Treatment of non-small cell lung cancer with 6-THIO-dg and cemiplimab

Abstract

Disclosed herein are methods for treating non-small cell lung cancer (NSCLC) using a sequential therapy of 6-thio-dG followed by the PD-1 checkpoint inhibitor cemiplimab (Libtayo®).

Description

TREATMENT OF NON-SMALL CELL LUNG CANCER WITH 6-THIO-dGAND CEMIPLIMABFIELD OF THE INVENTION

[0001] The present disclosure relates to the fields of medicine, pharmacology, molecular biology and oncology. More particularly, the disclosure relates to methods for treating non-small cell lung cancer (NSCLC) using a sequential therapy of 6-thio-dG followed by the PD-1 checkpoint inhibitor cemiplimab (Libtayo®).BACKGROUND OF THE DISCLOSURE

[0002] Immunotherapies have revolutionized the treatment of many cancers in the immuno-oncology field (Brahmer et al., 2012; Hodi et al., 2010; Ribas and Wolchok, 2018; Topalian et al., 2012). The most commonly used immunotherapies are PD-L1 / PD-1 checkpoint blockades that have been approved by the FDA for advanced cancers such as melanoma, non-small cell lung cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, Hodgkin lymphoma, liver, cancer, lung cancer, renal cell cancer, stomach cancer, rectal cancer, and any solid tumor that is not able to repair errors in its DNA that occur during replication (Garon et al., 2015; Ribas et al., 2016; Rizvi et al., 2015b; Socinski et al., 2018; National Cancer Institute). Despite the success of immunotherapies, many patients do not respond well to these therapies due to the immune suppressive tumor microenvironment, tumor immunogenicity and the emergence of primary and adaptive resistance (Chen and Han, 2015; Gide et al., 2018). Although recent studies show that the abundance of tumor mutations and neoantigens partially dictate cancer patient responses to checkpoint blockade, there are still considerable numbers of patients with high mutations and neoantigens that do not respond well (Le et al., 2017; Mandal et al., 2019; Rizvi et al., 2015a), suggesting neoantigens are not sufficient for provoking anti-tumor immune responses. Therefore, there is an urgent need to identify other factors for better immune responses and to develop new approaches to improve patient overall survival.

[0003] The generation of effective anti-tumor adaptive immune responses require tumor antigen presentation by antigen presenting cells, whose activation heavily rely on adequate innate sensing. Innate sensing is often provided by danger signals such as high mobility group box 1 protein, extracellular ATP and tumorDNAs released from stressed tumor cells (Kroemer et al., 2013; Pitt et al., 2017). Recent studies highlight the importance of cytosolic DNA sensing in radiation and DNA damaging therapies (Deng et al., 2014; Sen et al., 2019). The presence of DNA in the cytoplasm, for example, in the form of micronuclei (small DNA containing organelles) that lose nuclear envelop membranes can trigger immune responses. Micronuclei are the products of chromosome damage as a result of genotoxic stress and chromosome mis-segregation during cell division (Fenech et al., 2011). The cytosolic DNA sensor cGAS recognizes micronuclei and converts GTP (guanosine triphosphate) and ATP (adenosine triphosphate) into second messenger cGAMP (cyclic GMP-AMP) (Wu et al., 2013). Then the adaptor protein Stimulator of IFN Gene (STING) binds to cGAMP (Ablasser et al., 2013; Diner et al., 2013; Gao et al., 2013; Zhang et al., 2013). This complex process activates TANK-binding kinase 1 (TBK1) and IFN regulatory factor 3 (IRF3) (Liu et al., 2015; Tanaka and Chen, 2012) and further activates the downstream transcription of type I IFNs and other cytokines (reviewed in (Li and Chen, 2018)), which ultimately increases innate sensing.

[0004] Eukaryotic linear chromosomes are capped by special structures called telomeres (TTAGGG), which are essential to maintain chromosomal stability (reviewed in (Blackburn, 1991)). Telomeres constitute the final ~10 kb of all human chromosomes and the final 12-80 kb of all mouse chromosomes (Lansdorp et al., 1996; Zijlmans et al., 1997). In all somatic human cells, telomeres shorten with each cell division due to the end replication problem and the absence of a telomere maintenance mechanism (reviewed in (Greider, 1996)). However, unicellular eukaryotes, germline cells and immortal cancer cells maintain their telomeres at a constant length almost always by activating the enzyme telomerase (Greider and Blackbum, 1985; McEachern and Blackbum, 1996; Morin, 1989; Nakamura et al., 1997; Singer and Gottschling, 1994; Yu et al., 1990). Telomerase is a reverse transcriptase enzyme that elongates telomeres by adding TTAGGG repeats to the ends of chromosomes and is expressed in ~90% of human tumors, but not in most normal cells (Shay and Bacchetti, 1997). Therefore, telomerase is an attractive target to develop anti-cancer therapies.

[0005] The nucleoside analogue, 6-thio-2’-deoxyguanosine (6-thio-dG), is a new and effective therapeutic approach in the cancer field. Its incorporation into de novo synthesized telomeres by telomerase is known to induce damage on telomeric DNA (Mender et al., 2015a). This results in rapid tumor shrinkage or growth arrestin many tumor-derived xenograft models with minimal side effects (Mender et al., 2018; Sengupta et al., 2018; Zhang et al., 2018). The most important advantage of this telomere-targeted therapy over direct telomerase inhibitors is that 6-thio-dG does not have a long lag period for tumor killing effects. Additionally, it does not directly inhibit telomerase but is preferentially recognized by telomerase over other polymerases and incorporated into the telomeres resulting in an immediate DNA chain termination. Importantly, its effect is independent of initial telomere length by hijacking tumor telomerase to make unstable telomeres (Mender et al., 2015b).SUMMARY OF THE INVENTION

[0006] In one aspect of the disclosure, provided herein are methods of treating non-small cell lung cancer (NSCLC) in a subject involving administering to said subject an effective amount of 6-thio-2’-deoxyguanosine (6-thio-dG) followed by treatment with the PD-1 checkpoint inhibitor cemiplimab (Libtayo®).

[0007] In some embodiments of the methods disclosed herein, 6-thio-dG is administered at a dose of about 60 mg, 180 mg, 360 mg, or 540 mg.

[0008] In some embodiments the 6-thio-dG is administered at a dose of about 180 mg.

[0009] In some embodiments the 6-thio-dG is administered at a dose of about 60 mg.

[0010] In some embodiments the is 6-thio-dG is administered at a dose of about 180 mg.

[0011] In some embodiments the is 6-thio-dG is administered at a dose of about 360 mg.

[0012] In some embodiments the 6-thio-dG is administered at a dose of about 540 mg.

[0013] In some embodiments the 6-thio-dG is administered at a dose of about 60 mg to about 540 mg.

[0014] In some embodiments the 6-thio-dG is administered at a dose of about 60 mg to about 360 mg.

[0015] In other embodiments, 6-thio-dG is administered for about 1 to about 3 days per therapeutic cycle followed by cemiplimab.

[0016] In some embodiments, the 6-thio-dG is administered for 3 days followed by cemiplimab.

[0017] In some embodiments, cemiplimab is administered on day 5.

[0018] In some embodiments of the methods disclosed herein, administration of 6-thio-dG and cemiplimab results in inhibition of tumor growth.

[0019] In some embodiments of the methods disclosed herein, administration of 6-thio-dG followed by cemiplimab results in remission of the NSCLC treated.

[0020] In some embodiments of the methods disclosed herein, administration of 6-thio-dG followed by cemiplimab results in reduction in NSCLC tumor burden.

[0021] In some embodiments of the methods disclosed herein, administration of 6-thio-dG followed by cemiplimab results in disease free progression of NSCLC

[0022] In some embodiments of the methods disclosed herein, administration of 6-thio-dG followed by cemiplimab results in disease free progression of NSCLC from about 6 months to greater than about one year.

[0023] In some embodiments, the subject being treated has an NSCLC that is resistant to check point inhibitors.

[0024] In some embodiments, the NSCLC treated with THIO plus cemiplimab results in the production of telomere dysfunction-induced foci (TIF)- positive circulating tumor cells.BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 shows THIO-101 Study Schema

[0026] Figure 2 shows efficacy findings in patients receiving THIO as 3L treatment, 180 mg dose (N=8)*- Legend-the dark bars represent treatment, the lighter bars represent Follow-up; diamonds represent stable disease; squares represent partial response, triangles represent progressive disease; hexagons represent patient deaths; circles represent patient withdrawals and unfilled triangles equal others.

[0027] Figure 3 shows efficacy findings in patients receiving THIO as 3L treatment, all dose levels; Legend- dark bars represent treatment, the lighter bars represent Follow-up; diamonds represent stable disease; squares represent partial response, triangles represent progressive disease; hexagons represent patient deaths; circles represent patient withdrawals and unfilled triangles equal others.

[0028] Figure 4 shows efficacy findings in patients receiving THIO as 2L treatment, all dose levels (n=45); Legend the dark bars represent treatment, thelighter bars represent Follow-up; diamonds represent stable disease; squares represent partial response, triangles represent progressive disease; hexagons represent patient deaths; circles represent patient withdrawals and unfilled triangles equal others.

[0029] Figure 5- shows TIFSs fraction in CTCs (paired samples at each dose level)

[0030] Figure 6- shows TIFs fraction in CTCs (patient Status).DETAILED DESCRIPTION

[0031] Resistance to immunotherapy develops in most advanced nonsmall-cell lung cancer (NSCLC) patients treated with immune checkpoint inhibitors (ICI) with very limited treatment options for patients who progress. Ramucirumab and docetaxel combination in second line (2L) showed an improvement in Disease Control Rate (DCR) (64%) and Overall Response Rate (ORR) (23%) compared to docetaxel (DCR (53%) and ORR (14%) respectively). In addition, ramucirumab- docetaxel combination showed OS improvement of 1.4 months (10.5 vs 9.1 months) and PFS improvement of 1.5 months (4.5 vs 3.0 months) over docetaxel single agent. However, 79% of patients treated in the combination arm developed Grade > 3 TEAE1, therefore there’s a high need for new safer and more active treatments.

[0032] In third line (3L), currently there is no standard of care (SoC) thus, various chemotherapy agents are being utilized, with reported DCRs in the 25-35% range(Matsumoto H et al. Transl Lung Cancer Res. 2021 May; 10(5): 2278-2289). In NSCLC IL with PD-L1 expression >50%, the use of Keytruda resulted in a 71% disease control rate (DCR) in KEYNOTE-0243.

[0033] THIO (6-thio-2'-deoxyguanosine) is a small molecule, first-in class direct cancer telomere targeting agent which selectively kills cancer cells that are telomerase positive (TERT+). Over 80% of all cancers are TERT+ and approximately 85% of all NSCLC types (A Survey of Telomerase Activity in Human Cancer - JW Shay, S Bacchetti - European Journal of Cancer, 33,5,787- 791, 1997; Telomerase Activity in Human Liver Tissues; H Tahara, et al; Cancer Research 55, 2734-2736,1995).

[0034] THIO is incorporated de novo in synthesized telomeres leading to chromatin uncapping, DNA damage signals generation and rapid apoptosis

[0035] Preclinical data in NSCLC indicates that low doses of THIO induce sensitivity to immune check point inhibitors when administered prior to an ICI in tumors which otherwise are resistant or do not respond to an ICI (Mender I, Zhang A, Ren Z, et al. Cancer Cell. 2020;38:400-411).

[0036] THIO targets and selectively kills cancer cells through 2 key mechanisms (Id.): a) Induction of DNA damage pathway results in the formation of Telomere Dysfunction Induced Foci (TIF), followed by rapid G2 / M arrest or cell death of telomere-positive cancer cells; and 2) Activation of cGAS-STING- dependent innate and adaptive immune responses upon release of intracellular and extracellular THIO-containing telomeric DNA fragments.

[0037] Sequential treatment of THIO and immune check point inhibitors showed a potent and durable antitumor activity in preclinical models (Mender et al.).

[0038] DCR has been demonstrated to be a better predictor of OS advantage compared to ORR in NSCLC post first line of therapy (Matsumoto et al.).

[0039] THIO (6-thio-2'-deoxyguanosine) is a small molecule, first-in class direct cancer telomere targeting agent which selectively kills cancer cells that are telomerase positive (TERT+). Over 80% of all cancers are TERT+ and approximately 85% of all NSCLC types (Shay et al; H. Tahara et al.).

[0040] THIO is incorporated de novo in synthesized telomeres leading to chromatin uncapping, DNA damage signals generation and rapid apoptosis

[0041] Preclinical data (Mender et al.) in NSCLC indicates that low doses of THIO induce sensitivity to immune check point inhibitors when administered prior to an ICI in tumors which otherwise are resistant or do not respond to an ICI

[0042] THIO targets and selectively kills cancer cells through 2 key mechanisms (Mender et al.): Induction of DNA damage pathway results in the formation of Telomere Dysfunction Induced Foci (TIF), followed by rapid G2 / M arrest or cell death of telomere-positive cancer cells, and Activation of cGAS- STING-dependent innate and adaptive immune responses upon release of intracellular and extracellular THIO-containing telomeric DNA fragments.

[0043] Sequential treatment of THIO and immune check point inhibitors showed a potent and durable antitumor activity in preclinical models (Mender et al).

[0044] Telomerase is almost universally expressed in tumor cells. The telomerase-mediated telomere-targeted drug, 6-thio-dG, reduces the lag timebetween the initial treatment and response to therapy by directly inducing telomere damage in telomerase-positive cancer cells but not in normal telomerase silent cells. In this study, the inventors aimed to explore whether 6-thio-dG that induces telomere stress in telomerase-positive cancer cells could initiate rapid DNA damage for innate sensing. They used syngeneic wild-type and genetic deficient mice to evaluate how 6-thio-dG triggers innate sensing and how it contributes to host antitumor immunity. Importantly, they demonstrate that 6-thio-dG overcomes PD-L1 blockade resistance in advanced tumors. Unexpectedly, 6-thio-dG induced DNA mediated innate sensing and activation of immune responses in a host STING- dependent manner, leading to improved anti-tumor efficacy. Moreover, 6-thio-dG sequentially followed by anti-PD-Ll therapy can completely eliminate advanced tumors. Thus, 6-thio-dG is a tumor-targeting and immune-stimulating drug that can benefit telomerase-positive and PD-L1 resistant cancer patients in the clinic. These and other aspects of the disclosure are described in detail below.I. Telomeres, Telomerase and Telomere Dysfunction

[0045] During mitosis, cells make copies of their genetic material. Half of the genetic material goes to each new daughter cell. To make sure that information is successfully passed from one generation to the next, each chromosome has a special protective cap called a telomere located at the end of its “arms.” Telomeres are controlled by the presence of the enzyme telomerase.

[0046] A telomere is a repeating DNA sequence (for example, TTAGGG) at the end of the body's chromosomes. The telomere can reach a length of 15,000 base pairs. Telomeres function by preventing chromosomes from losing base pair sequences at their ends. They also stop chromosomes from fusing to each other. However, each time a cell divides, some of the telomere is lost (usually 25-200 base pairs per division). When the telomere becomes too short, the chromosome reaches a “critical length” and can no longer replicate. This means that a cell becomes old and dies by a process called apoptosis or undergoes senescence. Telomere activity is controlled by two mechanisms: erosion and addition. Erosion, as mentioned, occurs each time a cell divides due to the failure of lagging strand DNA synthesis to be completed all the way to the end. Addition is determined by the activity of telomerase.

[0047] Telomerase, also called telomere terminal transferase, is an enzyme made of protein and RNA subunits that elongates chromosomes by addingTTAGGG sequences to the end of existing chromosomes. Telomerase is found in fetal tissues, adult germ cells, and also tumor cells. Telomerase activity is regulated during development and has a very low, almost undetectable activity in somatic (body) cells. Because these somatic cells do not regularly use telomerase, they age. The result of aging cells is an aging body. If telomerase is activated in a cell, the cell will continue to grow and divide. This “immortal cell” theory is important in two areas of research: aging and cancer.

[0048] Cellular aging, or senescence, is the process by which a cell becomes old and stops growing or dies. It is due to the shortening of chromosomal telomeres to the point that the chromosome reaches a critical length. Cellular aging is analogous to a wind-up clock. If the clock stays wound, a cell becomes immortal and constantly produces new cells. If the clock winds down, the cell stops producing new cells and undergoes what is termed replicative senescence or dies. Cells are constantly aging. Being able to make the body's cells extend their replication ability certainly creates some exciting possibilities especially for disease associated with genetic inheritance of short telomeres (termed telomeropathies or telomere spectrum disorders). Telomerase research could therefore yield important discoveries related to the aging process.

[0049] Cancer cells have escaped the normal short telomere aging phenomenon and become malignant cells. The malignant cells multiply until they form a tumor that grows uncontrollably and spreads to distant tissue throughout the human body. Telomerase has been detected in almost all human cancer cells. This provides a selective growth advantage to many types of tumors. If telomerase activity was to be turned off, then telomeres in cancer cells would progressively shorten, just like they do in normal body cells. This would prevent the cancer cells from dividing uncontrollably in their early stages of development. In the event that a tumor has already thoroughly developed, it may be removed and anti-telomerase therapy could be administered to prevent relapse. In essence, preventing telomerase from performing its function would change cancer cells from immortal to mortal. However, direct telomerase inhibitors require a lag period from initiation of treatment until tumor shrinkage occurs and have not progressed well in clinical development due to increased toxicities. Thus, the present invention provides methods to reduce the lag period but require telomerase activity to be effective and potentially reduce side effects.II. Treating CancerA. Therapeutic Agents for Sequential Therapy

[0050] Cemiplimab, sold under the brand name Libtayo®, is a monoclonal antibody medication for the treatment of squamous cell skin cancer, basal cell carcinoma skin cancer, and non-small Cell Lung Cancer. Cemiplimab belongs to a class of drugs that binds to the programmed death receptor- 1 (PD- 1), blocking the PD-1 / PD-L1 pathway. In September 2018, it was approved by the U.S. Food and Drug Administration (FDA) for treating people with metastatic cutaneous squamous cell carcinoma (CSCC) or locally advanced CSCC who are not candidates for curative surgery or curative radiation. Cemiplimab is being investigated for the treatment of melanoma cervical cancer, brain cancer, head and neck cancer, renal cell carcinoma, and Hodgkin’s lymphoma. Cemiplimab is described in U.S. Patent No. 9987500.

[0051] Thiopurines, such as 6-thioguanine and 6-mercaptopurine, are currently used as anti-inflammatory, antileukemic, and immunosuppressive agents in clinical practice. In activation reactions, 6-thioguanine is converted to 6- thioguanosine monophosphate by the hypoxanthine guanine phosphoribosyltransferase (HPRT) enzyme. Then, 6-thioguanosine monophosphate is further metabolized to 6-thio-2 '-deoxyguanosine 5 '-triphosphate by kinases and RNA reductases, which may eventually be incorporated into DNA strands during DNA replication. DNA-incorporated 6-thioguanine may also generate reactive oxygen species, which may cause additional damage to DNA, proteins, and other cellular macromolecules, and thus block cellular replication. Although the thiopurines are in clinical use for the treatment of some types of leukemia, their utility for solid tumor treatment has been limited, in part, due to increased toxicities and the development of other therapies.

[0052] One particular thiopurine is 6-thio-dG. This compound is a nucleoside analog and has proven to be a telomerase-mediated telomere disrupting compound. As such, cancer cells are very sensitive to 6-thio-dG with observed IC50 values ranging from 0.7-2.9 pM, depending on cell type, even including therapy resistant cancers (Mender et al., 2018). The structure is shown below:B. Treatment Regimens

[0053] The present disclosure provides for sequential treatment of NSCLC using 6-thio-dG treatment followed by cemiplimab. The periods for each treatment may vary, for example, the gap prior to cemiplimab treatment should be at least one day and may be up 14 days, for example, the 6-thio-dG treatment may be as little as 2 days but may be 3, 4 or more days, including about 2-4 days, 1-5 days, 2-5 days, 3-5 days, 4-5 days, etc. It is contemplated that a short gap between treatments will be advantageous. The gap prior to cemiplimab treatment should be at least one day.

[0054] The daily dosage of 6-thio-dG will be 60 mg, 180 mg, 360 mg or 560mg. The dose of cemiplimab will be consistent with approved current dosing schedules, for example, 350mg.C. Telomerase-Positive Cancers

[0055] Telomerase-positive cancers are far more susceptible to the methods of the present disclosure than are telomerase-negative cancers. Therefore, testing a biopsy to determine whether the cancer is or is not telomerase-positive is highly useful, though not required.

[0056] The most common methods for detecting telomerase activity are telomeric repeat amplification protocols (TRAPs), which allow one to perform semi-quantitative and quantitative analyses, using some of their modifications (called ddTRAP for droplet digital TRAP). Among these modifications are the scintillation proximity assay, hybridization protection assay, transcription amplification assay, and the magnetic bead-based extraction assay.

[0057] The telomeric repeat amplification protocol can be subdivided into three main stages: primer elongation, amplification of telomerase-synthesizedDNA, and finally its detection. At the elongation stage, telomeric repeats are added to the telomere-imitating oligonucleotide by telomerase present in the cell extract. PCR-amplification of telomerase-synthesized DNA is carried out with telomereimitating and reverse primers. Different labels can be incorporated into the telomerase-synthesized DNA. This stage is then followed by detection (e.g, electrophoretic separation and imaging of PCR products).

[0058] Still other methods involve the quantitative isolation of telomerase, and the subsequent measurement of the overall activity of the telomerase from a given cell quantity, which can be compared to appropriate standards. A wide variety of labeling and detection methodologies can be employed once telomerase has been isolated and tested in vitro.III. Pharmaceutical Formulations and Routes of Administration

[0059] Where clinical applications are contemplated, pharmaceutical compositions will be prepared in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.

[0060] One will generally desire to employ appropriate salts and buffers to render drugs stable and allow for uptake by target cells. Aqueous compositions of the present disclosure comprise an effective amount of the drug dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The phrase “pharmaceutically or pharmacologically acceptable” refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, “pharmaceutically acceptable carrier” includes solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like acceptable for use in formulating pharmaceuticals, such as pharmaceuticals suitable for administration to humans. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients of the present disclosure, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions, provided they do not inactivate the agents of the compositions.

[0061] The active compositions of the present disclosure may include classic pharmaceutical preparations. Administration of these compositionsaccording to the present disclosure may be via any common route so long as the target tissue is available via that route, but generally including systemic administration. This includes oral, nasal, or buccal. Alternatively, administration may be by intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection, or intratumoral or regional to a tumor, such as in the tumor vasculature. Such compositions would normally be administered as pharmaceutically acceptable compositions, as described supra.

[0062] The active compounds may also be administered parenterally or intraperitoneally. By way of illustration, solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations generally contain a preservative to prevent the growth of microorganisms.

[0063] The pharmaceutical forms suitable for injectable use include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Generally, these preparations are sterile and fluid to the extent that easy injectability exists. Preparations should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Appropriate solvents or dispersion media may contain, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0064] Sterile injectable solutions may be prepared by incorporating the active compounds in an appropriate amount into a solvent along with any other ingredients (for example as enumerated above) as desired, followed by filteredsterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the desired other ingredients, e.g., as enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation include vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient(s) plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0065] The compositions of the present disclosure generally may be formulated in a neutral or salt form. Pharmaceutically acceptable salts include, for example, acid addition salts (formed with the free amino groups of the protein) derived from inorganic acids (e.g., hydrochloric or phosphoric acids, or from organic acids (e.g, acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups of the protein can also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides) or from organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine and the like.

[0066] Upon formulation, solutions are preferably administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations may easily be administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like. For parenteral administration in an aqueous solution, for example, the solution generally is suitably buffered and the liquid diluent first rendered isotonic for example with sufficient saline or glucose. Such aqueous solutions may be used, for example, for intravenous, intramuscular, subcutaneous and intraperitoneal administration. Preferably, sterile aqueous media are employed as is known to those of skill in the art, particularly in light of the present disclosure. By way of illustration, a single dose may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.

[0067] Suitable subjects / patients according to the present disclosure include mammalian subjects. Mammals according to the present disclosure include, but are not limited to, canine, feline, bovine, caprine, equine, ovine, porcine, rodents, lagomorphs, primates, and the like, and encompass mammals in utero. In one embodiment, humans are suitable subjects. Human subjects may be of either gender, and at any stage of development.

[0068] The skilled artisan is directed to “Remington’s Pharmaceutical Sciences” 15th Edition, Chapter 33, in particular pages 624-652. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.V. Examples

[0069] The following Examples section provides further details regarding examples of various embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques and / or compositions discovered by the inventors to function well. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure. These examples are illustrations of the methods and systems described herein and are not intended to limit the scope of the disclosure. Non-limiting examples of such include but are not limited to those presented below.Example 1 -Methods

[0070] THIO-101 is a Phase 2, randomized, dose-optimization clinical study in adults with advanced NSCLC who either progressed or relapsed after first line (IL) treatment with immune checkpoint inhibitors (ICI alone) or in combination with chemotherapy. Using a modified 3+3 design, the safety lead-in (Part A) enrolled 10 patients who received THIO, 360 mg IV [120 mg QD, Dl-3], followed by 350 mg cemiplimab on D5, Q3W. The trial is testing the hypothesis that low doses of THIO administered prior to cemiplimab (Libtayo®) will enhance and prolong immune response in patients with advanced NSCLC who previously didnot respond or developed resistance and progressed after first-line treatment regimen containing another checkpoint inhibitor. The trial design has two primary objectives: (1) to evaluate the safety and tolerability of THIO administered as an anticancer compound and a priming immune activator (2) to assess the clinical efficacy of THIO using Overall Response Rate (ORR) as the primary clinical endpoint.

[0071] Following completion of Part A, enrollment was opened in the dosefinding portion of the study (Part B).

[0072] Using a Simon 2-stage design, a total of 79 patients (41 patients / arm) were assigned to one of the THIO doses: 360, 180, or 60 mg followed by cemiplimab Q3W for up to 1 year in part B . Disease status is assessed at Cycle 3 Day 1, Cycle 5 Day 1 and every 9 -12 weeks thereafter. We report here data from the 79 patients enrolled on the study, who received at least one dose of the treatment (see Fig. 1-which shows the Study Schema).Baseline Characteristics (See Table 1):

[0073] At the time of cutoff (16 September 2024), 79 subjects with advanced NSCLC received greater than one dose of THIO.

[0074] All subjects had previously failed at least 1 prior line of ICI + / - chemotherapy in the advanced setting and had documented disease progression at study entry

[0075] 34% of patients had 2 or more prior treatments lines at study entry.Table 1. Study Baseline CharacteristicsSex, n (%) i

[0076] DCR, [defined as CR, PR, or SD per RECIST 1.1 at Cycle 3 Day 1 (6 weeks)] met the pre-determined statistical requirements per protocol to proceed to Stage 2. However, the data is not mature yet to assess the ORR.

[0077] Disease control was reported in 32 of 35 subjects (91%) with at least one post baseline assessment, including 17 of 17 subjects (100%) in 2nd line and 14 of 16 subjects (88%) in 3rd line (Table 2).

[0078] By dose level, Disease control was reported in 11 of 13 subjects (85%) assigned THIO 360 mg / cycle, 10 of 10 subjects (100%) assigned THIO 180 mg / cycle, and 11 of 12 subjects (92%) assigned THIO 60 mg / cycle.

[0079] Safety Findings- Tables 2 and 3 below show related Treatment Emergent Adverse Event (TEAEs) by level reported in greater than 2 patientsTable 2- Related TEAEs by dose level reported in >2 patientsTable 3. Related Grade > 3 TEAEsCerebellar and ischemic strokes reported refer to the same event. The medical monitor assessment was unrelated

[0080] Overall, THIO + cemiplimab has been generally well tolerated in a heavily pre-treated population, with most events being Grade 1-2 in severity.

[0081] Most TEAEs were laboratory value elevations, except nausea (12.7% overall and 2.4% at the 180 mg dose) and decreased appetite (3.8% overall and 2.4% at the 180 mg dose).

[0082] No DLTs have been reported in the Part A safety lead in.

[0083] A related Grade >3 ALT increase was reported in 9 patients (11.4%), including 2 patients receiving 360 mg, 4 at 180 mg, and 3 at 60 mg. No clinical symptoms were associated with the elevated laboratory values, and all returned to baseline or normal without sequelae.

[0084] All other related Grade >3 events occurred in <5% of patients. Following an event of Grade 4 LFT elevation in a patient receiving 360 mg in Part B, enrollment into the 360 mg arm was paused.

[0085] Enrollment was completed in Part B at the selected dose of 180 mg / cycle in February 2024.

[0086] THIO mechanism of action allows for more selective targeting of cancer cells, potentially reducing the frequency of adverse events relative to nontargeted therapies.Efficacy Findings:

[0087] 69 evaluable patients had completed >1 post-baseline assessment at the time of data cut-off (45 in 2L, 20 in 3L, 4 in 4L+). Partial Responses (PRs) RECIST 1.1 were reported for 9 subjects (6 in 2L, 3 in 3L), with 7 PRs confirmed by a 2nd scan per Investigators' assessment (4 in 2L, 3 in 3L). 19 patients with survival follow-up above 12 months: 9 in 2L, 9 ongoing follow-up: 10 in 3L, 8 ongoing follow-up; and 1 patient has received 25 cycles of therapy

[0088] In the 3L setting (n=20): DCR was 85% for THIO vs. standard of care 25-35% for chemotherapy (Matsumoto H, et al. Transl Lung Cancer Res 2021;10:2278-89; 14 / 20 (70%) patients crossed the 5.8-month OS threshold (Girard N, et al. J Thorac One 2009;12:1544-1549);17 / 20 (85%) patients crossed the 2.5- month PFS threshold (Shepherd F, et al. N Engl J Med 2005;353:123-132; and Fossella F, et al. J Clin Oncol 2000;18(12):2354-62). The median survival followup time is currently 11.5 months (n=20).

[0089] In the 3L setting with THIO at 180 mg (n=8) the Median PFS was: 5.5 months (24.1 weeks); the OS rate at 6 months was 75%; the ORR was 38% (3 / 8) versus standard of care 6-10% for chemotherapy (Girard N, et al. J ThoracOnc 2009;12:1544-154. Still further, 6 / 8 (75%) patients crossed the 5.8-month OS threshold (Id.). 7 / 8 (88%) patients crossed the 2.5-month PFS threshold (Shepherd F, et al. N Engl J Med 2005;353:123-132; and Fossella F, et al. J Clin Oncol 2000;18(12):2354-62); and the median survival follow-up time is currently 11.4 months (n=8).Conclusions:

[0090] The combination of THIO + cemiplimab has durable activity in this hard-to-treat patient population (CPI resistant and chemotherapy resistant progressors).

[0091] The ORR in the 3L setting with the 180 mg dose is 38% (3 / 8), which compares favorably with response rates reported of ~6% for other currently available treatments for heavily pre-treated patients.

[0092] Median survival follow-up in the 3L setting has surpassed 11.5 months.

[0093] Induction of TIFs in CTCs from patients treated with THIO + cemiplimab shows on-target effect. These findings suggest a potential link between biomarker TIF positivity and more favorable clinical outcomes.

[0094] THIO + cemiplimab has so far been generally well-tolerated in a heavily pre-treated population.

[0095] Treatment has the potential to be given for longer, which usually translates into longer survival.

[0096] The ongoing Phase 2 study selected the best dose of THIO in November 2023. The 180 mg dose has shown better safety and superior efficacy compared with other doses: to date, 9.8% of patients receiving the 180 mg dose reported related Grade >3 AEsBiomarker Findings- Induction of Telomere dysfunction-induced foci (TIF)- positive CTCs:

[0097] Induction of Telomere dysfunction-induced foci (TIF)-positive CTCs were identified from patients treated with THIO and cemiplimab which shows on-target effect. Telomere dysfunction-induced foci (TIF)-positive CTCs were characterized as TRFl+ / gammaH2AX+fraction in CD326+ / PanKRT+ CTCs. TIF (Telomere dysfunction-induced Foci) analysis demonstrated the intended on- target mechanism of action: modification of telomeres in circulating tumor cells (CTCs) by THIO (Figure 5).

[0098] On average, patients in the Stable Disease (SD) and Partial Response (PR) groups showed increased levels of TIF biomarker, whereas the Progressive Disease (PD) group did not demonstrate a statistically significant increase in the TIF biomarker (Figure 6).

[0099] These data indicate that TIF formation in CTCs was shown to be a good biomarker of on-target activity.

[0100] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of particular embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims

Claims

WHAT IS CLAIMED IS:

1. A method of treating a non-small cell lung cancer (NSCLC) in a subject comprising administering to said subject 6-thio-2’ -deoxyguanosine (6-thio-dG) followed by treatment with cemiplimab (Libtayo®).

2. The method according to claim 1, wherein 6-thio-dG is administered at a dose of about 60 mg, 180 mg, 360 mg, or 540 mg.

3. The method according to claim 1, wherein 6-thio-dG is administered at a dose of about 60 mg.

4. The method according to claim 1, wherein 6-thio-dG is administered at a dose of about 180 mg.

5. The method according to claim 1, wherein 6-thio-dG is administered at a dose of about 360 mg.

6. The method according to claim 1, wherein 6-thio-dG is administered at a dose of about 540 mg.

7. The method according to claim 1, wherein 6-thio-dG is administered at a dose of about 60 mg to about 540 mg.

8. The method according to claim 1, wherein 6-thio-dG is administered at a dose of about 60 mg to about 360 mg.

9. The method according to claim 1, wherein 6-thio-dG is administered at a dose of about 60 mg-180 mg.

10. The method according to claim 1, wherein 6-thio-dG is administered at a dose of about 180 mg to about 360mg.

11. The method according to claim 1, wherein 6-thio-dG is administered at a dose of about 180 mg to about 540 mg.

12. The method according to claim 1, wherein 6-thio-dG is administered at a dose of about 360 mg to about 540 mg.

13. The method according to claim any one of claims 1-12, wherein 6-thio-dG is administered on days 1-3 followed by cemiplimab.

14. The method according to claim 13, wherein 6-thio-dG is administered for 3 days followed by cemiplimab.

15. The method according to any one of claims 1-12, wherein cemiplimab is administered on day 5.

16. The method according to any one of claims 1-15 wherein the administration of 6-thio-dG and cemiplimab results in inhibition of tumor growth.

17. The method according to any one of claims 1-15 wherein the administration of 6-thio-dG and cemiplimab results in remission of the NSCLC.

18. The method according to any one of claims 1-15 wherein the administration of 6-thio-dG and cemiplimab results in reduction in NSCLC tumor burden.

19. The method according to any one of claims 1-15 wherein the administration of 6-thio-dG and cemiplimab results in disease free progression of NSCLC.

20. The method according to any one of claims 1-15 wherein the administration of 6-thio-dG and cemiplimab results in disease free progression of NSCLC from about 6 months to greater than about one year.

21. The method of claim 1, wherein the subject being treated has an NSCLC that is resistant to check point inhibitors.

22. The methods of any one of claims 1-21, wherein the NSCLC treated with THIO plus cemiplimab results in the production of telomere dysfunction-induced foci (TIF)-positive circulating tumor cells.

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