COMBINED THERAPY AGAINST CANCER

Abstract

The present invention relates to a pharmaceutical composition comprising a LCoR activator and an immune checkpoint inhibitor (ICI), to its use in the treatment of cancer and to the use of LCoR as marker of response to immunotherapy in cancer treatment.

Claims

1. A pharmaceutical composition comprising: a. an LCoR activator; and b. at least one immune checkpoint inhibitor (ICI) wherein said LCoR activator is other than doxorubicin.

2. The pharmaceutical composition according to the preceding claim, wherein the LCoR activator is an agent that induces LCoR expression, or an agent that increases the amount of LCoR.

3. The pharmaceutical composition according to any one of claim 1 or 2, wherein the LCoR activator is a nucleic acid comprising a codifying sequence for SEQ ID NO: 1, or for SEQ ID NO: 2 or for SEQ ID NO: 3.

4. The pharmaceutical composition according to the preceding claim, wherein the nucleic acid is comprised in exosomes.

5. The pharmaceutical composition according to any one of the preceding claims, wherein the at least one ICI inhibits CTLA-4, PD-1, PD-L1, Lag-3, Tim-3, TIGIT or a combination thereof, preferably inhibits PD-1 or PD-L1, more preferably inhibits PD-1.

6. The pharmaceutical composition according to any one of the preceding claims, wherein the ICI is selected between ipilimumab, tremelimumab, nivolumab, dostarlimab, pembrolizumab, pidilizumab, MEDI0680, atezolizumab, BMS-936559, MEDI4736 (durvalumab), MSB0010718C (avelumab), TSR-022 (cobolimab), BMS-986016, TSR-033, BMS-986207, and combinations thereof, preferably is selected between pembrolizumab, atezolizumab, dostarlimab and combinations thereof.

7. The composition according to any one of the preceding claims for use in the treatment of cancer.

8. The composition for use according to the preceding claim, wherein the cancer is selected from chondrosarcoma, Ewing's sarcoma, malignant fibrous histiocytoma of bone/osteosarcoma, osteosarcoma, rhabdomyosarcoma, heart cancer, astrocytoma, brainstem glioma, pilocytic astrocytoma, ependymoma, primitive neuroectodermal tumor, cerebellar astrocytoma, cerebral astrocytoma, glioma, medulloblastoma, neuroblastoma, oligodendroglioma, pineal astrocytoma, pituitary adenoma, visual pathway and hypothalamic glioma, breast cancer, invasive lobular carcinoma, tubular carcinoma, invasive cribriform carcinoma, medullary carcinoma, male breast cancer, phyllodes tumor, inflammatory breast cancer, adrenocortical carcinoma, islet cell carcinoma (endocrine pancreas), multiple endocrine neoplasia syndrome, parathyroid cancer, pheochromocytoma, thyroid cancer, Merkel cell carcinoma, uveal melanoma, retinoblastoma, anal cancer, appendix cancer, cholangiocarcinoma, carcinoid tumor, colon cancer, extrahepatic bile duct cancer, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, hepatocellular cancer, pancreatic cancer, islet cell, rectal cancer, bladder cancer, cervical cancer, endometrial cancer, extragonadal germ cell tumor, ovarian cancer, ovarian epithelial cancer (surface epithelial-stromal tumor), ovarian germ cell tumor, penile cancer, renal cell carcinoma, renal pelvis and ureter, transitional cell cancer, prostate cancer, testicular cancer, gestational trophoblastic tumor, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Wilms tumor, esophageal cancer, head and neck cancer, nasopharyngeal carcinoma, oral cancer, oropharyngeal cancer, paranasal sinus and nasal cavity cancer, pharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, basal cell carcinoma, squamous cell carcinoma, skin adnexal tumors (e.g. sebaceous carcinoma), melanoma, sarcomas of primary cutaneous origin (e.g. dermatofibrosarcoma protuberans), lymphomas of primary cutaneous origin (e.g. mycosis fungoides), bronchial adenomas/carcinoids, small cell lung cancer, mesothelioma, non-small cell lung cancer, pleuropulmonary blastoma, laryngeal cancer, thymoma and thymic carcinoma, Kaposi sarcoma, epithelioid hemangioendothelioma (EHE), desmoplastic small round cell tumor, liposarcoma, preferably the cancer is selected from lung cancer, melanoma, renal carcinoma, bladder carcinoma, prostate cancer, mesothelioma, glioblastoma and breast cancer, more preferably the cancer is selected from melanoma, bladder cancer and breast cancer, preferably triple-negative breast cancer.

9. The composition for use according to the preceding claim, wherein the LCoR activator and the ICI are administered simultaneously or sequentially.

10. A prognostic method of the response to immunotherapy in a subject afflicted from cancer comprising: (a) determining the presence of LCoR, LCoR expression levels and/or amount of LCoR protein in a biological sample from said subject, (b) determining the presence of LCoR, LCoR expression levels and/or amount of LCoR protein in a control, (c) comparing the presence of LCoR, LCoR expression levels and/or amount of LCoR protein determined in steps (a) and (b), wherein the presence of LCoR versus its absence or higher LCoR expression levels or amount of LCoR protein in the subject biological sample relative to the control indicates that the subject afflicted from cancer would respond to immunotherapy.

11. The prognostic method according to the preceding claim, wherein the biological sample is from the cancerous tissue of the subject afflicted from cancer.

12. The prognostic method according to any one of the two preceding claims, wherein the expression levels of LCoR or the amount of LCoR protein are determined and compared in steps (a) to (c).

13. The prognostic method according to any one of the three preceding claims, wherein the expression levels of LCoR or the amount of LCoR protein are determined by qPCR, in situ hybridization, Western blot or immunohistochemistry (IHC).

14. The prognostic method according to any one of the four preceding claims, wherein the immunotherapy is the administration of at least one ICI inhibits CTLA-4, PD-1, PD-L1, Lag-3, Tim-3, TIGIT or a combination thereof, preferably the at least one ICI inhibits PD-1 or PD-L1, more preferably it inhibits PD-1.

15. The prognostic method according to any one of the five preceding claims, wherein the cancer is selected from chondrosarcoma, Ewing's sarcoma, malignant fibrous histiocytoma of bone/osteosarcoma, osteosarcoma, rhabdomyosarcoma, heart cancer, astrocytoma, brainstem glioma, pilocytic astrocytoma, ependymoma, primitive neuroectodermal tumor, cerebellar astrocytoma, cerebral astrocytoma, glioma, medulloblastoma, neuroblastoma, oligodendroglioma, pineal astrocytoma, pituitary adenoma, visual pathway and hypothalamic glioma, breast cancer, invasive lobular carcinoma, tubular carcinoma, invasive cribriform carcinoma, medullary carcinoma, male breast cancer, phyllodes tumor, inflammatory breast cancer, adrenocortical carcinoma, islet cell carcinoma (endocrine pancreas), multiple endocrine neoplasia syndrome, parathyroid cancer, pheochromocytoma, thyroid cancer, Merkel cell carcinoma, uveal melanoma, retinoblastoma, anal cancer, appendix cancer, cholangiocarcinoma, carcinoid tumor, colon cancer, extrahepatic bile duct cancer, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, hepatocellular cancer, pancreatic cancer, islet cell, rectal cancer, bladder cancer, cervical cancer, endometrial cancer, extragonadal germ cell tumor, ovarian cancer, ovarian epithelial cancer (surface epithelial-stromal tumor), ovarian germ cell tumor, penile cancer, renal cell carcinoma, renal pelvis and ureter, transitional cell cancer, prostate cancer, testicular cancer, gestational trophoblastic tumor, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Wilms tumor, esophageal cancer, head and neck cancer, nasopharyngeal carcinoma, oral cancer, oropharyngeal cancer, paranasal sinus and nasal cavity cancer, pharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, basal cell carcinoma, squamous cell carcinoma, skin adnexal tumors (e.g. sebaceous carcinoma), melanoma, sarcomas of primary cutaneous origin (e.g. dermatofibrosarcoma protuberans), lymphomas of primary cutaneous origin (e.g. mycosis fungoides), bronchial adenomas/carcinoids, small cell lung cancer, mesothelioma, non-small cell lung cancer, pleuropulmonary blastoma, laryngeal cancer, thymoma and thymic carcinoma, Kaposi sarcoma, epithelioid hemangioendothelioma (EHE), desmoplastic small round cell tumor, liposarcoma, preferably the cancer is selected from lung cancer, melanoma, renal carcinoma, bladder carcinoma, prostate cancer, mesothelioma, glioblastoma and breast cancer, more preferably the cancer is selected from melanoma, bladder cancer and breast cancer, preferably triple-negative breast cancer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1. LCoR induces the antigen processing machinery and antigen presentation in breast cancer cells. A. Relative mRNA levels of PSMB-8, HLA-C, PSMB-9, TAP1 and B2M in control and MDA-MB-231 breast cancer cells overexpressing LCoR. B. Relative mRNA levels of PSMB-8, HLA-C, PSMB-9, TAP1 and B2M in control and MDA-MB-231 breast cancer cells where LCoR was knocked down. C. Flow cytometry analysis of OVA in PyMT8119-OVA cells and human breast cancer MDA-MB-231-OVA cells with ectopic LCoR-OE increased the OVA positive cells D. The cell viability of PyMT8119-OVA cells co-cultured with CD8+ T-cells is reduced with LCoR-OE and prevented with LCoR-KD.

[0027] FIG. 2. LCoR induces PDL1 expression and T-cell killing of LCoR expressing cells in combination with immunotherapy. A. Relative mRNA levels of PD-L1 in the indicated mouse breast cancer cells, control and with ectopic LCoR-OE. B. Flow cytometry analysis of PD-L1 in the indicated mouse breast cancer cells, control and with ectopic LCoR-OE. C. Correlation analysis of LCoR and PD-L1 mRNA levels in 1200 breast cancer patients of the TCGA database. D. Cytotoxic T-cell lymphocyte (CTL) assay cocultured with PyMT8119 mouse breast cancer cells at the indicated ratios (T: tumor; E: effector T cell). Control and LCoR overexpression conditions are compared for viability with/out anti-PD-L1 treatment (Atezolizumab).

[0028] FIG. 3. LCoR expression induce and predicts immunotherapy response. A. Waterfall plot of in vivo tumor growth in 4T07 mouse breast cancer cells, control and LCoR overexpression conditions treated with anti-PD-L1 (Atezolizumab). Represents the percentual change of tumor growth upon onset of the treatment until day 20. B. Growth curves of the same experiment in A C. mRNA LCoR levels comparing patients that do not respond to ICI and patients that respond in renal carcinoma and melanoma of the indicated studies.

EXAMPLES

[0029] The following examples are provided to further illustrate, but not to limit this invention.

Example 1

LCoR Increased Antigen Presentation in Tumor Cells

[0030] We analyzed the transcriptomic profiles ectopic LCoR overexpression (OE), and LCoR knock-down (KD) in MDA-MB-231 breast cancer cells (FIGS. 1A and B). We confirmed by qPCR that AMP genes are also upregulated in breast cancer MDA-MB-231 cells overexpressing LCoR (FIG. 1A).

[0031] We used different breast cancer cell lines transduced with the pLEX-OVA-IRES-mCherry (pLEX-OVAiC) plasmid, that overexpress the ovalbumin (OVA). The intracellular proteolytic processing of OVA generates the OVA derived peptide SIINFEKL (OVA 257-264) that is presented in the context of MHC class-I molecule H2Kb specific of the C57BL/6 background. We used this system to measure the antigen presentation ability of tumor cells by fluorescence-activated cell sorting (FACS) with a specific anti-SIINFEKL antibody.

[0032] PyMT8119-OVA cells with ectopic LCoR-OE increased the OVA positive cells from 18% to 53% of cells (FIG. 1 C). In human breast cancer MDA-MB-231 cells transduced with the pLEX-OVAiC, we also observed a strong increase the OVA presentation (FIG. 1 C). These results show that LCoR strongly induces cellular immunogenicity.

[0033] We also found higher immune detection and killing promoted by LCoR: we performed cytotoxic Tcell lymphocyte assays (CTL) by co-culture of CD8+T-cells with tumor cell assays in vitro. The transgenic OT-I mouse model strain contain the specific TCR to detect SIINFEKL MHC-I presented. PyMT8119-OVA cells were cocultured with previously activated CD8 T-cells isolated from the spleen of OT-I mice in a 0.5 ratio of effector T-cells:tumor cells (E:T). Tumor cells with LCoR-OE were more sensitive to the immune detection and killing than the control cells, instead the LCoR-KD cells were not killed (FIG. 1 D).

Example 2

Doxorubicin Treatment in Cancer Cells Increased LCoR Expression

[0034] Low dose treatment of breast cancer cells (360 nM during 48 hours) induced an increase of 2.3 fold change mRNA expression of LCoR in MDA-MB-231 cells.

Example 3

LCoR Increased PD-L1 Expression in Tumor Cells and Response to Anti-PDL-L1 In Vitro

[0035] Using qPCR and FACS we observed that the overexpression of LCoR also induced the PD-L1 expression and membrane localization in different breast cancer cell lines (FIGS. 2A and B). In addition, analyzing the TOGA dataset of 1200 breast cancer samples, we observed a positive coexpression of LCoR and PD-L1 in breast cancer, reinforcing this regulatory loop as clinically relevant (FIG. 2 C).

[0036] These paradoxical effects of LCoR, increased immunogenicity vs. increased PD-L1, poses LCoR expressing cells as largely responsive cells to anti-PD-L1 immune-checkpoint blockade therapy. The CTL co-culture assay with OT-I CD8+ T-cells and LCoR-OE tumor cells in combination with anti-PD-L1 (Atezolizumab) showed an enhanced sensitivity to immune detection and killing (FIG. 2 D), proving that LCoR is an ideal therapeutic partner of anti-PD-L1 agents.

Example 4

LCoR Leads to Complete Tumor Regression in Combination with Anti-PD-L1 Therapy In Vivo

[0037] The therapeutic effect of the combination of LCoR with anti-PD-L1 therapy in preclinical settings was tested as follows: we performed mammary fat pad (MFP) injections of mouse 4TO7 breast cancer cells and let the tumors grow to 0.5 cm of diameter. Strikingly, 20 out of 21 4TO7 tumor with ectopic LCoR expression completely disappeared with a regime of twice a week anti-PD-L1 treatment. Instead, none of the wild type 4TO7 tumors regressed, 20 out 20 wt tumors progressed and mice were sacrificed once the tumor burden limit was reached (FIG. 3A). Remarkably, these 4TO7 control cells were not responsive to anti-PD-L1 therapy (FIG. 3 B), indicating that LCoR ectopic expression converted them from immunotherapy resistant state to immunotherapy sensitive. In addition, we used exosomes as vehicles for therapeutic delivery of nucleic acids codifying for LCoR. Using this strategy, we were able to therapeutically deliver LCoR in MFP 4TO7 tumors. The combination of exosome-LCOR delivery with anti-PDL1 treatment also showed a robust reduction of tumor growth in all tumors (n=8/condition) and stopped the tumor progression, showing that using LCOR in combination with anti-PDL1 is a therapeutic feasible strategy.

[0038] To further exploit the potential therapeutic effects of this combination, luciferase labeled 4TO7 cells (4TO7-luc) were injected through the tail-vein to generate lung metastasis, monitored by bioluminescence (BLI) corresponding to luciferase positive cells. After 2 weeks of injection, mice with developed lung metastasis determined by BLI, were treated with anti-PD-L1 twice a week. Remarkably, metastasis of 4TO7-LCoROE cells were totally regressed and cured. However, the 4TO7-wt metastasis progressed normally until the mice were sacrificed.

[0039] Therefore, we were able to cure experimental primary breast cancer and advanced metastatic breast cancer with the combination of ectopic LCoR expression and anti-PD-L1 treatment in preclinical settings.

Example 5

LCoR Correlates with Immunotherapy Response in Different Cancers

[0040] We analyzed all the clinical public available datasets GSE67501 (Ascierto et al. (2016) Cancer Immunol Res. 2;4(9):726-33) GSE79691 (Ascierto et al. (2017) Clin Cancer Res 3(12):3168-3180) GSE78220 (Hugo et al. (2016) Cell 165(1):35-44) and phs001919 (Abril-Rodriguez et al. (2020) Nature Cancer volume 1, 46-58) of immunotherapy treated patients with mRNA gene expression data. Samples were from primary tumors of melanoma and bladder cancer patients before they were subjected to immune-based therapy. Despite the low number of patients of these datasets, we found that LCoR expression levels were increased in the responsive patients versus the non-responsive patients (FIG. 3 C). These results support that LCoR is a good predictive biomarker of immune-checkpoint blockade inhibitor response. In addition, immunohistochemistry of LCoR in human triple-negative breast cancer samples of 17 patients from different clinical trials showed a trend of LCoR to predict response to anti-PD1/PDL1, in which among the high LCoR expressing patients, 75% responded to the treatment.

[0041] Methods

[0042] Animal studies. Animal procedures were approved by the PRBB animal facility policy and La Generalitat de Catalunya; Departament de Territori i Sostenibilitat. Tumor formation experiment for orthotopic mammary fat pad (MFP) injections, 10 mice or glands were used for each experimental group and the primary tumors were monitored twice weekly by palpation. Tumors were measured by calipers for calculation of tumor volumes (p×length×width2/6). For lung colonization metastasis experiments, 4TO7 luciferase labeled cells were used for tail vein injections of 50,000 cells performed in Balb/c mice.

[0043] Development of metastases was monitored by measuring photon flux of metastatic lesions based on bioluminescence imaging (BLI) as previously described. For the anti-PD-L1 treatment experiment, subcutaneously injection of 10 mg/kg of monoclonal InVivoMab anti-mouse PD-L1 (BioXcell) once a week. Exosomes secreted from 4TO7 cells were packed with synthetized LCoR mRNA. The delivery was done every 3 days through intraperitoneal injections and tumors were isolated to validate the expression of ectopic LCoR tagged with HA.

[0044] The transgenic OT-I mouse model strain with specific TCR for the OVA-derived peptide SIINFEKL MHC-I presented, were used to isolate splenocytes and CD8+T-cells that specifically recognize the SIINFEKL peptide (OVA 257-264). 9-week OT-I mice were sacrificed to obtain the spleen. Using mechanical dissociation of the spleen over a sterile 40 μm mesh screen. Total splenocytes were plated in six well plates and non-attached T cells were separated from adherent dendritic cells (DC) after 24 hours. CD8+T-cells were isolated by FACS.

[0045] Cell lines, culture conditions and treatments. All cell lines used in the study, including mammary epithelial cell lines (human HMLE), breast cancer cell lines (human MDA-MB-231 and mouse 4TO7; PyMT8119; AT3 cell lines) and other cell lines (HEK293T, H29 and HeLa), were cultured using the standard conditions according the American Type Culture Collection (ATCC) instructions. Doxorrubicin treatment was performed at 360 nM during 48 hours in MDA-MB-231 cells.

[0046] Viral production and infection of cell lines. Lentiviral plasmids were transfected into HEK293T cells together with the envelope plasmid (VSVG) and gag-pol plasmid (pCMV-dR8.91) following the standard lentiviral packaging protocol to generate lentiviruses. For established cell lines, cells were transduced in culture and selected with the corresponding antibiotic resistance. For ectopic expression of human and mouse LCoR, we used the previously generated overexpressing constructs pLEX-LCoR-HA and pLEXLcor (Celia-Terrassa et al. 2017), respectively. For gene knock-down studies, shRNA lentiviral vectors were purchased from Sigma-Aldrich for targeting mouse genes Lcor (TRCN0000085107) and human LCoR (TRCN0000016306 and TRCN0000436034). For OVA ectopic expression, lentiviral particles were generated using the pLEX-OVA-IRES-mCherry (pLEX-OVAiC) plasmid to overexpress the ovalbumin (OVA) in PyMT8119, HMLE, MDA-MB-231 and AT3 cells. Proteolytic processing of OVA generated the OVA-derived peptide SIINFEKL (OVA 257-264) that is presented in the context of MHC class I molecule H2Kb specific of C57BL/6 background.

[0047] Flow cytometry analysis and cell sorting. Cultured cells were lifted using trypsin (Gibco), resuspended in FACS buffer (PBS supplemented with 5%newborn calf serum) and filtered through 70 mm nylon cell strainers. Samples were stained with anti- SIINFEKL-APC antibody (1:200, Cell Signaling), and anti- PD-L1-PE antibody (1:200, Cell Signaling, 3199) for 30 minutes. Cells were analyzed on a FACSort LSRII instrument (BD Biosciences) and analyzed with FlowJo vX. CD8+ T-cell isolation was performed staining with 1:500 of the anti-mouse CD8-PE antibody (Biolegend) using the FACSAria.

[0048] Cytotoxic T-cell lymphocyte (CTL) co-culture assays. CD8+ T-cells were isolated from the spleen of OT-I mice and activated in culture with OVA peptide during 48 hours. Activated CD8+ T-cells were cocultured in 24-well plates with PyMT8119-OVA cells in different ratios of effector T-cells:tumor cells (E:T). After 5 days of coculture, T-cells and debris were removed, washed with media and stained with Sulforhodamine B (SRB) for measuring cytotoxicity (Vichai and Kirtikara 2006). Briefly, the SRB colorimetric assay was performed by fixing the remaining cell with 10% trichloroacetic acid (TCA) and stained with SRB for 30 minutes, washed with 1% acetic acid and dissolve with 10 mM Tris-base solution. Measure OD at 510 nm with spectrometer.

[0049] qRT-PCR analyses. Total mRNA was isolated using the RNAeasy isolation Kit (QIAGEN). mRNA reverse transcription was done using Applied Biosystems reactants and real-time quantitative PCR performed using the Power SYBR green PCR master mix (Applied Biosystems). All analyses were performed using an ABI 7900HT PCR machine. mRNA expression was normalized by the expression of GAPDH in each sample.

[0050] Immunohistochemistry. Immunohistochemical (IHC) staining was performed at the Barcelona MAR Health Park Histopathology Core using a Dako PT link pre-treatment system with anti-LCoR (Novus Biologicals, NBP1-83477; 1:100 dilution). Nuclear immunoreactive score (nIRS) were done for LCoR valuation. Slides were visualized and analyzed using QuPath-0.2.0 software. For immune infiltration, we consider up to 1 mm of positive cells surrounding tumors. Necrotic areas were not considered in the quantification.

[0051] Statistical analysis. Results were represented as indicated in figure legends, generally as mean±s.e. (standard error). For experiments with two groups, a small sample size (less than 30), and normally distributed data, the significance was evaluated using a two-tailed unpaired Student's t-test under the assumption of unequal variance. Asterisks will denote p-value significance: p<0.05*; p<0.01**; p<0.005***.

[0052] For multiple independent groups, one-way ANOVA was evaluated. Non-parametric data sets were evaluated using the Mann-Whitney-Wilcoxon U test. For correlation analysis of clinical samples, Pearson correlation was used to assess significance. All of the experiments with images (BLI and FACS) were repeated >3 times and representative images are shown.

[0053] Analyzed datasets. Clinical transcriptomic data of breast cancer patients (1200 patients) from TCGA dataset were download using TCGAbiolinks package in Rstudio (Colaprico A. et al 2015; Silvia, CT et al 2016; Mounir M. et al 2019). Levels of gene expression were measured by RNA-Seq by Expectation-Maximization (RSEM) and transformed to log2(RSEM). Co-expression analysis of PD-L1 and LCOR expression was performed using Spearman's correlation. P-value and Rho coefficient was calculated.