SEROLOGIC TEST FOR THERAPY CONTROL OF HPV16 POSITIVE CARCINOMA

Abstract

A method for therapy control of HPV16 positive carcinoma, an antibody for use in the corresponding diagnostic method as well as a test for performing the method. In particular, a serologic method for monitoring the development of the amount of antibodies in samples, which were taken from a patient before and after the treatment of a HPV16 positive carcinoma over a predetermined period of time. In addition, an immunologic test in the form of a kit, with which the method can be performed.

Claims

1. An in vitro method for therapy control after treatment of HPV16-positive carcinoma comprising the steps of (a) contacting a sample from a patient with an HPV16-positive carcinoma, said patient having been administered an anti-cancer therapy, with a plurality of antigens comprising a conformational epitope of HPV16 L1 capsid or capsomer, wherein said epitope is not present in monomeric and/or denatured HPV16 L1, under conditions at which antibodies present in the sample can bind to the antigens; and (b) detecting the binding of antibodies in the sample to the antigens, wherein the binding of said antibodies bears a negative correlation with the success of anti-cancer therapy in the patient.

2. The method according to claim 1, wherein the binding of antibodies in the sample to the antigens is detected by contacting the sample with labeled antibodies that specifically bind to the conformational epitope of the HPV16 L1 capsid or capsomer.

3. The method according to claim 1, wherein the binding of said antibodies is compared with a reference level of binding.

4. The method according to claim 2, wherein the labeled antibodies are present in mobile form on a test strip, and wherein complexes of an antigen and a labeled antibody are detected by binding to a subsequent antibody.

5. The method according to claim 3, wherein a patient identified as having a recurrence of the HPV16 positive carcinoma is administered an anti-cancer therapy.

6. An in vitro method for therapy control after treatment of HPV16-positive carcinoma comprising: i) mixing a sample of a patient with a plurality of antigens, wherein the antigens present HPV16 L1 capsid or capsomer structures, which have conformational epitopes not present in monomeric and/or denatured HPV16 L1, under conditions at which antibodies present in the sample can bind to the HPV16 L1 antigens, ii) contacting the mixture of step i) with labeled antibodies, which specifically bind to the conformational epitopes of the HPV16 L1 capsid or capsomer structure presenting antigens, particularly with labeled antibodies, which are obtained from the hybridoma cell line with the deposit number DSM ACC3306, iii) quantifying the labeled antibodies and/or the antibodies of the sample, which have bound to the HPV16 L1 capsid or capsomer-structure-presenting antigens, respectively, iv) repeating steps i) to iii) one or more times with samples taken from the same patient in predetermined time intervals so that a trend of the amount of antibodies, that bind to the HPV16 L1 capsid or capsomer structure presenting antigens, in the patient is tracked based on the samples over a predetermined period of time, and v) determining the amount of antibodies that bind to the HPV16 L1 capsid or capsomer structure presenting antigens in the samples to observe a decrease of the amount after successful therapy, and/or vi) determining the amount of antibodies that bind to the HPV16 L1 capsid or capsomer structure presenting antigens in the samples to observe a reoccurrence of a HPV16-positive carcinoma, if the amount of antibodies that bind to the HPV16 L1 capsid or capsomer structure presenting antigens increases again in the sample within the predetermined period of time.

7. The method according to claim 1, wherein the antigens are not immobilized but are provided in a liquid phase, to which the patient sample is added.

8. The method according to claim 1, wherein the patient sample is simultaneously contacted or mixed with the antigens and contacted with the labeled antibodies.

9. The method according to claim 6, wherein in step ii) the mixture runs across a test strip, on which the labeled antibodies are present in mobile form, and wherein in step iii) complexes of antigen and labeled antibody are detected by binding to another antibody.

10. A method of treating a patient that has previously been administered at least one anti-cancer therapy targeting an HPV16-positive carcinoma comprising: (A) requesting a test providing results of an analysis to determine whether the patient has an increase in antibodies that bind to a conformational epitope of HPV16 L1 capsid or capsomer over a predetermined time period; and (B) administering an additional anti-cancer therapy targeting HPV16-positive carcinoma if an increase in antibodies is detected in the patient.

11. An antibody, which specifically binds to conformational epitopes of HPV16-L1 capsid or capsomer structure presenting antigens, which is obtained from the hybridoma cell line with the deposit number DSM ACC3306.

12. The antibody according to claim 11, wherein the antibody is configured for use in a diagnostic method for determining a reoccurrence of a HPV16-positive carcinoma after treatment.

13. An antigen, which presents HPV16-L1 capsid or capsomer structures, or virus like particle, which presents HPV16 L1 capsid or capsomer structures.

14. The antigen according to claim 13, wherein the antigen is configured for use in a diagnostic method for determining a reoccurrence of a HPV16-positive carcinoma after treatment.

15. A kit for determining an amount of antibodies in a sample of a patient comprising: I) a composition comprising antigens presenting conformational epitopes of HPV16 L1 capsid or capsomer structures, and II) a composition comprising labeled antibodies, which specifically bind to conformational epitopes of HPV16 L1 capsid or capsomer structure presenting antigens.

16. The kit as defined in claim 15, wherein the labeled antibodies are obtained from the hybridoma cell line with the deposit number DSM ACC3306.

17. The method according to claim 2, wherein the labeled antibodies are obtained from the hybridoma cell line with the deposit number DSM ACC3306

18. The method according to claim 3, wherein the binding of said antibodies is compared with the binding of said antibodies in one or more samples taken from said patient at predetermined time intervals, wherein a decrease in said binding over a predetermined time indicates successful anti-cancer therapy, and wherein an increase in said binding over time indicates a recurrence of the HPV16-positive carcinoma in said patient.

19. The method according to claim 4, wherein subsequent antibody is also one that are obtained from the hybridoma cell line with the deposit number DSM ACC3306.

20. The method according to claim 9, wherein other antibodies are also ones that are obtained from the hybridoma cell line with the deposit number DSM ACC3306.

21. The antigen or virus like particle according to claim 13, wherein the HPV16 L1 capsid or capsomer structures have conformational epitopes, which specifically bind to an antibody which is obtained from the hybridoma cell line with the deposit number DSM ACC3306.

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0070] FIG. 1 shows the development of the amount of antibodies in the serum of a patient with a positive therapy course over 27 weeks after primary therapy of a HPV16 positive carcinoma. On the X axis, the weeks starting from 0 at the point in time of the primary therapy are shown, on the Y axis, the concentration of the antibody is shown in ng/ml.

[0071] FIG. 2 shows the development of the amount of antibodies in the serum of a patient, in which after about 35 weeks after the primary therapy of a HPV16 positive carcinoma, a relapse occurred. Up to week 27, a continuous decrease of the amount of antibodies was observed. Beginning in week 31, the amount of antibodies increased again. The increase continued until week 35, at which time the clinical correlation was found. On the X axis, the weeks starting from 0 at the point in time of the primary therapy are shown, on the Y axis, the concentration of the antibody is shown in ng/ml.

DETAILED DESCRIPTION

Example 1

Screening for the Antibodies and Antigens

[0072] Preparation of papillom virus like particles (VLPs): The L1 gene of HPV16 (GenBank: K02718.1) was amplified by PCR and cloned into the transfer vector pVL1392. The recombinant vectors were introduced in Sf9 cells together with BaculoGold DNA (Pharmingen) using calcium phosphate precipitation. Recombinant viruses were amplified and purified by plaque assay according to manufacturer's instructions.

[0073] Virus like particles (VLPs) were purified according to Volpers et al. (Volpers, C., P. Schirmacher, R. E. Streeck, and M. Sapp. 1994. Assembly of the major and the minor Kapsid protein of human papilloma virus type 33 into virus-like particles and tubular structures in insect cells. Virology 200:504-512).

[0074] Production, screening and cloning of the monoclonal antibodies. BALB/c mice were subcutaneously immunized with 20 g of intact HPV16 VLPs dissolved in phosphate buffered salt solution (PBS), after these had been mixed with complete Freund's adjuvant. The immunization was repeated after one month and after three months.

[0075] Three days after the third immunization the spleen was taken out and a single cell suspension was produced. The spleen cells were fused with the mouse myeloma cell line X63Ag8.653 using polyethylene glycol 2500 (Boehringer Mannheim) and cultured in Iscoves modified Eagle Medium (IMDM) in the presence of 10% fetal calf serum in 96-well plates. Fused cells were selected with azaserine and hypoxanthine. After 6 to 8 days, the supernatant of the cells was tested for secretion of HPV16 L1 specific antibodies using ELISA. Denatured L1 protein, as well as VLPs of HPV-6, HPV-11, HPV-18, HPV-31, HPV-33 and HPV-39 served as controls to exclude unspecific reactivities.

Example 2

Observation of the Decrease of the Amount of Antibodies in a Patient after Successful Therapy

[0076] Male Patient, age 53, with oncologic combination therapy (surgery/radio-chemotherapy) for a HPV16 positive tonsillar carcinoma. On the day before the therapy began, 5 ml blood were taken from the patient to obtain patient serum. Testing of the serum at the beginning of the therapy gave an antibody concentration of 13200 ng/ml.

[0077] Six weeks after the primary therapy, 5 ml blood were taken from the patient again to obtain serum. An antibody concentration of 5600 ng/ml was measured. This corresponds to a decrease of the antibody concentration of over 50% within 6 weeks.

[0078] With the decrease of the amount of antibodies, a successful therapy can be controlled because the tumor antigen HPV16 L1 forming tumor cells were successfully removed and the tumor antigen (HPV16 L1 protein) does not induce the immune system anymore to form HPV16 L1 specific antibodies.