Pharmaceutical combination and uses thereof

Abstract

Provided is a pharmaceutical combination, comprising (E)-N-hydroxy-3-(l-(phenylsulfonyl)- indolin-5-yl)-acrylamide, a vascular endothelial growth factor (VEGF) inhibitor, and a programmed cell death protein 1 (PD-l) / programmed cell death ligand 1 (PD-L1) pathway inhibitor. Also provided is a method for treating cancer using the pharmaceutical combination. The pharmaceutical combination has been demonstrated to synergistically enhance tumor growth inhibition, improve objective response rates (ORR), and achieve long-term survival rates in cancers refractory to existing therapies, including microsatellite-stable (MSS) colorectal cancer and hepatocellular carcinoma.

Description

PHARMACEUTICAL COMBINATION AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority of U.S. Provisional Application No. 63 / 762,081, filed on February 23, 2025, the content of which is incorporated herein in its entirety by reference.FIELD

[0002] The present disclosure relates to cancer treatment, and more particularly to a pharmaceutical combination comprising (E)-N-hydroxy-3-(l-(phenylsulfonyl)-indolin-5-yl)-acrylamide, a vascular endothelial growth factor (VEGF) inhibitor, and a programmed cell death protein 1 (PD-l) / programmed cell death ligand 1 (PD-L1) pathway inhibitor, as well as a method of treating cancers using the pharmaceutical combination.BACKGROUND

[0003] Cancer therapy has been transformed by immune checkpoint blockade targeting the PD-1 receptor or its ligand, PD-L1. PD-1 is primarily expressed on the surface of activated T cells, B cells, and myeloid cells, and functions as an immune checkpoint that limits autoimmunity. Its ligand, PD-L1, is frequently upregulated on the surface of tumor cells and various cells within the tumor microenvironment (TME). The interaction between PD-1 and PD-L1 transmits an inhibitory signal that suppresses T-cell activation and proliferation, allowing tumor cells to evade immune surveillance. Various immune checkpoint inhibitors (ICIs) have been developed to block the PD-1 / PD-L1 interaction, and some have been approved for cancer treatment. However, durable responses to these therapies remain limited to a subset of patients. Many tumors exhibit primary resistance or develop adaptive resistance during treatment, which has been associated with impaired antigen presentation, exclusion of effector T cells, and the establishment of an immunosuppressive TME. In particular, tumors characterized by poor immune infiltration or dysfunctional interferon signaling frequently demonstrate reduced sensitivity to PD-1 / PD-L1 inhibition despite adequate target engagement.

[0004] Epigenetic regulation plays a central role in tumor immune recognition and cellulardifferentiation. Histone deacetylase (HD AC) inhibitors have been reported to alter transcriptional programs involved in antigen processing, cytokine signaling, and immune cell function. While HD AC inhibition in certain cancers has been reported to modulate tumor immunogenicity, such effects, even when combined with immune checkpoint blockade, are often constrained by independent resistance factors, such as aberrant tumor vasculature and persistent hypoxia, which shield the tumor from immune attack.

[0005] The VEGF pathway is another key regulator of tumor angiogenesis. Although therapeutic inhibition of VEGF signaling, achieved with either antibody-based agents or smallmolecule inhibitors of VEGF receptor activity, has been shown to remodel tumor vasculature, such anti-angiogenic effects alone typically yield limited clinical benefit. Importantly, overcoming vascular barriers through VEGF inhibition does not inherently reverse the underlying immunosuppressive conditions within the tumor microenvironment, often resulting in persistent immune evasion despite improved immune infiltration.

[0006] Accordingly, there remains a need for therapeutic approaches that can simultaneously modulate the complex and interconnected networks of immune checkpoint signaling, epigenetic reprogramming, and vascular biology to overcome immune resistance refractory to existing combination therapies.SUMMARY OF THE INVENTION

[0007] The present disclosure concerns a combination therapy that concurrently addresses immune checkpoint signaling, epigenetic regulation, and VEGF-mediated tumor biology, as well as its application in treating cancers refractory to existing therapies, including tumors resistant to immune checkpoint inhibition, anti-angiogenic therapy, or combinations thereof.

[0008] One aspect of the present disclosure is to provide a pharmaceutical combination comprising (E)-N-hydroxy-3-(l-(phenylsulfonyl)-indolin-5-yl)-acrylamide, avascular endothelial growth factor (VEGF) inhibitor, and a programmed cell death protein 1 (PD-l) / programmed cell death ligand 1 (PD-L1) pathway inhibitor. (E)-N-hydroxy-3-(l-(phenylsulfonyl)-indolin-5-yl)-acrylamide (with the structure of Formula (I)), also known as imofinostat, is an HD AC inhibitorwith demonstrated activity against multiple HD AC isoforms and antineoplastic properties. Certain crystalline forms of this compound, termed ABT-301, have been described with enhanced stability suitable for pharmaceutical development.

[0009] In certain embodiments, the VEGF inhibitor is a VEGF receptor tyrosine kinase inhibitor, an antibody or an antigen-binding fragment thereof capable of binding to VEGF or a VEGF receptor, or a VEGF-binding protein. In certain embodiments, the VEGF inhibitor is selected from the group consisting of sunitinib, sorafenib, regorafenib, cabozantinib, axitinib, pazopanib, lenvatinib, bevacizumab, ranibizumab, brolucizumab, ramucirumab, aflibercept, and conbercept.

[0010] In certain embodiments, the PD-1 / PD-L1 pathway inhibitor is an anti-PD-1 antibody or an antigen-binding fragment thereof, an anti-PD-Ll antibody or an antigen-binding fragment thereof, a small-molecule PD-1 inhibitor, or a small-molecule PD-L1 inhibitor. In certain embodiments, the PD-1 / PD-L1 pathway inhibitor is selected from the group consisting of sintilimab, camrelizumab, pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab, toripalimab, tislelizumab, cosibelimab, RMP1-14, atezolizumab, sugemalimab, envafolimab, avelumab, durvalumab, INCB086550, GS-4224, and BMS-1166.

[0011] In certain embodiments, the VEGF inhibitor and the PD-1 / PD-L1 pathway inhibitor are provided as a multi-specific antibody comprising a VEGF-binding domain and a PD-1 or PD-L1 -binding domain.

[0012] In another aspect, the present disclosure provides a method for treating cancer in a subject in need thereof using the pharmaceutical combination disclosed herein. The method comprises administering to the subject a therapeutically effective amount of (E)-N-hydroxy-3-(l-(phenylsulfonyl)-indolin-5-yl)-acrylamide, a therapeutically effective amount of a VEGF inhibitor, and a therapeutically effective amount of a PD-1 / PD-L1 pathway inhibitor.

[0013] In certain embodiments, the cancer is colorectal cancer (CRC). In certain embodiments, the cancer is liver cancer, such as hepatocellular carcinoma (HCC), the most common type of primary liver cancer. In certain embodiments, the cancer is resistant to a prior treatment with an immune checkpoint inhibitor, such as a PD-1 inhibitor or a PD-L1 inhibitor.

[0014] In certain embodiments, the (E)-N-hydroxy-3-(l-(phenylsulfonyl)-indolin-5-yl)-acrylamide is administered in an amount of about 0.5 mg / kg to about 500 mg / kg, and / or the VEGF inhibitor is administered in an amount of about 0.5 mg / kg to about 100 mg / kg, and / or the PD-1 / PD-L1 pathway inhibitor is administered in an amount of about 0.1 mg / kg to about 50 mg / kg.

[0015] In certain embodiments, the VEGF inhibitor and the PD-1 / PD-L1 pathway inhibitor are provided as a multi-specific antibody comprising a VEGF-binding domain and a PD-1 or PD-L1 -binding domain, and the multi-specific antibody is administered in a therapeutically effective amount.

[0016] The pharmaceutical combination and treatment strategy disclosed herein are designed to address the clinical challenge of suboptimal responses to current cancer therapies. By combining ABT-301 with a VEGF inhibitor and a PD-1 / PD-L1 pathway inhibitor, this triple combination therapy demonstrates a synergistic antitumor effect that promotes tumor suppression across various malignancies. Experimental data indicates that the present combination can enhance tumor response rates and prolong survival in different advanced tumor models, suggesting its efficacy in modulating the tumor microenvironment. Accordingly, this therapeutic approach offers a viable option for cancers that are resistant to conventional immune checkpoint inhibitor monotherapies or standard dual-combination regimens.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The present disclosure will be apparent to those skilled in the art from the following detailed description, with reference to the accompanying drawings, where:

[0018] FIG. 1A illustrates changes in tumor volume in HT-29 tumor-bearing micefollowing administration of the indicated treatments for 27 days; data are presented as mean ± SEM; n = 5-8 per group; * P < 0.05, ** P < 0.01, *** P < 0.005 by t-test;

[0019] FIG. IB illustrates individual tumor volumes and group means in HT-29 tumorbearing mice on day 36 following administration of the indicated treatments; data are presented as mean ± SEM; n = 5-8 per group; * P < 0.05, ** P < 0.01, *** P < 0.005 by t-test;

[0020] FIG. 2A illustrates changes in tumor volume in CT26 tumor-bearing mice following administration of the indicated treatments for 14 days; data are presented as mean ± SEM; n = 10 for the control group; n = 9 for all other groups;

[0021] FIG. 2B shows individual tumor volumes and group means in CT26 tumor-bearing mice on day 14 following administration of the indicated treatments; data are presented as mean ± SEM; n = 10 for the control group; n = 9 for all other groups; *** P < 0.001, **** P < 0.0001 compared with the control group by t-test;#P < 0.05 compared with the PD-li + VEGFi group by t-test;

[0022] FIG. 3 illustrates changes in tumor volume in CT26 tumor-bearing mice following administration of the indicated treatments for 37 days; data are presented as mean ± SEM;

[0023] FIGs. 4A-4H illustrate individual relative tumor volume changes in CT26 tumorbearing mice following the indicated treatments for 37 days;

[0024] FIG. 5 is a Kaplan-Meier plot illustrating the percent survival of CT26 tumor-bearing mice receiving the indicated treatments; P < 0.05, P < 0.01, P < 0.001 compared with the control group by Log-rank test; and##P <0.01 between the PD-li and ABT-301 + PD-li groups by Log-rank (Mantel-Cox) test;

[0025] FIGs. 6A and 6B illustrate changes in tumor volume in CT26 tumor-bearing mice following administration of the indicated treatments for 11 days; data are presented as mean ± SEM; n = 11 for the control group; n = 7 for the ABT-301 + PD-li group; n = 8 for all other groups;

[0026] FIGs. 7A and 7B illustrate changes in tumor volume in CT26 tumor-bearing mice following administration of the indicated treatments for 36 days; data are presented as mean ±SEM; n = 11 for the control group; n = 7 for the ABT-301 + PD-li group; n — 8 for all other groups;

[0027] FIGs. 8A-8G illustrate individual relative tumor volume changes in CT26 tumorbearing mice following administration of the indicated treatments for 36 days;

[0028] FIGs. 9A and 9B are Kaplan-Meier plots illustrating the percent survival of CT26 tumor-bearing mice receiving the indicated treatments; P < 0.05, P < 0.01, P < 0.001 compared with the PD-li group by Log-rank (Mantel-Cox) test;

[0029] FIG. 10A illustrate changes in tumor volume in Hepa 1-6 tumor-bearing mice following administration of the indicated treatments for 47 days; data are presented as mean ± SEM; n = 6 for the control group; n = 8 for all other groups;

[0030] FIG. 10B illustrates individual tumor volumes and group means in Hepa 1 -6 tumorbearing mice on day 47 following administration of the indicated treatments; data are presented as mean ± SEM; *** P < 0.05; and

[0031] FIGs. 11A-11F illustrate individual relative tumor volume changes in Hepa 1-6 tumor-bearing mice following administration of the indicated treatments for 47 days.DETAILED DESCRIPTION

[0032] The present invention is further explained in the following embodiments and examples. It is understood that the examples given below do not limit the scope of the invention, and it will be evident to those skilled in the art that modifications can be made without departing from the scope of the appended claims.

[0033] Unless defined otherwise, all technical and scientific terms and abbreviations used herein have the same meaning as commonly understood by a person skilled in the art to which this invention pertains.

[0034] Definition

[0035] As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly indicates otherwise. For example, reference to “an inhibitor” includes a mixture of multiple types of inhibitors.

[0036] Numerical quantities given herein are approximate, and experimental values may vary within 20 percent, preferably within 10 percent, or most preferably within 5 percent. Thus, the terms “about” and “approximately” refer to within 20 percent, preferably within 10 percent, or most preferably within 5 percent of a given value or range.

[0037] As used herein, the term “(E)-N-hydroxy-3-(l-(phenylsulfonyl)-indolin-5-yl)-acrylamide” is used interchangeably with “ABT-301.”

[0038] Pharmaceutical combination

[0039] The present disclosure provides a pharmaceutical combination comprising (E)-N-hydroxy-3-(l-(phenylsulfonyl)-indolin-5-yl)-acrylamide (ABT-301), a vascular endothelial growth factor (VEGF) inhibitor, and a programmed cell death protein 1 (PD-l) / programmed cell death ligand 1 (PD-L1) pathway inhibitor.

[0040] ABT-301 is a selective HDAC inhibitor targeting multiple HDACs, including HDAC1, HDAC2, HDAC3 and HDAC6. The term “HDAC inhibitor” refers to any compound or agent capable of inhibiting the enzymatic activity of one or more histone deacetylases. Unless otherwise specified, references to “ABT-301” herein encompass ABT-301 in any pharmaceutically acceptable form, including its pharmaceutically acceptable salts. By inhibiting HDAC activity, HDAC inhibitors promote hyperacetylation of histones and non-histone proteins, leading to chromatin relaxation, altered gene transcription, and modulation of various cellular processes, including cell cycle arrest, apoptosis, differentiation, angiogenesis inhibition, and immune modulation.

[0041] The term “VEGF inhibitor” as used herein refers to any agent that interferes with the vascular endothelial growth factor (VEGF) signaling pathway by blocking VEGF ligands, VEGF receptors, or downstream signaling cascades. By inhibiting VEGF signaling, VEGF inhibitors suppress tumor angiogenesis, reduce vascular permeability, and normalize tumor vasculature, thereby enhancing immune cell infiltration and potentiating the efficacy of immune checkpoint inhibitors. VEGF inhibitors include, but are not limited to: (i) VEGF receptor tyrosinekinase inhibitors (VEGFR-TKIs), which are small molecule compounds that inhibit the tyrosine kinase activity of VEGFR-1, VEGFR-2, and / or VEGFR-3; (ii) antibodies or antigen-binding fragments thereof capable of binding to VEGF or VEGF receptors, which are immunoglobulins or fragments thereof that specifically bind to VEGF ligands (e.g., VEGF-A, VEGF-B, P1GF) or VEGF receptors (e.g., VEGFR-1, VEGFR-2, VEGFR-3); and (iii) VEGF-binding proteins, which are recombinant fusion proteins comprising VEGF receptor extracellular domains fused to an immunoglobulin Fc region, functioning as soluble decoy receptors. Examples of VEGFR-TKIs include sorafenib, sunitinib, lenvatinib, regorafenib, axitinib, pazopanib, and cabozantinib. Examples of anti-VEGF / VEGFR antibodies include bevacizumab (anti-VEGF-A), ramucirumab (anti- VEGFR-2), ranibizumab, and brolucizumab. Examples of VEGF-binding proteins include aflibercept and conbercept.

[0042] The term “PD-1 / PD-L1 pathway inhibitor” as used herein refers to any agent that disrupts the interaction between programmed cell death protein 1 (PD-1) and programmed cell death-ligand 1 (PD-L1), thereby preventing PD-1 / PD-L1 -mediated immune suppression and restoring anti-tumor T cell activity. PD-1 / PD-L1 pathway inhibitors include, but are not limited to: (i) anti-PD-1 antibodies or antigen-binding fragments thereof, which are immunoglobulins or fragments thereof that specifically bind to PD-1 and block the interaction between PD-1 and its ligands, including PD-L1 and PD-L2; (ii) anti-PD-Ll antibodies or antigen-binding fragments thereof, which are immunoglobulins or fragments thereof that specifically bind to PD-L1 and block its interaction with PD-1 and CD80; (iii) small-molecule PD-1 inhibitors, which are non-antibody compounds that bind to PD-1 and disrupt the interaction between PD-1 and its ligands, including PD-L1 and PD-L2; and (iv) small-molecule PD-L1 inhibitors, which are non-antibody compounds that bind to PD-L1 and disrupt the PD-L1 / PD-1 interaction. Examples of anti-PD-1 antibodies include pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab, toripalimab, tislelizumab, sintilimab, camrelizumab, cosibelimab, and RMP1-14. Examples of anti-PD-Ll antibodies include atezolizumab, durvalumab, avelumab, sugemalimab, and envafolimab.Examples of small-molecule PD-1 inhibitors include GS-4224 and other investigational agents. Examples of small-molecule PD-L1 inhibitors include INCB086550, BMS-1166, and other investigational agents.

[0043] The term “antigen-binding fragment” encompasses Fab, Fab', F(ab')2, Fv, scFv, single-domain antibodies (nanobodies), diabodies, and other antibody fragments that retain the ability to specifically bind to the indicated antigen.

[0044] The pharmaceutical combination may be provided in various physical forms and packaging configurations to facilitate administration, storage, and patient compliance. In some embodiments, each of the three active agents (ABT-301, the VEGF inhibitor, and the PD-l / PD-L1 pathway inhibitor) is formulated and packaged separately as distinct pharmaceutical compositions. Each component may be in its own container with appropriate pharmaceutical excipients and in a dosage form suitable for its route of administration. For example, ABT-301 or pharmaceutically acceptable salts thereof may be formulated as tablets, capsules, or oral suspension in a first container. The VEGF inhibitor may be formulated as tablets or capsules (if a VEGFR-TKI) in a second container, or as a sterile solution (if an antibody) for injection in a vial or a pre-filled syringe. The PD-1 / PD-L1 pathway inhibitor, e.g., an antibody, may be formulated as a sterile solution for injection in a vial, a pre-filled syringe, or an intravenous (IV) infusion bag in a third container. The separately packaged components may be administered sequentially or concurrently according to a prescribed treatment regimen.

[0045] In other embodiments, the pharmaceutical combination is provided as a kit or package comprising two or more separate containers, each containing one of the active agents in a pharmaceutically acceptable formulation. The kit may comprise: (a) a first container comprising ABT-301 or a pharmaceutically acceptable salt thereof, optionally in a solid dosage form (e.g., tablet); (b) a second container comprising a VEGF inhibitor, which may be (i) a small-molecule VEGF inhibitor in a solid dosage form (e.g., tablet), or (ii) an antibody or VEGF-binding protein in a sterile liquid formulation (e.g., vial, pre-filled syringe, IV bag); and (c) a third containercomprising a PD-1 / PD-L1 pathway inhibitor, which may be (i) a small-molecule PD-1 or PD-L1 inhibitor in a solid dosage form, or (ii) an anti-PD-1 or anti-PD-Ll antibody in a sterile liquid formulation. The kit may further include instructions for administration, dosing schedules, and ancillary materials (diluents, syringes, IV sets).

[0046] In some embodiments, two of the three active agents are formulated together in a single unit dosage form, while the third agent is provided separately. In some embodiments, ABT-301 and a VEGF receptor tyrosine kinase inhibitor (e.g., regorafenib) are formulated together in a single oral dosage form, and the PD-1 / PD-L1 pathway inhibitor (e.g., an antibody) is provided separately, typically as an injectable formulation. In some embodiments, ABT-301 is coformulated with a small-molecule PD-1 / PD-L1 inhibitor (e.g., GS-4224) in a single oral dosage form, and the VEGF inhibitor would be provided separately.

[0047] In some embodiments, the VEGF inhibitor and the PD-1 / PD-L1 pathway inhibitor are provided in a multi-specific format, such as a bispecific antibody. In certain embodiments, the VEGF inhibitor and PD-1 / PD-L1 pathway inhibitor are combined as a single antibody that specifically binds to a VEGF pathway target (e.g., VEGF-A or VEGFR-2) and a PD-1 / PD-L1 pathway target (e.g., PD-1 or PD-L1). One example of a bispecific antibody that binds PD-1 and VEGF is ivonescimab, which has been reported to exhibit both immune checkpoint inhibitory activity and anti-angiogenic activity (Xiong A. et al. Ivonescimab versus pembrolizumab for PD-L1 -positive non-small cell lung cancer (HARMONi-2): a randomised, double-blind, phase 3 study in China. Lancet. 2025; 405: 839-49). The bispecific antibody may be in formats such as IgG-based bispecifics (e.g., knobs-into-holes, CrossMab), tandem scFvs, bispecific T-cell engager, dual-affinity re-targeting proteins (DARTs), or other bispecific architectures. Example binding specificities include (i) anti- VEGF- A x anti-PD-1, (ii) anti- VEGF- A x anti-PD-Ll, (iii) anti-VEGFR-2 x anti-PD-1; and (iv) anti- VEGFR-2 x anti-PD-Ll. The term “multi-specific” as used herein refers to a molecule capable of specifically binding to at least two different target molecules. Multi-specific antibodies include, but are not limited to, bispecific or trispecific antibodies andantigen-binding fragments thereof.

[0048] Methods of treatment

[0049] The present disclosure is also directed to methods of treating cancer by administering the disclosed pharmaceutical combination to a subject in need thereof. As used herein, the term “treatment’ may refer to a curative or palliative measure. The term “treating” includes partially or completely inhibiting tumor growth, reducing tumor size, delaying tumor progression, inducing tumor regression, reducing tumor burden, prolonging survival, and / or preventing metastasis. Treating may also include partially or completely alleviating, ameliorating, relieving, delaying the onset of, inhibiting progression of, reducing severity of, and / or reducing incidence of one or more symptoms, secondary disorders, or features associated with cancers. For example, symptoms, secondary disorders, and / or conditions associated with colorectal cancer include, but are not limited to, fever, weakness, fatigue, weight loss, pain, cough, bleeding, skin change, diarrhea or constipation, nausea, vomiting, and loss of appetite.

[0050] Treatment may be administered to a subject who exhibits early signs of cancer or minimal tumor burden to reduce the risk of disease progression or development of cancer-associated symptoms or complications. Treatment is generally considered effective if tumor growth is inhibited, tumor burden is reduced, disease progression is delayed, objective tumor response is improved, and / or one or more cancer-associated symptoms or conditions are alleviated.

[0051] The subject treatable by the present pharmaceutical combination or the present method is a mammal, for example, a human, a mouse, a rat, a guinea pig, a hamster, a monkey, a pig, a dog, a cat, a horse, a sheep, a goat, a cow, and a rabbit. In certain embodiments, the subject is a human.

[0052] The pharmaceutical combination of the present disclosure has demonstrated antitumor activity in colorectal cancer and liver cancer models. While specific examples are provided for selected tumor types, the therapeutic effects arise from the coordinated modulation of biological processes implicated in tumor progression, such as epigenetic dysregulation,pathological angiogenesis, and immune evasion. Accordingly, the combination is expected to be therapeutically effective in various cancers characterized by one or more of the following features: aberrant histone deacetylase activity, VEGF-driven angiogenesis, or impaired antitumor T-cell responses. In certain embodiments, the cancer exhibits resistance or a suboptimal response to prior treatment with an immune checkpoint inhibitor. Representative cancers that may benefit include, without limitation, solid tumors exhibiting angiogenic activity or activation of an immune checkpoint pathway.

[0053] The term “therapeutically effective amount” (also referred to as “effective amount”) refers to the quantity of an active agent that is sufficient to achieve a desired therapeutic response when administered to a subject in need thereof. The desired therapeutic response includes a reduction in tumor size or burden, inhibition of tumor growth, delay in tumor progression, prolongation of survival, restoration of antitumor immune activity, improvement in disease parameters, and symptom alleviation. The specific effective amount will vary depending on various factors, including but not limited to: the particular condition being treated and its severity, the physical condition of the patient (e.g., body weight, age, gender, hepatic function), the type of subject being treated (e.g., human, mouse), the duration of the treatment, the nature of concurrent therapy (if any), the specific formulations employed, the route of administration, and the pharmacokinetic and pharmacodynamic properties of the active agent. The effective amount may be expressed, for example, in grams, milligrams, or micrograms, or as milligrams per kilogram of body weight (mg / kg).

[0054] The therapeutically effective amount varies depending on the subject being treated (e.g., human, mouse, rat) and is adjusted based on body weight, body surface area, and speciesspecific pharmacokinetic differences. In certain embodiments, ABT-301 is administered in an amount of about 0.5 mg / kg to about 500 mg / kg, and / or the VEGF inhibitor is administered in an amount of about 0.5 mg / kg to about 100 mg / kg, and / or the PD-1 / PD-L1 pathway inhibitor is administered in an amount of about 0.1 mg / kg to about 50 mg / kg.

[0055] In certain embodiments, for preclinical animal models (e.g., mice, rats), ABT-301 may be administered in an amount of about 5 mg / kg to about 500 mg / kg per dose, including about 5 mg / kg to about 25 mg / kg, about 10 mg / kg to about 50 mg / kg, about 25 mg / kg to about 100 mg / kg, about 50 mg / kg to about 200 mg / kg, about 100 mg / kg to about 300 mg / kg, or about 200 mg / kg to about 500 mg / kg per dose. The anti-PD-1 or anti-PD-Ll antibody may be administered in an amount of about 0.5 mg / kg to about 50 mg / kg per dose, including about 0.5 mg / kg to about 2 mg / kg, about 1 mg / kg to about 5 mg / kg, about 2 mg / kg to about 10 mg / kg, about 5 mg / kg to about 20 mg / kg, about 10 mg / kg to about 30 mg / kg, or about 20 mg / kg to about 50 mg / kg per dose. The VEGFR-TKI may be administered in an amount of about 2 mg / kg to about 100 mg / kg per dose, including about 2 mg / kg to about 10 mg / kg, about 5 mg / kg to about 20 mg / kg, about 10 mg / kg to about 40 mg / kg, about 20 mg / kg to about 60 mg / kg, about 40 mg / kg to about 80 mg / kg, or about 60 mg / kg to about 100 mg / kg per dose. The anti-VEGF or anti-VEGFR antibody may be administered in an amount of about 0.5 mg / kg to about 50 mg / kg per dose, including about 0.5 mg / kg to about 2 mg / kg, about 1 mg / kg to about 5 mg / kg, about 2 mg / kg to about 10 mg / kg, about 5 mg / kg to about 20 mg / kg, about 10 mg / kg to about 30 mg / kg, or about 20 mg / kg to about 50 mg / kg per dose.

[0056] For human subjects, therapeutically effective doses can be calculated based on human equivalent dose (HED) conversions from animal efficacious doses. In certain embodiments, ABT-301 may be administered in an amount of about 0.5 mg / kg to about 100 mg / kg per dose, including about 0.5 mg / kg to about 2 mg / kg, about 1 mg / kg to about 5 mg / kg, about 2 mg / kg to about 10 mg / kg, about 5 mg / kg to about 25 mg / kg, about 10 mg / kg to about 50 mg / kg, or about 25 mg / kg to about 100 mg / kg per dose. The anti-PD-1 or anti-PD-Ll antibody may be administered in an amount of about 0.1 mg / kg to about 20 mg / kg per dose, including about 0.1 mg / kg to about 0.5 mg / kg, about 0.2 mg / kg to about 1 mg / kg, about 0.5 mg / kg to about 2 mg / kg, about 1 mg / kg to about 5 mg / kg, about 2 mg / kg to about 10 mg / kg, or about 5 mg / kg to about 20 mg / kg per dose. The anti-VEGF or anti-VEGFR antibody may be administered in an amount ofabout 0.5 mg / kg to about 40 mg / kg per dose, including about 0.5 mg / kg to about 2 mg / kg, about 1 mg / kg to about 5 mg / kg, about 2 mg / kg to about 10 mg / kg, about 5 mg / kg to about 20 mg / kg, about 10 mg / kg to about 30 mg / kg, or about 20 mg / kg to about 40 mg / kg per dose. The VEGFR-TKI may be administered in an amount of about 0.5 mg / kg to about 20 mg / kg per dose, including about 0.5 mg / kg to about 2 mg / kg, about 1 mg / kg to about 5 mg / kg, about 2 mg / kg to about 10 mg / kg, or about 5 mg / kg to about 20 mg / kg per dose.

[0057] Each component in the pharmaceutical combination may be administered at a frequency appropriate for its pharmacokinetic and pharmacodynamic properties. In certain embodiments, ABT-301 may be administered once daily, twice daily, or on an intermittent schedule (e.g., once daily for 5 consecutive days followed by 2 days off each week). For VEGF inhibitors, the VEGFR-TKI may be administered once daily (e.g., discontinuous or continuous dosing), twice daily, or on an intermittent schedule (e.g., once daily for 21 days followed by 7 days off each 28-day cycle). The anti- VEGF or anti-VEGFR antibody or the VEGF-binding protein may be administered once or twice every week, once every 2 weeks, or once every 3 weeks. For PD-1 / PD-L1 pathway inhibitors, the anti-PD-1 or anti-PD-Ll antibody may be administered once or twice every week, once every 2 weeks, once every 3 weeks, or once every 4 to 6 weeks. The small-molecule PD-1 / PD-L1 inhibitors may be administered once daily or twice daily. Bispecific antibodies may be administered once every 2 weeks, once every 3 weeks, or once every 4 weeks. The dosing frequency for each component may be adjusted based on clinical response, tolerability, pharmacokinetic parameters, and patient-specific factors.

[0058] The pharmaceutical combination may be administered using various dosing regimens to optimize therapeutic efficacy and safety. The choice of regimen depends on the pharmacokinetic properties of each active agent, the disease being treated, prior therapies, and patient tolerance. In certain embodiments, all components are administered simultaneously, either as a single combined formulation or as separate compositions within the same treatment session or on the same day. In certain embodiments, the components are administered in a definedsequence. For example, ABT-301 may be administered as a priming agent for 3-7 days, prior to administration of the VEGF inhibitor and the PD-1 / PD-L1 pathway inhibitor. Alternatively, the VEGF inhibitor may be administered first to normalize tumor vasculature, followed by the PD-1 / PD-L1 pathway inhibitor and ABT-301.

[0059] In certain embodiments, the components are administered on different schedules such that their pharmacological activities overlap in the subject. For example, ABT-301 is administered daily or on a 5-days-on / 2-days-off schedule; the VEGF inhibitor is administered weekly or every 2-3 weeks; and the PD-1 / PD-L1 pathway inhibitor is administered weekly or every 2-6 weeks. Even though the actual administration times differ, the agents exert concurrent biological effects, ensuring continuous multi-pathway modulation of the tumor microenvironment.

[0060] In certain embodiments, one or more components may be administered intermittently or in cycles. For instance, ABT-301 is administered daily for 21 days, followed by a 7-day off. The VEGF inhibitor is administered daily for 21 days, followed by 7 days off. The PD-1 / PD-L1 pathway inhibitor is administered every 2-4 weeks continuously.

[0061] Each component is administered via a route suitable for its formulation. Oral administration is suitable for ABT-301, VEGFR-TKIs (e.g., regorafenib), and small-molecule PD-1 / PD-L1 inhibitors. Intravenous administration is the standard route for therapeutic antibodies or proteins, including anti-VEGF antibodies (e.g., bevacizumab), anti-VEGFR antibodies (e.g., ramucirumab), VEGF-binding proteins (e.g., aflibercept), anti-PD-1 antibodies (e.g., nivolumab), anti-PD-Ll antibodies (e.g., atezolizumab), and bispecific antibodies. Subcutaneous administration may be used for certain antibody formulations designed for subcutaneous delivery. Intratumoral or intra-arterial infusion may be employed for liver tumors (e.g., hepatocellular carcinoma). Intraperitoneal administration may be used for peritoneal tumors. Transdermal, rectal, or mucosal routes may be employed for specific formulations as appropriate.

[0062] Examples

[0063] The following examples evaluate the antitumor therapeutic efficacy of variouscombinations comprising ABT-301, a VEGF inhibitor (hereinafter referred to as VEGFi), and a PD-1 / PD-L1 pathway inhibitor (hereinafter referred to as PD-li for PD-1 inhibitors or PD-Lli for PD-L1 inhibitors) using various tumor animal models. As used in the examples, VEGFi may include VEGF-binding proteins (e.g., aflibercept), antibodies targeting VEGF (e.g., bevacizumab), or multi-kinase inhibitors with anti-angiogenic activity (e.g., regorafenib); and PD-li / PD-Lli may include antibodies targeting the PD-1 receptor (e.g., RMP1-14 or nivolumab) or its ligand PD-LI (e.g., atezolizumab). These studies are designed to demonstrate the potential and therapeutic breadth of the triple combination across diverse cancer types.

[0064] Data are generally presented as mean ± standard deviation, unless otherwise specified. Statistical analysis was performed using GraphPad Prism. Statistical significance was analyzed using Student's t-test or one-way ANOVA. P-value <0.05 indicates a statistically significant difference.

[0065] Example 1 Antitumor efficacy in HT-29 CRC model

[0066] In this study, the potential of a triple combination therapy comprising ABT-301, a PD-1 inhibitor (e.g., nivolumab, an anti-PD-1 monoclonal antibody), and a VEGF inhibitor (e.g., aflibercept, a recombinant protein that binds to VEGF) to overcome cancer immune resistance was evaluated. The study employed a humanized PBMC (hu-PBMC) xenograft mouse model bearing microsatellite-stable (MSS) HT-29 colorectal tumors to simulate a human tumor microenvironment that is typically non-responsive to conventional immune checkpoint blockade.

[0067] Female immunodeficient mice (NOD.Cg- rfccfc^ H2r^mIVstlN3 , NPG) aged 6 weeks were purchased from BioLASCO (Taipei, Taiwan) and inoculated with 5xl06HT-29 cells (human colon adenocarcinoma cells; ATCC No. HTB-38) via subcutaneous (SC) injection on the back for tumor metastasis. The first day of mice receiving tumor cell inoculation was denoted as Day 0. After four days (Day 4), mice were inoculated with human PBMC (CTG Global) by intravenous (IV) injection to establish the HT-29 tumor-bearing hu-PBMC mouse model. These mice (weighting about 20 to 25 g) were randomly divided into five groups and administered asindicated on Day 10 (TABLE 1). Specifically, ABT-301 (Anbogen Therapeutics), prepared with 0.5% carboxymethyl cellulose and 0.1% Tween 80 in 5% dextrose, was administered at 50 mg / kg orally (PO) once daily (QD). Nivolumab (Ono Pharmaceutical Co. Ltd.) was administered at 100 pg by intraperitoneal (IP) injection in 0.1 ml phosphate-buffered saline (PBS) twice per week (BIW). Aflibercept (Ono Pharmaceutical Co. Ltd.) was administered at 100 pg by IP injection in 0.1 ml PBS twice per week, alternating with the administration schedule of Nivolumab. The control group mice received a vehicle (0.5% carboxymethyl cellulose and 0.1% Tween 80 in 5% dextrose). During the dosing period, body weight and tumor volume were monitored twice a week. Tumor volume (mm3) was measured using a caliper and calculated using the formula [(LxW2) / 2], where L and W represent the longest diameter and the shortest diameter in mm, respectively. Tumor growth inhibition (TGI) was calculated as follows: TGI (%) = [1 - (final tumor volume -initial tumor volume for each treated group) / (final tumor volume - initial tumor volume for the control group)] x 100. The study was terminated (on Day 36) when the tumor diameter was over 1 cm, and all mice were euthanized. To further monitor the status of human immune cells in mice, mouse blood was collected on Day 36 to detect CD45+, CD3+, and CD14+cells using flow cytometry analysis.

[0068] TABLE 1 Treatment regimen in HT-29 tumor-bearing mice

[0069] As shown in FIGs. 1A-1B and TABLE 2, rapid tumor growth was observed in control mice, whereas the HT-29 tumor growth was inhibited in mice treated with antitumor agents.Specifically, mean tumor volumes on Day 36 were as follows: 821.3 mm3in the control group, 384.8 mm3in the ABT-301 group, 500.8mm3in the PD-li group, 216.9 mm3in the dual-combo group, and 75.2 mm3in the tri-combo group. On the other hand, the mean TGIs of mice treated with ABT-301, PD-li, dual-combo (ABT-301 combined with PD-li), or tri-combo (ABT-301 combined with PD-li and VEGFi) were 58.2%, 39.1%, 75.3%, and 92.3%, respectively. The dualcombo treatment exhibited significantly higher antitumor activity over the ABT-301 or PD-li single treatment. Surprisingly, the tri-combo treatment achieved a TGI of 92.3%, which was significantly higher than the TGI of the dual-combo treatment (75.3%), leaving the residual tumor burden of the tri-combo group about one-third of that of the dual-combo group.

[0070] TABLE 2 Antitumor efficacy and human immune cell distribution in HT-29 tumorbearing mice following administration of the indicated treatments for 27 days

[0071] Throughout the study, there was no apparent body weight loss in the control, PD-li, and ABT-301 groups (data not shown), suggesting that mouse health was not affected. The dual-combo and tri-combo groups experienced slight body weight loss but maintained their immune profiles (as described below).

[0072] Human immune cell engraftment was verified by flow cytometry analysis of CD45+, CD3+, and CD14+cell populations. CD45+cells represent total human leukocytes. CD3+cells represent human T lymphocytes. CD14+cells represent human monocytes. Verification of these cell populations (TABLE 2) confirmed successful humanization of the mouse model and the presence of functional human immune cells capable of responding to immune checkpoint inhibitor therapy. The immunological data also revealed a surprising effect of the tri-combo treatment onthe immune status. While treatment with PD-li alone resulted in a drastic reduction of leukocytes (CD45+) to about 32.223% and a significant decrease of the T-cell population (CD3+) to about 33.337%, the combination with ABT-301 and VEGFi in the tri-combo group successfully restored and maintained the immune profile. Specifically, the tri-combo group maintained the levels of leukocytes and T lymphocytes comparable to those in the control group. Although the T-cell population remained comparable between the dual-combo and tri-combo groups, the tri-combo treatment effectively reduced the residual tumor burden by nearly three folds relative to the dualcombo treatment, indicating that the combined use of ABT-301 with a PD-1 / PD-L1 pathway inhibitor (nivolumab) and a VEGF inhibitor (aflibercept) not only maintains systemic immune stability, but synergistically enhances the antitumor quality of immune cells.

[0073] Example 2 Antitumor efficacy in CT26 CRC model

[0074] In this study, the antitumor efficacy of a triple combination therapy comprising ABT-301, a PD-1 inhibitor (e.g., RMP1-14, a surrogate anti-mouse PD-1 antibody), and a VEGF inhibitor (e.g., aflibercept) was further evaluated. The study employed a CT26 allograft mouse model, established with CT26.WT cells (ATCC No. CRL-2638). CT26.WT (abbreviated as CT26) is a mouse colon carcinoma cell line, which is highly metastatic and usually classified as microsatellite stable.

[0075] Female BALB / c mice (BALB / cByJNarl) aged 7 weeks were subcutaneously inoculated with 2><105CT-26 cells in the right flank. When a mean tumor volume reached approximately 157 mm3, the tumor-bearing mice (weighing about 19 to 25 g) were randomized to treatment, and the day of treatment initiation was designated Day 0. The mice were divided into eight groups and administered as indicated (TABLE 3). Specifically, ABT-301 (Anbogen Therapeutics), prepared with 0.5% carboxymethyl cellulose and 0.1% Tween 80 in 5% dextrose, was administered at 50 mg / kg orally once daily. RMP1-14 (Leinco Technologies) was administered at 200 pg by IP injection twice per week. Aflibercept (Zaltrap®; Sanofi) was administered at 200 pg by IP injection twice per week. The control group mice received a vehicle(0.5% carboxymethyl cellulose and 0.1% Tween 80 in 5% dextrose) and a rat IgG2a (abbreviated as IgG) as an isotype control antibody. IgG, RMP1-14, and aflibercept were prepared with PBS. Treatments were given for the indicated period or until the humane endpoint (tumor volume > 2000 mm3, defined as death). During the dosing period, body weight and tumor volume were monitored two to three times per week. Tumor volume (mm3) was measured using a caliper and calculated using the formula [(L*W2) / 2], where L and W represent the longest diameter and the shortest diameter in mm, respectively. Tumor growth inhibition (TGI) was calculated as follows: TGI (%) = [1 - (final tumor volume - initial tumor volume for each treated group) / (final tumor volume - initial tumor volume for the control group)] x 100. Based on changes in tumor size, tumor responses were evaluated using Response Evaluation Criteria in Solid Tumors (RECIST). Complete response (CR) is defined as the complete disappearance of measurable or evaluable tumor lesions; partial response (PR) is defined as a reduction in the size of measurable tumor lesions by at least 30% (> 30%) compared to the original tumor size; stable disease (SD) is defined as a reduction in the size of measurable tumor lesions by less than 30% (< 30%) or enlargement of the size of measurable tumor lesions by less than 20% (< 20%) compared to the original tumor size; and progressive disease (PD) is defined as more than 20% (> 20%) increase in the tumor volume compared to the original tumor size or development of new lesions. Objective response rate (ORR) was calculated as the percentage of evaluable subjects exhibiting CR or PR within one group.

[0076] TABLE 3 Treatment regimen in CT26 tumor-bearing mice

[0077] FIG. 2 A shows the growth of CT26 tumors in mice following treatments for 14 days; FIG.2B illustrates individual tumor volumes and group means in mice on Day 14. According to FIGs. 2A-2B and TABLE 4, the triple combination (ABT-301 + PD-li + VEGFi) treatment exhibited the most potent initial tumor suppression among all tested groups. By Day 14, the triple combination treatment achieved the highest mean TGI (92.70%) and the lowest mean tumor volume (272.09 mm3), indicating a superior early-stage tumor-suppressing effect compared with both monotherapies and dual combination therapies.

[0078] TABLE 4 Initial (early-stage) antitumor efficacy in CT26 tumor-bearing mice following administration of the indicated treatments for 14 daysData are presented as mean ± SEM. n = 10 for the control group; n = 9 for all other groups. *** P < 0.001, **** P < 0.0001 compared with the control group by t-test.#P < 0.05 compared with the PD-li + VEGFi group by t-test.

[0079] At the end of the study (Day 37), the tumor burden among surviving mice was assessed. In FIG. 3 and TABLE 5, the triple combination group maintained the lowest mean tumor volume of 704.38 mm3until Day 37. Although the VEGFi single treatment and the ABT-301 + VEGFi dual combination showed the second-highest initial antitumor efficacy (TGIs of 87.15% and 91.69%), the triple combination significantly limited tumor progression, suggesting long-termtumor-suppressing capability.

[0080] TABLE 5 Long-term antitumor efficacy in CT26 tumor-bearing mice following administration of the indicated treatments for 37 daysData are presented as mean ± SEM.

[0081] Throughout the study period, mice in all groups maintained mean relative body weights (normalized to Day 0) between approximately 97% and 107%. Although individual subjects in the PD-li + VEGFi and ABT-301 + PD-li + VEGFi groups exhibited body weight loss greater than 10%, these cases were sporadic, and no obvious abnormalities were observed in these specific subjects, indicating that these treatments were well-tolerated at the administered doses.

[0082] The relative tumor volume changes in mice receiving various treatments are shown in FIGs. 4A-4H. After 37 days of treatment, all mice in the control group or receiving monotherapies exhibited progressive disease (PD). In the ABT-301 + PD-li group, 7 of 9 mice exhibited PD, and 2 of 9 mice showed partial response (PR). In the ABT-301 + VEGFi group, 8 of 9 mice showed PD, and 1 of 9 mice showed PR. In the PD-li + VEGFi group, 8 of 9 mice exhibited PD, and 1 of 9 mice showed stable disease (SD). In the triple combination group, 5 of 8 mice exhibited PD, 1 of 8 mice showed SD, and 2 of 8 mice showed PR. The tumor response data (summarized in TABLE 6) further demonstrated the therapeutic potential of the triple combination, which achieved the highest objective response rate (ORR) of 25.0%. Notably, while the dual combination of PD-li and VEGFi failed to induce any partial or complete response (i.e., ORR 0%), the addition of ABT-301 in the triple combination successfully sensitized the tumors, leading toan improved response profile.

[0083] TABLE 6 Tumor response in CT26 tumor-bearing mice following administration of the indicated treatments for 37 days

[0084] FIG. 5 and TABLE 7 present survival outcomes and median survival in CT26 tumor-bearing mice treated with different regimens. The median survival of mice treated with ABT-301, PD-li, or VEGFi was prolonged compared with the control group. The median survival in the dual- or triple-combination groups exceeded 37 days and was not reached during the study period, suggesting that the combined therapy of ABT-301 + PD-li, ABT-301 + VEGFi, PD-li + VEGFi, or ABT-301 + PD-li + VEGFi improved both ORR (TABLE 6) and overall survival. The triple combination of ABT-301 + PD-li + VEGFi was the only treatment group to achieve a 100% survival rate, providing a noticeable improvement over the dual combinations of ABT-301 + PD-li (55.6% survival) or PD-li + VEGFi (66.7% survival). These results demonstrated the critical therapeutic benefit of integrating a VEGF inhibitor into combination therapy with ABT-301 and a PD-1 / PD-LI pathway inhibitor. The triple combination therapy not only maximizes early-stage tumor suppression and objective response rates but also ensures 100% long-term survival, effectively overcoming the immune resistance typically observed in MSS-type colorectal cancers.

[0085] TABLE 7 Survival outcomes and median survival of CT26 tumor-bearing mice following administration of the indicated treatments for 37 daysIn the ABT-301 + PD-li + VEGFi group, one mouse was found dead on Day 30 due to nontreatment-related causes as confirmed by necropsy. Consequently, this mouse was excluded from the tumor response and survival analyses.

[0086] Example 3 Antitumor efficacy comparison of HD AC inhibitors in CT26 model

[0087] In this study, the antitumor activity of ABT-301 combined with a PD-1 inhibitor (e.g., RMP1-14) and either a VEGF inhibitor (e.g., regorafenib) or 5-fluorouracil (5-FU) was evaluated in a CT26 allograft mouse model and compared with chidamide-based combinations. Chidamide is a benzamide-class HD AC inhibitor that selectively inhibits HDAC1, HDAC2, HDAC3, and HDAC10.

[0088] Female BALB / c mice (BALB / cByJNarl) aged 5 to 8 weeks were subcutaneously inoculated with 5xl05CT-26 cells in the right flank. When a mean tumor volume reached approximately 260 mm3, the tumor-bearing mice (weighing about 18 to 24 g) were randomized to treatment, and the day of treatment initiation was designated Day 0. The mice were divided into seven groups and administered as indicated (TABLE 8). Specifically, ABT-301 (Anbogen Therapeutics), prepared with 0.5% carboxymethyl cellulose and 0.1% Tween 80 in 5% dextrose, was administered at 50 mg / kg orally once daily. RMP1-14 (Leinco Technologies) was administered at 200 pg by IP injection twice per week. Chidamide (Cayman Chemical), preparedwith 0.2% carboxymethyl cellulose and 0.1% Tween 80 in double-distilled water, was administered at 20 mg / kg orally daily. Regorafenib (China Anfen Group Limited), prepared with Cremophor EL, ethanol, and 0.9% sodium chloride solution (12.5%:12.5%:75%, v:v:v), was administered at 30 mg / kg orally daily. 5 -fluorouracil (5-FU; China Anfen Group Limited), prepared in saline with 3% Tween 80, was administered at 25 mg / kg by IP injection thrice weekly (TIW). The control group mice received a rat IgG2a (abbreviated as IgG) as an isotype control antibody. IgG and RMP1-14 were prepared with PBS. Treatments were given for the indicated period or until the humane endpoint (tumor volume > 2000 mm3, defined as death). During the dosing period, body weight and tumor volume were monitored two to three times per week. Tumor volume (mm3) was measured using a caliper and calculated using the formula [(LxW2) / 2], where L and W represent the longest diameter and the shortest diameter in mm, respectively. Tumor growth inhibition (TGI) was calculated as follows: TGI (%) = [1 - (final tumor volume - initial tumor volume for each treated group) I (final tumor volume - initial tumor volume for the control group)] x 100. Tumor responses in this study were evaluated using the same criteria as described in Example 2.

[0089] TABLE 8 Treatment regimen in CT26 tumor-bearing mice

[0090] FIGs. 6A-6B show the growth of CT26 tumors in mice following treatments for 11 days; and TABLE 9 presents the mean tumor volumes and mean TGI on Day 11. As demonstratedin TABLE 9, the triple combination of ABT-301 + PD-li + VEGFi exhibited the most potent initial tumor suppression. Notably, this triple combination achieved a mean TGI of 85.88%, which was significantly higher than the TGI of 51.98% achieved by the triple combination utilizing chidamide (another HD AC inhibitor). Furthermore, the ABT-301 -based VEGF-targeted triple-combination therapy demonstrated an early-stage inhibitory effect comparable to or even superior to the ABT-301-based chemo-immunotherapy (ABT-301 + PD-li + 5-FU), which achieved a mean TGI of 69.40%. The results indicated the unique antitumor potency of the combination of ABT-301, a PD-1 inhibitor, and a VEGF inhibitor.

[0091] TABLE 9 Initial (early-stage) antitumor efficacy in CT26 tumor-bearing mice following administration of the indicated treatments for 11 daysData are presented as mean ± SEM. n = 10 for the control group; n = 7 for the ABT-301 + PD-li group; n = 8 for all other groups. The mouse in the control group that reached the humane endpoint on Day 6 was excluded. * P <0.05 compared with the PD-li group by t-test.

[0092] At the end of the study (Day 36), the tumor burden among surviving subjects was assessed (FIGs. 7A-7B and TABLE 10). The triple combination of ABT-301 + PD-li + VEGFi maintained a larger population of survivors (n = 7) compared to the dual combination of ABT-301 + PD- 1 i (n = 5) . Although mean tumor volumes on Day 36 varied among triple combination groups due to differences in tumor regression, the consistent survival profile and controlled tumor growth across the ABT-301 -based treatment groups indicate a long-term tumor-suppressing effect when ABT-301 is used as a component of triple combination therapy.

[0093] TABLE 10 Long-term antitumor efficacy in CT26 tumor-bearing mice following administration of the indicated treatments for 36 daysData are presented as mean ± SEM.

[0094] Throughout the study period, mice in all groups maintained mean relative body weights (normalized to Day 0) between approximately 97% and 120%. No obvious abnormalities were observed in any group, indicating that these treatments were well-tolerated at the administered doses.

[0095] The relative tumor volume changes in mice receiving various treatments are shown in FIGs. 8A-8G, and the tumor response data are summarized in TABLE 11. After 36 days of treatment, all mice in the control group and the PD-li monotherapy group exhibited progressive disease (PD). In the ABT-301 + PD-li group, 3 of 7 mice exhibited PD, 2 of 7 mice showed partial response (PR), and 2 of 7 mice achieved complete response (29% CR rate). In the chidamide + PD-li group, 5 of 8 mice exhibited PD, and 3 of 8 mice showed PR (0% CR rate). In the ABT-301 + PD-li + VEGFi group, 3 of 8 mice exhibited PD, and 5 of 8 mice achieved CR (62.5% CR rate). In the chidamide + PD-li + VEGFi group, 3 of 8 mice exhibited PD, 2 of 8 mice showed PR, and 3 of 8 mice achieved CR (37.5% CR rate). In the ABT-301 + PD-li + 5-FU group, 3 of 8 mice exhibited PD, 2 of 8 mice showed PR, and 3 of 8 mice achieved CR (37.5% CR rate). The observation that all mice in the PD-li monotherapy group exhibited PD highlights the inherent resistance of this tumor model to conventional immunotherapy. The triple combination of ABT-301 + PD-li + VEGFi effectively overcame this resistance, yielding a 62.5% CR rate, demonstrating that this combination is particularly effective for treating cancers that are resistantto immune checkpoint inhibitor monotherapies.

[0096] Moreover, comparison of ABT-301 -based and chidamide-based combinations revealed superior therapeutic depth for the ABT-301 -based combinations; for example, the dual combination of ABT-301 + PD-li yielded 2 CRs, whereas the chidamide + PD-li combination failed to induce any CR. Notably, although several combined regimens achieved an ORR of 62.5%, the ABT-301 + PD-li + VEGFi group induced the highest CR rate (5 / 8 mice, 62.5%), and the median time to CR was 34 days, indicating rapid and durable tumor regression. These results indicate that the integration of ABT-301 with VEGFi (regorafenib) specifically enhances therapeutic efficacy, leading to complete tumor eradication.

[0097] TABLE 11 Tumor response in CT26 tumor-bearing mice following administration of the indicated treatments for 36 days

[0098] FIGs. 9A-9B and TABLE 12 present survival outcomes and median survival in CT26 tumor-bearing mice treated with different regimens. The median survival in the dual- or triple-combination groups was significantly prolonged compared with the control group. The ABT-301 + PD-li + VEGFi combination achieved a survival rate of 87.5%, surpassing the chidamide + PD-li + VEGFi regimen (75.0% survival rate). Similar results were observed in the dual-combination groups, with the ABT-301 + PD-li combination (71% survival rate) outperforming the chidamide + PD-li combination (50.0% survival rate). These results demonstrate that ABT-301, when combined with VEGF and PD-1 / PD-L1 inhibitors, enables superior tumor eradication and long-term protection that is not readily achievable by other HD ACinhibitors.

[0099] TABLE 12 Survival outcomes and median survival of CT26 tumor-bearing mice following administration of the indicated treatments for 36 days

[0100] Example 4 Antitumor efficacy in Hepa 1-6 HCC model

[0101] The antitumor efficacy of a triple combination therapy comprising ABT-301 , a PD-L1 inhibitor (e.g., atezolizumab, an anti-PD-Ll monoclonal antibody), and a VEGF inhibitor (e.g., bevacizumab, an anti- VEGF monoclonal antibody) was assessed in an HCC allograft mouse model. The model was established using Hepa 1-6 cells (ATCC No. CRL-1830), a highly malignant mouse hepatoma cell line used to model advanced human hepatocellular carcinoma and the associated immune-resistant tumor microenvironment.

[0102] Female C57BL / 6 mice aged 6 to 8 weeks were subcutaneously inoculated with 4*105Hepa 1-6 cells in the left flank. When a mean tumor volume reached approximately 100 mm3, the tumor-bearing mice (weighing about 18 to 23 g) were randomized to treatment, and the day of treatment initiation was designated Day 0. The mice were divided into six groups and administered as indicated (TABLE 13). Specifically, ABT-301 (Anbogen Therapeutics), prepared with 0.5% carboxymethyl cellulose and 0.1% Tween 80 in 5% dextrose, was administered at 100 mg / kg orally on a 5-day-on, 2-day-off weekly schedule (5-on / 2-off schedule). Atezolizumab (Novus Biologicals) was administered at 200 pg by IP injection twice per week. Bevacizumab (Leinco Technologies) was administered at 200 pg by IP injection twice per week. The controlgroup mice received a vehicle (0.5% carboxymethyl cellulose and 0.1% Tween 80 in 5% dextrose). Atezolizumab was prepared with PBS. During the dosing period, body weight and tumor volume were monitored twice a week. Tumor volume (mm3) was measured using a caliper and calculated using the formula [(L*W2) / 2], where L and W represent the longest diameter and the shortest diameter in mm, respectively. Tumor growth inhibition (TGI) was calculated as follows: TGI (%) = [1 - (final tumor volume - initial tumor volume for each treated group) / (final tumor volume -initial tumor volume for the control group)] * 100. Tumor responses in this study were evaluated using the same criteria as described in Example 2.

[0103] TABLE 13 Treatment regimen in Hepa 1-6 tumor-bearing mice

[0104] FIG. 10A shows the growth of Hepa 1-6 tumors in mice following treatments for 47 days; FIG. 10B illustrates individual tumor volumes and group means in mice on Day 47. As shown in TABLE 14, all combination treatment groups exhibited tumor growth inhibition relative to the control group. By Day 47, the mean tumor volume of the triple combination group (ABT-301 + PD-Lli + VEGFi) was significantly reduced to 140.07 ± 14.89 mm3, compared with 371.62 ± 26.24 mm3for the PD-Lli monotherapy and 347.10 ± 18.78 mm3for the dual combination of PD-Lli + VEGFi. Notably, the triple combination achieved the highest TGI of 94.2%, which was markedly superior to the PD-Lli + VEGFi dual combination (51.5%), a combination representing the clinical standard of care. These results indicate a potent synergistic effect of adding ABT-301 to a VEGF-targeted immunotherapy regimen, compared with all tested dual combinations ormonotherapies.

[0105] TABLE 14 Antitumor efficacy in Hepa 1-6 tumor-bearing mice following administration of the indicated treatments for 47 daysData are presented as mean or mean ± SEM. n = 6 for the control group; n = 8 for all other groups.

[0106] Throughout the 47-day study period, mice in all groups maintained mean relative body weights (normalized to Day 0) between approximately 94% and 113%. No significant body weight loss was observed in any treatment group, indicating that the triple combination therapy was well-tolerated even with prolonged administration.

[0107] The relative tumor volume changes in mice receiving various treatments are shown in FIGs. 11A-1 IF, and the tumor response data are summarized in TABLE 15 The response data further highlight the immunomodulatory role of ABT-301 in overcoming treatment resistance. In this Hepa 1-6 model, which exhibited intrinsic resistance to both PD-Lli monotherapy (100% PD) and the standard dual combination of PD-Lli + VEGFi (100% PD), the triple combination successfully controlled tumor growth, with 5 out of 8 mice achieving stable disease (SD). These findings demonstrate that the integration of ABT-301 into a VEGF-targeted immunotherapy is crucial for overcoming primary resistance to current immune-checkpoint-based therapies in hepatocellular carcinoma.

[0108] TABLE 15 Tumor response in Hepa 1-6 tumor-bearing mice following administration of the indicated treatments for 47 days

[0109] The present disclosure demonstrates, through multiple in vivo studies, that the pharmaceutical combination comprising ABT-301 , a VEGF inhibitor, and a PD-1 / PD-L1 pathway inhibitor yields significant and synergistic antitumor efficacy. Compared to well-known monotherapies or dual combination therapies employing immune checkpoint blockade, antiangiogenesis, or epigenetic regulation, the triple combination therapy disclosed herein exhibited superior therapeutic outcomes, including long-term tumor suppression, enhanced objective response rates and complete response rates, and prolonged overall survival. Such effects were observed across various tumor models representing different histological origins and distinct immune microenvironments. Accordingly, the pharmaceutical combination of the present disclosure can be utilized to treat various cancers, particularly those that are resistant, refractory, or non-responsive to conventional immunotherapies.

Claims

WHAT IS CLAIMED IS:

1. A pharmaceutical combination, comprising:(E)-N-hydroxy-3-(l-(phenylsulfonyl)-indolin-5-yl)-acrylamide;a vascular endothelial growth factor (VEGF) inhibitor; anda programmed cell death protein 1 (PD-l) / programmed cell death ligand 1 (PD-L1) pathway inhibitor.

2. The pharmaceutical combination of claim 1, wherein the VEGF inhibitor is a VEGF receptor tyrosine kinase inhibitor, an antibody or an antigen-binding fragment thereof capable of binding to VEGF or a VEGF receptor, or a VEGF-binding protein.

3. The pharmaceutical combination of any of claims 1 to 2, wherein the VEGF inhibitor is selected from the group consisting of sunitinib, sorafenib, regorafenib, cabozantinib, axitinib, pazopanib, lenvatinib, bevacizumab, ranibizumab, brolucizumab, ramucirumab, aflibercept, and conbercept.

4. The pharmaceutical combination of any of claims 1 to 3, wherein the PD-1 / PD-L1 pathway inhibitor is an anti-PD-1 antibody or an antigen-binding fragment thereof, an anti-PD-Ll antibody or an antigen-binding fragment thereof, a small-molecule PD-1 inhibitor, or a smallmolecule PD-L1 inhibitor.

5. The pharmaceutical combination of any of claims 1 to 4, wherein the PD-1 / PD-L1 pathway inhibitor is selected from the group consisting of sintilimab, camrelizumab, pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab, toripalimab, tislelizumab, cosibelimab, RMP1-14, atezolizumab, sugemalimab, envafolimab, avelumab, durvalumab, INCB086550, GS-4224, and BMS-1166.

6. The pharmaceutical combination of claim 1, wherein the VEGF inhibitor and the PD-1 / PD-L1 pathway inhibitor are provided as a multi-specific antibody comprising a VEGF-binding domain and a PD-1 or PD-L1 -binding domain.

7. A method for treating cancer in a subject in need thereof, comprising administering tothe subject a therapeutically effective amount of (E)-N-hydroxy-3-(l-(phenylsulfonyl)-indolin-5-yl)-acrylamide, a therapeutically effective amount of a VEGF inhibitor, and a therapeutically effective amount of a PD-1 / PD-L1 pathway inhibitor.

8. The method of claim 7, wherein the cancer is colorectal cancer or liver cancer.

9. The method of any of claims 7 to 8, wherein the cancer is resistant to a prior treatment with an immune checkpoint inhibitor.

10. The method of any of claims 7 to 9, wherein the (E)-N-hydroxy-3-(l-(phenylsulfonyl)-indolin-5-yl)-acrylamide is administered in an amount of about 0.5 mg / kg to about 500 mg / kg, and / or the VEGF inhibitor is administered in an amount of about 0.5 mg / kg to about 100 mg / kg, and / or the PD-1 / PD-L1 pathway inhibitor is administered in an amount of about 0.1 mg / kg to about 50 mg / kg.

11. The method of any of claims 7 to 10, wherein the VEGF inhibitor is a VEGF receptor tyrosine kinase inhibitor, an antibody or an antigen-binding fragment thereof capable of binding to VEGF or a VEGF receptor, or a VEGF -binding protein.

12. The method of any of claims 7 to 11, wherein the VEGF inhibitor is selected from the group consisting of sunitinib, sorafenib, regorafenib, cabozantinib, axitinib, pazopanib, lenvatinib, bevacizumab, ranibizumab, brolucizumab, ramucirumab, aflibercept, and conbercept.

13. The method of any of claims 7 to 12, wherein the PD-1 / PD-L1 pathway inhibitor is an anti-PD-1 antibody or an antigen-binding fragment thereof, an anti-PD-Ll antibody or an antigenbinding fragment thereof, a small-molecule PD-1 inhibitor, or a small-molecule PD-L1 inhibitor.

14. The method of any of claims 7 to 13, wherein the PD-1 / PD-L1 pathway inhibitor is selected from the group consisting of sintilimab, camrelizumab, pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab, toripalimab, tislelizumab, cosibelimab, RMP1-14, atezolizumab, sugemalimab, envafolimab, avelumab, durvalumab, INCB086550, GS-4224, and BMS-1166.

15. The method of any of claims 7 to 9, wherein the VEGF inhibitor and the PD-1 / PD-L1pathway inhibitor are provided as a multi-specific antibody comprising a VEGF -binding domain and a PD-1 or PD-L1 -binding domain, and the multi-specific antibody is administered in a therapeutically effective amount.

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