USE OF THE CD71 RECEPTOR IN THE PROGNOSIS AND TREATMENT OF ENDOMETRIOSIS

Abstract

The CD71 receptor is used as a target in the prognosis and/or treatment of endometriosis, and to a test for the prognosis or therapeutic monitoring of endometriosis, targeting this receptor.

Claims

1.-10. (canceled)

11. A method of treating endometriosis, comprising administering to a subject having endometriosis, an anti-CD71 antibody or a molecule targeting the CD71 receptor.

12. A method for the prognosis of endometriosis, or for predicting or monitoring success of a treatment of endometriosis, in a subject having endometriosis, comprising contacting an anti-CD71 antibody with a biological sample of said subject.

13. The method according to claim 12, for the prognosis of a development of an hyperproliferative endometriosis overexpressing the CD71 receptor.

14. The method according to claim 12, for predicting success of a treatment of endometriosis overexpressing the CD71 receptor.

15. A prognostic, predictive or therapeutic monitoring kit, comprising an anti-CD71 antibody.

16. The kit according to claim 15, further comprising antibodies targeting the KI67 marker and/or the Bcl2 protein.

17. An immunohistochemistry kit according to claim 15.

18. A method for the prognosis of endometriosis, according to claim 12, which is for in vitro detection of the degree/severity of endometriosis by comparative measurement of the concentration/density/expression of CD71 between a healthy subject and a subject having endometriosis by immunohistochemistry implementing an anti-CD71 antibody.

Description

EXAMPLE 1: PROOF OF CONCEPT OF THE USE OF THE ANTI-CD71 ANTIBODY AS A POSSIBLE TREATMENT OF ENDOMETRIOSIS

[0067] 1) Validation of the Presence of Transferrin Receptors on Retrospective Endometriotic Lesions Embedded in Paraffin

[0068] This step was conducted on endometriosis samples embedded in paraffin (n=44) thanks to the immunohistochemistry (IHC) technique, a technique that Endodiag routinely uses. Each series of sample blocks was representative of a stage of the pathology.

[0069] In parallel with the validation of the presence of CD71 at the level of the endometriosis lesions, IHCs targeting the proliferation marker KI67 and the oncogene Bcl2 were carried out. Indeed, the anti-CD71 antibody preferably targets cells with a high rate of proliferation and induces their apoptosis by iron deprivation. The cells targeted by the anti-CD71 antibody, in addition to expressing CD71, are positive for the proliferation factor KI67 and the anti-apoptotic marker Bcl2.

[0070] FIG. 1 shows: [0071] a) anti CD71/transferrin receptor IHC, [0072] b) anti KI67 (proliferation marker) IHC, [0073] c) anti Bcl2 (anti-apoptotic marker) IHC.

[0074] The IHCs were carried out on 44 biopsies of endometriotic lesions. The results demonstrated that the intensity of CD71 staining is correlated with that of KI67. Thus, the higher the KI67 staining (gland and/or stroma), the higher the CD71 staining will be (tissue No. 1). Conversely, for lesions having low KI67 staining, CD71 staining is also barely intense (tissue No 3).

[0075] There is also a correlation between the KI67 and CD71 stainings and that of Bcl2. The higher the KI67 or CD71 staining, the higher the bcl2 staining will be. This confirms the proliferative status of endometriotic cells (high proliferation and inhibited apoptosis).

[0076] The most proliferative endometriotic lesions (associated with high KI67) also overexpress the CD71 receptor. There is a correlation between the expression of these two markers. This first result makes it possible to assume that, like cancer cells, these proliferative lesions would be sensitive to the targeted action of the anti-CD71 antibody.

[0077] These tests made it possible to validate the interest of testing the anti-CD71 antibody in the treatment of endometriosis.

EXAMPLE 2: DEMONSTRATION OF THE CORRELATION BETWEEN THE CD71 DENSITY AT THE SURFACE OF ENDOMETRIOTIC CELLS AND THE THERAPEUTIC EFFECTIVENESS OF THE ANTI-CD71 ANTIBODY

[0078] a) The first part begins during in vitro tests carried out on primary cultures of cells originating from endometriotic lesions. It consists in testing the effect of the anti-CD71 antibody on these cultures (test of proliferation, apoptosis and invasiveness, dose-dependent) and checking on the anti-proliferative effect of the anti-CD71 antibody. Immunocytochemistry with the anti-CD71 antibody is carried out in parallel to quantify the CD71 receptors at the cell surface. Flow cytometry experiments are also carried out to quantify the CD71 receptors at the cell surface. These data are analyzed in parallel with the effect observed on the cells in vitro and a correlation between CD71 density and the anti-CD71 antibody efficiency is looked for.

[0079] b) Secondly, this correlation is demonstrated during in vivo tests on a mouse model of endometriotic cell xenograft. The different tests carried out on this model make it possible to highlight the effectiveness of the anti-CD71 antibody on the endometriotic cells. These results are then correlated with the CD71 receptors density at the level of the endometriotic lesions in the xenografted mouse and are analyzed by IHC at the end of the experiment.

EXAMPLE 3: IN VIVO TESTS

[0080] Once the in vivo technique has been validated, endometriotic cells originating from 30 dissociated fresh lesions are injected and a series of tests is carried out using this model with the use of multiple doses of the anti-CD71 antibody, ranging from 1, 5, 10, 20 and 40 mg/kg in one single peritoneal injection, as we previously validated for hematological malignancies. Then several modes of administration are tested, intraperitoneal (I.P) and intravenous (I.V) as well as several doses of this treatment: once a week to 3 times a week. This study allows us to validate the use of the anti-CD71 antibody in vivo for the treatment of endometriosis and to determine a theoretical dose to be used in the first toxicology tests in monkeys. In order to be able to accurately quantify the inhibition of cell proliferation following the treatment with the anti-CD71 antibody, a Xenolight RediJect 2-DG-750 probe that allows visualizing the glucose metabolism within the tumor (synonym of proliferation) is injected to monitor cell proliferation in vivo. Fluorescence is measured using Kodak's imagine FX pro in vivo detector and images are analyzed by Carestream MI software (FIG. 2). Experimental studies on animals are subject to referral to the ethics committee and are validated by Paris V university regulatory authorities.

[0081] FIG. 2 shows an example of following-up the subcutaneous development of xenografts in NSG mouse by microscopic imaging. Differences in cell proliferation are accounted by subtracting fluorescences between treated and control animals

EXAMPLE 4: DESCRIPTION OF THE KIT

[0082] The kit is an immunohistochemistry (IHC) kit based on the use of the anti-CD71 antibody, thus guaranteeing sensitivity and selectivity. The anti-CD71 antibody intended for the diagnosis use differs from the anti-CD71 antibody intended for therapeutic use only by the production method that will not be done under the strict framework of GMP (Good Manufacturing Practices). The clones producing the anti-CD71 will nevertheless be identical.

[0083] The anti-CD71 IHC kit makes it possible to carry out between 30 and 50 tests, which is generally proposed for commercially available IHC kits.

[0084] It comprises all the reagents necessary for carrying out the test as well as the positive and negative control slides and a complete protocol with the elements necessary for the analysis of the result.

EXAMPLE 5: OPTIMIZATION OF THE TEST

[0085] It consists in defining the optimal conditions and the limiting conditions of use of the kit. This phase requires the use of twenty fresh specimens of endometriosis lesions.

[0086] This step covers respectively: [0087] the pre-analytical steps that represent all the steps of the process preceding the analysis of the tissues namely, tissue sampling, transport, fixation, inclusion, cutting thickness, slide type, etc. until the completion of the section of the paraffin embedded block. [0088] The actual steps of the analysis namely all steps of the IHC protocol, staining and mounting of the slides [0089] The post-analytical steps which comprise the interpretation and the renderings of results.

[0090] During the pre-analytical phases allow defining the following criteria and variables: [0091] Method for sampling tissue [0092] Fixation (time, method) [0093] Decalcification (time, method)

[0094] These steps are critical because the lesions sampling conditions can vary significantly from one surgeon to another and lead to sample degradation. For example, the use of pliers and scissors makes the samples unanalyzable in 40% of cases.

[0095] These phases have already been optimized, thanks to the development of an endometriosis biopsy gun and a sampling kit containing all the elements necessary for the rapid fixation of biopsies (see WO2013/034573).

[0096] The analytical phases make it possible to define the following criteria and variables: [0097] Thickness of cut [0098] Slide type [0099] dewaxing method [0100] antigen unmasking method [0101] visualization system [0102] reagents [0103] controls [0104] antibodies [0105] method for carrying out the test (automatic, manual) [0106] staining method [0107] type of lamella mounting

[0108] In more detail, it is a question of validating all the conditions of completion of the test and the reagents which are used during the latter in order to obtain a satisfactory staining, a properly colored and mounted tissue and therefore optimally analyzable. For each parameter to be adjusted, the various methods commonly used are tested and the most appropriate one is selected.

[0109] Thus, the following data are evaluated: [0110] the impact of the thickness of the cut on the results, [0111] the impact of the slide type (simple slide, superfrost+ slide, FLEX slide), [0112] the impact of the dewaxing method (time, temperature, material, coupled or not with the antigen unmasking), [0113] the impact of the visualization system; kit based on biotin/streptavidin or on a polymer, type of chromogen used (DAB, AEC), type of enzyme (HRP or AP), etc., [0114] the impact of the reagents (washing buffer with or without detergent, blocking of endogenous peroxidase, etc.), [0115] appropriate controls (positive and negative internal and/or external controls), [0116] The concentration and optimal antibody staining time required to obtain high quality detection and staining and the most appropriate diluent [0117] the stability of the antibody from 0 to 6 months [0118] the stability of the other selected reagents [0119] the used method, manual or using an IHC automaton [0120] the time and method of counterstaining (hematoxylin from Mayer, Harris) [0121] the type of coverslip mounting (permanent, aqueous, according to the selected chromogen)