NON-HUMAN ANIMAL MODELS OF SÉZARY SYNDROME

Abstract

Sézary syndrome is a rare, aggressive, and leukemic form of cutaneous T-cell lymphoma (CTCL) characterized by erythroderma associated with generalized peripheral lymphadenopathy and circulating clonal malignant T cells called Sézary cells. Current animal models of Sézary syndrome are not satisfactory since no cutaneous symptoms or occurrence of metastases could be observed. Now the inventors developed a new non-human animal model that repeat the major cutaneous symptoms of the human disease. This model could be suitable for screening new drugs and biomarkers of the disease.

Claims

1. A process of producing an animal model of Sézary syndrome comprising the steps of i) engrafting an amount of peripheral blood mononuclear cells (PBMC) obtained from a patient suffering from the disease in an immunodeficient non-human animal and ii) promoting the expansion and maintaining the survival of tumor cells by weekly administering to the animal an amount of IL-2 and IL-7.

2. The process of claim 1 wherein the PBMC are obtained from a patient that presents a % of tumor cells ≥95% among their CD4+ T cell population.

3. The process of claim 1 wherein the immunodeficient non-human animal is is a NOD SCID gamma (NSG) mouse.

4. The process of claim 1 wherein an amount of about 1×10.sup.6 PBMC cells, 2×10.sup.6 PBMC cells, 3×10.sup.6 PBMC cells, 4×10.sup.6 PBMC cells, 5×10.sup.6 PBMC cells, 6×10.sup.6 PBMC cells, 7×10.sup.6 PBMC cells, 8×10.sup.6 PBMC cells, 9×10.sup.6 PBMC cells, 10×10.sup.6 PBMC cells, 11×10.sup.6 PBMC cells, 12×10.sup.6 PBMC cells, 13×10.sup.6 PBMC cells, 14×10.sup.6 PBMC cells, 15×10.sup.6 PBMC cells, 16×10.sup.6 PBMC cells, 17×10.sup.6 PBMC cells, 18×10.sup.6 PBMC cells, 19×10.sup.6 PBMC cells, 20×10.sup.6 PBMC cells, 21×10.sup.6 PBMC cells, 22×10.sup.6 PBMC cells, 23×10.sup.6 PBMC cells, 24×10.sup.6 PBMC cells, 25×10.sup.6 PBMC cells, 26×10.sup.6 PBMC cells, 27×10.sup.6 PBMC cells, 28×10.sup.6 PBMC cells, 29×10.sup.6 PBMC cells, or 30×10.sup.6 PBMC cells is used.

5. The process of claim 1 wherein an amount of about 20×10.sup.6 of PBMC is engrafted in the immunodeficient animal.

6. The process of claim 1 wherein an amount of IL-2 of about 80 UI/ml, 81 UI/ml, 82 UI/ml, 83 UI/ml, 84 UI/ml, 85 UI/ml, 86 UI/ml, 87 UI/ml, 90 UI/ml, 91 UI/ml, 92 UI/ml, 93 UI/ml, 94 UI/ml, 95 UI/ml, 96 UI/ml, 97 UI/ml, 98 UI/ml, 99 UI/ml, 100 UI/ml, 101 UI/ml, 102 UI/ml, 103 UI/ml, 1040 UI/ml, 105 UI/ml, 106 UI/ml, 107 UI/ml, 108 UI/ml, 109 UI/ml, 110 UI/ml, 111 UI/ml, 112 UI/ml, 113 UI/ml, 114 UI/ml, 115 UI/ml, 116 UI/ml, 117 UI/ml, 118 UI/ml, 119 UI/ml, or 120 UI/ml is used.

7. The process of claim 1 wherein an amount of IL-2 of about 100 UI/ml is used.

8. The process of claim 1 wherein an amount of IL-7 of about 10 ng/ml, 11 ng/ml, 12 ng/ml, 13 ng/ml, 14 ng/ml, 15 ng/ml, 16 ng/ml, 17 ng/ml, 18 ng/ml, 19 ng/ml, or 20 ng/ml is used.

9. The process of claim 1 wherein an amount of IL-7 of about 15 ng/ml is used.

10. A non-human animal model of Sézary syndrome obtainable by a process of producing an animal model, said process comprising the steps of i) engrafting an amount of peripheral blood mononuclear cells (PBMC) obtained from a patient suffering from the disease in an immunodeficient non-human animal and ii) promoting the expansion and maintaining the survival of tumor cells by weekly administering to the animal an amount of IL-2 and IL-7.

11. A process for screening drugs or biomarkers comprising the step of administering to the animal model of claim 10 an amount of a test substance.

12. A process for screening a drug suitable for the treatment of Sézary syndrome comprising the steps of i) administering the animal model of claim 10 with an amount of a test substance, and ii) selecting the test substance that is able to kill or to reduce the amount of the tumor cells in said animal.

13. The process of claim 12, wherein the survival of tumor cells in the animal administered with the test substance is compared with the survival of tumor cells in an animal that was not administered with the test substance, wherein a higher survival observed in the animal administered with the test substance than the survival observed with the animal that was not administered with the test substance indicates that the test substance is useful for killing or reducing the amount of tumor cells.

14. A process for screening potential treatments for Sézary syndrome in a subject, comprising producing the animal model of claim 10; administering a candidate treatment to the animal before, during, or after the step of engrafting; and evaluating at least one parameter of the tumor cells that is associated with cancer treatment efficacy or lack of efficacy.

15. A process for treating Sézary syndrome in a subject, comprising selecting a candidate treatment from among a plurality of candidate treatments, and administering the selected treatment to the subject, wherein the selected candidate treatment has been determined to be effective in treating Sézary syndrome in the non-human animal model of claim 10.

Description

FIGURES

[0035] FIG. 1: H&E coloration of skin biopsies from human and mouse. Cryosections were prepared from skin fragments of a healthy donor (A), Sézary patient (B), untreated NSG mouse (C) or NSG mouse engrafted with Sézary patient PBMC (D) and subjected to H&E coloration.

[0036] FIG. 2: Flow cytometry analysis of the T cell tumor burden pre- and post-injection. Immunostaining was performed on Sézary patient blood (A) or on cells extracted from the skin of the corresponding recipient NSG mouse (B). Tumor CD4.sup.+ T cells are identified through expression of KIR3DL2 and TCR-Vβ clonality.

EXAMPLE

[0037] Material and Methods:

[0038] Cells

[0039] PBMC were isolated from Sézary syndrome (SS) patients heparinized venous blood by gradient centrifugation on lymphocytes separation medium (LSM; EuroBio). Cells were washed once in phosphate buffer saline (PBS; Invitrogen) and resuspended at a concentration of 20×10.sup.6 cells/200 μl of saline solution (0.9% NaCl).

[0040] Generation of a Sézary Mouse Model

[0041] Sézary patient tumor burden was first evaluated by flow cytometry. Only patients who presented a % of tumor cells ≥95% among their CD4.sup.+ T cell population were selected for the next experimental steps. PBMC were then prepared and immediately processed for engraftment. Four to twelve-week old NOD/SCID/gamma (NSG) female mice were engrafted by caudal intravenous injection of 20×10.sup.6 Sézary patient PBMC. IL-2 and IL-7 (cytokine mix; Peprotech) were added to the cells at concentrations of 100 UI/ml and 15 ng/ml, respectively. Once per week, a fresh cytokine mix was re-injected.

[0042] Mice well-being was monitored each other day in terms of weight, behaviour and skin appearance (after hair removal with a depilatory cream). Mice were sacrificed when abnormal behaviour, skin damages and/or inherent itching became incompatible with the animal well-being.

[0043] Lymphocytes Extraction from Skin Biopsies

[0044] Biopsies were dilacerated and skin fragments were incubated in RPMI 1640 culture medium supplemented with 2 mg/ml of collagenase II (Sigma-Aldrich) at 37° C. for 30 min. After washes, skin debris were eliminated by passing the mixture through a 100 μm nylon cell strainer and the collected cells were analyzed by flow cytometry as described below.

[0045] Flow Cytometry

[0046] Blood or extracted cutaneous cells were immunolabeled with the following mix of fluorochrome-conjugated antibodies to allow detection of the malignant CD4.sup.+ T cells: TCRVβ-FITC/KIR3DL2-PE/CD3-PC5/CD4-PC7/CD45-Pacific Blue. Tumor cells were identified as CD3.sup.+ TCRVβ.sup.+ CD45.sup.+ CD4.sup.+ KIR3DL2.sup.+ cells. Cells were acquired on a cytometer (CytoFlex; Beckman Coulter) and data analyzed using FlowJo software.

[0047] Haematoxylin & Eosin (H&E) Coloration

[0048] Immediately after isolation, skin biopsies were immersed in 4% paraformaldehyde, included in paraffin and stored at 4° C. until use. Five μm cryosections were cut using a cryostat, air-dried, fixed in acetone, washed and incubated sequentially in haematoxylin solution, 0.08% NH.sub.4OH and 0.2% eosin solution. Washes were performed between each step of the procedure. After a final wash in ethanol, pictures were acquired on a Leica DMRB microscope.

[0049] Results

[0050] Sézary syndrome is an advanced and aggressive form of cutaneous T cell lymphoma characterized by the presence of tumor T cells in the blood and skin. In patients' skin, the presence of malignant T cell infiltrates results in keratinocytes hyper-proliferation leading to epidermis thickening (FIGS. 1A and 1B). A similar cutaneous pattern was observed in NSG mice following injection of Sézary patient PBMC when compared to non-treated mice (FIGS. 1C and 1D). In addition, cells extraction performed on skin biopsies from engrafted mice led to the detection of malignant CD4.sup.+ T cells exhibiting TCR-Vβ rearrangement and KIR3DL2-positivity identical to the one detected on the tumor CD4.sup.+ T cell clone present within the originally inoculated PBMC (FIGS. 2A and 2B). This clearly indicates that the majority of the human T lymphocytes encountered in the skin of the engrafted mice corresponded to the patient malignant T cell clone. Finally, in some mice, the presence of circulating tumor cells was also observed in the blood stream at the time of sacrifice (data not shown). In our 5 sets of experiments performed on a total of 31 mice, only one mouse showed a GVHD reaction while all other animals developed cutaneous manifestations (development of patches, plaques and/or erythrodermia). However, no metastases were observed in the main organs (lung, liver or heart), while a mild splenomegaly was evidenced in some subjects (n=10/31) at sacrifice.

REFERENCES

[0051] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.