Treating triple negative breast cancer (TNBC) with combinations of immunotherapy and acylfulvenes

Abstract

The disclosure provides combination regimens and compositions for the treatment of triple-negative breast cancer (TNBC). A subject in need thereof is administered a therapeutically effective amount of hydroxyureamethyl acylfulvene and a therapeutically effective amount of an immunotherapy such as a PD-1 or PD-L1 checkpoint inhibitor. Also disclosed are pharmaceutical compositions and kits containing the acylfulvene, the immunotherapy component, or both in admixture with pharmaceutically acceptable carriers.

Description

Treating Triple Negative Breast Cancer (TNBC) with Combinations of Immunotherapy and AcylfulvenesPRIOR RELATED APPLICATION DATA

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 674,475, filed July 23, 2024, which is incorporated by reference herein in its entirety.TECHNICAL FIELD

[0002] This application relates to cancer treatments and more specifically this application relates to cancer treatments using a combination therapy including immunotherapy and acylfulvene.BACKGROUND

[0003] Triple-negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer and is characterized by the absence of estrogen receptors, progesterone receptors, and HER2 protein. TNBC often has a poorer prognosis compared to other breast-cancer subtypes, partly due to the lack of targeted therapies. Resistance to anticancer treatments is common, and the prognosis of advanced TNBC remains poor. There are several potential sources of cancer-drug resistance, including alterations to drug transporters, the suppression of apoptosis, mitochondrial alterations, the promotion of DNA-damage repair, autophagy, epithelial-mesenchymal transition, and cancer stem cells (CSCs). Appropriate strategies that consider these mechanisms are necessary to treat TNBC effectively.

[0004] Combination-therapy treatments for cancer have become more common, in part due to the perceived advantage of attacking the disease via multiple avenues. Although many effective combination-therapy treatments have been identified over the past few decades; in view of the continuing high number of deaths each year resulting from TNBC, a continuing need exists to identify effective therapeutic regimens for use in anticancer treatment.

[0005] Accordingly, there is always a need for improved methods to treat TNBC.BRIEF DESCRIPTION OF THE FIGURES

[0006] FIG. 1 shows a graph of mean tumor volume versus time in the syngeneic Til TNBC model, comparing vehicle + isotype, anti-PD-1 alone, LP-184 alone, and the LP-184 + anti-PD-1 combination.

[0007] FIG. 2 shows a scatter (or box-and-whisker) plot of individual tumor volumes at the day- 22 end-point for the four Til treatment cohorts of FIG. 1.

[0008] FIGs. 3A and 3B show flow-cytometry bar charts quantifying total intratumoral macrophages (CDllb+F4 / 80+) across the four TH treatment groups, showing an overall macrophage reduction after LP-184 administration.

[0009] FIG. 3C shows bar charts or stacked plot depicting the relative proportions of Ml (CD86+) versus M2 (CD206+) macrophages from FIG. 3 A.

[0010] FIG. 3D shows bar charts showing absolute numbers (or percentages) of CD8+and CD4+T cells infiltrating Ti l tumors.

[0011] FIG. 4 shows histogram or flow-cytometry overlay demonstrating increased Granzyme B and TNF-a expression in intratumoral CD8+T cells after combination treatment.

[0012] FIG. 5A shows graph of mean tumor volume versus time in Til mice treated on a synchronized, low-intensity schedule.

[0013] FIG. 5B shows corresponding tumor-volume graph for the 4T1-PTGR1 TNBC model under the same treatment schedule.

[0014] FIG. 6 shows an immunofluorescence micrographs of MDA-MB-231 cells treated with LP-184.

[0015] FIG. 7 shows Single-cell RNA-seq heatmap (or dot plot) of 4T1 tumor myeloid clusters after treatments.

[0016] FIG. 8 shows heatmap of tumor-cell transcript levels showing up-regulation of majorhistocompatibility-complex class I and II genes and induction of type-I and type-II interferonresponsive programs in tumors receiving the LP-184 + anti-PD-1 combination.

[0017] FIG. 9 illustrates the enhanced activation of interferon signaling pathways in tumor cells following combination therapy.SUMMARY

[0018] This application discloses the discovery that treatment of cancer in a subject with an combination of an illudin or an illudin analog (e.g., acylfulvene) and immunotherapy has greater effects (i.e., greater than the effects of each added together) than those provided by either agent alone. For example, the cytotoxicity delivered from treating a cancer with a combination of illudin or acylfulvene and immunotherapy is unexpectedly greater compared to the cytotoxicity delivered when treating the cancer with illudin or acylfulvene or immunotherapy, alone (or greater than the cytotoxicity of both added together). In addition, it was discovered that the combination therapy results in more rapid killing of cancer cells and more rapid tumor shrinkage than was found when either therapy, alone, was used.

[0019] One aspect of this application includes a combination therapy for treating cancers. In embodiments, the therapy includes administering a combination of active agents including an illudin or illudin analog (e.g., acylfulvene), and immunotherapy.

[0020] Another aspect of this application provides pharmaceutical compositions comprising an illudin or illudin analog (e.g., acylfulvene) and immunotherapy, or pharmaceutically acceptable salts thereof, mixed with pharmaceutically suitable carriers or excipient(s) at doses to treat or prevent cancer. The pharmaceutical compositions can also be administered in combination with other therapeutic agents or therapeutic modalities simultaneously, sequentially, or in alternation.

[0021] Another aspect of this application includes therapeutically effective amounts of both illudin or acylfulvene and immunotherapy, which, when used in combination, will be lower than the amounts required for monotherapy with each agent alone. Such dose reduction can lessen toxicity.

[0022] Another aspect of this application includes use of (-)-hydroxyureamethyl acylfulvene as the acylfulvene component of the therapy.

[0023] Another aspect of this application includes the therapy including an acylfulvene that is Irofulven.

[0024] Another aspect of this application includes treatments of solid tumors, and hematological malignancies. Tumors such as, but not limited to, neoplastic disease, such as a carcinoma, sarcoma, or mixed type cancer, including breast, colon, rectal, endometrial, gastric, prostate, or brain, mesothelioma, ovarian, lung or pancreatic cancer can be targeted for therapy.DETAILED DESCRIPTION

[0025] This application provides a combination therapy for treating TNBC. In embodiments, the therapy includes administering a combination of active agents including an illudin or an illudin analog (e.g., acylfulvene) and immunotherapy. In other embodiments, the therapy includes administering a combination of other therapies. In other embodiments, the therapy includes the combination therapy can be used to treat biochemical occurrence and recurrence of TNBC in which an acylfulvene (e.g., hydroxyureamethyl acylfulvene) or a salt thereof and immunotherapy are administered in a therapeutically effective amount to the patient.111 nd in or Acylfulvene

[0026] In one embodiment, this application includes the use of an illudin or an illudin analog (e.g., acylfulvene). Acylfulvenes are a class of cytotoxic semi -synthetic derivatives of illudin, a natural product that can be extracted from the jack o'lantern mushroom (Omphalotus olearius). Acylfulvene, derived from the sesquiterpene illudin S by treatment with acid (reverse Prins reaction), is far less reactive to thiols than illudin S.

[0027] In one example, the acylfulvene is (-) - hydroxyureamethyl acylfulvene (termed LP- 184 by Lantern Pharma Inc.), which shifts light negatively, is shown below:Combination Therapy

[0028] In one embodiment, acylfulvene or hydroxyureamethyl acylfulvene or its salt may be administered either prior to, concomitantly with, or subsequent to the administration of immunotherapy. In one embodiment, an acylfulvene — such as hydroxyureamethyl acylfulvene —or a pharmaceutically acceptable salt thereof is administered in a flexible sequence relative to the immunotherapy component to maximize synergy while preserving tolerability. Administration can (a) precede immunotherapy by an interval sufficient to create DNA lesions that enhance tumor immunogenicity — typically 2 to 48 hours before infusion of the checkpoint inhibitor; (b) occur concomitantly — for example, on the same day or within a 2-hour window — to ensure overlapping systemic exposure and cooperative engagement of DNA-damage-response and immune-activation pathways; or (c) follow immunotherapy — generally 24 hours to 7 days later — to exploit immunotherapy-induced down-regulation of nucleotide-excision-repair proteins (e.g., XPB / ERCC3) and thereby heighten tumor susceptibility to acylfulvene-mediated cytotoxicity. The precise sequencing, dose, and interval are selected according to tumor burden, pharmacokinetic profiles, and patient-specific factors such as renal and hepatic function, with the goal of achieving a pharmacodynamic window in which circulating acylfulvene concentrations overlap with peak cytotoxic T-cell activity while maintaining acceptable safety margins.Immunotherapies

[0029] Immunotherapies can be treatments that use the body's immune system to fight cancer They work by stimulating or restoring the immune system's natural ability to detect and destroy cancer cells. There are several types of immunotherapies used in cancer treatment, including checkpoint inhibitors, adoptive cell transfer, monoclonal antibodies, and cancer vaccines. Checkpoint inhibitors, such as pembrolizumab (Keytruda) and nivolumab (Opdivo), block proteins that prevent immune cells from attacking cancer cells. Adoptive cell transfer involves modifying a patient's T cells to enhance their cancer- fighting capabilities before reinfusing them into the patient. Monoclonal antibodies, like trastuzumab (Herceptin), are designed to target specific antigens on cancer cells, marking them for destruction by the immune system. Cancer vaccines, such as sipuleucel-T (Provenge), stimulate the immune system to attack cancer cells by presenting them with cancer-specific antigens.

[0030] Checkpoint Inhibitors used with breast cancer include Pembrolizumab (Keytruda), Atezolizumab (Tecentriq), Nivolumab (Opdivo) Monoclonal Antibodies include Trastuzumab (Herceptin), Pertuzumab (Peg eta), Trastuzumab deruxtecan (Enhertu) and Atezolizumab (Tecentriq) in combination with nab-paclitaxel. Other can be antibody-drug conjugates. Other canbe cancer vaccines such as E75 (NeuVax), GP2 vaccine. Immune Modulators include Interleukin- 2 (IL-2), Interferon-alpha (IFN-a)

[0031] One aspect of this application includes a method of treating TNBC in a subject in need thereof The method involves administering to the subject an effective amount of immunotherapy and an effective amount of an acylfulvene. Immunotherapy may be administered prior to or concomitantly with an acylfulvene for optimal synergistic effects. Immunotherapy degrades the key Nucleotide Excision Repair (NER) protein XPB / ERCC3 (reference: https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC7277409 / ) and leads to NER deficiency. Subjects with NER deficiency are more sensitive to an illudin- based anti -cancer agent.

[0032] Another embodiment includes a pharmaceutical composition having a therapeutically effective amount of an illudin or an illudin analog thereof, derivative, or a pharmaceutically acceptable salt thereof; and a therapeutically effective amount of immunotherapy or an analog, derivative, or a pharmaceutically acceptable salt thereof. The illudin analog can be HydroxyUreaMethylAcylfulvene.

[0033] In another embodiment, a kit for the treatment of TNBC in a subject includes a therapeutically effective amount of an illudin or an illudin analog thereof, derivative, or a pharmaceutically acceptable salt thereof; and a therapeutically effective amount of immunotherapy or an analog, derivative, or a pharmaceutically acceptable salt thereof:

[0034] In another embodiment, the second therapeutic is one or more chemotherapeutic agents selected from camptothecin derivatives, paclitaxel, docetaxel, epothilone B, 5-FU, gemcitabine, oxaliplatin, cisplatinum, carboplatin, melphalan, dacarbazine, temozolomide, doxorubicin, imatinib, erlotinib, bevacizumab, cetuximab, and a Raf kinase inhibitor.

[0035] In another embodiment, the second therapeutic is one or more chemotherapeutic agents selected from paclitaxel or cisplatinum.

[0036] The term “combination therapy” can include or includes the administration of the therapeutic agents as described above in further combination with other biologically active ingredients and non-drug therapies (e.g., surgery or radiation treatment). Where the combination therapy further comprises a non-drug treatment, the non-drug treatment may be conducted at any suitable time so long as a beneficial effect from the co-action of the combination of the therapeutic agents and non-drug treatment is achieved. For example, in appropriate cases, the beneficial effectis still achieved when the non-drug treatment is temporally removed from the administration of the therapeutic agents, perhaps by days or even weeks.

[0037] In another aspect, a composition or combination therapy herein, or a pharmaceutically acceptable salt or solvate thereof, may be administered in combination with radiation therapy. Radiation therapy can also be administered in combination with a composition of the present invention and another chemotherapeutic agent described herein as part of a multiple agent therapy.

[0038] Combination therapy can be achieved by administering two or more agents, e.g., an acylfulvene, immunotherapy, and one or more other therapeutic agents, each of which is formulated and administered separately, or by administering two or more agents in a single formulation. Other combinations are also encompassed by combination therapy. For example, two agents can be formulated together and administered in conjunction with a separate formulation containing a third agent. While the two or more agents in the combination therapy can be administered simultaneously, they need not be. For example, administration of a first agent (or combination of agents) can precede administration of a second agent (or combination of agents) by minutes, hours, days, or weeks. Thus, the two or more agents can be administered within minutes of each other or within 1, 2, 3, 6, 9, 12, 15, 18, or 24 hours of each other or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 days of each other or within 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks of each other. In some cases, even longer intervals are possible. While in many cases it is desirable that the two or more agents used in combination therapy be present within the patient's body at the same time, this need not be so.

[0039] The methods of combination therapy may or should result in a synergistic or additive effect, wherein the effect of a combination of compounds or other therapeutic agents is greater than the sum of the effects resulting from the administration of any of the compounds or other therapeutic agents as single agents. A synergistic effect may also be an effect that cannot be achieved by administration of any of the compounds or other therapeutic agents as single agents. The synergistic effect may include, but is not limited to, an effect of treating TNBC by reducing tumor size, inhibiting tumor growth, or increasing the survival of the subject. The synergistic effect may also include reducing cancer cell viability, inducing cancer cell death, and inhibiting or delaying cancer cell growth.

[0040] Therapeutically effective doses can vary, as recognized by those skilled in the art, depending on the diseases treated, the severity of the disease, the route of administration, the ageand general health condition of the patient, excipient usage, the possibility of co- usage with other therapeutic treatments such as the use of other agents, and the judgment of the treating physician. For example, guidance for selecting an effective dose can be determined by reference to the prescribing information for hydroxyureamethyl acylfulvene or journal discussions of the same.

[0041] The term “effective amount” as used herein refers to the amount of an agent needed to alleviate at least one or more symptoms of the disease or disorder and relates to a sufficient amount of a pharmacological composition to provide the desired effect. The term “therapeutically effective amount” therefore refers to an amount of the agent that is sufficient to provide a particular effect when administered to a typical subject. An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom of the disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease. Thus, it is not generally practicable to specify an exact “effective amount”. However, for any given case, an appropriate “effective amount” can be determined by one of ordinary skill in the art using only routine experimentation.

[0042] The dosage ranges for the administration of an agent according to the methods described herein depend upon, for example, the form of the agent, its potency, and the extent to which symptoms, markers, or indicators of a condition described herein are desired to be reduced, for example, the percentage reduction desired for tumor growth. The dosage should not be so large as to cause adverse side effects. Generally, the dosage will vary with the age, condition, and sex of the patient and can be determined by one of skill in the art. The dosage can also be adjusted by the individual physician in the event of any complication.

[0043] The term “therapeutically effective amount”, as used herein, refers to an amount of a pharmaceutical agent to treat, ameliorate, or prevent an identified disease or condition, or to exhibit a detectable therapeutic or inhibitory effect. The effect can be detected by any assay method known in the art. The precise effective amount for a subject will depend upon the subject's body weight, size, and health; the nature and extent of the condition; and the therapeutic or combination of therapeutics selected for administration. Therapeutically effective amounts for a given situation can be determined by routine experimentation that is within the skill and judgment of the clinician. In a preferred aspect, the disease or condition to be treated is TNBC. In another aspect, the disease or condition to be treated is a cell proliferative disorder.

[0044] The efficacy of an agent described herein in, e.g., the treatment of a condition described herein, or to induce a response as described herein (e.g., TNBC) can be determined by the skilled clinician. However, a treatment is considered “effective treatment” (or a “therapeutically effective treatment”) as the term is used herein, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced e.g., by at least 10% following treatment according to the methods described herein. Therapeutic effectiveness refers to the assessment of the observable consequences of a therapeutic intervention in relation to its intended aim. Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and / or the incidence of a condition treated according to the methods described herein or any other measurable parameter appropriate, e.g., tumor size and / or growth rate. Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization or the need for medical interventions (i.e., progression of the disease is halted). Methods of measuring these indicators are known to those of skill in the art and / or are described herein Treatment includes any treatment of a disease in an individual or an animal (some non- limiting examples include a human or an animal) and includes: (1) inhibiting the disease, e.g., preventing a worsening of symptoms (e.g., pain or inflammation); or (2) relieving the severity of the disease, e.g., causing regression of symptoms. An effective amount for the treatment of a disease means that amount which, when administered to a subject in need thereof, is sufficient to result in effective treatment as that term is defined herein, for that disease. Efficacy of an agent can be determined by assessing physical indicators of a condition or desired response. It is well within the ability of one skilled in the art to monitor the efficacy of administration and / or treatment by measuring any one of such parameters, or any combination of parameters. Efficacy can be assessed in animal models of a condition described herein, for example, treatment of TNBC in a mouse model. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change in a marker is observed, e.g., tumor size and / or growth rate. In some embodiments, the therapeutically effective amount of hydroxyureamethyl- acylfulvene, acylfulvenes, or Irofulven, or a pharmaceutically acceptable salt thereof is selected from the group consisting of 0.5 mg / day, 1 mg / day, 2.5 mg / day, 5 mg / day, 10 mg / day, 20 mg / day, 30 mg / day, 60 mg / day, 90 mg / day, 120 mg / day, 150 mg / day, 180 mg / day, 210 mg / day, 240 mg / day, 270 mg / day, 300 mg / day, 360 mg / day, 400 mg / day, 440 mg / day,480 mg / day, 520 mg / day 580 mg / day, 600 mg / day, 620 tng / day, 640 mg / day, 680 mg / day, and 720 mg / day.

[0045] The administration dose should be adjusted for the requirement of the individual in need For example, in humans, the administration of acylfulvene daily for the treatment of TNBC can be based on established clinical guidelines and adjusted according to the patient's response and tolerability. For illustration, acylfulvene dose concentrations that can be used range from 5 to 25 pM, 25 to 100 rng / kg in mice (i.p.), and 20 to 200 mg daily (oral) clinically in humans. The dose may vary based on weight or other physical characteristics of the patient.

[0046] The term “treat” is used and includes both therapeutic treatment and prophylactic treatment (reducing the likelihood of development). Both terms mean to decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease (e g., TNBC), lessen the severity of the disease, or improve the symptoms associated with the disease.

[0047] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0048] The composition of the present invention is capable of further forming salts. The composition of the present invention can form more than one salt per molecule, e g., mono-, di-, tri-. All of these forms are also contemplated within the scope of the claimed invention.

[0049] As used herein, “pharmaceutically acceptable salts” refer to derivatives of the compounds of the present invention wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic acids, and the like. For example, such conventional non -toxic salts include, but are not limited to, those derived from inorganic and organic acids selected from 2- acetoxybenzoic, 2- hydroxyethane sulfonic, acetic, ascorbic, benzene sulfonic, benzoic, bicarbonic, carbonic, citric, edetic, ethane disulfonic, 1,2-ethane sulfonic, fumaric, glucoheptonic, gluconic, glutamic, glycolic, glycollyarsanilic, hexylresorcinic, hydrabamic, hydrobromic, hydrochloric, hydroiodic, hydroxy maleic, hydroxy naphthoic, isethionic, lactic, lactobionic, lauryl sulfonic, maleic, malic, mandelic, methane sulfonic, napsylic, nitric, oxalic, pamoic, pantothenic, phenylacetic, phosphoric, polygalacturonic, propionic, salicylic, stearic, subacetic, succinic, sulfamic, sulfanilic, sulfuric, tannic, tartaric, toluene sulfonic, and the commonly occurring amine acids, e.g., glycine, alanine, phenylalanine, arginine, etc.

[0050] Other examples of pharmaceutically acceptable salts include hexanoic acid, cyclopentane propionic acid, pyruvic acid, malonic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, 4- chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4- toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo-[2.2.2]-oct-2-ene-l-carboxylic acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, muconic acid, and the like. The present invention also encompasses salts formed when an acidic proton in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like.

[0051] It should be understood that all references to pharmaceutically acceptable salts include solvent addition forms (solvates).

[0052] As used herein, the term “selectively” means tending to occur at a higher frequency in one population than in another population. The compared populations can be cell populations. Preferably, a compound of the present invention, or a pharmaceutically acceptable salt or solvate thereof, acts selectively on a cancer or precancerous cell but not on a normal cell. Preferably, a compound of the present invention, or a pharmaceutically acceptable salt or solvate thereof, acts selectively to modulate one molecular target (e.g., nucleotide excision repair (NER) players ERCC3). The invention also provides a method for selectively inhibiting the activity of an enzyme, such as NER proteins. Preferably, an event occurs selectively in population A relative to population B if it occurs greater than two times more frequently in population A as compared to population B. An event occurs selectively if it occurs greater than five times more frequently in population A An event occurs selectively if it occurs greater than ten times more frequently in population A; more preferably, greater than fifty times; even more preferably, greater than 100 times; and most preferably, greater than 1000 times more frequently in population A as compared to population B. For example, cell death would be said to occur selectively in cancer cells if it occurred greater than twice as frequently in cancer cells as compared to normal cells.

[0053] The composition or pharmaceutically acceptable salts or solvates thereof, can be administered orally, nasally, transdermally, pulmonarily, inhalationally, buccally, sublingually, intraperitoneally, subcutaneously, intramuscularly, intravenously, rectally, intrapleurally, intrathecally, and parenterally. In one embodiment, the compound is administered orally. One skilled in the art will recognize the advantages of certain routes of administration.

[0054] The dosage regimen utilizing the compounds is selected in accordance with a variety of factors including type, species, age, weight, sex, and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt thereof employed. An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to prevent, counter, or arrest the progress of the condition.

[0055] Techniques for formulation and administration of the disclosed compounds of the invention can be found in Remington: the Science and Practice of Pharmacy, 19.sup.th edition, Mack Publishing Co., Easton, Pa. (1995). In an embodiment, the compounds described herein, and the pharmaceutically acceptable salts thereof, are used in pharmaceutical preparations in combination with a pharmaceutically acceptable carrier or diluent. Suitable pharmaceutically acceptable carriers include inert solid fillers or diluents and sterile aqueous or organic solutions. The compounds will be present in such pharmaceutical compositions in amounts sufficient to provide the desired dosage amount in the range described herein.

[0056] All percentages and ratios used herein, unless otherwise indicated, are by weight. Other features and advantages of the present invention are apparent from the different examples. The provided examples illustrate different components and methodology useful in practicing the present invention. The examples do not limit the claimed invention. Based on the present disclosure, the skilled artisan can identify and employ other components and methodology useful for practicing the present invention.

[0057] As used herein, a “subject in need thereof is a subject having a precancerous condition. Preferably, a subject in need thereof has TNBC. A “subject” includes a mammal. The mammal can be, e.g., any mammal, e.g., a human, primate, bird, mouse, rat, dog, cat, cow, horse, goat, camel, sheep, or a pig. Preferably, the mammal is a human. The subject of the present invention includes any human subject who has been diagnosed with, has symptoms of, or is at risk of developing TNBC or a precancerous condition.

[0058] A subject in need thereof may have refractory or resistant TNBC. “Refractory or resistant TNBC” means TNBC that does not respond to treatment. The TNBC may be resistant at the beginning of treatment, or it may become resistant during treatment. In some embodiments, the subject in need thereof has TNBC recurrence following remission on most recent therapy. In some embodiments, the subject in need thereof received and failed all known effective therapies forTNBC treatment. In some embodiments, the subject in need thereof received at least one prior therapy. In certain embodiments, the prior therapy is monotherapy. In certain embodiments, the prior therapy is combination therapy.

[0059] In some embodiments, a subject in need thereof may have a secondary cancer as a result of a previous therapy. “Secondary cancer” means cancer that arises due to or as a result of previous carcinogenic therapies, such as chemotherapy.

[0060] TNBC is a group of diseases that may cause almost any sign or symptom. The signs and symptoms will depend on where the TNBC is, the size of the TNBC, and how much it affects the nearby organs or structures. If TNBC spreads (metastasizes), then symptoms may appear in different parts of the body.

[0061] Treating TNBC can result in a reduction in size of a tumor. A reduction in size of a tumor may also be referred to as “tumor regression”. Preferably, after treatment, tumor size is reduced by 5% or greater relative to its size prior to treatment; more preferably, tumor size is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced by greater than 75% or greater. Size of a tumor may be measured by any reproducible means of measurement. The size of a tumor may be measured as a diameter of the tumor.

[0062] Treating TNBC results in a decrease in the number and size of tumors. Preferably, after treatment, tumor number or size is reduced by 5% or greater relative to that prior to treatment; more preferably, by 10% or greater; more preferably, by 20% or greater; more preferably, by 30% or greater; more preferably, by 40% or greater; even more preferably, by 50% or greater; and most preferably, by greater than 75%. The number of tumors may be measured by any reproducible means. The number of tumors may be measured by counting tumors visible to the naked eye or at a specified magnification. Preferably, the specified magnification is 2x, 3*, 4x, 5x;iox, or 50*.

[0063] Treating TNBC can result in a decrease in the number of metastatic lesions in other tissues or organs distant from the primary tumor site. Preferably, after treatment, the number of metastatic lesions is reduced by 5% or greater relative to the number prior to treatment; more preferably, the number of metastatic lesions is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced by greater than75%. The number of metastatic lesions may be measured by any reproducible means of measurement. The number of metastatic lesions may be measured by counting metastatic lesions visible to the naked eye or at a specified magnification. Preferably, the specified magnification is 2x, 3x, 4x, 5x, lOx, or 50x.

[0064] Treating TNBC can result in an increase in the average survival time of a population of treated subjects in comparison to a population receiving the carrier alone Preferably, the average survival time is increased by more than 30 days; more preferably, by more than 60 days; more preferably, by more than 90 days; and most preferably, by more than 120 days. An increase in the average survival time of a population may be measured by any reproducible means. An increase in the average survival time of a population may be measured, for example, by calculating for a population the average length of survival following initiation of treatment with an active compound. An increase in the average survival time of a population may also be measured, for example, by calculating for a population the average length of survival following completion of a first round of treatment with an active compound.

[0065] Treating TNBC can result in an increase m the average survival time of a population of treated subjects in comparison to a population of untreated subjects. Preferably, the average survival time is increased by more than 30 days; more preferably, by more than 60 days; more preferably, by more than 90 days; and most preferably, by more than 120 days. An increase in the average survival time of a population may be measured by any reproducible means. An increase in the average survival time of a population may be measured, for example, by calculating for a population the average length of survival following initiation of treatment with an active compound. An increase in the average survival time of a population may also be measured, for example, by calculating for a population the average length of survival following completion of a first round of treatment with an active compound.

[0066] Treating TNBC can result in an increase m the average survival time of a population of treated subjects in comparison to a population receiving monotherapy with a drug that is not a compound of the present invention, or a pharmaceutically acceptable salt or solvate thereof. Preferably, the average survival time is increased by more than 30 days; more preferably, by more than 60 days; more preferably, by more than 90 days; and most preferably, by more than 120 days. An increase in the average survival time of a population may be measured by any reproducible means. An increase in the average survival time of a population may be measured, for example,by calculating for a population the average length of survival following initiation of treatment with an active compound. An increase in the average survival time of a population may also be measured, for example, by calculating for a population the average length of survival following completion of a first round of treatment with an active compound.

[0067] Treating TNBC can result in a decrease in the mortality rate of a population of treated subjects in comparison to a population receiving the carrier alone. Treating TNBC can result in a decrease in the mortality rate of a population of treated subjects in comparison to an untreated population. Treating TNBC can result in a decrease in the mortality rate of a population of treated subjects in comparison to a population receiving monotherapy with a drug that is not a compound of the present invention, or a pharmaceutically acceptable salt or solvate thereof. Preferably, the mortality rate is decreased by more than 2%; more preferably, by more than 5%; more preferably, by more than 10%; and most preferably, by more than 25%. A decrease in the mortality rate of a population of treated subjects may be measured by any reproducible means. A decrease in the mortality rate of a population may be measured, for example, by calculating for a population the average number of disease-related deaths per unit time following initiation of treatment with an active compound. A decrease in the mortality rate of a population may also be measured, for example, by calculating for a population the average number of disease-related deaths per unit time following completion of a first round of treatment with an active compound.

[0068] Treating TNBC can result in a decrease in the tumor growth rate. Preferably, after treatment, the tumor growth rate is reduced by at least 5% relative to the number prior to treatment; more preferably, the tumor growth rate is reduced by at least 10%; more preferably, reduced by at least 20%; more preferably, reduced by at least 30%; more preferably, reduced by at least 40%; more preferably, reduced by at least 50%; even more preferably, reduced by at least 50%; and most preferably, reduced by at least 75%. Tumor growth rate may be measured by any reproducible means of measurement Tumor growth rate can be measured according to a change in tumor diameter per unit time.

[0069] Treating TNBC can result in a decrease in tumor regrowth. After treatment, tumor regrowth can be less than 5%; more preferably, tumor regrowth can be less than 10%; more preferably, less than 20%; more preferably, less than 30%; more preferably, less than 40%; more preferably, less than 50%; even more preferably, less than 50%; and most preferably, less than 75%. Tumor regrowth may be measured by any reproducible means of measurement. Tumor regrowth ismeasured, for example, by measuring an increase in the diameter of a tumor after a prior tumor shrinkage that followed treatment. A decrease in tumor regrowth is indicated by the failure of tumors to reoccur after treatment has stopped.

[0070] Treating or preventing a cell proliferative disorder can result in a reduction in the rate of cellular proliferation. Preferably, after treatment, the rate of cellular proliferation is reduced by at least 5%; more preferably, by at least 10%; more preferably, by at least 20%; more preferably, by at least 30%; more preferably, by at least 40%; more preferably, by at least 50%; even more preferably, by at least 50%; and most preferably, by at least 75%. The rate of cellular proliferation may be measured by any reproducible means of measurement. The rate of cellular proliferation is measured, for example, by measuring the number of dividing cells in a tissue sample per unit time.

[0071] Treating or preventing a cell proliferative disorder can result in a reduction in the proportion of proliferating cells. Preferably, after treatment, the proportion of proliferating cells is reduced by at least 5%; more preferably, by at least 10%; more preferably, by at least 20%; more preferably, by at least 30%; more preferably, by at least 40%; more preferably, by at least 50%; even more preferably, by at least 50%; and most preferably, by at least 75%. The proportion of proliferating cells may be measured by any reproducible means of measurement. Preferably, the proportion of proliferating cells is measured, for example, by quantifying the number of dividing cells relative to the number of nondividing cells in a tissue sample. The proportion of proliferating cells can be equivalent to the mitotic index.

[0072] Treating or preventing a cell proliferative disorder can result in a decrease in the size of an area or zone of cellular proliferation. Preferably, after treatment, the size of an area or zone of cellular proliferation is reduced by at least 5% relative to its size prior to treatment; more preferably, reduced by at least 10%; more preferably, reduced by at least 20%; more preferably, reduced by at least 30%; more preferably, reduced by at least 40%; more preferably, reduced by at least 50%; even more preferably, reduced by at least 50%; and most preferably, reduced by at least 75%. The size of an area or zone of cellular proliferation may be measured by any reproducible means of measurement. The size of an area or zone of cellular proliferation may be measured as a diameter or width of an area or zone of cellular proliferation.

[0073] Treating or preventing a cell proliferative disorder can result in a decrease in the number or proportion of cells having an abnormal appearance or morphology. Preferably, after treatment, the number of cells having an abnormal morphology is reduced by at least 5% relative to its sizeprior to treatment; more preferably, reduced by at least 10%; more preferably, reduced by at least 20%; more preferably, reduced by at least 30%; more preferably, reduced by at least 40%; more preferably, reduced by at least 50%; even more preferably, reduced by at least 50%; and most preferably, reduced by at least 75%. An abnormal cellular appearance or morphology may be measured by any reproducible means of measurement. An abnormal cellular morphology can be measured by microscopy, e.g., using an inverted tissue culture microscope. An abnormal cellular morphology can take the form of nuclear pleiom orphism

[0074] Administering a composition of the present invention to a cell or a subject in need thereof can result in modulation (i.e., stimulation or inhibition) of an activity of a protein methyltransferase of interest.

[0075] Treating TNBC or a cell proliferative disorder can result in cell death, and preferably, cell death results in a decrease of at least 10% in the number of cells in a population. More preferably, cell death means a decrease of at least 20%; more preferably, a decrease of at least 30%; more preferably, a decrease of at least 40%; more preferably, a decrease of at least 50%; most preferably, a decrease of at least 75%. The number of cells in a population may be measured by any reproducible means. Several cells in a population can be measured by fluorescence activated cell sorting (FACS), immunofluorescence microscopy, and light microscopy Methods of measuring cell death are as shown in Li et al., Proc. Natl. Acad. Sci. USA. 100(5): 2674-8, 2003. In an aspect, cell death occurs by apoptosis.

[0076] Preferably, an effective amount of a composition of the present invention, or a pharmaceutically acceptable salt or solvate thereof, is not significantly cytotoxic to normal cells A therapeutically effective amount of a compound is not significantly cytotoxic to normal cells if administration of the compound in a therapeutically effective amount does not induce cell death in greater than 10% of normal cells. A therapeutically effective amount of a compound does not significantly affect the viability of normal cells if the administration of the compound in a therapeutically effective amount does not induce cell death in greater than 10% of normal cells. In an aspect, cell death occurs by apoptosis.

[0077] Contacting a cell with a composition of the present invention, or a pharmaceutically acceptable salt or solvate thereof, can induce or activate cell death selectively in cancer cells Administering to a subject in need thereof a compound of the present invention, or a pharmaceutically acceptable salt or solvate thereof, can induce or activate cell death selectively incancer cells. Contacting a cell with a composition of the present invention, or a pharmaceutically acceptable salt or solvate thereof, can induce cell death selectively in one or more cells affected by a cell proliferative disorder. Preferably, administering to a subject in need thereof a composition of the present invention, or a pharmaceutically acceptable salt or solvate thereof, induces cell death selectively in one or more cells affected by a cell proliferative disorder.

[0078] This application relates to a method of treating or preventing TNBC by administering a composition described herein, or a pharmaceutically acceptable salt or solvate thereof, to a subject in need thereof, where administration results in one or more of the following: prevention of cancer cell proliferation by accumulation of cells in one or more phases of the cell cycle (e g., Gl, Gl / S, G2 / M); induction of cell senescence; promotion of tumor cell differentiation; promotion of cell death in cancer cells via cytotoxicity, necrosis, or apoptosis without significant death in normal cells; or antitumor activity in animals with a therapeutic index of at least 2.

[0079] The term “kit” means a combination of partners as defined above can be dosed independently or by use of different fixed combinations with distinguished amounts of the combination partners, i.e., simultaneously or at different time points. The parts of the kit of parts can then, e.g., be administered simultaneously or chronologically staggered, that is at different time points and with equal or different time intervals for any part of the kit of parts. The ratio of the total amounts of the combination partners to be administered in the combined preparation can be varied. The combination partners can be administered by the same route or by different routes

[0080] One skilled in the art may refer to general reference texts for detailed descriptions of known techniques discussed herein or equivalent techniques. These texts can, of course, also be referred to in making or using an aspect of the invention.EXAMPLESExample 1

[0081] To evaluate whether an acylfulvene can act synergistically or at least additively with immune-checkpoint blockade in triple-negative breast cancer (TNBC), (-) - hydroxyureamethyl acylfulvene or LP-184 was tested in the immunocompetent T11 mouse model. Once orthotopically implanted tumors reached approximately 50 mm3(day 7), the animals were divided into four treatment cohorts. One group received only vehicle plus an isotype control antibody; a second received anti-PD-1 antibody (10 mg kg1, clone RMP 1-14); a third received LP-184 alone(4 mg kg1); and the fourth received the combination of LP-184 with anti-PD-1. LP-184 was administered intraperitoneally on days 7, 10, and 13, whereas anti-PD-1 (or isotype) was given on the same three days and then again on days 15, 17, 20, and 22.

[0082] As shown in FIG. 1, tumor volumes were recorded throughout the study. Whereas LP-184 alone slowed growth by roughly 60 % and anti-PD-1 alone produced only a modest delay, the combination caused a striking 89 % inhibition, with several tumors regressing to near-complete remission by day 22. FIG. 2 shows individual end-point volumes to further show this effect: three of five tumors in the combination arm shrank to < 50 mm3, whereas none in the monotherapy groups did so. Analysis of tumor volume change trend and terminal tumor volumes confirmed that the dual regimen out-performed either agent used singly, indicating at least an additive interaction..

[0083] Flow-cytometric profiling offered mechanistic insight. As summarized in FIGs. 3A, 3B, 3C and 3D, LP-184 alone reduced the overall macrophage compartment, with a disproportionate fall in immunosuppressive M2 (CD206+) cells. When combined with anti-PD-1, this macrophage shift was accompanied by a three-fold increase in intratumoral CD8+T cells and a greater than two-fold rise in CD4+T cells.

[0084] FIG. 4 shows that functional analysis of these lymphocytes revealed heightened expression of Granzyme B and TNF-a, pointing to an activated cytotoxic phenotype.

[0085] The short-term exposure to LP-184 generated DNA double-strand breaks that activated the cGAS-STING pathway, evidenced by phosphorylation of TBK1 and IRF3 in tumor lysates. This innate-immune signaling is believed to drive the chemokine surge (CXCL10, CCL5) that attracts T cells and to cooperate with PD-1 blockade. Importantly, the regimen was well tolerated: transient, self-resolving neutropenia was the only notable systemic effect, and no animal experienced > 10 % body -weight loss.

[0086] The example shows that administering LP-184 at 4 mg kg1on an every -three-day schedule, together with anti-PD-1 at 10 mg kg ', transforms a PD-1 -refractory T 11 TNBC tumor into an immune-responsive state, leading to deep and durable tumor control. The data illustrate how acylfulvene-induced DNA damage can remodel the tumor microenvironment — diminishing M2 macrophages, activating cGAS-STING signaling, and amplifying effector-T-cell function — to unlock the full potential of checkpoint inhibition.Example 2

[0087] Acylfulvene LP-184 reproducibly could amplify immune-checkpoint blockade in triple-negative breast cancer by testing a synchronized, low-intensity schedule in two immunocompetent mouse models: Til and 4T1-PTGR1. Beginning on day 7 — when tumors measured roughly 50 mm3— mice received LP-184 intraperitoneally at 4 mg kg1on days 7, 10, and 13, an anti-PD-1 antibody (clone RMP1-14) at 10 mg kg1on the same three days, either agent alone, or matched vehicle and isotype controls, as shown in FIG 5.

[0088] Antitumor activity was strikingly consistent across models. In the TH cohort, LP-184 monotherapy inhibited tumor growth by 51 %, whereas anti-PD-1 alone achieved just 17 %. When combined, the two agents suppressed growth by 72 % at the day-22 endpoint (Table 1, FIG. 5 A).TABLE 1

[0089] The notoriously refractory 4T1-PTGR1 line echoed this pattern: LP-184 alone produced 42 % inhibition and anti-PD-1 22 %, yet the combination curtailed growth by 78 % by day 15 (Table 2, FIG. 5B). Bliss-independence analysis verified genuine synergy rather than mere additivity in both settings.TABLE 2

[0090] Mechanistic read-outs linked these efficacy gains to innate- and adaptive-immune remodeling. As shown in FIG. 6, immunofluorescence imaging of MDA-MB-231 cells showedthat LP-184 accumulated single-stranded DNA in the cytoplasm; DNase I abolished the signal, confirming that authentic DNA fragments were present and poised to activate the cGAS-STING pathway. In 4T1 tumors, single-cell RNA-sequencing revealed that the combination treatment shifted macrophages from an immunosuppressive M2 phenotype toward a pro-inflammatory Ml state, marked by diminished expression of Mrcl and Argl and elevated Nos2 and Tnf. At the same time, chemokines such as Ccl5, Cxcl9, and CxcllO rose, while suppressive cytokines Tgfbl and 1110 fell — changes collectively creating a microenvironment more hospitable to effector T cells, as shown in FIG. 7. Tumor cells themselves also became more visible to the immune system: combination therapy up-regulated transcripts encoding both MHC-I and MHC-II components and triggered robust type-I and type-II interferon programs, as shown in FIG. 8. FIG. 9 illustrates the enhanced activation of interferon signaling pathways in tumor cells following combination therapy. The analysis focused on two key immune pathways: the Type I interferon (IFN-I, top panel) and Type II interferon (IFN-II, bottom panel) responses. The analysis focused on two key immune pathways: the Type I interferon (IFN-I, top panel) and Type II interferon (IFN-II, bottom panel) responses. Both pathways exhibited increased activation scores in the combination treatment group, reflecting a heightened immune response. Notably, IFN-II activation was more pronounced, suggesting stronger engagement of the IFN-y axis, which is known to play a critical role in stimulating antigen presentation and orchestrating anti -tumor immunity. This upregulation implies that the combination therapy boosts the immunogenicity of tumor cells by improving their visibility to the immune system through enhanced MHC expression and downstream signaling cascades.

[0091] As such, LP-184 causes transcription-coupled DNA damage that generates cytosolic ssDNA, igniting cGAS-STING signaling; innate activation then recruits and energizes T cells, while interferons drive MHC up-regulation on cancer cells. Checkpoint blockade with anti-PD-1 removes inhibitory brakes at precisely this moment, allowing the newly primed immune milieu to exert maximal pressure. The net result — 72-78 % tumor-growth inhibition in two distinct syngeneic models — demonstrates that acylfulvene-induced DNA damage and PD-1 blockade can cooperate powerfully, transforming otherwise “cold” TNBC tumors into immunologically engaged lesions. These findings provide a strong preclinical rationale for advancing LP-184 / checkpoint-inhibitor combinations into clinical testing for patients with triple-negative breast cancer.

[0092] While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions, and subcombinations thereof. It is therefore intended that the following appended claims and claims hereafter are interpreted to include all such modifications, permutations, additions, and sub- combinations as are within their true spirit and scope.

Claims

CLAIMS1. A method of treating triple-negative breast cancer (TNBC) in a subject in need thereof, comprising: administering to the subject a therapeutically effective amount of hydroxyureamethyl acylfulvene; and administering to the subject a therapeutically effective amount of an immunotherapy, wherein the combination of the hydroxyureamethyl acylfulvene and immunotherapy are therapeutically effective in treating TNBC.

2. The method of Claim 1, wherein the hydroxyureamethyl acylfulvene is selected from the group consisting of (-)-hydroxyureamethyl acylfulvene.

3. A pharmaceutical composition for treating TNBC, comprising: a therapeutically effective amount of hydroxyureamethyl acylfulvene; and a therapeutically effective amount of an immunotherapy, wherein the combination of the illudin or illudin analog and immunotherapy provides a therapeutic effect in treating TNBC.

4. The pharmaceutical composition of Claim 3, wherein the acylfulvene is (-)- hydroxyuream ethyl acylfulvene.

5. A kit for the treatment of TNBC in a subject in need thereof, comprising: a. a therapeutically effective amount of hydroxyureamethyl acylfulvene; and b. a therapeutically effective amount of an immunotherapy, wherein the combination of the hydroxyuream ethyl acylfulvene and immunotherapy provides a therapeutic effect in treating TNBC.

6. The method of Claim 5, further comprising administering to the subj ect one or more additional chemotherapeutic agents selected from the group consisting of camptothecin derivatives, paclitaxel, docetaxel, epothilone B, 5-FU, gemcitabine, oxaliplatin, cisplatinum,carboplatin, melphalan, dacarbazine, temozolomide, doxorubicin, imatinib, erlotinib, bevacizumab, cetuximab, and a Raf kinase inhibitors.

7. A method of treating triple-negative breast cancer (TNBC) in a mammalian subj ect comprising administering: a therapeutically effective amount of an acylfulvene compound selected from hydroxyuream ethyl acylfulvene, Irofulven, or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of an immune-checkpoint inhibitor that blocks the PD-1 / PD-L1 pathway, wherein the acylfulvene compound and the immune-checkpoint inhibitor are administered on overlapping schedules and the combined administration produces tumor-growth inhibition.

8. The method of claim 7, wherein the acylfulvene compound is hydroxyuream ethyl acylfulvene (LP-184).

9. The method of claim 7, wherein the immune-checkpoint inhibitor is an anti-PD-1 monoclonal antibody selected from pembrolizumab, nivolumab, or a rat IgG2a clone RMP1-14.

10. The method of claim 7, wherein the acylfulvene compound is administered intraperitoneally at a dose of 2-6 mg kg1per injection.

11. The method of claim 10, wherein the acylfulvene compound is administered at 4 mg kg1on a schedule of every 72 ± 6 hours for three doses.

12. The method of claim 7, wherein the immune-checkpoint inhibitor is administered intraperitoneally at 8-12 mg kg1per injection.

13. The method of claim 1, wherein the immune-checkpoint inhibitor is administered on the same calendar days as the acylfulvene compound.

14. The method of claim 1, wherein the subject’s tumor is refractory to prior anti-PD-1 monotherapy before treatment.

15. The method of claim 1, further comprising monitoring a biomarker of cGAS- STING pathway activation selected from phosphorylation of TBK1, phosphorylation of IRF3, or increased CXCL10 expression.

16. The method of claim 1, wherein treatment reduces M2-polarized macrophages and increases CD8+T-cell infiltration in the tumor microenvironment.

17. The method of claim 1, wherein treatment up-regulates both class I and class II major-histocompatibility-complex gene expression in tumor cells.

18. The method of claim 1, wherein the combined administration results in at least a 70 % reduction in tumor volume relative to baseline within 15-22 days.

19. The method of claim 1, wherein the therapeutically effective amount of the acylfulvene compound is 25-50 % lower than a monotherapy amount that produces equivalent tumor-growth inhibition.

20. A pharmaceutical kit comprising(i) a first container holding hydroxyureamethyl acylfulvene, formulated for administration;(ii) a second container holding an anti-PD-1 antibody formulated for parenteral administration; and(iii) printed instructions directing sequential or concomitant use of the first and second containers to treat TNBC, wherein the instructions specify that the acylfulvene compound is administered at 2-6 mg kg1every 48-96 hours for at least two doses.

21. The kit of claim 20, wherein the first container comprises a lyophilized dose of hydroxyuream ethyl acylfulvene and the second container comprises nivolumab.

22. The kit of claim 20, further comprising a reagent for detecting cytosolic single-stranded DNA in patient biopsy samples.

23. The kit of claim 20, wherein the printed instructions recommend initiating the anti-PD-1 antibody on the same day as the first acylfulvene dose.

24. The kit of claim 20, wherein the acylfulvene compound is supplied in a ready -to-use solution of 10 % ethanol, 40 % polyethylene glycol 400, and 50 % saline.

25. The kit of claim 20, wherein the instructions state that combined therapy is expected to produce synergistic tumor regression evidenced by at least a 50 % reduction in tumor volume by day 22 of treatment.

26. A use of an acylfulvene compound for the manufacture of a medicament that, when administered in combination with an anti-PD-1 antibody, (i) decreases expression of M2-associated genes Mrcl and Argl in tumor-associated macrophages and (ii) increases expression of MHC-I genes H2-K1 and H2-D1 in breast-cancer cells.

27. The use of claim 26, wherein the medicament comprises hydroxyuream ethyl acylfulvene.

28. The use of claim 26, wherein the medicament is administered to a subject whose tumor exhibits low baseline PD-L1 expression.

29. The use of claim 26, wherein the medicament is configured for intraperitoneal, intravenous, or subcutaneous administration.

30. The use of claim 26, wherein the medicament further comprises a pharmaceutically acceptable carrier selected to permit bolus injection.

31. The use of claim 26, wherein co-admini strati on with the anti-PD-1 antibody increases intratumoral CD8+Granzyme B+T cells by at least two-fold relative to antibody alone.

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