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 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.

2. The antibody according to claim 1, wherein the antibody is configured for use in a diagnostic method for determining a reoccurrence of a HPV16-positive carcinoma after treatment.

3. 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 that specifically bind to conformational epitopes of HPV16 L1 capsid or capsomer structure presenting antigens, wherein the labeled antibodies are obtained from the hybridoma cell line with the deposit number DSM ACC3306.

4. The kit as defined in claim 3, wherein labeled antibodies are configured for use in a diagnostic method for determining a reoccurrence of a HPV16-positive carcinoma after treatment.

5. The kit as defined in claim 3, wherein the antigens are configured for use in a diagnostic method for determining a reoccurrence of a HPV16-positive carcinoma after treatment.

6. The kit according to claim 3, wherein the antigens are virus like particles.

7. The kit according to claim 6, wherein the antigens are configured for use in a diagnostic method for determining a reoccurrence of a HPV16-positive carcinoma after treatment.

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

(1) 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.

(2) 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

(3) 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.

(4) 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).

(5) Production, screening and cloning of the monoclonal antibodies. BALB/c mice were subcutaneously immunized with 20 μg 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.

(6) 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

(7) 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.

(8) 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.

(9) 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.