NEW METHOD TO TREAT CUTANEOUS T-CELL LYMPHOMAS AND TFH DERIVED LYMPHOMAS

Abstract

The present invention relates to the treatment of cutaneous T-cell lymphomas (CTCL) and T.sub.FH derived lymphomas. In this study, the inventors showed the expression of ICOS by tumor cells in the skin of patients with MF and SS (CTCL) at different stages of the disease, and in the blood of patients with SS. The idea was thus to kill these tumor cells using ADC-antibodies specifics to ICOS. Thanks to cell lines murine xenograft models and Patient Derived Xenografts (PDXs), they showed the efficacy of such anti-ICOS ADCs on T.sub.FH-derived lymphomas, such as CTCL and AITL. Thus, the present invention relates to an anti-ICOS antibody for use in the treatment of a cutaneous T-cell lymphomas (CTCL) and/or a TFH derived lymphoma in a subject in need thereof.

Claims

1. A method of treating a cutaneous T-cell lymphomas (CTCL) and/or a T.sub.FH derived lymphoma in a subject in need thereof comprising, administering to the subject a therapeutically effective amount of an anti ICOS antibody.

2. The method according to the claim 1, wherein the T.sub.FH derived lymphoma is an angioimmunoblastic T-cell Lymphoma (AITL).

3. The method according to claim 1, wherein the CTCL is a mycosis fungoides or a Sézary syndrome.

4. The method according to claim 1, wherein the antibody is the 53.3 mab, the 88.2 mab, the 92.17 mab, the 145.1 mab or the 314.8 mab.

5. The method according to claim 1, wherein the antibody is used in an antibody-drug conjugate (ADC) or antibody-dependent cell-mediated cytotoxicity (ADCC) and/or antibody-dependent cellular phagocytosis (ADCP).

6. The method according to claim 1, wherein said antibody is conjugated to a cytotoxic moiety.

7. The method according to claim 6 wherein said cytotoxic moiety is selected from the group consisting of taxol; cytochalasin B; gramicidin D; ethidium bromide; emetine; mitomycin; etoposide; tenoposide; vincristine; vinblastine; colchicin; doxorubicin; daunorubicin; dihydroxy anthracin dione; a tubulin-inhibitor; an antimitotic agent; dolastatin 10 or 15; irinotecan; mitoxantrone; mithramycin; actinomycin D; 1-dehydrotestosterone; a glucocorticoid; procaine; tetracaine; lidocaine; propranolol; puromycin; calicheamicin; an antimetabolite; an alkylating agent; a platinum derivative; duocarmycin A, duocarmycin SA, rachelmycin (CC-1065; an antibiotic; pyrrolo[2,1-c][1,4]-benzodiazepines (PDB); diphtheria toxin, ricin toxin, cholera toxin, a Shiga-like toxin, LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas exotoxin, alorin, saporin, modeccin, gelanin, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins, Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, and enomycin toxins; ribonuclease (RNase); DNase I, Staphylococcal enterotoxin A; pokeweed antiviral protein; and an amatoxin.

8. The method according to claim 7, wherein said cytotoxic moiety is MMAE.

9. (canceled)

10. (canceled)

11. The method of claim 7, wherein the tubulin-inhibitor is maytansine; the antimitotic agent is monomethyl auristatin E or F (MMAE or MMAF); the antimetabolite is methotrexate, 6 mercaptopurine, 6 thioguanine, cytarabine, fludarabin, 5 fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, or cladribine; the alkylating agent is mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine or mitomycin C; the platinum derivative is cisplatin or carboplatin; the antibiotic is dactinomycin, bleomycin, daunorubicin, doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin or anthramycin (AMC); the ricin toxin is ricin A or a deglycosylated ricin A chain toxin; the Shiga-like toxin is SLT I, SLT II, SLT of IIV; the Phytolacca americana protein is PAPI, PAPII or PAP-S; and the amatoxin is α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanullin, amanullinic acid, amaninamide, amanin or aroamanullin.

Description

FIGURES

[0214] FIG. 1: Anti-ICOS ADCs have a specific in vitro efficacy in ICOS-expressing cell lines. A. Anti-ICOS ADCs have a specific in vitro efficacy in ICOS-expressing cell lines. (A-E) Percentage of cell viability in increasing ADC concentrations, assessed with alamarBlue™ (mean of 16 replicates), on MyLa cells (A), MJ cells (B), HUT78 cells (C), Jurkat cells (D) and Jurkat-ICOS cells (E). Anti-HER2 ADCs were used as negative control, whereas anti-CD30 ADCs (BV) were positive control. ***: p<0.001; **: p=0.001-0.01; *: p=0.01-0.05; ns: not significant.

[0215] FIG. 2. Evaluation of the in vivo efficacy of anti-ICOS-MMAE ADCs in a mouse xenograft model with MyLa cells. (A) Twenty-one mice were engrafted with 8.106 MyLa cells each, which were subcutaneously injected with 200 μL of PBS and no basement membrane matrix. Mice were then randomly assigned to three groups were monitored for tumor volume after two treatments administered 4 days apart (D10 and D14 after engraftment) of either anti-HER-2, anti-CD30, or anti-ICOS ADCs. (B) Overall survival curves (Kaplan-Meier) comparing the effect of anti-ICOS and anti-CD30 ADCs. The difference between the two curves is significant (p=0.0006). (C-E) Detection of the development of bioluminescent MyLa metastases in 26 mice assigned to three groups and treated with either anti-HER2, anti-CD30, or anti-ICOS ADCs, in lungs (C), spleen (D) and liver (E).

[0216] FIG. 3. In vivo efficacy of anti-ICOS-MMAE ADCs on ICOS+ PDXs. (A-C) Fourteen mice were engrafted with 5.105 cells of PDXs from patients with SS and assigned in two groups (anti-ICOS-MMAE ADC group and the anti-HER2 ADC control group). Both treatments were injected at D55, D58, D62 and D65 at the dose of 3 mg/kg IV. Mice were then sacrificed at D69 and organs were removed, dissociated and analyzed by flow cytometry (A: in the blood; B: in the bone marrow; C: in the spleen). (D) Thirty mice were engrafted with 5.105 cells of PDXs from patients with AITL, and were assigned in three groups of 10 mice. Treatment began at D22, when the earliest blasts were detected in the mice' blood (approximately 0.2 blasts/μl). Anti-ICOS ADC and saline serum (NaC 0.96) were injected at D22, D25, D38 and D43, at the dose of 3 mg/kgs IV. Vincristine were administered at D22, D29 and D38 at 0.25 mg/kgs IP. *: p=0.01 to 0.05. ***: p0.001.

[0217] FIG. 4. MAB-Zap assay allows the identification of ICOS clones that would be the best candidates for the development of anti-ICOS ADCs. (A) Schematic representation of the way MAB-Zap operates. (B) Percentage of cell viability in increasing ADC 10 concentrations on MJ, assessed with AlamarBllue™. Note that anti-ICOS 53.3-MMAE and anti-ICOS 92.17-MMAE are more effective than anti-ICOS 314.8-MMAE.

[0218]

TABLE-US-00006 TABLE 3 Summary table of IC50 values expressed in ng/ml of all the ADCs ICOS-MMAE ICOS-PBD CD30-MMAE Myla 8.2 1.2 30.6 MJ 36.2 0.8 6.5 HUT78 9 251.9 Jurkat 733 Jurkat-ICOS 6.7 0.7 128

TABLE-US-00007 TABLE 4 Efficacy of each anti-ICOS mAbs, expressec as IC50, to act as ADCs with MAB-Zap assay. MyLa MJ Efficacy with Efficacy with ICOS clones MAB-Zap (IC50) MAB-Zap (IC50) 314.8 0.05 0.84 92.17 0.19 0.46 53.3 0.1 0.27 293.1 0.09 0.23 88.2 0.12 0.32 279.1 >1000 0.38 145.1 >1000 >1000 121.4 >1000 >1000

EXAMPLE

Example 1: Use of an Anti-ICOS Antibody-Drug Conjugates (ADC)

[0219] Material & Methods

[0220] Study Design and Population

[0221] We conducted a prospective multicenter study between November 2017 and October 2018. Patients were >18 years old and signed written informed consent forms prior to the initiation of any procedure related to the study. The diagnosis of CTCL was carried out by a clinician and a pathologist, both members of the French Cutaneous Lymphoma Group (GFELC: Groupe Frangais d'Etude des Lymphomes Cutanes). We characterized each patient according to the 2018 WHO-EORTC diagnosis and classification criteria.25 We then performed clinical staging according to the revised staging system for CTCL, based on the tumor-node-metastasis-blood (TNMB) classification system.26 To confirm a diagnosis of SS, the patient had to meet the criteria of group B2 of the TNMB classification. For functional tests, patients with SS were included either at initial diagnosis or at clinical and biological relapse (B2 criteria). We excluded patients undergoing treatment with immunotherapy or in a therapeutic trial.

[0222] Skin samples from 52 patients with CTCL at diagnosis (38 patients) or in relapse (14 patients) were obtained by 4-mm punch biopsy under local anesthesia then fixed with formaldehyde and embedded in paraffin. Blood samples from 13 patients with SS consisted of 15 mL of whole blood in EDTA tubes. Skin samples from 12 patients with B-cell lymphoma, 14 with CD30+ lymphoproliferative disease (LPD) (cutaneous anaplastic large cell lymphoma and lymphomatoid papulosis), 12 with PCSMLPD and 13 with AITL were used as control. The clinical characteristics of patients with CTCL and controls are summarized in Supplementary Table S1. The healthy volunteers were blood donors at the Etablissement Frangais du Sang (EFS).

[0223] All patient tissue collection and research use adhered to protocols approved by the Institutional Review and Privacy Boards at Institut Paoli-Calmettes (ICOS-LYMPH-IPC2018003), Saint-Louis Hospital, and the Henri-Mondor Hospital, in accordance with the Declaration of Helsinki.

[0224] Generation of mAbs

[0225] For the generation of anti-ICOS ADCs, a purified murine anti-ICOS antibody generated in our laboratory 27 was sent to Levena Biopharma and Concortis Biotherapeutics (San Diego, Calif., USA) for coupling to MMAE and pyrrolobenzodiazepine (PBD).

[0226] BV (anti-CD30-MMAE) and ado-trastuzumab emtansine (anti-HER2-MMAE) were provided by our hospital pharmacy.

[0227] Cell Culture

[0228] We used three CTCL cell lines: MyLa (from: Pr N. Ortonne, Department of Pathology, Henri-Mondor Hospital, Creteil, France), MJ (from: American Type Culture Collection [ATCC], VA, USA) and HUT78 (from: ATCC). Myla and MJ are MF cell lines, while HUT78 is a SS cell line. MyLa and HUT78 cells were cultured in RPMI 1640 medium (Life Technologies) supplemented with 10% fetal calf serum (FCS), 2% L-glutamine, 1% pyruvate; MJ in Iscove's modified Dulbecco's medium (IMDM) (Life Technologies) supplemented with 20% FCS. The diffuse large B-cell lymphoma (Daudi, ATCC CCL-213) and T-cell leukemia (Jurkat, ATCC TIB-152) cell lines were also purchased from ATCC and were cultured in the same way as MyLa and HUT78 cells. The Jurkat cell line that was transfected to express the ICOS receptor was named Jurkat-ICOS. The MyLa cell line transfected to express luciferase (infection with lentivirus vector expressing LUC2) was named MyLa-Luciferase.

[0229] Patient-derived xenografts (PDXs) of AITL (DFTL 78024V1) and SS (DFTL 90501V3) were obtained from the Dana-Farber Cancer Institute, Boston (MA, USA) 28.

[0230] Flow Cytometry and Immunochemistry

[0231] We used rabbit anti-ICOS antibodies (rabbit polyconal antibody from Spring Biosciences [Abcam, Cambridge, UK] for immunohistochemistry, and the SP98 rabbit monoclonal antibody, Spring Biosciences, with anti-rabbit Alexa488 secondary antibodies), as well as mouse antibodies to PD-1 (NAT105, Abcam), CD4 (4B12, Novocastra) (Leica Biosystems, Wetzlar, Germany), CD8 (C8/144B, Dako) (Agilent Technologies, Santa Clara, Calif., USA), and FoxP3 (236A/E7, Abcam) for fluorescent multiplex stainings using anti-mouse Texas red secondary antibodies and DAPI for nuclear stainings. All staining experiments were done on 3 m thick sections from formalin fixed paraffin embedded skin and node biopsies, either manually or using the Bond Max device (Leica Microsystems). The expression of ICOS and all other markers was scored semi-quantitatively and divided into four categories, based on the proportion of positive cells within the tumoral T-cell infiltrate (0: no staining, low expression: <5%, moderate expression: 5-50%, high expression: >50%).

[0232] For flow cytometry and functional tests, we used anti-ICOS 314.8 antibodies generated in our laboratory (for details, see Le et a127). Other antibodies were purchased from Beckman Coulter (BC) (Brea, Calif., USA), Becton-Dickinson (BD) (Franklin Lakes, N.J., USA), Miltenyi Biotech (Bergisch Gladbach, Germany), and eBioscience (San Diego, Calif., USA): CD45 KO (BC), CD3 percpCy5.5 (BD), CD4 Pacblue (BD), CD7 FITC (BD), CD26 APC (Miltenyi), CD14 APCH7 (BD), CD158e/k PE Vio770 (Miltenyi), CD52 PE (Miltenyi), CD56 APC-Vio770 (Miltenyi), CD19 APC (BD), CD20 PE (BC), CD25 PE-Cf 594 (BD), and FoxP3 FITC (eBioscience).

[0233] Flow cytometric analyses were performed on a FACS Canto II (BD Biosciences, San Jose, Calif., USA) cytometer. The raw data generated were analyzed with the DIVA FACS Canto II software version 8.0.1.

[0234] Measurement of Cell Line Viability in the Presence of ADCs

[0235] Cell viability was measured with alamarBlue™ (Biosource, Carlsbad, Calif., USA). After 4 to 5 days of cell exposure to ADCs, alamarBlue™ was added. After 4 hours of incubation at 37° C., fluorescence was measured by a luminometer (OPTIMA, BMG Labtech) at a wavelength of 560 nm, as recommended by the manufacturer.

[0236] Animals and Xenograft Models

[0237] All experiments were done in agreement with the French Guidelines for animal handling, the ARRIVE guidelines and approved by local ethics committee (Agreement no. APAFIS #6069-2016071216263470 v3).

[0238] Non-obese diabetic severe combined immunodeficiency gamma (NSG/NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) male mice of 6-8 weeks of age were used for mouse studies and were obtained from Charles Rivers (l'Arbresle, France). Mice were housed under sterile conditions with sterilized food and water provided ad libitum and were maintained on a 12-h light and 12-h dark cycle and under temperature and humidity control. Cages contained an enriched environment with bedding material.

[0239] Mice were given subcutaneous injections of 8 million Myla or MylaLuc cells in PBS. Tumor growth was monitored by measuring with a digital caliper and calculating tumor volume (length×width2×7π/6). When tumors reached an average size close to 100 mm3, mice were randomized (n=7 per group) and used to determine the treatment response. Treatments with ADCs were injected intravenously into the caudal vein. BV and anti-ICOS ADCs were administered at the same dose (3 mg/kg) and ado-trastuzumab emtansine at 10 mg/kg. Bioluminescence analysis was performed using a PhotonIMAGER (Biospace Lab, Nesles-la-Vallée, France) following addition of endotoxin-free luciferin (30 mg/kg). After completion of the analysis, mice autopsies were performed, and organ luminescence was assessed. Daily monitoring of mice for symptoms of disease (tumor volume >1500 mm3, significant weight loss, ruffled coat, hunched back, weakness, and reduced mobility) determined the time of killing for injected animals with signs of distress. Survival curves were estimated by the Kaplan-Meier method and compared using the log-rank test.

[0240] To explore the efficiency of ADC treatments on lymphoma progression, we utilized PDXs of AITL (DFTL 78024V1) and SS (DFTL 90501V3). For each PDX, 100,000-500,000 cells from the PDXs were injected intravenously into the caudal vein of NSG mice without prior in vitro culture. When mice were engrafted (hCD45.sup.+ cells detected in peripheral blood by flow cytometry), NSG mice were treated in the same manner as previously described.

[0241] Statistical Analysis

[0242] All data were analyzed with the GraphPad Prism program (GraphPad Software, San Diego, Calif., USA). An unpaired non-parametric Student's t-test with level of significance set at p<0.05 was used to compare the in vitro efficacy of the antibody of interest and its control. The grouped efficacy analyses were performed with a two-way analysis of variance (ANOVA) test. IC50 (median inhibitory dose) was calculated with non-linear regression. The in vivo survival curves were compared with the log-rank test (Kaplan-Meier).

[0243] Results

[0244] ICOS is Widely Expressed by Malignant Cells in the Skin of Patients with MF and SS

[0245] We used immunohistochemistry to study ICOS expression in skin biopsies of 52 patients with CTCL at diagnosis (38 patients) or in relapse (14 patients). In 5 patients with SS, we also analyzed concomitant core biopsies from histologically proven involved nodes with tumor T-cell invasion at histological evaluation (pN3). We measured the ICOS expression of the CD3.sup.+ tumoral T-cell population which was characterized morphologically (nuclear atypias) and phenotypically (pan-T-cell antigen loss amongst CD2, CD5, CD7; PD-1 expression for SS samples; CD30 expression for primary cutaneous CD30.sup.+ T-cell lymphoproliferative disorders [LPD]).

[0246] Atypical lymphocytic infiltrates in 61% of 23 patients with early-stage MF (stages IA to IIA, without large cell transformation) showed moderate to high ICOS expression. Tumoral cells from 75% of the 12 patients with transformed MF had moderate to high expression of ICOS. Finally, ICOS was highly expressed by 15/17 (88%) of the skin biopsies of patients with SS (data not shown). As expected, ICOS was poorly expressed in B-cell lymphoma and widely expressed in PCSMLPD and AITL tumoral infiltrates. Interestingly, tumoral cells of CD30.sup.+ LPD exhibited a low expression of ICOS. Moreover, ICOS was expressed by atypical lymphocytes in all the five nodes with SS involvement, being highly expressed in four of them. Therefore, ICOS expression increases with the progression of the disease and becomes widely expressed in SS, both in the skin and nodes.

[0247] Double staining experiments were performed in both skin and lymph node samples from these five patients to further characterize ICOS expression by neoplastic T-cells and by the microenvironment (data not shown). We observed that most atypical CD4.sup.+ T-cells (>50%) expressed ICOS, as well as most PD-1.sup.+ atypical cells, except for one patient with a low to moderate ICOS expression. In the latter, all ICOS.sup.− lymphocytes co-expressed PD-1. A high PD-1 expression was found in all skin and node sample, and ICOS.sup.+PD-1.sup.− lymphocytes appeared to be absent or very rare (<5%). Only very few (<5%) ICOS.sup.+CD8.sup.+ T-cells could be identified in the tumor microenvironment in 3 node samples. Few to moderate amounts of CD4.sup.+ T-cells appeared to be ICOS− in the skin and node samples. A low proportion of FoxP3.sup.+ Tregs lymphocytes were identified in 3 cases, both in the skin and lymph nodes for two and only in the node for one. A low to moderate proportion of them expressed ICOS (data not shown). Thus, ICOS expression appears mainly restricted to neoplastic CD4.sup.+ T-cells, with rare ICOS.sup.+CD8.sup.+ T-cells or FoxP3.sup.+ Tregs in the tumor micro-environment.

[0248] ICOS is Widely Expressed by Malignant Cells in the Blood of Patients with SS

[0249] ICOS expression by circulating malignant cells was then evaluated using flow cytometry. To ensure the most specific selection of Sézary cells, we considered CD4.sup.+KIR3DL2.sup.+ T-cells with loss of either CD7 or CD26 to be malignant cells. Data shows the distribution of lymphocyte populations in 13 patients compared to 12 healthy volunteers. In patients, the median percentage of malignant CD4.sup.+ T-cells (Sézary cells) among all lymphoid cells was 53.1% (35.9-71), meaning that 64% of all CD4.sup.+ T-cells in patients were malignant cells. Tregs (CD4.sup.+CD25.sup.+FoxP3.sup.+) accounted for 2% of all lymphocytes, i.e. 4.3% of non-tumoral lymphocytes; this was 3.4% in healthy donors. In addition, NK lymphocytes made up 2.4% of all lymphocytes in patients (5% of non-tumoral lymphocytes), compared to 5.5% in healthy donors. NK lymphocytes did not express ICOS (data not shown).

[0250] Expression of ICOS by circulating tumor cells was found in all patients. The expression was strong: 69±7.3% of tumor cells expressed ICOS versus 38.8±7.1% of non-tumoral CD4.sup.+ cells in patients (p<0.009; 95% confidence interval [CI95%]: 8.654-51.55); and 31±3.2% of CD4+ cells in healthy volunteers (p<0.0001; CI95%: 20.29-46.34) (data not shown). In patients, 14.4±2.7% of Foxp3.sup.+CD25.sup.+CD4.sup.+ Tregs expressed ICOS, compared to 5.6±1.2% in healthy volunteers (p=0.04) (data not shown).

[0251] Anti-ICOS ADCs Mediate Killing of MyLa, MJ and HUT78 Cell Lines

[0252] We first tested anti-ICOS ADCs on MF (MyLa and MJ) and SS (HUT78) cell lines to ensure their functionality. ICOS expression was strong on MyLa (MFI ratio=143.9) and MJ (MFI ratio=96) but low on HUT78 (MFI ratio=4.5). CD30 was strongly expressed on all 3 cell lines (data not shown).

[0253] We observed a significant dose-dependent decrease in cell viability in the presence of anti-ICOS-MMAE ADCs in the MyLa and MJ cell lines (FIGS. 1A-B). In the MyLa cell line, anti-ICOS-MMAE ADCs had a better but not statistically significant different IC50 than BV (respectively 8.2 ng/ml and 30.6 ng/ml). In MJ cells, the anti-ICOS-MMAE ADCs tended to be less effective than BV. This difference could be explained by the fact that anti-ICOS mAbs 10 were internalized more in MyLa than in MJ cells, while the opposite occurred for anti-CD30 mAbs (data not shown).

[0254] In HUT78 cells, BV is less effective than in MyLa and MJ (IC50=251.9 ng/ml) and anti-ICOS-MMAE ADCs exhibit no activity (FIG. 1C). Indeed, HUT78 cell line displays resistance to MMAE, as IC50 of free-MMAE is respectively of 8.2e-007 μM and 0.001 μM in MyLa and HUT78 (data not shown). However, anti-ICOS-PBD ADCs mediate potent killing of the cells, suggesting that anti-ICOS ADCs coupled with a well-adapted drug could be effective even with low levels of ICOS expression.

[0255] Finally, we assessed the specificity of ADCs by testing the anti-ICOS ADCs on Jurkat and Jurkat-ICOS cells (FIGS. 1D-E). IC50 values of all the ADCs are summarized in Table 3.

[0256] In Vivo, Anti-ICOS-MMAE ADCs are Superior to BV in Terms of Overall Survival and Prevents the Development of Metastases

[0257] Mice subcutaneously engrafted with 8.106 MyLa cells were randomly assigned to three groups: an anti-ICOS-MMAE ADC group, BV group, anti-HER2 (ado-trastuzumab-emtansine) ADC group.

[0258] Mice treated with anti-HER2 ADCs died between day (D)10 and D12. A rapid decline in tumor volume occurred after treatment with anti-ICOS-MMAE ADCs or BV (FIG. 2A).

[0259] Subcutaneous tumor volumes were no longer noticeable from the fifteenth day after the first injection, with no significant difference between the two treatments. Tolerance was excellent, with no evidence of ADC toxicity in treated mice. Interestingly, anti-ICOS-MMAE ADCs provided a longer overall survival (OS) than BV (HR=15.2; CI95%: 3.2-71.1; p<0.0006) (FIG. 2B). The median survival in the BV group was 35 days and was not reached in the anti-ICOS ADC group.

[0260] In a second experiment, we aimed to monitor the development of metastases using MyLa-Luciferase cells. Twenty-seven mice were engrafted and treated under the same conditions as in the first experiment. On D25, 7 mice from each group were sacrificed and their organs were scanned with the luminometer to detect the presence of metastases. The other mice were maintained until D40 to detect in vivo the onset of subcutaneous recurrence. On D25, all mice in the anti-HER2 group had metastases in the lungs, liver, and spleen. In the BV group, around 50% of mice had at least one metastasis in one of these three organs. In the anti-ICOS-MMAE group, the organs did not exhibit significant bioluminescence (FIGS. 2C,D,E). On D40, subcutaneous recurrence was perceived in vivo in mice of the BV group, while mice in the anti-ICOS group were still in remission (data not shown).

[0261] Anti-ICOS-MMAE ADCs have a Potent In Vivo Efficacy in PDXs of ICOS+ Lymphomas

[0262] To improve the predictive value of our preclinical model, we assessed the efficacy of anti-ICOS-MMAE ADCs in ICOS.sup.+PDXs from patients with SS and AITL.

[0263] ICOS.sup.+PDXs from patients with SS were intravenously injected into fourteen NSG mice. On D40 after engraftment, we observed a brutal and rapid increase in the number of Sézary cells. We took blood samples from each mouse and quantified the number of circulating tumor cells to evenly distribute the living mice into two groups of 7 mice: the anti-ICOS-MMAE ADC group and the anti-HER2 ADC control group. Fifteen days after treatment, the mice were sacrificed, and we quantified the number of malignant cells in the blood and organs by flow cytometry. We observed a reduced number of tumor cells in the blood, bone marrow, and spleen of the anti-ICOS ADC group (FIG. 3A,B,C). Anti-ICOS ADCs here show a rapid and significant efficacy, suggesting that this therapeutic strategy could be used in patients with advanced SS.

[0264] In a second experiment, ICOS.sup.+PDXs from patients with AITL were intravenously 25 injected into NSG mice. We subsequently took blood samples to detect tumor cells by flow cytometry. The first tumor cells were detected on D21 after transplantation, so treatments began on D22. Mice were treated with anti-ICOS-MMAE ADCs, vincristine (positive control, with the same mode of action as MMAE), or saline solution (NaCl à.9%). Median survival in the negative control and vincristine group was D67 and D68, respectively. Median survival in the anti-ICOS group was not reached. The better survival of mice treated with anti-ICOS ADC compared to those receiving saline solution was highly significant (p<0.0001) (FIG. 3D). No evidence of ADC toxicity was observed in treated mice. On D120, the mice treated with anti-ICOS ADCs were in complete remission since no blasts were more detectable. (data not shown).

Example 2: Use of Others Anti-ICOS Antibodies-Drug Conjugates (ADC)

[0265] Material & Methods

[0266] To assess the ability of different anti-ICOS antibodies to act as ADCs, we used MAB-Zap (Advanced Targeting System, San Diego, USA), which is a secondary anti-murine IgG antibody coupled to saporin, a ribosome inhibitor (FIG. 4A). The MAB-Zap recognizes the Fc fragment of our antibody of interest, then the MAB-Zap-Antibody complex binds to the surface antigen and is internalized. Saporin is released into the cytosol and inhibits the ribosome, stopping protein synthesis and resulting in cell death. The commercial kit also includes a negative control corresponding to serum polyclonal Ig, IgG-SAP, also coupled with saporin.

[0267] In 96-well round-bottomed plates, cells are exposed to purified antibodies at increasing concentrations from OnM to 40 nM. MAB-Zap is added at a concentration of 4.5 nM (manufacturer's recommendations), as well as IgG-SAP in the control wells. After 3 days incubation at 37° C., AlamarBlue is added to each well (10% of the total well volume) and the fluorescence is read with OPTIMA luminometer.

[0268] Results

[0269] We tested on MyLa and MJ the 9 following anti-ICOS mAbs: 314.8, 92.17, 53.3, 298.1, 88.2, 279.1, 145.1, 121.4. All these mAbs showed an ability to act as ADCs using the MAB-Zap assay except for 3 on MyLa (279.1, 145.1 and 121.4) and 2 on MJ (145.1 and 121.4). The efficacy of each mAbs, expressed as IC50, is shown in Table 4. To confirm these results, we coupled 53.3, 92.17 and 145.1 anti-ICOS mAbs to MMAE, and compared them to our first 314.8 anti-ICOS ADCs (FIG. 4B).

REFERENCES

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