Patent Application
20230082929
The immune system is critical in fighting cancer. However, at certain times, the immune system has trouble finding and/or recognizing the cancer cells that should be eliminated. These cancer cells are “cold” to the immune system, i.e., difficult to be located and destroyed.
Cellular senescence is a bona fide tumor suppression mechanism that can be induced by a number of stresses including chemotherapeutics such as cisplatin (Herranz and Gil, 2018). Therapy-induced senescence is tumor suppressive by triggering a stable cell growth arrest (Herranz and Gil, 2018). Senescent cells also have non-cell autonomous activities exemplified by secretion of inflammatory cytokines and chemokines, which is termed the senescence-associated secretory phenotype (SASP)(Coppe et al., 2008).
Immune checkpoint blockades (ICBs) such as monoclonal antibodies targeting the PD-1/PD-L1 axis have demonstrated striking clinical benefit in several cancer types (Darvin et al., 2018). However, despite this important advance, the majority of cancers show unacceptably low response rates to ICB (O'Donnell et al., 2017). Therefore, new therapeutic strategies are urgently needed to expand the utility of ICBs through sensitizing ICBs resistant tumors. Ovarian cancer remains the most lethal gynecological malignancy in the developed world. Tumor-infiltrating lymphocytes positively correlate with ovarian cancer patient survival, which is recognized as a predictive biomarker for immunotherapy and chemotherapy responses (Zhang et al., 2003). Notably, CD8+ T cells are important antitumor effectors in ovarian cancer (Sato et al., 2005). However, objective response rates to ICB in ovarian cancer range from 5.9 to 15% (Wang et al., 2019). Therefore, sensitizing ICB resistant ovarian cancer to ICB remains an unmet clinical need. Harnessing the SASP to help transform a “cold” tumor to a “hot” tumor would appear to be an effective strategy in targeting elusive cancer cells. However, accumulating evidence shows that senescent cells can have deleterious effects on the tissue microenvironment. The most significant of these effects is the acquisition of the SASP which turns senescent fibroblasts into proinflammatory cells that have the ability to promote tumor progression.
Therefore, what is needed is a method to harness the power of the SASP in turning cancer cells from “cold” to “hot” without the detrimental effects associated with the phenotype.
Provided herein are compositions and methods for treating cancer in a subject in need thereof. The compositions and methods described herein harness the power of the SASP to “light up” cancer cells, making them visible to the immune system. As described herein, ex vivo therapy-induced senescent (TIS) cells are able to home to the residual cancer cells from which the TIS cells originated, lighting up residual cancer and allowing the immune system to target and destroy these cells.
In one aspect, a method of treating cancer in a subject in need thereof is provided. In one embodiment, the method includes administering therapy-induced senescent (TIS) cells and an immune checkpoint inhibitor to the subject. In another embodiment, the method includes obtaining cancer cells from the subject; treating the cancer cells with a chemotherapeutic agent or radiation and a TOP inhibitor to induce senescence; optionally, confirming senescence and/or sorting senescent cells from non-senescent cells; administering the senescent cells to the subject; and administering a checkpoint inhibitor to the subject. In certain embodiments, the checkpoint inhibitor is a PD-1 or PD-L1 inhibitor or CTLA4 inhibitor.
In another aspect, a pharmaceutical composition is provided. The composition includes therapy induced senescent cells, as described herein, and a pharmaceutically acceptable carrier, diluent, or excipient.
In another aspect, provided herein is a pharmaceutical composition produced by a method that includes the following: obtaining cancer cells from a subject; treating the cancer cells ex vivo with a chemotherapeutic agent and an inhibitor of TOP1, TOP2, or both to produce therapy induced senescent (TIS) cells; and optionally, confirming senescence and/or sorting senescent cells from non-senescent cells.
Other aspects and advantages of the invention will be readily apparent from the following detailed description of the invention.
As disclosed herein, methods and compositions are described which are useful in treatment of cancer in a mammalian subject. The inventors have shown that treatment of cancer cells with a chemotherapeutic agent and topoisomerase inhibitor induces senescence and SASP resulting in therapy-induced senescent cells. The TIS cells are then administered to the subject whereby the TIS cells home to any remaining cancer cells. The cytokines and/or chemokines released by the TIS cells “light up” the cancer cells, where, especially in conjunction with a checkpoint inhibitor, the immune system is able to locate and eradicate the cancer cells. This strategy is particularly effective in sensitizing the cancer cells to other therapies, such as checkpoint therapy.
Unless defined otherwise in this specification, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the fields of biology, biotechnology and molecular biology and by reference to published texts, which provide one skilled in the art with a general guide to many of the terms used in the present application. The definitions herein are provided for clarity only and are not intended to limit the claimed invention.
“Patient” or “subject” as used herein means a mammalian animal, including a human, a veterinary or farm animal, a domestic animal or pet, and animals normally used for clinical research. In one embodiment, the subject of these methods and compositions is a human.
As used herein, the term “treatment of cancer” or “treating cancer” can be described by a number of different parameters including, but not limited to, reduction in the size of a tumor in an animal having cancer, reduction in the growth or proliferation of a tumor in an animal having cancer, preventing, inhibiting, or reducing the extent of metastasis, and/or extending the survival of an animal having cancer compared to control.
As used herein for the described methods and compositions, the term “antibody” refers to an intact immunoglobulin having two light and two heavy chains or fragments thereof capable of binding to a biomarker protein or a fragment of a biomarker protein. Thus, a single isolated antibody or an antigen-binding fragment thereof may be a monoclonal antibody, a synthetic antibody, a recombinant antibody, a chimeric antibody, a humanized antibody, a human antibody, or a bi-specific antibody or multi-specific construct that can bind two or more target biomarkers.
The term “antibody fragment” as used herein for the described methods and compositions refers to less than an intact antibody structure having antigen-binding ability. Such fragments, include, without limitation, an isolated single antibody chain or an scFv fragment, which is a recombinant molecule in which the variable regions of light and heavy immunoglobulin chains encoding antigen-binding domains are engineered into a single polypeptide. Other scFV constructs include diabodies, i.e., paired scFvs or non-covalent dimers of scFvs that bind to one another through complementary regions to form bivalent molecules. Still other scFV constructs include complementary scFvs produced as a single chain (tandem scFvs) or bispecific tandem scFvs.
Other antibody fragments include an Fv construct, a Fab construct, an Fc construct, a light chain or heavy chain variable or complementarity determining region (CDR) sequence, etc. Still other antibody fragments include monovalent or bivalent minibodies (miniaturized monoclonal antibodies) which are monoclonal antibodies from which the domains non-essential to function have been removed. In one embodiment, a minibody is composed of a single-chain molecule containing one VL, one VH antigen-binding domain, and one or two constant “effector” domains. These elements are connected by linker domains. In still another embodiment, the antibody fragments useful in the methods and compositions herein are “unibodies”, which are IgG4 molecules from with the hinge region has been removed.
The terms “analog”, “modification” and “derivative” refer to biologically active derivatives of the reference molecule that retain desired activity as described herein. In general, the term “analog” refers to compounds having a native polypeptide sequence and structure with one or more amino acid additions, substitutions (generally conservative in nature) and/or deletions, relative to the native molecule, so long as the modifications do not destroy activity and which are “substantially homologous” to the reference molecule as defined herein. Preferably, the analog, modification or derivative has at least the same desired activity as the native molecule, although not necessarily at the same level. The terms also encompass purposeful mutations that are made to the reference molecule. Particularly preferred modifications include substitutions that are conservative in nature, i.e., those substitutions that take place within a family of amino acids that are related in their side chains. Specifically, amino acids are generally divided into four families: acidic, basic, non-polar and uncharged polar. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. For example, it is reasonably predictable that an isolated replacement of leucine with isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar conservative replacement of an amino acid with a structurally related amino acid, will not have a major effect on the biological activity. For example, the molecule of interest may include up to about 5-20 conservative or non-conservative amino acid substitutions, so long as the desired function of the molecule remains intact. One of skill in the art can readily determine regions of the molecule of interest that can tolerate change by reference to Hopp/Woods and Kyte Doolittle plots, well known in the art.
TOP1 inhibitors are clinically used for cancer therapy. As demonstrated herein, TOP inhibitors have additional applications to sensitize tumors to immunotherapy, especially targeting cancer cells that become resistant or senescent in response to therapies such as chemotherapy or radiotherapy. Here we show that topoisomerase 1-DNA covalent cleavage complex (TOP1cc) is both necessary and sufficient for cGAS-mediated cytoplasmic chromatin recognition and SASP during senescence. TOP Ice localizes to cytoplasmic chromatin and TOP1 interacts with cGAS to enhance the binding of cGAS to DNA. Retention of TOP1cc to cytoplasmic chromatin depends on its stabilization by the chromatin architecture protein HMGB2. Functionally, the HMGB2-TOP1cc-cGAS axis determines the response of orthotopically transplanted ex vivo therapy-induced senescent cells to immune checkpoint blockade in vivo. Together, these findings establish a HMGB2-TOP1cc-cGAS axis that enables cytoplasmic chromatin recognition and response to immune checkpoint blockade.
cGAS is essential for the antitumor effect of immune checkpoint blockades such as anti-PD-L1 antibody19. Here it is described that TOP Ice plays a critical role in mediating recognition of CCF (cytoplasmic chromatin fragments) by cGAS and the associated SASP during senescence. Mechanistically, HMGB2 stabilizes TOP1cc to enhance the binding of cGAS to dsDNA. Indeed, the HMGB2-TOP1cc-cGAS axis determines the response of orthotopically transplanted ex vivo therapy-induced senescent cells to immune checkpoint blockade in vivo. Also described herein is the use of TOP inhibitors in inducing senescence in tumor cells and using these cells to treat cancer.
Provided herein, in one aspect, is a method of treating cancer in a subject in need thereof. The method includes administering therapy-induced senescent (TIS) cells and an immune checkpoint inhibitor to the subject. In one embodiment, the method includes sensitizing cancer cells to checkpoint therapy using TIS cells.
Cancer therapy has traditionally relied on cytotoxic treatment strategies on the assumption that complete cellular destruction of tumors optimizes the potential for patient survival. This view has limited the treatment options that oncologists have at their disposal to toxic compounds and high dose radiation. These approaches may produce complete cell death within a solid tumor and can cause severe side effects in patients. Such cancers often develop resistance to treatment and recur or progress to advanced primary and metastatic tumors. An alternative strategy is the induction of cytostasis, which permanently disables the proliferative capacity of cells without inducing cancer cell death. Initial clinical studies utilizing cytostatic treatments have yielded promising preliminary results, suggesting that these treatments may be as effective as cytotoxic therapies in preventing continued tumor growth. This approach to treatment could provide equivalent or prolonged survival with fewer and less severe side effects related to cytotoxicity and may provide a more realistic goal for the chronic management of some cancers. See, Ewald et al, Therapy-Induced Senescence in Cancer, J Natl Cancer Inst. 2010 Oct. 20; 102(20): 1536-1546, which is incorporated herein by reference.
A promising approach to induction of cytostasis in tumor cells is therapy-induced senescence. Senescent cells remain viable and metabolically active but are permanently growth arrested. In contrast to cells undergoing apoptosis or mitotic catastrophe in response to conventional cytotoxic drugs, senescent cells may persist indefinitely. As described herein, senescent cells can be exploited to “light up” remaining tumor cells, making those residual cells more susceptible to treatment and immune response.
Topoisomerase 1 (TOP1) is responsible for relaxing higher order topological DNA structures during DNA replication and gene transcription18. TOP1 forms a stable protein-DNA cleavage complex (TOP1cc) through its enzymatic activity and TOP1 becomes covalently bound to the catalytically generated DNA strand break18. Trapped or persistent TOP Ice induced by TOP1 inhibitors such as camptothecin are harmful to normal cellular function because they block both DNA and RNA polymerases18. However, the role of TOP1cc in senescence has never been explored.
In one embodiment, the method of treating cancer includes obtaining cancer cells from a subject; treating the cancer cells ex vivo with an effective amount of a chemotherapeutic agent, and an inhibitor of TOP1, TOP2, or both, to produce therapy induced senescent (TIS) cells; and administering the TIS cells to the same subject. In another embodiment, the cancer cells are treated ex vivo with an effective amount of an inhibitor of TOP1, TOP2, or both, to produce therapy induced senescent (TIS) cells.
In one embodiment, the TIS cells are derived from the subject who will be receiving the therapy. In one embodiment, the cells are removed from the subject prior to induction of senescence. The TIS cells may be derived from cancer cells from the subject, e.g., such as tumor cells from an excised or biopsied tumor, or blood cancer cells.
In one embodiment, the cells are collected from the patient. The cells may be pooled, concentrated, enriched or expanded to increase the number of cells available for treatment, using techniques known in the art, and described herein.
The term “cancer” or “proliferative disease” as used herein means any disease, condition, trait, genotype or phenotype characterized by unregulated cell growth or replication as is known in the art. A “cancer cell” is cell that divides and reproduces abnormally with uncontrolled growth. This cell can break away from the site of its origin (e.g., a tumor) and travel to other parts of the body and set up another site (e.g., another tumor), in a process referred to as metastasis. A “tumor” is an abnormal mass of tissue that results from excessive cell division that is uncontrolled and progressive, and is also referred to as a neoplasm. Tumors can be either benign (not cancerous) or malignant. In various embodiments of the methods and compositions described herein, the cancer can include, without limitation, breast cancer, lung cancer, prostate cancer, colorectal cancer, brain cancer, esophageal cancer, stomach cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, acute and chronic lymphocytic and myelocytic leukemia, myeloma, Hodgkin's and non-Hodgkin's lymphoma, and multidrug resistant cancer. In certain embodiments, the cancer treated includes, but is not limited to, a solid tumor, a hematological cancer (e.g., leukemia, lymphoma, myeloma, e.g., multiple myeloma), and a metastatic lesion. In one embodiment, the cancer is a solid tumor. Examples of solid tumors include malignancies, e.g., sarcomas and carcinomas, e.g., adenocarcinomas of the various organ systems, such as those affecting the lung, breast, ovarian, lymphoid, gastrointestinal (e.g., colon), anal, genitals and genitourinary tract (e.g., renal, urothelial, bladder cells, prostate), pharynx, CNS (e.g., brain, neural or glial cells), head and neck, skin (e.g., melanoma or Merkel cell carcinoma), and pancreas, as well as adenocarcinomas which include malignancies such as colon cancers, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell lung cancer, cancer of the small intestine, cancer of the esophagus. The cancer may be at an early, intermediate, late stage or metastatic cancer.
In one embodiment, the cancer is chosen from a lung cancer (e.g., a non-small cell lung cancer (NSCLC) (e.g., a NSCLC with squamous and/or non-squamous histology, or a NSCLC adenocarcinoma)), a skin cancer (e.g., a Merkel cell carcinoma or a melanoma (e.g., an advanced melanoma)), a kidney cancer (e.g., a renal cancer (e.g., a renal cell carcinoma (RCC) such as a metastatic RCC or clear cell renal cell carcinoma (CCRCC)), a liver cancer, a myeloma (e.g., a multiple myeloma), a prostate cancer (including advanced prostate cancer), a breast cancer (e.g., a breast cancer that does not express one, two or all of estrogen receptor, progesterone receptor, or Her2/neu, e.g., a triple negative breast cancer), a colorectal cancer, a pancreatic cancer, a head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC), a brain cancer (e.g., a glioblastoma), an endometrial cancer, an anal cancer, a gastro-esophageal cancer, a thyroid cancer (e.g., anaplastic thyroid carcinoma), a cervical cancer, a neuroendocrine tumor (NET) (e.g., an atypical pulmonary carcinoid tumor), a lymphoproliferative disease (e.g., a post-transplant lymphoproliferative disease) or a hematological cancer, T-cell lymphoma, B-cell lymphoma, a non-Hodgkin lymphoma, or a leukemia (e.g., a myeloid leukemia or a lymphoid leukemia). In one embodiment, the cancer is ovarian cancer. In another embodiment, the cancer is melanoma.
The cells that are derived from the subject to be treated are then treated ex vivo to induce senescence. In one embodiment, the cancer cells are treated ex vivo with a chemotherapeutic agent or radiation to induce senescence resulting in TIS cells. In one embodiment, the chemotherapeutic agent is a TOP1 or TOP2 inhibitor, or both. In another embodiment, the TOP1 or TOP2 inhibitor, or both, is administered in addition to another chemotherapeutic agent, or agents.
Chemotherapeutic agents (e.g., anti-cancer agents) are well known in the art and include, but are not limited to, anthracenediones (anthraquinones) such as anthracyclines (e.g., daunorubicin (daunomycin; rubidomycin), doxorubicin, epirubicin, idarubicin, and valrubicin), mitoxantrone, and pixantrone; platinum-based agents (e.g., cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin, nedaplatin, triplatin, and lipoplatin); tamoxifen and metabolites thereof such as 4-hydroxytamoxifen (afimoxifene) and N-desmethyl-4-hydroxytamoxifen (endoxifen); taxanes such as paclitaxel (taxol) and docetaxel; alkylating agents (e.g., nitrogen mustards such as mechlorethamine (HN2), cyclophosphamide, ifosfamide, melphalan (L-sarcolysin), and chlorambucil); ethylenimines and methylmelamines (e.g., hexamethylmelamine, thiotepa, alkyl sulphonates such as busulfan, nitrosoureas such as carmustine (BCNU), lomustine (CCNLJ), semustine (methyl-CCN-U), and streptozoein (streptozotocin), and triazenes such as decarbazine (DTIC; dimethyltriazenoimidazolecarboxamide)); antimetabolites (e.g., folic acid analogues such as methotrexate (amethopterin), pyrimidine analogues such as fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluorodeoxyuridine; FUdR), and cytarabine (cytosine arabinoside), and purine analogues and related inhibitors such as mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; 6-TG), and pentostatin (2′-deoxycofonnycin)); natural products (e.g., vinca alkaloids such as vinblastine (VLB) and vincristine, epipodophyllotoxins such as etoposide and teniposide, and antibiotics such as dactinomycin (actinomycin D), bleomycin, plicamycin (mithramycin), and mitomycin (mitomycin Q); enzymes such as L-asparaginase; biological response modifiers such as interferon alpha); substituted ureas such as hydroxyurea; methyl hydrazine derivatives such as procarbazine (N-methylhydrazine; MIH); adrenocortical suppressants such as mitotane (o,p′-DDD) and aminoglutethimide; analogs thereof derivatives thereof and combinations thereof. In one embodiment, chemotherapeutic agents include topoisomerase inhibitors. In one embodiment, the topoisomerase inhibitor is a topoisomerase 1 (also called TOP1) inhibitor. In another embodiment, the topoisomerase inhibitor is a topoisomerase 2 (also called TOP2) inhibitor. In one embodiment, the topoisomerase inhibitor is a pan-topoisomerase 1 (also called pan-TOP) inhibitor. In another embodiment, the chemotherapeutic agent is cisplatin.
Inhibitors of TOP1 include, but are not limited to irinotecan; irinotecan hydrochloride; camptothecin; topotecan; topotecan hydrochloride (Hycamtin, commercially available); indimitecan (LMP776) (Purdue); indotecan (LMP400) (Purdue); Genz-644282 (Genzyme); ONZEALD (etirinotecan pegol) (Nektar Therapeutics); gimatecan (Lee's Pharmaceutical Holdings, LLC); PEG-irinotecan (3SBio Inc.); belotecan hydrochloride (Chong Kun Dang Pharmaceutical Corp); IT-141 (Intezyne Inc); TBX.CE+irinotecan hydrochloride (irinotecan hydrochloride) (TheraBiologics Inc); PLX-038(ProLynx LLC); LMP-744 (Gibson Oncology LLC); Sinotecan (Jiangsu Chia-tai Tianqing Pharmaceutical Co Ltd); 2X-131 (Oncology Venture U.S. Inc); HSSYO-001 (Sichuan Sinovation Bio-technology Co Ltd); AR-67 (Vivacitas Oncology Inc); rubitecan (Vivacitas Oncology Inc); NK-012 (Nippon Kayaku Co Ltd); simmitecan hydrochloride (Shanghai Haihe Biopharma Co Ltd); ATT-1 IT (Aposense Ltd); BACPT-DP (DEKK-TEC Inc); APH-0201 (Aphios Corp); CZ-150729 (NMT Pharmaceuticals Pte Ltd); SNB-101 (SN BioScience); Small Molecule to Inhibit DNA Topoisomerase I for Lung Cancer (CAO Pharmaceuticals Inc); TRX-920 (TaiRx Inc); CBX-12 (Cybrexa Inc); moeixitecan (Jiangsu Chia-tai Tianqing Pharmaceutical Co Ltd), BAX 2398; BAX-2398; BAX2398; irinotecan hydrochloride nanoliposomal; irinotecan nydrochloride; Irinotecan liposome injection; irinotecan sucrosofate liposomal; irinotecan; liposomal irinotecan sucrosulfate; MM 398; MM-398; MM398; nal-IRI; nanoliposomal irinotecan hydrochloride; nanoliposomal irinotecan; onivyde; PEP 02; PEP-02; PEP02; SHP 673; SHP-673; SHP673; PEG-irinotecan; CZ-48; IT-141; NLG-207; PEN-866; BO-1978; LMP-135; PCS-11T (SN-38 prodrug); ZBH-01; DFP 13318; DFP-13318; DFP13318; PEG-SN-38 conjugate; Pegylated form of SN38; PEGylated-SN-38 conjugate; PL 0264; PL-0264; PL0264; PLX 0264; PLX 038; PLX-0264; PLX0264; PLX038; and Ultra-long acting PEG-SN-38. See also, e.g., Burton et al. Clin Cancer Res. 2018 Dec. 1; 24(23):5830-5840, which is incorporated herein by reference.
Inhibitors of TOP2 include, without limitation, teniposide, daunorubicin, aurintricarboxylic acid, HU-331, etoposide, doxorubicin, mitoxantrone, dexrazoxane, aclarubicin, amsacrine, and ellipticine. Other suitable TOP2 inhibitors include, without limitation, iodoquinol+metronidazole, iodoquinol+sulfaguanidine, amsacrine, fleroxacin, idarubicin hydrochloride, teniposide, 2X-111 (Glutathione-enhanced, PEGylated Liposomal Doxorubicin, previously named 2B3-101) (Allarity Therapeutics A/S), Aldoxorubicin (ImmunityBio Inc), Annamycin (Moleculin Biotech Inc), Idronoxil (NXP-001) (Noxopharm Ltd), epirubicin, camsirubicin (MNPR-201; GPX-150; 5-imino-13-deoxydoxorubicin; analog of doxorubicin) (Monopar Therapeutics Inc), pegylated doxorubicin (GP Pharm SA), pirarubicin, pixantrone dimaleate (Servier Laboratories Ltd), Razoxane (TRP-1001) (Tryp Therapeutics Inc), SQ-3370 (Shasgi Inc), and Vosaroxin (DB106) (Denovo Biopharma LLC). See also, e.g., Saleh et al, Reversibility of chemotherapy-induced senescence is independent of autophagy and a potential model for tumor dormancy and cancer recurrence, BioRxiv, doi: 10.1101/099812, posted Jan. 11, 2017. This document is incorporated herein by reference.
In one embodiment, the cancer cells are treated with the chemotherapeutic agent prior to treatment with the TOP inhibitor. In another embodiment, the cancer cells are treated with the chemotherapeutic agent after treatment with the TOP inhibitor. In yet another embodiment, the cancer cells are treated with the chemotherapeutic agent essentially simultaneously with treatment with the TOP inhibitor, or the treatment periods overlap.
In one embodiment, the cancer cells are treated with more than one chemotherapeutic agent, optionally in conjunction with radiation. The cells are treated with an effective amount of the chemotherapeutic agent or agents, and/or radiation, suitable to produce senescence in the cancer cells, resulting in TIS cells. In one embodiment, the cells are treated ex vivo with a combination of an inhibitor of TOP1, TOP2, or both and cisplatin. In one embodiment, the TOP1 inhibitor is irinotecan. In one embodiment, the TOP1 inhibitor is camptothecin. In one embodiment, the TOP2 inhibitor is etoposide.
The cells are treated with an effective amount of the chemotherapeutic agent and the inhibitor of TOP1, TOP2, or both. It should be understood that the “effective amount” for the chemotherapeutic agent or agents or TOP inhibitor(s) may vary depending upon the agent(s) selected for use in the method, and may be determined by the person of skill in the art. In one embodiment an effective amount for the chemotherapeutic agent or TOP inhibitor includes without limitation about 0.1 μM to about 100 μM. In one embodiment, the range of effective amount is 0.001 to 0.01 μM. In another embodiment, the range of effective amount is 0.001 to 0.1 μM. In another embodiment, the range of effective amount is 0.001 to 1 μM. In another embodiment, the range of effective amount is 0.001 to 10 μM. In another embodiment, the range of effective amount is 0.001 to 20 μM. In another embodiment, the range of effective amount is 0.01 to 25 μM. In another embodiment, the range of effective amount is 0.01 to 0.1 μM. In another embodiment, the range of effective amount is 0.01 to 1 μM. In another embodiment, the range of effective amount is 0.01 to 10 μM. In another embodiment, the range of effective amount is 0.01 to 20 μM. In another embodiment, the range of effective amount is 0.1 to 25 μM. In another embodiment, the range of effective amount is 0.1 to 1 μM. In another embodiment, the range of effective amount is 0.1 to 10 μM. In another embodiment, the range of effective amount is 0.1 to 20 μM. In another embodiment, the range of effective amount is 1 to μM. In another embodiment, the range of effective amount is 1 to μM. In another embodiment, the range of effective amount is 1 to 10 μM. In another embodiment, the range of effective amount is 1 to 20 μM. In another embodiment, the range of effective amount is 5 to 15 μM. In another embodiment, the range of effective amount is about 10 μM. Still other doses falling within these ranges are expected to be useful. The effective amount of the chemotherapeutic agent(s) and TOP inhibitor(s) may be individually chosen based on the agents selected and other factors, e.g., number of cells being treated, type of cancer, etc.
In one embodiment, the cells which have been removed from the subject are contacted with a chemotherapeutic agent for a time sufficient to induce senescence. The time may range from minutes to days or weeks. In one embodiment, the cells are contacted with the chemotherapeutic agent for about 5 minutes to about 4 weeks. In another embodiment, the cells are contacted with the chemotherapeutic agent for about 1 hour to about 2 weeks. In one embodiment, the cells are contacted with the chemotherapeutic agent for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. In another embodiment, the cells are contacted with the chemotherapeutic agent for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 days. The chemotherapeutic agent may be administered more than once in the treatment period.
In one embodiment, the cells which have been removed from the subject are contacted with a TOP1 inhibitor, a TOP2 inhibitor, or both for a time sufficient to induce the release of cytokines, chemokines, and/or other factors associated with senescence. The time may range from minutes to days or weeks. In one embodiment, the cells are contacted with the TOP1 inhibitor, a TOP2 inhibitor, or both for about 5 minutes to about 4 weeks. In another embodiment, the cells are contacted with the TOP1 inhibitor, a TOP2 inhibitor, or both for about 1 hour to about 2 weeks. In one embodiment, the cells are contacted with the TOP1 inhibitor, a TOP2 inhibitor, or both for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. In another embodiment, the cells are contacted with the TOP1 inhibitor, a TOP2 inhibitor, or both for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 days. The TOP inhibitor may be administered more than once in the treatment period.
In one embodiment, the methods described herein include identifying whether the ex vivo treated cells have obtained senescence. In one embodiment, cells are sorted into senescent and non-senescent cells. Such techniques include sorting by size and/or granularity (Meng et al, Radiation-inducible Immunotherapy for Cancer: Senescent Tumor Cells as a Cancer Vaccine Molecular Therapy, 20(5):1046-1055, May 2012) (larger size than senescent cells), cyclin A expression (negative for senescent cells), SASP expression (increased for senescent cells), and/or TOP1cc level (increased for senescent cells).
In one embodiment, that involves detection of a senescence-associated secretory phenotype (SASP) in the treated cells. The SASP includes several families of soluble and insoluble factors including, those in the table below. Detection of the SASP may include detection of one or more of the following factors: TECK, ENA-78, I-309, I-TAC, GM-CSE, G-CSE, IFN-γ, BLC, MIF, amphiregulin, epiregulin, heregulin, EGF, bFGF, HGF, KGF (FGF7), VEGF, angiogenin, SCF, SDF-1, PIGF, NGF, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6, IGFBP-7, MMP-1, MMP-3, MMP-10, MMP-12, MMP-13, MMP-14, TIMP-1, TIMP-2, PAI-1, PAI-2, tPA, uPA, cathepsin B, ICAM-1, ICAM-3, OPG, sTNFRI, TRAIL-R2, Fas, and sTNFRII. See, e.g., Coppe et al, The Senescence-Associated Secretory Phenotype: The Dark Side of Tumor Suppression, Annu Rev Pathol. 2010; 5: 99-118, which is incorporated by reference herein.
In another embodiment, the level of TOPcc is detected in the cells wherein an increase in TOPcc levels is indicative of senescence in the cells. In yet another embodiment, the treated cancer cells are assayed for SA-β-Gal to detect senescence. It is contemplated that in some of the methods, a portion of the treated cells are used as a measurement to determine whether senescence has been obtained. In some embodiments, these cells may be stained and/or fixed, and thus, not suitable for readministration into the subject. In other embodiments, the cells are stained and/or sorted, and administered to the subject.
The TIS cells or a pharmaceutical composition containing the TIS cells are then administered to the subject. The cells may be administered using any suitable route of administration. For example, compositions may be administered via intravenous, parenteral, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisteral, intraperitoneal, intranasal, or aerosol administration. The route of administration may be determined by the person of skill based on various factors including without limitation the type of cancer being treated. In one embodiment, the TIS cells are injected in the area where the cancer is/was located. In another embodiment, the TIS cells are administered intravenously.
In one embodiment, the effective amount of the TIS cells ranges from about 1 cell to about 100,000,000 cells, including all integers or fractional amounts within the range. In one embodiment, the effective amount of the TIS cells ranges from about 10,000 cells to about 10,000,000 cells, including all integers or fractional amounts within the range. In one embodiment, the effective amount of the TIS cells ranges from about 100,000 cells to about 5,000,000 cells, including all integers or fractional amounts within the range. Other ranges and dosages may be determined by the person of skill taking into account various factors including, without limitation, the type of cancer, the size of the subject, etc.
In one embodiment, the subject is administered a checkpoint inhibitor in addition to the TIS cells. Immune checkpoints represent significant barriers to activation of functional cellular immunity in cancer, and antagonistic antibodies specific for inhibitory ligands on T cells including CTLA4 and programmed death-1 (PD-1) are examples of targeted agents being evaluated in the clinics. In one embodiment, the subject has previously received checkpoint therapy, prior to receiving TIS cell therapy. The subject may, in some embodiments, receive the same or different checkpoint therapy after administration of the TIS cells.
Immune checkpoint molecules that may be targeted for blocking or inhibition include, but are not limited to, CTLA-4, 4-1BB (CD137), 4-1BBL (CD137L), PDL1, PDL2, PD1, CD134, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, 2B4, CD160 (also referred to as BY55) and CGEN-15049. In one embodiment, the checkpoint inhibitor is PD-1 inhibitor. In another embodiment, the checkpoint inhibitor is PD-L1 inhibitor.
Immune checkpoint inhibitors include antibodies, or antigen binding fragments thereof, or other binding proteins, that bind to and block or inhibit the activity of one or more of CTLA-4, PDL1, PDL2, PD1, CD134, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, 2B4, CD160 and CGEN-15049. Suitable immune checkpoint inhibitors include those that block PD-1, such as pembrolizumab, nivolumab, AGEN 2034, BGB-A317, BI-754091, CBT-501 (genolimzumab), MEDI0680, MGA012, PDR001, PF-06801591, REGN2810 (SAR439684), and TSR-042. MK-3475 (PD-1 blocker) Nivolumab, CT-011 Immune checkpoint inhibitors also include those that block PD-L1, such as durvalumab, atezolizumab, avelumab, and CX-072. Other suitable inhibitors include Anti-B7-H1 (MEDI4736), AMP224, BMS-936559, MPLDL3280A, and MSB0010718C.
Suitable immune checkpoint inhibitors include those that block CTLA-4, such as AGEN 1884, ipilimumab, and tremelimumab.
In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, an anti-CD28 antibody, an anti-TIGIT antibody, an anti-LAGS antibody, an anti-TIM3 antibody, an anti-GITR antibody, an anti-4-1BB antibody, or an anti-OX-40 antibody. In some embodiments, the additional therapeutic agent is an anti-TIGIT antibody. In some embodiments, the additional therapeutic agent is an anti-LAG-3 antibody selected from the group consisting of: BMS-986016 and LAG525. In some embodiments, the additional therapeutic agent is an anti-OX-40 antibody selected from: MEDI6469, MEDI0562, and MOXR0916. In some embodiments, the additional therapeutic agent is the anti-4-1BB antibody PF-05082566. The present disclosure provides compositions and methods that include blockade of immune checkpoints. Immune checkpoints are molecules in the immune system that either turn up a signal (e.g., co-stimulatory molecules) or turn down a signal. Inhibitory checkpoint molecules that may be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer-cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAGS), programmed death 1 (PD-1), T-cell immunoglobulin domain and mucin domain 3 (TIM-3) and V-domain Ig suppressor of T cell activation (VISTA). In particular, the immune checkpoint inhibitors target the PD-1 axis and/or CTLA-4.
The immune checkpoint inhibitors may be drugs such as small molecules, recombinant forms of ligand or receptors, or, in particular, are antibodies, such as human antibodies (e.g., International Patent Publication WO2015016718; Pardoll, Nat Rev Cancer, 12(4): 252-64, 2012; both incorporated herein by reference). Known inhibitors of the immune checkpoint proteins or analogs thereof may be used, in particular chimerized, humanized or human forms of antibodies may be used. As the skilled person will know, alternative and/or equivalent names may be in use for certain antibodies mentioned in the present disclosure. Such alternative and/or equivalent names are interchangeable in the context of the present invention. For example, it is known that lambrolizumab is also known under the alternative and equivalent names MK-3475 and pembrolizumab.
In addition, more than one immune checkpoint inhibitor (e.g., anti-PD-1 antibody and anti-CTLA-4 antibody) may be used in combination with the TIS cells.
The subject is treated with an effective amount of the checkpoint inhibitor. It should be understood that the “effective amount” for the checkpoint inhibitor may vary depending upon the agent(s) selected for use in the method, and may be determined by the person of skill in the art. In one embodiment an effective amount for the checkpoint inhibitor includes without limitation about 1 μg to about 25 mg. In one embodiment, the range of effective amount is 0.001 to 0.01 mg. In another embodiment, the range of effective amount is 0.001 to 0.1 mg. In another embodiment, the range of effective amount is 0.001 to 1 mg. In another embodiment, the range of effective amount is 0.001 to 10 mg. In another embodiment, the range of effective amount is 0.001 to 20 mg. In another embodiment, the range of effective amount is 0.01 to 25 mg. In another embodiment, the range of effective amount is 0.01 to 0.1 mg. In another embodiment, the range of effective amount is 0.01 to 1 mg. In another embodiment, the range of effective amount is 0.01 to 10 mg. In another embodiment, the range of effective amount is 0.01 to 20 mg. In another embodiment, the range of effective amount is 0.1 to 25 mg. In another embodiment, the range of effective amount is 0.1 to 1 mg. In another embodiment, the range of effective amount is 0.1 to 10 mg. In another embodiment, the range of effective amount is 0.1 to 20 mg. In another embodiment, the range of effective amount is 1 to 25 mg. In another embodiment, the range of effective amount is 1 to 5 mg. In another embodiment, the range of effective amount is 1 to 10 mg. In another embodiment, the range of effective amount is 1 to 20 mg. Still other doses falling within these ranges are expected to be useful. The effective amount of the checkpoint inhibitor may be individually chosen based on the agent selected and other factors, e.g., size of the patient, type of cancer, etc.
In one embodiment, the TIS cells and checkpoint inhibitor are administered approximately simultaneously. In another embodiment, the TIS cells are administered prior to checkpoint inhibitor. In another embodiment, the TIS cells are administered subsequent to the checkpoint inhibitor.
In another embodiment, the method includes administering a chemotherapeutic agent to the subject in addition to the TIS cells, and optionally with a checkpoint inhibitor.
The chemotherapeutic agents, TIS cells and checkpoint inhibitors may be administered using any suitable route of administration. For example, compositions may be administered via intravenous, parenteral, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisteral, intraperitoneal, intranasal, or aerosol administration. The route of administration for each composition (e.g., TIS cells, chemotherapeutic agents, checkpoint inhibitors) may be determined individually and may be the same or different.
The compositions described herein (e.g., TIS cells, chemotherapeutic agents, checkpoint inhibitors) are administered in an amount that is sufficient to treat or prevent the disease or disorder, or to treat the symptoms of the disease or disorder, in a subject. The combination of substances (or compounds) is preferably a synergistic combination. Synergy, as described for example by Chou and Talalay, Adv. Enzyme Regul., 22:27 (1984), occurs when the effect of the compounds when administered in combination is greater than the additive effect of the compounds when administered alone as a single agent. In general, a synergistic effect is most clearly demonstrated at suboptimal concentrations of the compounds. Synergy can be in terms of lower cytotoxicity, increased activity, or some other beneficial effect of the combination compared with the individual components.
Other therapeutic benefits or beneficial effects provided by the methods described herein may be objective or subjective, transient, temporary, or long-term improvement in the condition or pathology, or a reduction in onset, severity, duration or frequency of an adverse symptom associated with or caused by cell proliferation or a cellular hyperproliferative disorder such as a neoplasia, tumor or cancer, or metastasis. A satisfactory clinical endpoint of a treatment method in accordance with the invention is achieved, for example, when there is an incremental or a partial reduction in severity, duration or frequency of one or more associated pathologies, adverse symptoms or complications, or inhibition or reversal of one or more of the physiological, biochemical or cellular manifestations or characteristics of cell proliferation or a cellular hyperproliferative disorder such as a neoplasia, tumor or cancer, or metastasis.
A therapeutic benefit or improvement therefore may be a cure, such as destruction of target proliferating cells (e.g., neoplasia, tumor or cancer, or metastasis) or ablation of one or more, most or all pathologies, adverse symptoms or complications associated with or caused by cell proliferation or the cellular hyperproliferative disorder such as a neoplasia, tumor or cancer, or metastasis. However, a therapeutic benefit or improvement need not be a cure or complete destruction of all target proliferating cells (e.g., neoplasia, tumor or cancer, or metastasis) or ablation of all pathologies, adverse symptoms or complications associated with or caused by cell proliferation or the cellular hyperproliferative disorder such as a neoplasia, tumor or cancer, or metastasis. For example, partial destruction of a tumor or cancer cell mass, or a stabilization of the tumor or cancer mass (in terms of size or cell numbers) by inhibiting progression or worsening of the tumor or cancer, can reduce mortality and prolong lifespan even if only for a few days, weeks or months, even though a portion or the bulk of the tumor or cancer mass remains.
A reduction or inhibition of cancer can be measured relative to the incidence observed in the absence of the treatment and, in further testing, inhibits tumor growth. The tumor inhibition can be quantified using any convenient method of measurement. Tumor growth can be reduced by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater.
In another aspect, a pharmaceutical composition comprising the TIS cells as described herein, is provided. In one embodiment, the pharmaceutical composition includes a pharmaceutically acceptable carrier, excipient, adjuvant, or diluent. The pharmaceutical composition may further comprise an immune checkpoint inhibitor or chemotherapeutic agent, as described herein. Any reference herein to administration of TIS cells also refers, in one embodiment, to administration of a pharmaceutical composition comprising TIS cells.
In yet another aspect, a pharmaceutical composition produced by the following method is provided. The method includes obtaining cancer cells from a subject; and treating the cancer cells ex vivo with an inhibitor of TOP1, TOP2, or both to produce therapy induced senescent (TIS) cells, as described more fully herein. In one embodiment, the method includes obtaining cancer cells from a subject; and treating the cancer cells ex vivo with a chemotherapeutic agent and an inhibitor of TOP1, TOP2, or both to produce therapy induced senescent (TIS) cells, as described more fully herein. The method includes optionally sorting the cells for senescence or confirming senescence. In one embodiment, the cells are sorted for senescence by size and/or granularity. See, e.g., Meng et al, Radiation-inducible Immunotherapy for Cancer: Senescent Tumor Cells as a Cancer Vaccine Molecular Therapy, 20(5):1046-1055, May 2012, which is incorporated herein by reference.
In one embodiment, the cells are collected from the patient. The cells may be pooled, concentrated, enriched or expanded to increase the number of cells available for treatment, using techniques known in the art, and described herein.
By “pharmaceutically acceptable carrier, excipient, or diluent” is meant a solid and/or liquid carrier, in in dry or liquid form and pharmaceutically acceptable. The compositions are typically sterile solutions or suspensions. Examples of excipients which may be combined with the TIS cells include, without limitation, solid carriers, liquid carriers, adjuvants, amino acids (glycine, glutamine, asparagine, arginine, lysine), antioxidants (ascorbic acid, sodium sulfite or sodium hydrogen-sulfite), binders (gum tragacanth, acacia, starch, gelatin, polyglycolic acid, polylactic acid, poly-d,l-lactide/glycolide, polyoxaethylene, polyoxapropylene, polyacrylamides, polymaleic acid, polymaleic esters, polymaleic amides, polyacrylic acid, polyacrylic esters, polyvinylalcohols, polyvinylesters, polyvinylethers, polyvinylimidazole, polyvinylpyrrolidon, or chitosan), buffers (borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids), bulking agents (mannitol or glycine), carbohydrates (such as glucose, mannose, or dextrins), clarifiers, coatings (gelatin, wax, shellac, sugar or other biological degradable polymers), coloring agents, complexing agents (caffeine, polyvinylpyrrolidone, β-cyclodextrin or hydroxypropyl-β-cyclodextrin), compression aids, diluents, disintegrants, dyes, emulsifiers, emollients, encapsulating materials, fillers, flavoring agents (peppermint or oil of wintergreen or fruit flavor), glidants, granulating agents, lubricants, metal chelators (ethylenediamine tetraacetic acid (EDTA)), osmo-regulators, pH adjustors, preservatives (benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, hydrogen peroxide, chlorobutanol, phenol or thimerosal), solubilizers, sorbents, stabilizers, sterilizer, suspending agent, sweeteners (mannitol, sorbitol, sucrose, glucose, mannose, dextrins, lactose or aspartame), surfactants, syrup, thickening agents, tonicity enhancing agents (sodium or potassium chloride) or viscosity regulators. See, the excipients in “Handbook of Pharmaceutical Excipients”, 5th Edition, Eds.: Rowe, Sheskey, and Owen, APhA Publications (Washington, D.C.), 2005 and U.S. Pat. No. 7,078,053, which are incorporated herein by reference. The selection of the particular excipient is dependent on the nature of the compound selected and the particular form of administration desired.
Solid carriers include, without limitation, starch, lactose, dicalcium phosphate, microcrystalline cellulose, sucrose and kaolin, calcium carbonate, sodium carbonate, bicarbonate, lactose, calcium phosphate, gelatin, magnesium stearate, stearic acid, or talc. Fluid carriers without limitation, water, e.g., sterile water, Ringer's solution, isotonic sodium chloride solution, neutral buffered saline, saline mixed with serum albumin, organic solvents (such as ethanol, glycerol, propylene glycol, liquid polyethylene glycol, dimethylsulfoxide (DMSO)), oils (vegetable oils such as fractionated coconut oil, arachis oil, corn oil, peanut oil, and sesame oil; oily esters such as ethyl oleate and isopropyl myristate; and any bland fixed oil including synthetic mono- or diglycerides), fats, fatty acids (include, without limitation, oleic acid find use in the preparation of injectables), cellulose derivatives such as sodium carboxymethyl cellulose, and/or surfactants.
Pharmaceutical compositions may be formulated for any appropriate route of administration. For example, compositions may be formulated for intravenous, parenteral, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisteral, intraperitoneal, intranasal, or aerosol administration. In some embodiments, pharmaceutical compositions are formulated for direct delivery to the tumor (intratumoral) or to the tumor environment. In another embodiment, pharmaceutical compositions are formulated for delivery to the lymph nodes.
Pharmaceutical compositions may be in the form of liquid solutions or suspensions (as, for example, for intravenous administration, for oral administration, etc.). Alternatively, pharmaceutical compositions may be in solid form (e.g., in the form of tablets or capsules, for example for oral administration). In some embodiments, pharmaceutical compositions may be in the form of powders, drops, aerosols, etc. Methods and agents well known in the art for making formulations are described, for example, in “Remington's Pharmaceutical Sciences,” Mack Publishing Company, Easton, Pa. Formulations may, for example, contain excipients, diluents such as sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes.
In one embodiment, the effective amount of the TIS cells ranges from about 1 cell to about 100,000,000 cells, including all integers or fractional amounts within the range. In one embodiment, the effective amount of the TIS cells ranges from about 10,000 cells to about 10,000,000 cells, including all integers or fractional amounts within the range. In one embodiment, the effective amount of the TIS cells ranges from about 100,000 cells to about 5,000,000 cells, including all integers or fractional amounts within the range. Other ranges and dosages may be determined by the person of skill taking into account various factors including, without limitation, the type of cancer, the size of the subject, etc.
In one embodiment, the above amounts represent a single dose. In another embodiment, the above amounts define an amount delivered to the subject per day. In another embodiment, the above amounts define an amount delivered to the subject per day in multiple doses. In still other embodiments, these amounts represent the amount delivered to the subject over more than a single day.
Throughout this specification, the words “comprise”, “comprises”, and “comprising” are to be interpreted inclusively rather than exclusively. The words “consist”, “consisting”, and its variants, are to be interpreted exclusively, rather than inclusively. It should be understood that while various embodiments in the specification are presented using “comprising” language, under various circumstances, a related embodiment is also be described using “consisting of” or “consisting essentially of” language.
The term “a” or “an”, refers to one or more, for example, “a biomarker,” is understood to represent one or more biomarkers. As such, the terms “a” (or “an”), “one or more,” and “at least one” are used interchangeably herein.
As used herein, the term “about” means a variability of 10% from the reference given, unless otherwise specified.
The invention is now described with reference to the following examples. These examples are provided for the purpose of illustration only. The compositions, experimental protocols and methods disclosed and/or claimed herein can be made and executed without undue experimentation in light of the present disclosure. The protocols and methods described in the examples are not considered to be limitations on the scope of the claimed invention. Rather this specification should be construed to encompass any and all variations that become evident as a result of the teaching provided herein. One of skill in the art will understand that changes or variations can be made in the disclosed embodiments of the examples, and expected similar results can be obtained. For example, the substitutions of reagents that are chemically or physiologically related for the reagents described herein are anticipated to produce the same or similar results. All such similar substitutes and modifications are apparent to those skilled in the art and fall within the scope of the invention.
Cyclic cGMP-AMP synthase (cGAS) is a pattern recognition cytosolic DNA sensor that is essential for cellular senescence. cGAS promotes inflammatory senescence-associated secretory phenotype (SASP) through recognizing cytoplasmic chromatin during senescence. cGAS-mediated inflammation is essential for the antitumor effects of immune checkpoint blockade. However, the mechanism by which cGAS recognizes cytoplasmic chromatin is unknown. Here we show that topoisomerase 1-DNA covalent cleavage complex (TOP Ice) is both necessary and sufficient for cGAS-mediated cytoplasmic chromatin recognition and SASP during senescence. TOP1cc localizes to cytoplasmic chromatin and TOP1 interacts with cGAS to enhance the binding of cGAS to DNA. Retention of TOP1cc to cytoplasmic chromatin depends on its stabilization by the chromatin architecture protein HMGB2. Functionally, the HMGB2-TOP1cc-cGAS axis determines the response of orthotopically transplanted ex vivo therapy-induced senescent cells to immune checkpoint blockade in vivo. Together, these findings establish a HMGB2-TOP1cc-cGAS axis that enables cytoplasmic chromatin recognition and response to immune checkpoint blockade.
IMR90 human diploid fibroblasts were cultured according to American Type Culture Collection (ATCC) under low oxygen tension (2%) in DMEM (4.5 g per liter glucose) supplemented with 10% fetal bovine serum (FBS), L-glutamine (Thermo Fisher. Cat. No: 25030081), sodium pyruvate (Thermo Fisher. Cat. No: 11360070), nonessential amino acid (Thermo Fisher. Cat. No: 11140-050), sodium bicarbonate (Gibco. Cat. No: 25080094) and 1% penicillin/streptomycin (Corning. Cat. No: 30-002-CI). All experiments were performed on IMR90 fibroblasts cultured between the population doubling of 25 and 35. Human ovarian cancer cell line OVCAR3 obtained from ATCC and mouse ovarian cancer cell line ID8-Defb29 Vegf-a gifted by Dr. Jose R. Conejo-Garcia were cultured in RPMI 1640 supplemented with 10% FBS and 1% penicillin/streptomycin. The mouse ovarian cancer cell lines UPK10 and ID8 were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. These cell lines are authenticated at The Wistar Institute's Genomics Facility using short tandem repeat DNA profiling. Regular mycoplasma testing was performed using the LookOut Mycoplasma polymerase chain reaction (PCR) detection (Sigma, Cat. No: MP0035).
Etoposide was purchased from Sigma (Cat. No: E1383). Cisplatin was purchased from Selleck (Cat. No: S1166). Doxycycline was purchased from Selleck (Cat. No: S4163). Camptothecin was purchased from Selleck (Cat. No: S1288). 4′ 6-Diamidino-2-phenylindole dihydrochloride (DAPI) was purchased from Sigma (Cat. No: D9542). Cytochalasin B was purchased from Sigma (Cat. No: C6762). Spermidine was purchased from Sigma (Cat. No: S2626). Formaldehyde solution was purchased from Sigma (Cat. No: F8775). Paraformaldehyde (PFA) was purchased from Sigma (Cat. No: 158127). The DNA ladder was purchased from ThermoFisher (Cat. No: SM1333). Benzonase was purchased from Sigma (Cat. No: E1014).
The pMSCVpuro-eGFP-hcGAS, pBABE-puro-H-RASG12V, pBABE-puro-Empty and pGEX6P1-GST-cGAS plasmids were obtained from Addgene. pLKO.1-shHMGB2 (shHMGB2 #1: TRCN000000150009; shHMGB2 #2: TRCN0000019011) and pLKO.1-shTOP1 (TRCN0000059090) were obtained from the Molecular Screening Facility at the Wistar Institute. pLKO.1-shcGAS short hairpins were purchased from Sigma (TRCN0000146282, TRCN0000149984).pLentiCRISPR-HMGB2 was constructed by inserting the HMGB2 guide RNA (gRNA; 5′-AACACCCTGGCCTATCCA TT-3′(SEQ ID NO: 1)) as we previously published15. Tet-pLKO-puro-shHMGB2 was constructed using the Tet-pLKO-puro backbone (Addgene. Cat. No: 21915) and shHMGB2 sequence (forward: 5′-CCGGGCTCAACATTAGCTTCAGTATCTCGAGATAC TGAAGCTAATGTTGAGCTTTTTG-3′ (SEQ ID NO: 2); reverse: 5′-AATTCAAAAAGCTCAACATTAGCTTCAGTATCTCGA GATACTGAAGCTAATGTTGAGC-3′(SEQ ID NO: 3)).
Recombinant cGAS protein was purchased from Cayman (Cat. No: 22810). Recombinant HMGB2 protein was purchased from Prospec (Cat. No: PRO-888). Recombinant TOP1 protein was purchased from Prospec (Cat. No: ENZ-306). Recombinant TOP1 Y723F mutant protein was purchased from Speed Biosystems (Cat. No: OP10402). Recombinant his-tagged TOP1 protein was purchased from Sino Biological (Cat. No: 17455-H07B). ATP Solution (100 mM) was purchased from Thermo Fisher (Cat. No: R0441). GTP Solution (100 mM) was purchased from Thermo Fisher (Cat. No: R0461). SYBR™ Green I Nucleic Acid Gel Stain was purchased from Thermo Fisher (Cat. No: S7563). (ISD)2 interferon stimulatory double strand DNA (dsDNA) was purchased from InvivoGen (Cat. No: tlrl-isdn).
The following antibodies were purchased from the indicated suppliers and used for immunoblotting or immunostaining at the indicated concentrations: mouse monoclonal anti-γH2AX (clone JBW301) (Millipore. Cat. No: 05-636), 1:500 for immunofluorescence; rabbit monoclonal anti-γH2AX (20E3) (Cell Signaling Technology. Cat. No: 9718), 1:500 for immunofluorescence; Alexa Fluor® 594 anti-γH2AX (2F3) (Biolegend. Cat. No: 613410), 1:200 for immunofluorescence; rabbit polyclonal anti-HMGB2 (Abcam. Cat. No: 67282), 1:1000 for immunoblotting and 1:500 for immunofluorescence; mouse monoclonal anti-cGAS (D9) (Santa Cruz. Cat. No: sc-515777), 1:200 for immunofluorescence, 1:1000 for immunoblotting; rabbit monoclonal anti-cGAS (D1D3G) (Cell Signaling Technology. Cat. No: 15102), 1:200 for immunofluorescence, 1:1000 for immunoblotting; rabbit monoclonal anti-STING (D2P2F) (Cell Signaling Technology. Cat. No: 13647S), 1:1000 for immunoblotting, rabbit polyclonal anti-Cyclin A (H432) (Santa Cruz. Cat. No: sc-751), 1:1000 for immunoblotting; mouse monoclonal anti-RAS (BD Biosciences. Cat. No: 610001), 1:1000 for immunoblotting; mouse monoclonal anti-P16 (JC8) (Santa Cruz. Cat. No: sc-56330), 1:1000 for immunoblotting; mouse monoclonal anti-P21 (187) (Santa Cruz. Cat. No: sc-817), 1:1000 for immunoblotting; mouse monoclonal anti-β-actin (Sigma. Cat. No: A2228), 1:10000 for immunoblotting; rabbit polyclonal anti-TOP1 (Proteintech. Cat. No: 20705-1-AP), 1:1000 for immunoblotting and 1:200 for immunofluorescence; mouse monoclonal anti-Topoisomerase I-DNA Covalent Complexes (TOP1cc) (clone 1.1A) (Millipore. Cat. No: MABE1084), 1:1000 for slot blot and 1:200 for immunofluorescence;
For flow cytometric analysis, APC/CY7 anti-CD69 (Cat. No: 104525), APC anti-CD4 (Cat. No: 100516), PE anti-CD8 (Cat. No: 100708), FITC anti-Granzyme B (Cat. No: 372206), PE/Cy7 anti-interferon-gamma (Cat. No: 505825) antibodies were purchased from Biolegend and used at 1:150 dilutions. Zombie yellow dye (Biolegend. Cat. No: 423103, 1:200) was used as a viability staining.
Retrovirus production and transduction were performed using Phoenix cells to package the infection viruses (Dr. Gary Nolan, Stanford University) (Nacarelli et al., 2019). Lentivirus was produced using the ViraPower kit (Invitrogen) based on manufacturer's instructions in the 293FT human embryonal kidney cell line by Lipofectamine 2000 transfection (Thermo Fisher. Cat. No: 11668019). Lentivirus was harvested and filtered with 0.45 μm filter 48 hours post transfection. Cells infected with lentiviruses were selected in 1 μg/ml puromycin 48 hours post infection.
For oncogene-induced senescence, IMR90 cells were infected with retrovirus produced by pBABE-puro-H-RASG12V (Addgene) at 37° C. for 24 hr. A second round of infection was performed on the same target cells. Infected cells were drug-selected using 3 μg/ml puromycin (Nacarelli et al., 2019). For Etoposide-induced senescence, IMR90 or OVCAR3 cells at approximately 60-70% confluency were treated with 50 μM or 2 μM Etoposide for 48 hours. The treated cells were cultured in fresh medium and harvested at day 8. For Cisplatin-induced senescence, OVCAR3 or ID8-Defb29/Vegf-a cells at approximately 60-70% confluency were treated with 2 μM Cisplatin for 48 hours. The treated cells were cultured in fresh medium and harvested at day 8.
SA-β-Gal staining was performed as previously described (Nacarelli et al., 2019). Briefly, cells were fixed for 5 mins at room temperature in 2% formaldehyde/0.2 glutaraldehyde in PBS. After washing twice with PBS, cells were stained at 37° C. overnight in a non-CO2 incubator in staining solution (40 mM Na2HPO4, pH 6.0, 150 mM NaCl, 2 mM MgCl2, 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, and 1 mg/ml X-gal. After counterstaining with Nuclear Fast Red solution (Ricca, Cat. No: R5463200500), slides were subjected to an alcohol dehydration series and mounted with Permount (FisherScientific. Cat. No: SP15-100). Slides were examined using a Zeiss AxioImager A2.
UPK10 and ID8 cells were treated with 10 M Cisplatin, 10 M Irinotecan, or a combination for three days. The drugs were then released from drug treatment and cultured for three days or extended period as indicated. The senescent cells were labelled with SPiDER-0 Gal Cellular Senescence Detection Kit (Dojindo, Cat. No: SG02-10) following the manufacture's instruction. Both senescent and non-senescent cells were sorted using flow cytometry.
For cytokine-array analysis, cells were washed once and cultured in serum-free medium for 48 hours (Nacarelli et al., 2019). Conditioned medium was filtered (0.2 μm) and then subjected to cytokine-array assay using Human Cytokine Array C1 kit (RayBiotech. Cat. No: AAH-CYT-1-2) following the manufacturer's guidelines. After collection of conditional media, the cell number of each sample was counted. The intensities of array dots were visualized on film after incubation with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific. Cat. No: 34580). The integrated density was measured using Image J and normalized to the cell number from which the conditioned medium was generated.
Mouse Cytokine Array C1 kit (RayBiotech. Cat. No: AAM-CYT-1-2) was used for cytokine analysis following the manufacturer's guidelines. Briefly, cells were washed once and cultured in serum-free medium for 48 hrs. Conditioned medium was filtered (0.2 μm) and then subjected to cytokine-array analysis. After collection of conditional media, the cell number of each sample was counted. The intensities of array dots were visualized on film after incubation with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific. Cat. No: 34580). The integrated density was measured using Image J and normalized to the cell number from which the conditioned medium was generated.
2′ 3′-cGAMP Measurement
2′ 3′-cGAMP ELISA Kit (Cayman chemical. Cat. No:501700) was used to analyze the endogenous level of 2′ 3′-cGAMP following the manufacturer's instructions. 1×105 IMR90 cells were incubated in 200 μL lysis buffer (ThermoFisher, Cat. No: 78501) on ice for 30 minutes. The 2′ 3′-cGAMP ELISA was performed following the manufacturer's instructions.
Cells were fixed with 4% paraformaldehyde (PFA) for 15 mins at room temperature followed by permeabilization with 0.2% Triton X-100 in PBS for 5 min. For DNase I digestion, after fixation and permeabilization, cells were treated with 500 units/mL RNase-Free DNase I (Qiagen, Cat. No: 79254) for one hour at 37° C. After blocking with 1% BSA in PBS, cells were incubated with primary antibody overnight at 4° C. and Alexa-Fluor conjugated secondary antibody (Life Technologies). Fluorescent images were captured using Leica TCS SP5 II scanning confocal microscope.
Cells were lysed in 1× sample buffer (2% SDS, 10% glycerol, 0.01% bromophenol blue, 62.5 mM Tris, pH 6.8, and 0.1 M DTT) and heated to 95° C. for 10 min. Protein concentrations were determined using the protein assay dye (Bio-Rad. Cat. No: #5000006) and Nanodrop. An equal amount of total protein was resolved using SDS-PAGE gels and transferred to PVDF membranes at 110 V for 2 hours at 4° C. Membranes were blocked with 5% nonfat milk in TBS containing 0.1% Tween 20 (TBS-T) for 1 hour at room temperature. Membranes were incubated overnight at 4° C. in the primary antibodies in 4% BSA/TBS+0.025% sodium azide. Membranes were washed four times in TBS-T for 5 min at room temperature, after which they were incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology. Cat. No: 7076S, 7074S) for 1 hour at room temperature. After washing four times in TBS-T for 5 min at room temperature, proteins were visualized on film after incubation with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific. Cat. No: 34580).
For immunoprecipitation, cells were collected and washed once with ice-cold PBS. Whole-cell extracts were lysed with RIPA buffer (50 mM Tris-HCl pH 7.4, 1% NP-40, 150 mM NaCl, 1 mM EDTA, 10% sodium deoxycholate, freshly added with 1 mM phenylmethylsulfonyl fluoride (PMSF), and cOmplete™, EDTA-free Protease Inhibitor Cocktail (Roche. Cat. No: C762Q77)). After 12,000×g centrifuge for 15 min, the supernatant was collected and incubated with antibody or isotype IgG control (5 μg per sample) at 4° C. overnight, followed by addition of 10 μL of protein A/G-conjugated agarose beads mixture (ThermoFisher. Cat. No: 10002D and 10004D). The precipitates were washed 4 times with ice-cold RIPA buffer, resuspended in 2×Laemmle buffer, and resolved by SDS-PAGE followed by immunoblotting.
GST pull-down assay was carried out by incubating equal amounts of GST or GST-tagged cGAS (Addgene, Cat. No: 108676) that are immobilized on glutathione-sepharose beads (GE Healthcare Cat. No: 17-0756-01) with in vitro translated His-tagged TOP1 (Sino Biological, Cat. No: 17455-H07B) at 4° C. for 16 hours. Precipitated proteins were washed 3 times with elution buffer including 150 mM NaCl, eluted with SDS sample buffer, and subjected to immunoblot analysis.
Quantification PCR with Reverse Transcription
Total RNA was isolated using Trizol (Invitrogen) according to the manufacturer's instruction. Extracted RNAs were used for reverse-transcriptase PCR (RT-PCR) with High-Capacity cDNA Reverse Transcription Kit (Thermo fisher, Cat. No: 4368814). Quantitative PCR (qPCR) was performed using iTaq™ Universal SYBR® Green Supermix (BIO-RAD, Cat. No: 1725121) and QuantStudio 3 Real-Time PCR System.
The primers sequences used for quantitative RT-PCR are as follows:
A CCF purification protocol was developed by modifying previous protocols (Shimizu et al., 1996; Ly et al., 2017). Briefly, 500 million senescent cells were collected, resuspended, and incubated in DMEM containing 10 μg/mL cytochalasin B for 30 minutes at 37° C. After wash once with ice-cold PBS, the cell pellet was gently dounce homogenized in ice-cold pre-chilled lysis buffer (10 mM Tris-HCl, 2 mM magnesium acetate, 3 mM CaCl2), 0.32 M sucrose, 0.1 mM EDTA, 1 mM DTT, 0.1% NP-40, 0.15 mM spermine, 0.75 mM spermidine, 10 μg/ml cytochalasin B, pH 8.5, 4° C.) with ten slow strokes of a loose-fitting pestle. Release of nuclei was confirmed by DAPI-staining and microscopy. The homogenate was fixed with 1% formaldehyde for 10 minutes, and mixed well with an equal volume of 1.6M sucrose buffer (10 mM Tris-HCl, 5 mM magnesium acetate, 0.1 mM EDTA, 1 mM DTT, 0.3% BSA, 0.15 mM spermine, 0.75 mM spermidine, pH 8.0, 4° C.). A 10 mL portion of homogenate was layered on the top of sucrose buffer gradient (20 mL and 15 mL containing 1.8M and 1.6M of sucrose, respectively) in a 50 mL tissue culture tube. The tubes were centrifuged at 1200×g for 20 minutes at 4° C. After centrifugation, the upper 3 mL of the gradient was discarded, and the next 15 mL containing CCFs was collected. The collected fraction was diluted with an equal volume of ice-cold PBS, and filtered through 5 μm low protein binding durapore (PVDF) membrane (Millipore. Cat. No: SLSV025LS) to remove the contaminated nuclei. DAPI-staining was performed at this step to confirm the clearance of contaminated nuclei. The CCF fractions were diluted 5-fold by adding ice-cold PBS, then centrifuged at 2000×g for 15 minutes at 4° C. Finally, the pellet was suspended in 200 μL ice-cold PBS buffer. The CCF samples were broken down by one pulse of bioruptor with high output. DNA concentration was measured using Nanodrop and 5 μg DNA was used for slot blot analysis.
Cells were plated on coverslips and labelled with 10 μg/ml BrdU for 24 hrs. Cells were fixed with 4% paraformaldehyde (PFA) for 15 mins at room temperature followed by permeabilization with 0.2% Triton X-100 in PBS for 5 min. Cells were incubated in 2.5M hydrochloric acid at 4° C. for 24 hrs. After blocking with 1% BSA in PBS, cells were incubated with primary antibody overnight at 4° C. and Alexa-Fluor conjugated secondary antibody (Life Technologies) for one hr. Fluorescent images were captured using Leica TCS SP5 II scanning confocal microscope.
SILAC DMEM Lysine (6) Arginine (10) Kit (Silantes. Cat No: 282986434) was used for the SILAC-MS analysis. Briefly, IMR90 cells were cultured in “heavy” medium containing 13C6 labeled lysine and 13C6, 15N4 labeled arginine, or “light” medium containing unlabeled lysine and arginine for at least four passages. The “heavy” labeled IMR90 cells were infected with short hairpin control lentivirus, and the “light” cells were infected with shHMGB2 short hairpin lentivirus (TRCN0000019011). After puromycin selection, the cells were treated with 50 μM Etoposide for 2 days. After washing off the drug with fresh medium, the treated cells were cultured for 6 days to induce senescence. The same numbers of both “heavy” and “light” labeled cells were mixed together and the CCF purification was performed. Purified CCF were mixed with 5×SDS sample buffer and boiled at 95° C. for 15 minutes.
LC-MS/MS analysis was performed using a Q Exactive HF mass spectrometer (ThermoFisher Scientific) coupled with a Nano-ACQUITY UPLC system (Waters). Samples were digested with trypsin and tryptic peptides were separated by reversed phase HPLC on a BEH C18 nanocapillary analytical column (75 μm i.d.×25 cm, 1.7 μm particle size; Waters) using a 240 min gradient formed by solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile). Eluted peptides were analyzed by the mass spectrometer set to repetitively scan m/z from 400 to 2000 in positive ion mode. The full MS scan was collected at 60,000 resolution followed by data-dependent MS/MS scans at 15,000 resolution on the 20 most abundant ions exceeding a minimum threshold of 10,000. Peptide match was set as preferred, exclude isotope option and charge-state screening were enabled to reject unassigned, and single charged ions. The sample was analyzed twice (technical replicate). Peptide sequences were identified using MaxQuant 1.6.2.329. MS/MS spectra were searched against a UniProt human protein database (October 2017) and a common contaminants database using full tryptic specificity with up to two missed cleavages, static carboxamidomethylation of Cys, and variable oxidation of Met, and protein N-terminal acetylation. Consensus identification lists were generated with false discovery rates set at 1% for protein and peptide identifications. The DAVID bioinformatics resources 6.8 was used for functional classification analysis. The protein list was further filtered to include only proteins classified as “nucleosome and chromosome related” and identified by at least two razor+unique peptides with a minimum absolute fold change of 1.2 in both replicates.
For chromatin fragments extraction, proliferating IMR90 cells were treated with 5 μM or 50 μM Camptothecin for 30 minutes to induce low or high levels of TOP1cc, respectively. Cells then were incubated with hypotonic buffer (10 mM Tris, pH 7.4, 30 mM NaCl, 3 mM MgCl2, 0.1% NP40), supplemented with protease inhibitor cocktail, on ice for 10 mins (Dou et al., 2017). The cells were then centrifuged at 300×g for 3 mins at 4° C. The supernatant was carefully removed, and the resulting pellets were incubated with benzonase buffer (50 mM Tris pH 7.5, 300 mM NaCl, 0.5% NP40, 2.5 mM MgCl2) with protease inhibitor cocktail, supplemented with 10 U of benzonase (Sigma Cat. No: E1014), on ice, for 30 min. The product was centrifuged again at 300×g for 3 mins at 4° C., and benzonase was inactivated by addition of 15 mM EDTA. The resulting supernatant contains chromatin fragments and soluble nuclear proteins. For the negative controls, buffer without benzonase was used and the resulting supernatant only contains soluble nuclear proteins without chromatin fragments. The product was then diluted 5 times with PBS. Slot blot were performed to confirm the TOP1cc level. The chromatin fragments or negative controls were transfected into proliferating IMR90 cells using lipofectamine 2000. Successful transfection was confirmed by immunofluorescence with DAPI staining. Transfected cells were harvested 4 days post transfection and were used for RT-qPCR or immunofluorescence analysis.
TOP1 ICE (In vivo Complex of Enzyme) Assay
Human Topoisomerase 1 ICE Assay Kit (TopoGEN. Cat. No:TG1020-1) was used to isolate protein-DNA samples which contain TOP1-DNA covalent complex (TOP Ice) for slot blot analysis. The isolation was performed following the manufacturer's guidelines. 5×105 cells were used for ICE assay and TOP1cc analysis. Purified CCF samples were sonicated and used for slot blot directly. Briefly, cells were lysed with 300 μL of room temperature buffer A, and then 115 μL buffer B was added to precipitate DNA. After washing with buffer C, DNA was dissolved in buffer D and buffer E. The DNA samples were kept in 37° C. to promote the recovery. Nano-Drop was used to measure the DNA concentration. 5 μg DNA was used for each slot blot analysis. Bio-Dot SF Microfiltration Apparatus (Bio Rad. Cat. No:1706542) was used for slot blot. Quantification was performed using NIH Image J software.
EMSA was performed as previously described (Li et al., 2013; Liu et al., 2019). Briefly, recombinant cGAS was incubated, in the presence or absence of recombinant TOP1 or TOP1Y723F mutant, with (ISD)2 dsDNA in the cGAMP synthesis buffer at 37° C. for 30 mins. The mixtures were loaded on 1% agarose gel using an electrophoresis buffer (40 mM Tris-HCl at pH 10.5). The gels were then stained with SYBR™ Green I Nucleic Acid Gel Stain and images were acquired using UV Transilluminator (Analytik Jena).
The protocols were approved by the Institutional Animal Care and Use Committee of the Wistar Institute. Results from in vitro experiments were used to determine the in vivo sample size. For orthotopic syngeneic model, luciferase expressing ID8-Defb29/Vegf-a with inducible shHMGB2 cells were pretreated with 2 μM Cisplatin for 48 hours to induce CCFs. 5×106 cells were i.p. injected into the peritoneal cavity of C57BL/6 mouse (female, 6-8 weeks old, CRL/NCI) (Zhu et al., 2016). Animals were randomly assigned to different treatment groups (10 mice/group). The mice in control groups were fed with control rodent diet (Fisher Scientific. Cat. No: 14-726-309). For the HMGB2 knockdown groups, mice were fed with Bio-Serv™ Doxycycline Grain-Based Rodent Diet (Fisher Scientific. Cat. No: 14-727-450) to induce HMGB2 knockdown. Tumor progression was monitored twice a week using a Xenogen IVIS Spectrum in vivo bioluminescence imaging system. Images were analyzed using Live Imaging 4.0 software. Tumor-bearing mice were treated by i.p. injection with isotype control IgG or anti-PD-L1 antibody (Bio X Cell, Cat. No: B7-H1, clone 10 F.9G2, 10 mg kg{circumflex over ( )}-1) every 3 days with or without simultaneous TOP1 inhibitor camptothecin treatment (Selleck Cat. No: S1288; 8 mg kg{circumflex over ( )}-1). For survival analysis, the Wistar Institute IACUC guideline was followed in determining the time for ending the survival experiments (tumor burden exceeds 10% of body weight).
For peritoneal wash, the peritoneal cavity of mice was washed three times with 5 ml PBS. Single-cell suspensions were prepared, and red blood cells were lysed using ACK Lysis Buffer (Thermo Fisher, Cat No: A1049201). Live/dead cell discrimination was performed using Zombie Yellow™ Fixable Viability Kit (Biolegend, Cat No: 423104). Cell surface staining was done for 30 mins at 4° C. using antibodies against CD3ε (Biolegend, Cat No: 423104), CD69 (Biolegend, 104525), CD8 (Biolegend, Cat No: 100708), CD4 (Biolegend, Cat No: 100516), Granzyme B (Biolegend, Cat No: 372206), and Interferon gamma (Biolegend, Cat No: 505825). Intracellular staining was done using an eBioscience fixation/permeabilization kit (Thermo Fisher, Cat No: 88-8824-00). All data acquisition was done using an LSR II (BD) or FACSCalibur (BD) and analyzed using FlowJo software (TreeStar) or the FlowCore package in the R language and environment for statistical computing.
The protocols were approved by the Institutional Animal Care and Use Committee of the Wistar Institute. 1×106 UPK10 cells were unilaterally injected into the ovarian bursa sac of C57BL/6 mouse (female, 6-8 weeks old, CRL/NCI). The orthotopically transplanted cells were allowed to form tumor for 15 days. Tumor-bearing mice were randomly assigned to different treatment groups. The mice were treated for two weeks. Specifically, the mice were pre-treated by i.p. injection (1×106cells per mouse) with control UPK10 cells (group 3), or senescent UPK10 cells sorted from cisplatin, irinotecan and cisplatin/irinotecan combination treated groups (group 4, 5 and 6), or 10 mg/kg DMXAA (group 8), or DMSO vehicle control (group 7). 24 hrs following the pre-treatment, the mice were treated by i.p. injection with anti-PD-1 antibody (Bio X Cell, Cat. No: BE0273, clone 29F.1A12, 10 mg/kg) or an isotype matched IgG control every 3 days. After two weeks of treatment, the tumors were collected and digested using mixture of 10 mg/mL Collagenase (Sigma, Cat No: C5138), 1 mg/mL Hyaluronidase (Sigma, Cat No: H3884) and 200 mg/mL DNase 1 (Sigma, Cat No: D5025) at 37° C. for 1 hr. Single-cell suspensions were prepared, and red blood cells were lysed using ACK Lysis Buffer (Thermo Fisher, Cat No: A1049201). Live/dead cell discrimination was performed using LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (Thermo Fisher, Cat No: L34968). Cell surface staining was done for 30 min at 4° C. All data acquisition was done using an LSR II (BD) or FACS Calibur (BD) and analyzed using FlowJo software (TreeStar) or the FlowCore package in the R language and environment for statistical computing. For survival analysis, the Wistar Institute IACUC guideline was followed in determining the time for ending the survival experiments (mice succumbed to the disease or tumor burden exceeds 10% of body weight).
Formalin-fixed, paraffin-embedded tumors were sectioned, and slides were deparaffinized and rehydrated. Antigen retrieval was performed by boiling for 40 mins in citrate buffer, pH6.0 (Thermo Fisher). Endogenous peroxidases were quenched with 3% hydrogen peroxide in methanol. Sections were then blocked with 5% BSA/PBS at room temperature for 1 hr. Sections were incubated with primary mouse anti-GFP (Santa Cruz, 1:400 dilution) or rabbit anti-mCherry (Proteintech, 1:200 dilution) antibodies at 4° C. overnight. Detection was performed using secondary Alexa Fluor 488-conjugated goat anti-mouse IgG (Thermo Fisher, 1:1000 dilution) and Alexa Fluor 555-conjugated goat anti-rabbit IgG (Thermo Fisher, 1:1000 dilution) at room temperature for 1 hr. The sections were counter stained with DAPI containing Duolink® in Situ mounting medium (Sigma Aldrich) and sealed. Samples were imaged on Leica TCS SP5 II Scanning Confocal Microscope.
Results are representative of a minimum of three independent experiments. All statistical analyses were conducted using GraphPad Prism 6 (GraphPad). The Student's t-test was performed to determine P values of the raw data unless otherwise stated, where P<0.05 was considered significant. Animal experiments were randomized. There was no exclusion from the experiments.
HMGB2 is Required for cGAS' Localization into CCF During Senescence
Since HMGB2 positively regulates SASP (Aird et al., 2016) and facilitates cytosolic nucleic-acid sensing (Yanai et al., 2009), we examined whether HMGB2 localized to the CCF during senescence. HMGB2 co-localized with γH2AX in the CCF in senescent OVCAR3 ovarian cancer cells induced by either cisplatin or etoposide (
cGAS Activation Requires TOP1cc During Senescence
We next determined the mechanism by which HMGB2 regulates cGAS' localization into CCF during senescence. Toward this goal, we developed a protocol to purify CCF from senescent cells (
We first validated the unbiased LC-MS results by showing that TOP1 localized to CCF and co-localized with γH2AX in both senescent IMR90 and OVCAR3 cells (
Since our results suggested that TOP1cc promotes SASP, we next directly examined whether induction of TOP1cc by CPT is sufficient to rescue the suppression of SASP induced by HMGB2 inhibition. Notably, CPT treatment restored the TOP1cc levels in the CCF isolated from HMGB2 knockdown or knockout senescent cells (
TOP1cc Enhances dsDNA Recognition by cGAS
Since HMGB2 positively regulates TOP1cc and HMGB2 inhibition decreases TOP1cc, we examined time-course kinetics of TOP1cc induction and stabilization in CPT-treated IMR90 cells with or without HMGB2 knockdown. Notably, HMGB2 knockdown did not affect the kinetics of TOP Ice formation (
Since SASP induction by TOP1cc is cGAS dependent, TOP1cc is a TOP1 covalently modified dsDNA complex (Pommier et al., 2016), and cGAS binds to dsDNA (Sun et al., 2013), we examined whether TOP1 interacts with cGAS by co-immunoprecipitation analysis. Indeed, TOP1 interacted with cGAS and there was an increase in their interaction in senescent compared with control cells (
We next sought to directly determine the effects of TOP1cc on the DNA binding affinity of cGAS. Electrophoretic mobility-shift assay (EMSA) showed high-molecular-weight cGAS bound (ISD)2 dsDNA complex in dose-dependent manner (
There is evidence to support that cGAS and its mediated expression of immune modulatory molecules such as SASP factors are essential for the antitumor effect of immune checkpoint blockade such as anti-PD-L1 antibody treatment (Xiang et al., 2017). To examine the relevance of the HMGB2-TOP1cc-cGAS pathway in immune checkpoint blockade treatment, we utilized an immune competent syngeneic ovarian cancer ID8-Defb29 Vegf-a mouse model (Zhu et al., 2016; Conejo-Garcia et al., 2004). Notably, the HMGB2-cGAS-TOP1cc axis is conserved in cisplatin-induced senescent ID8-Defb29/Vegf-a cells and cisplatin induced senescence in nearly 100% of the treated cells (
Consistent with previous reports (Aird et al., 2016), HMGB2 knockdown suppresses the growth of the tumor cells (
Here we identified the TOP1cc, a TOP1 covalently modified DNA complex, as a critical mediator of the recognition of CCF by cGAS through direct interaction between TOP1 and cGAS in a dsDNA dependent manner during senescence. In addition, we showed that HMGB2 functions upstream of the TOP1cc-cGAS axis by stabilizing TOP1cc. Thus, our studies provided additional mechanistic insights into how HMGB proteins boost cytosolic nucleic-acid sensing (Yanai et al., 2009). Finally, we show that the HMGB2-TOP1cc-cGAS axis functionally regulates SASP and the response to immune checkpoint blockade. These findings indicate that clinically applicable TOP1 inhibitors such as CPT can serve as a sensitizer to immune checkpoint blockade to target therapy-induced senescent cells. Notably, TOP1 inhibitors increase the sensitivity of patient-derived melanoma cell lines to T-cell-mediated cytotoxicity (McKenzie et al., 2018; Haggerty et al., 2011). This is consistent with our findings that TOP1 inhibitors-induced TOP1cc boosts cGAS-mediated inflammation and the associated immune checkpoint blockade treatment.
Therapy-induced senescence-associated secretory phenotype (SASP) correlates with overcoming resistance to immune checkpoint blockade (ICB). Intrinsic resistance to ICB is a major clinical challenge. For example, ovarian cancer is largely resistant to ICB. Here we show that adoptive transfer of SASP-boosted ex vivo therapy-induced senescent cells sensitizes ovarian tumors to ICB. Topoisomerase 1 (TOP1) inhibitors such as irinotecan enhance cisplatin-induced SASP, which depends on the TOP1 cleavage complex-regulated cGAS pathway. Transfer of cisplatin-induced, SASP-boosted senescent cells with irinotecan sensitizes ovarian tumor to anti-PD-1 antibody and improves the survival of tumor-bearing mice in an immunocompetent, syngeneic model. This correlates with the infiltration of transferred senescent cells in the established orthotopic tumors and an increase in the infiltration of activated CD8+ T cells and dendritic cells in the tumor bed.
To isolate senescent cells for adoptive transfer, we treated UPK10 mouse ovarian cancer cells with cisplatin to induce senescence as evidenced by induction of markers of senescence including senescence-associated β-galactosidase (SA-β-Gal) activity, p16 and γH2AX (
TOP1 Inhibitor Irinotecan Boosts SASP Through the cGAS Pathway
We next sought to characterize the sorted senescent cells from the different treatment groups. Compared with cisplatin-induced senescent cells, TOP1cc levels were increased by irinotecan addition (
We next sought to determine whether the observed enhancement of SASP by irinotecan is TOP1 and cGAS dependent. Toward this goal, we knocked down TOP1 or cGAS using two independent shRNAs to limit potential off-target effects (
Given the critical role played by cGAS in mediating ICB (Xiang et al., 2017) and the evidence that induction of inflammatory SASP correlates with sensitization of resistant melanomas to ICB (Jerby-Amon et al., 2018), we sought to explore the possibility of adoptive transfer of SASP-boosted senescent cells as a potential cell therapy to sensitize tumors to ICB. Toward this goal, we established a syngeneic, immunocompetent mouse ovarian tumor model using UPK10 cells (Scarlett et al., 2012). We orthotopically transplanted UPK10 into the mouse bursa and allowed the tumor to establish for two weeks (
Despite the fact that SASP-promoting cGAS is required for response to ICB (Xiang et al., 2017) and therapy-induced SASP correlates with overcoming resistance to ICBs (Jerby-Amon et al., 2018; Ruscetti et al., 2020), therapeutic approaches that leverage SASP of senescent cells to sensitize tumors to ICB have not been reported. Here we show that adoptive transfer of SASP-boosted, cisplatin-induced senescent cells using clinically applicable TOP1 inhibitor irinotecan sensitizes ovarian tumor to ICB. An advantage of this approach is that the treatment occurs ex vivo, which limits the potential systematic toxicity caused by direct treatment with these small molecules in vivo. Consistent with our findings, TOP1 inhibitors increase the sensitivity of patient-derived melanoma cell lines to T-cell-mediated cytotoxicity (Haggerty et al., 2011; McKenzie et al., 2018).
Notably, the observed sensitization correlates with infiltration of senescent cells into the tumor bed. Indeed, previous studies show that intravenously or subcutaneously injected ovarian cancer cells metastasize to ovary (Bai et al., 2019). This raised the possibility that transfer of SASP-boosted senescent cells may convert “cold” into “hot” tumors through infiltration of senescent cells into tumor bed and associated secretion of inflammatory SASP factors. However, we cannot exclude the possibility that the transferred senescent cells may localize to other areas. In addition, further studies are warranted to elucidate what SASP factors mediate the observed antitumor response and what cells are being impacted to regulate therapy response. Further, this approach in combination with subsequent ICB treatment allows for targeting and eradicating residual tumor nodules to prevent relapse, a major challenge in clinical management of ovarian cancer.
Although cisplatin-induced senescent cells are positive for SASP, their adoptive transfer was not sufficient to sensitize tumors to ICB. This supports the notion that levels of SASP dictate the outcome of adoptively transferred senescent cells. Consistently, STING agonist alone stimulated the expression of the SASP factors to a level that is comparable to those observed in cisplatin-induced senescent cells. However, this is not sufficient to sensitize tumors to ICB. Notably, UPK10 cells were isolated from mouse ovarian tumors developed from conditional activation of Kras and inactivation of Tp53 (Scarlett et al., 2012). In contrast, ID8 is wild-type for both Kras and Tp53. Given the fact that irinotecan boosted SASP induced by cisplatin in both UPK10 and ID8 cells, these findings suggest that the observed effects are independent of Kras or Tp53 status.
Each and every patent, patent application, and any document listed herein, and the sequence of any publicly available nucleic acid and/or peptide sequence cited throughout the disclosure, is/are expressly incorporated herein by reference in their entireties. U.S. Provisional Application No. 62/976,020, filed Feb. 13, 2020, is incorporated herein by reference in its entirety. Hao et al., Sensitization of ovarian tumor to immune checkpoint blockade by boosting senescence-associated secretory phenotype, iScience. 2020 Dec. 30; 24(1):102016 is incorporated by reference herein in its entirety. Embodiments and variations of this invention other than those specifically disclosed above may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims include such embodiments and equivalent variations.
This invention was made with government support under grant numbers R01CA160331, P01AG031862, and CA010815 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US21/17859 | 2/12/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62976020 | Feb 2020 | US |
