDVRDAWGRLPVDLAEELGHRDVAR;

(a ₇ ) GHRDVARYLRAAAGGTRGSNHARIDAAEGPSDIPD

(b) the functional equivalent of the fragment of (a) above which is still capable of inducing an immune response against p16 ^INK4a ; or

(c) a combination of the fragments of (a) and / or (b).

As used herein, the term "functional equivalent" refers to a fragment or variant of (a) that is still capable of inducing, for example, an immune response against ^p16INK4a , and thus is still effective as an effective vaccine. Variants are characterized by amino acid deletions, substitutions and / or additions. Amino acid differences are preferably due to one or more conservative amino acid substitutions. The term "conservative amino acid substitution" refers to substitution of the aliphatic or hydrophobic amino acids Ala, Val, Leu and Ile; Substitution of the hydroxyl residues Ser and Thr; Substitution of acidic residues Asp and GIu; Substitution of the amide residues Asn and Gln, substitution of the basic residues Lys, Arg and His; Substitution of aromatic residues Phe, Tyr and Trp, and substitution of small amino acids Ala, Ser, Thr, Met and Gly.

For the production of peptides showing a certain degree of identity, for example, genetic engineering can be used to introduce amino acid variations at specific positions in the cloned DNA sequence to identify sites critical for peptide function. For example, locating mutations or alanine-scanning mutations (introducing a single alanine mutation in all residues of the molecule) can be used (Cunningham and Wells, 1989). Immunogenicity tests can then be performed on the resulting mutant molecules using the test methods of the following Examples.

The variant preferably has aa substitution, deletion and / or addition of 8 or less, more preferably 6 or less, and even more preferably 4 or less.

Among the original p16 ^INK4a - fragments, it is preferred that at least 5 contiguous aa of a particular amino acid sequence remain, at least 10 contiguous aa remain, more preferably at least 15 contiguous aa remain, and at least 20 contiguous aa It is even more desirable that aa remains. These fragments can still elicit an immune response against p16 ^INK4a and thus can be used as an effective vaccine.

The invention also provides a nucleic acid encoding a fragment according to the invention or a vector comprising said nucleic acid. Direct injection of the genetic material into the living host produces small amounts of the introduced gene product. Inadequate gene expression within the host has important immunological consequences, resulting in a host specific immune activation of the gene transfer antigen. Direct injection of naked plasmid DNA results in a strong immune response against the antigen encoded by the gene vaccine. Once the plasmid DNA construct is injected, the host cell receives foreign DNA, expressing the viral gene and producing p16 ^INK4a inside the cell. This type of antigen presentation and treatment induces MHC and Class I and Class II to limit cellular humoral immune responses. The DNA vaccine generally consists of a vector containing two units: an antigen expression unit consisting of a promoter / enhancer sequence, followed by an antigen (FSP) -coding and polyadenylated sequence, and a sequence necessary for vector amplification and selection ≪ / RTI > The construction of a vector with a vaccine insert can be performed using recombinant DNA technology, and a person skilled in the art can be used in this approach. The efficacy of DNA immunization can be improved by stabilizing DNA against degradation and by increasing the DNA delivery effect into the antigen presenting cell. An example of such a method is a method of coating biodegradable cationic microparticles with DNA (such as poly (lactide-co-glycolide) produced with cetyltrimethylammonium bromide). Thus, DNA-coated microparticles can be effective in increasing CTL as much as recombinant vaccine virus, especially when mixed with alum. Particles with a diameter of 300 nm are shown to be most effective for absorption by antigen presenting cells.

For example, various expression vectors, such as plasmids or viral vectors, may be used to contain or express nucleic acid sequences encoding the FSPs of the present invention.

Preferred viral vectors include, but are not limited to, poxvirus, adenovirus, retrovirus, herpes virus or adeno-associated virus (AAV). The Fox virus is particularly preferably vaccinia virus, NYVAC, avian fox virus, canarypox virus, ALVAC, ALVAC (2), nailpox virus or TROVAC.

In addition, recombinant alphavirus-based vectors have also been used to enhance the efficacy of DNA vaccination. The gene encoding FSP is inserted into the alpha virus replicon, replacing the structural gene and leaving the unstructured replicase gene intact. Sindbis virus and Semliki forest virus have been used to construct recombinant alphavirus replicons. However, unlike conventional DNA vaccination, the alphavirus vector is only transiently expressed. Alphavirus replicons increase the immune response due to high concentration proteins expressed by such vectors, replicon-induced cytokine responses, or replicon-induced apoptosis, which enhances antigen uptake by dendritic cells.

The invention also provides a pharmaceutical composition comprising a fragment, nucleic acid sequence, or vector according to the invention in an amount suitable for the individual's immunization, preferably comprising at least one conventional adjuvant. Such fragments, nucleic acid sequences or vectors may be present as such or with the carrier. It is preferred that the carrier is not immune to the individual. Such a carrier may be a protein or foreign protein of the individual or a fragment thereof. Preferred are carriers such as serum albumin, fibrinogen or transferrin or fragments thereof. Such fragments may include an antigenic determinant recognized by cytotoxic T cells, such as CD8 ⁺ T cells or CD4 cells, and may induce an immune response. The antigen determining portion of such a cell cycle control protein can be determined by a person skilled in the art to which the present invention belongs. It may also be beneficial to have several fragments at the same time. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2 edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY (1989) can be cited as examples of the recombinant products of the fragment.

The invention also relates to the use of a fragment, nucleic acid sequence or vector according to the invention in the production of a vaccine for the prevention or treatment of p16 ^INK4a -expressing tumor formation, tumorigenesis or carcinoma (including advanced carcinoma).

Examples of such are ^HPV -induced, p16 ^INK4a -expressing anogenital carcinomas, particularly cervical cancer or head and neck tumors, and HPV-induced p16 ^INK4a -expressing tumors. Hyperplasia of papilloma, adenoma, epithelium or similar proliferation, benign transformation such as hepatic lobule or hematopoietic proliferation.

As used herein, the term "individual" means any entity that may become diseased with cancer. Examples of such individuals include humans, animals and their cells.

As used herein, the term " amount suitable for vaccination of an individual "is a concept that includes any amount of the fragments of the invention which allow the subject to be immune, to which the above description may apply accordingly. This amount depends on whether immunization is for preventive or therapeutic purposes. The age, sex, and weight of the individual also play an important role in determining the dosage. Depending on the method of administration, it is preferable to administer 100 μg to 1 g of the p16 fragment to the individual. The method of administration of the vaccine may be performed at various sites by intramuscular injection, subcutaneous injection, intradermal injection or any other administration mode. It may also be desirable to perform one or more "booster doses" with approximately the same amount. In this case, it is particularly preferred to use different fragments of each cell cycle control protein for individual scans.

The term " conventional adjuvant " as used herein in the context of the present invention refers to any adjuvant suitable for pharmaceutical compositions to immunize an individual. Such adjuvants include, for example, immunizing adjuvants such as GM-CSF or Freund's adjuvant, buffered common salt solution, emulsions such as water, oil / water emulsion, wetting agent, sterile solution, And the like.

According to the present invention, immunization of an individual, particularly a human or an animal, is possible. Immunization is caused by both the induction of antibodies and the stimulation of T cells. Thus, it can be applied to preventive and therapeutic stages for preliminary tumor formation, tumor formation and carcinoma.

Figure 1 shows ELISpot (interferon gamma) results of (A) and (B) before stimulating donor T cells with p16 ^INK4a peptide.
Spots were normalized by removing background spots detected in wells without peptide. In B, the increase in the number of intracellular spots stimulated with the peptides pl6lNK4a_37-63, pl6INK4a_51-80 and pl6INK4a_73-104 becomes apparent when compared to before stimulation (day 0). CEF = CMV, EBV, influenza (flu) peptide positive control.
Figure 2 shows ELISpot (interferon gamma) results of 23 patients (Tx and Fx) and 15 healthy controls (BCx) for a positive control virus mixture (CEF) and 7 30mer p16 ^INK4a peptides (Table 1).
In this result, the background was adjusted and only spots on the cut-off were considered (2 spots in the negative control well + 2 standard deviations of the reactivity to the p16 ^INK4a peptides, respectively). Only two of them showed CD4 ⁺ response and the rest showed CD8 + response. na = non-analytical.
Figure 3 shows the results of the Chromium release assay.
CD8 T cells collected from the cervical cancer patients, but induce cell lysis (lysis) of the cell B (black squares) that match the loaded histocompatibility antigen (HLA) with the p16 ^INK4a _37-63, the same without the p16 ^INK4a peptide Not so for B cells (open triplets). Cervical cancer cell line HeLa (pl6I ^NK4a +, HLA A68, B15, B95 and Cw7 Cwl2) and Caski (pl6 ^INK4a +, HLA A2, A3, B7, B37, Cw5 and Cw7) were lysed, whereas ME180 and K562 No dissolution was detected.
Figure 4 shows the proliferation of peripheral blood mononuclear cells collected from women with cervical dysplasias following stimulation with 7 p16 ^INK4a peptides (Table 1).
^Shows the optical density of the BrdU proliferation assay performed after culturing positive control (mitogen PHA and tetanus toxoid) and negative control (no antigen), p16 ^INK4a peptide and PBMCs. The dotted line indicates the cut-off for the positive control group. The asterisk indicates the proliferation inducing p16 ^INK4a peptide. Results from three reactive patients and one negative patient are shown.
Figure 5 shows the serological p16 ^INK4a reactivity in Western blot.
(a) (1) Silver satin of purified His-tagged p16 ^INK4a (2) Western blot of His-tagged p16 ^INK4a purified with monoclonal p16 ^INK4a antibody E6H4
(b) Six representative positive sera are shown. The protein size corresponds to the reaction of the monoclonal antibody (E6H4) to the recombinant protein.
(c) Examples of negative sera (1 and 2)
(d) p16 ^INK4a serum reactivity (1) and (2) before serum pre-incubation with recombinant p16 ^INK4a . (3) detection of p16 ^INK4a protein precipitated by serum from cervical cancer patients and detected directly by HRP-labeled monoclonal p16 ^INK4a antibody (Reuschenbach et al., 2008).

The present invention will be described in more detail with reference to the following examples.

Example  One

HPV -relation Tumor formation  have In the patient p16 ^INK4a  T cell reactivity to peptides

In order to assess to what extent and to what extent the patients with HPV-associated tumors increase and / or increase the T cell response to highly overexpressed p16 ^INK4a , another method is applied which allows for a careful characterization of the immune response to the p16 ^INK4a antigen Respectively. The discovery of a spontaneous immune response to p16 ^INK4a in cervical cancer patients generally demonstrates immunogenicity of antigen and specific p16 ^INK4a fragments and provides the logic for immunizing patients with p16 ^INK4a overexpressing tumors with p16 ^INK4a fragments.

Peripheral blood mononuclear cells (PBMCs) collected from 13 women with high-grade p16 ^INK4a -expressing cervical dysplasia (CIN2 / 3) were cultured with p16 ^INK4a peptide (Table 1) After testing with ^INK4a peptides, the proliferation of immune cells was determined by applying the BrdU test as a global measure to assess the likelihood of lymph ^vascular hyperplasia. The BrdU-test is a colorimetric immunoassay which is applied to quantitate cell proliferation based on measurement of thymidine-like 5-bromo-2'-deoxyuridine incorporation during DNA synthesis. PBMCs in IMDM medium supplemented with 10% human serum were seeded at a density of 150.000 cells / 50 μl / well in a 96-well-microtiter plate (flat bottom). Cells in each of the four replicate wells were incubated with tetanus ^metformin toxin (20 ng / ml, Calbiochem, La Jolla, Calif.) And mitogen PHA-L (5 μg / ml) as a positive control in the presence of 7 p16 ^INK4a peptides , Roche, Mannheim, Germany) and using non-antigen as a negative control for 6 days at 5% CO 2, 37 ° C. On the sixth day, 10 μl / well of the BrdU labeling solution (BrdU (colorimetric), Roche (Mannheim, Germany), Cell Proliferation ELISA was used for all BrdU test reagents) was diluted 1: 100 with IMDM medium + 10% , Added to each well, and further cultured at 37 ° C in 5% CO2 for 18 hours. On the 7th day, centrifugation was carried out at 1200 rpm for 30 minutes, and 50 μl of supernatant was removed and transferred to a new V-bottom plate and stored at -80 ° C for cytokine analysis. The remaining cells were dried for 15 minutes using a hair dryer, 150 μl / well of FixDenate solution was added to the cells, incubated at room temperature for 30 minutes, carefully decanted and patched to remove FixDenat. 100 μl / well anti-BrdU-POD working solution was added, incubated at room temperature for 90 minutes, then removed, replaced with 100 μl TMB substrate, and incubated at room temperature for 30 minutes. The enzyme reaction was stopped by adding 25 μl / well of 1N H ₂ SO ₄ and the optical density (OD) was measured at 450 nm (reference wavelength 620 nm). The cut-off for the positive reaction was set to three standard deviations of the ODs in the negative control negative wells. Three PBMCs from 13 tested females showed proliferation in response to p16 ^INK4a peptide indicating that culturing with the peptide activated the proliferative memory T cell response. Overall, the pattern of the peptide induction reaction is heterogeneous, indicating multiple T-cell antigenic determinants within the p16 antigen. One patient responded to the peptides pl6INK4a_18-44, pl6lNK4a_37-63, pl6INK4a_73-107 and pl6INK4a_123-156, one patient to the peptides pl6lNK4a_51-80 and pl6INK4a_98-128, and one patient to the peptides pl6INK4a_l-25 and pl6INK4a_37-63 (Fig. 4).

To demonstrate that certain p16 fragments can induce interferon gamma secretion as a signal for a Th1 response, we obtained from 23 patients with high grade ^metastatic stage lesions (CIN2 / 3) with high p16 ^INK4a overexpression and invasive cervical cancer T cells were tested for seven p16 ^INK4a peptides (Table 1) by the interferon gamma ELISpot assay.

T-cells were isolated from heparinized blood using plastic adhesive and antigen-coupled magnetic beads, Ficoll centrifugation. DCs were cultured with IL4 and GM-SCF (1000 U / ml) for 7 days to produce dendritic cells (CD11, CD16, CD19, CD36, CD56, Pan T cell isolation kit, ilteny, Bergisch Gladbach, Germany) , Which was used as an antigen presenting cell in an ELISpot.

Each 10 ⁵ T cell was tested after in vitro presentation for a short period (2 to 5 days) with representative peptides presented by 2 x 10 ⁴ dendritic cells. For seven cervical cancer patients, removal of the background (two spots in the negative control well + two standard deviations of reactivity to each p16 ^INK4a peptide) confirms T cell (CD4 or CD8) response to pl6INK4a_37-63 peptide (Fig. 2).

Example  2

p16 ^INK4a  Peptides in healthy donor T cells Invitro Priming

Seven long 25-35mer peptides covering the entire p16 ^INK4a amino acid sequence were tested in order to distinguish the p16 ^INK4a fragments capable of inducing interferon gamma secreting T cells from healthy donors by in vitro experiments, Have an amino acid overlap of 7-13 (Table 1). Table 1 shows seven overlapping p16 ^INK4a peptides used in the ^Invitro experiment.

The p16 ^INK4a fragment shows that in vitro stimulation of healthy donor T cells can stimulate interferon gamma, and the most immunogenic p16 ^INK4a To identify the inducible antigenic determinant, we examined whether T cells isolated from peripheral blood of healthy donors could be stimulated with these p16 ^INK4a peptides in vitro experiments. When p16 ^INK4a peptides are capable of inducing specific T cell responses in cell culture experiments, T cells secrete cytokines when tested in reactive ELISpot experiments with reactive p16 ^INK4a peptides. In the ELISpot assay, cytokine (interferon gamma) could be detected by a specific antibody as a subsequent color reaction.

Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood (100 ml) of healthy donors using Ficoll Plaqe density gradient centrifugation. 5 to 10 × 10 ⁷ PBMCs were separated into monocytic leukocytes and T cells by magnetic beads with a plastic adhesive and antigen binding (CD11, CD16, CD19, CD36, CD56, Pan T cell isolation kit, Milteny, Bergisch Gladbach, Germany). Mononuclear leukocytes were cultured for 7 days with GM-CSF and IL-4 (1000 U / ml each) to generate antigen-presenting dendritic cells.

culturing the 2 x 10 ⁷ T cells for 4 hours with the p16 ^INK4a peptide 2 x 10 ⁶ of dendritic cells treated with a first pulse (10μg / ml) was carried out for antigen presentation. 7 p16 ^INK4a Individual stimulation studies were conducted for each of the peptides.

T cells were re-stimulated with p16 ^INK4a peptide pulsed dendritic cells and treated with IL-2 and IL-7 (10 U / ml) every 7 days for 5 weeks.

p16 ^INK4a peptide-specific T cell responses were measured before (day 0) and after the final stimulation (day 35) by interferon gamma ELISpot assay. For the ELISpot assay, 96-well nitrocellulose plates (MAHA N4550 Millipore) were coated with anti-interferon gamma antibody 1-D1K (Mabtech, Nacka Strand, Sweden) at a concentration of 0.75 μg / well. Each 10 ⁵ T cell was tested with representative peptides presented by 2 x 10 ⁴ dendritic cells. After incubation at 37 ° C for 12 hours, biotinylated secondary anti-interferon gamma antibody 7BG-1 (Mabtech), streptavidin-alkaline phosphatase conjugate and BCIP / NBT substrate solution (Sigma Aldrich, St. Louis, USA) To detect secreted interferon gamma.

In addition to the virus peptide mixture (CEF = CMV & EBV & flu) used as a positive control, no reactivity was detected before stimulation (Day 0) on any of the seven p16 ^INK4a peptides, but on day 35 (day 35) When tested against target cells pulsed with a reactive p16 ^INK4a peptide, but not when tested against cells pulsed with the remaining p16 ^INK4a peptide, the T cells stimulated with the peptides pl6lNK4a_37-63, pl6INK4a_51-80 and pl6INK4a_73-104 The cells showed increased interferon gamma secretion in the ELISpot (Figure 1).

Example 3

p16 ^INK4a  Cervical cancer cell lines by reactive T cells Cell lysis ( lysis )

(Lyze) activation of p16 ^INK4a- expressing cervical cancer cells by chromium release assay using different cervical cancer cell lines, together with HLA-matched B cells loaded with p16 ^INK4a peptide as a target The ability of T cells to be tested was tested. 10x6 target cells were cultured with different ratios of T cells from a representative cervical cancer patient who responded to the target cells loaded with the peptide pl6lNK4a_37-63 in the ELIspot test with ⁵¹ Cr (100 mu Ci) for 1 hour. By detecting the emitted radiation, the specific solubility of target cells by T cells can be measured.

In the ELISpot test, CD8 + T cells in cervical cancer patients responding to the target cell loaded with peptide pl6INK4a_37-63 matched HLA as well as cervical cancer cell lines HeLa and Caski (both HPV and pl6INK positive) and pl6INK4a_37-63 Peptide-loaded B cells. ≪ / RTI > On the other hand, no solubility was detected for B cells matched with the same HLA without pl6INK4a_37-63 peptide and other HPV- and p16 ^INK4a negative cell lines (Fig. 3). These results demonstrate the cytotoxic activity of p16 ^INK4a peptide-specific T cells and indicate that the p16 ^INK4a -antigen present in the cervical cancer cells can be recognized by the patient's p16 ^INK4a reactive T cell clone.

Example 4

p16 ^INK4a  For Humoral  Immune response

In addition, the analysis of over 900 sera showed that the fractions of individual p16 ^INK4a (Fig. 4) (Reuschenbach et al., 2008). The ability of p16 ^INK4a to induce a humoral immune response has been shown to induce antibodies specifically binding to the derived antigenic determinant.

It has also been shown in vitro that antibodies can be detected in serum against the peptide pl6INK4a_37-63, which is the most potent inducer of interferon gamma by T cells. A total of 374 sera obtained from cervical cancer, small cell lung cancer, head and neck cancer patients and healthy individuals were tested with peptide ELISAs. The peptide (20 μg / ml) was coated overnight at 4 ° C. with a 96-well microtiter plate (Maxisorp, Nunc, Roskilde, Denmark), blocked with nonspecific binding sites with 0.5% casein, At a ratio of 1: 100.

The combined serum antibodies were detected with HRP-conjugated anti-human IgG antibody (Jackson Iramuno, West Grove, USA) and TMB substrate (Sigma Aldrich, St. Louis, USA). At the background cut-off normalized to an optical density of 0.3, 15% (56/374) of the tested sera had antibodies against pl6lNK4a_37-63.

These results show that patients with HR- ^HPV -induced ^dysplasia or tumorigenesis can develop an anti-p16 ^INK4a -immune response. If this immune response is capable of removing HR-HPV transformed cells, the p16 ^INK4a antigen is a very attractive candidate as a future vaccine for preventing or treating p16 ^INK4a -expressing tumor formation, particularly HR- ^HPV -induced tumors Lt; / RTI >

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<110> Ruprecht-Karls-Universitat Heidelberg <120> p16INK4a derived peptides for prophylaxis and therapy of          HPV-associated tumors and other p16INK4a expressing tumors <130> IP2015-040 <160> 7 <170> Kopatentin 2.0 <210> 1 <211> 25 <212> PRT <213> Homo sapiens <400> 1 Met Glu Pro Ala Ala Gly Ser Ser Met Glu Pro Ser Ala Asp Trp Leu   1 5 10 15 Ala Thr Ala Ala Ala Arg Gly Arg Val              20 25 <210> 2 <211> 27 <212> PRT <213> Homo sapiens <400> 2 Thr Ala Ala Arg Gly Arg Val Glu Glu Val Arg Ala Leu Leu Glu   1 5 10 15 Ala Gly Ala Leu Pro Asn Ala Pro Asn Ser Tyr              20 25 <210> 3 <211> 27 <212> PRT <213> Homo sapiens <400> 3 Leu Pro Asn Ala Pro Asn Ser Tyr Gly Arg Arg Pro Ile Gln Val Met   1 5 10 15 Met Met Gly Ser Ala Arg Val Ala Glu Leu Leu              20 25 <210> 4 <211> 30 <212> PRT <213> Homo sapiens <400> 4 Val Met Met Met Gly Ser Ala Arg Val Ala Glu Leu Leu Leu Leu His   1 5 10 15 Gly Ala Glu Pro Asn Cys Ala Asp Pro Ala Thr Leu Thr Arg              20 25 30 <210> 5 <211> 32 <212> PRT <213> Homo sapiens <400> 5 Ala Asp Pro Ala Thr Leu Thr Arg Pro Val His Asp Ala Ala Arg Glu   1 5 10 15 Gly Phe Leu Asp Thr Leu Val Val Leu His Arg Ala Gly Ala Arg Leu              20 25 30 <210> 6 <211> 31 <212> PRT <213> Homo sapiens <400> 6 His Arg Ala Gly Ala Arg Leu Asp Val Arg Asp Ala Trp Gly Arg Leu   1 5 10 15 Pro Val Asp Leu Ala Glu Glu Leu Gly His Arg Asp Val Ala Arg              20 25 30 <210> 7 <211> 35 <212> PRT <213> Homo sapiens <400> 7 Gly His Arg Asp Val Ala Arg Tyr Leu Arg Ala Ala Gly Gly Thr   1 5 10 15 Arg Gly Ser Asn His Ala Arg Ile Asp Ala Ala Glu Gly Pro Ser Asp              20 25 30 Ile Pro Asp          35

Claims (16)

a ^fragment of the following cyclin-dependent phosphorylase inhibitor p16 ^INK4a capable of inducing an immune response against p16 ^INK4a :
(a) to (a ₁₎ - amino acid, selected from the group consisting of (a ₇₎ SEQ ID NO:
(a ₁ ) MEPAAGSSMEPSADWLATAAARGRV;
(a ₂ ) TAAARGRVEEVRALLEAGALPNAPNSY;
(a ₃ ) LPNAPNSYGRRPIQVMMMGSARVAELL;
(a ₄ ) VMMMGSARVAELLLLHGAEPNCADPATLTR;
(a ₅ ) ADPATLTRPVHDAAREGFLDTLVVLHRAGARL;
(a ₆ ) HRAGARLDVRDAWGRLPVDLAEELGHRDVAR; And
(a ₇ ) GHRDVARYLRAAAGGTRGSNHARIDAAEGPSDIPD; or
(b) functional equivalents of (a) that are still capable of inducing an immune response against p16 ^INK4a
Lt; RTI ID = 0.0 &gt; p16 &lt; / RTI &gt; ^INK4a . A pharmaceutical composition for inducing an immune response to p16 ^INK4a comprising a nucleic acid sequence encoding a fragment according to claim 1. 3. The pharmaceutical composition according to claim 2, which comprises a vector containing the nucleic acid. 4. The pharmaceutical composition according to claim 3, wherein the vector is a plasmid or a viral vector. delete 5. The pharmaceutical composition according to any one of claims 1 to 4, further comprising an immunizing adjuvant. 7. A pharmaceutical composition according to claim 6 for use as a vaccine for the prevention or treatment of p16-expressing pre-neoplasia, tumorigenesis or tumor. delete 8. The pharmaceutical composition according to claim 7, wherein said p16-expressing tumor is advanced carcinoma. delete 8. The pharmaceutical composition according to claim 7, wherein the tumor is anogenital tumor or head and neck tumor. 8. The method of claim 7, wherein the tumor is an esophageal-gastrointestinal or hepatic-pancreatic-biliary tumor; Skin tumors or airway tumors; Kidney, urinary tract, breast, ovary, endometrium or prostate tumor; Neurological tumors including brain tumors; And &lt; RTI ID = 0.0 &gt; HPV-induced &lt; / RTI &gt; tumors. delete delete delete 12. The pharmaceutical composition according to claim 11, wherein the anal germ cell cancer is cervical cancer.

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