Combination therapy for tumors and methods of use

Abstract

Disclosed herein are treatment of cancer by combined administration of plinabulin and an immune checkpoint inhibitor.

Description

[0001] Cross-references to related applications This patent application claims priority to U.S. Provisional Application No. 63 / 523,609, filed June 27, 2023; U.S. Provisional Application No. 63 / 589,579, filed October 11, 2023; and U.S. Provisional Application No. 63 / 631,251, filed April 8, 2024. The entire contents of all of the foregoing applications are incorporated herein by reference for all purposes. Technical Field

[0002] This disclosure relates to the fields of chemistry and medicine, and specifically to a combination therapy for treating cancer that includes punabrine, immune checkpoint inhibitors, and radiation therapy. Background Technology

[0003] Human cancers carry multiple genetic and epigenetic alterations, producing neoantigens that may be recognized by the immune system (Sjoblom et al., 2006). The adaptive immune system, composed of T lymphocytes and B lymphocytes, has a powerful anti-tumor potential, responding to diverse tumor antigens with broad responsiveness and high specificity.

[0004] Recent research in cancer immunotherapy has focused heavily on enhancing anti-tumor immunity by mediating adaptive immune responses to relevant antigens and providing specific immunostimulants, such as immune checkpoint inhibitors. Although cancer remains incurable for the vast majority of patients, there is an urgent need to develop effective therapeutic agents and regimens for cancer treatment. Patients receiving immune checkpoint inhibitor therapy may initially respond to these inhibitors, but subsequently become unresponsive (resistant), or remain unresponsive from the start of administration (non-responders). Summary of the Invention

[0005] In a first aspect of this disclosure, a method for treating cancer in a subject is provided, the method comprising a treatment cycle including the following steps: (i) Administer one or more immune checkpoint inhibitors to the subject; (ii) administering punabrine or a pharmaceutically acceptable salt thereof to the subject; (iii) Obtaining biological samples from the subject; and (iv) Determine the levels of one or more biomarkers or one or more cells in the biological sample.

[0006] In some embodiments of the first aspect, treatment with the one or more immune checkpoint inhibitors and ponabulin is discontinued if the level of one or more biomarkers or one or more cells in the biological sample is higher or lower than a predetermined threshold level, or if a score determined by mathematically combining the levels of two or more biomarkers or two or more cells is higher or lower than a predetermined threshold.

[0007] In some embodiments of the first aspect, the one or more biomarkers are: CCR7, CD40, CD80, CD83, CD86, GEF-H1, IL-2, IFNγ, IL-6, IL-12p70, IL-12p40, IL-13, IL-17A, IL-23, G-CSF, PD-L1, IL-8, and IFN-β, or combinations thereof. In some embodiments, the one or more biomarkers include CCR7. In some embodiments, the one or more biomarkers include CD40. In some embodiments, the one or more biomarkers include CD80. In some embodiments, the one or more biomarkers include CD83. In some embodiments, the one or more biomarkers include CD86. In some embodiments, the one or more biomarkers include GEF-H1.

[0008] In some embodiments of the first aspect, the one or more immune checkpoint inhibitors are: pembrolizumab, nivolumab, cimiprimab, atezolizumab, avelumab, pildilizumab, ipilimumab, BMS 936559, durvalumab, camrelizumab, dotalipimab, tislelizumab, sintilimab, toripalimab, or combinations thereof. In some embodiments, the one or more immune checkpoint inhibitors are avelumab. In other embodiments, the one or more immune checkpoint inhibitors are atezolizumab. In still other embodiments, the one or more immune checkpoint inhibitors are durvalumab. In yet another embodiment, the one or more immune checkpoint inhibitors are nivolumab. In some embodiments, the one or more immune checkpoint inhibitors are pembrolizumab. In some embodiments, the one or more immune checkpoint inhibitors are camrelizumab. In some embodiments, the one or more immune checkpoint inhibitors are dotalipimab. In some embodiments, the one or more immune checkpoint inhibitors are tislelizumab. In some embodiments, the one or more immune checkpoint inhibitors are sintilimab. In some embodiments, the one or more immune checkpoint inhibitors are toripalimab.

[0009] In some embodiments of the first aspect, the treatment period is 21 days. In other embodiments, the treatment period is 28 days.

[0010] In some embodiments of the first aspect, the one or more immune checkpoint inhibitors are administered via intravenous injection.

[0011] In some embodiments of the first aspect, the one or more immune checkpoint inhibitors are administered on day 1 of the treatment cycle. In other embodiments, the one or more immune checkpoint inhibitors are administered on day 15 of the treatment cycle. In some embodiments, the dose of the one or more immune checkpoint inhibitors administered to the subject is from 50 mg to 2000 mg.

[0012] In some embodiments of the first aspect, the one or more immune checkpoint inhibitors are administered over a period of about 10 minutes to about 180 minutes. In some embodiments, the one or more immune checkpoint inhibitors are administered over a period of about 10 minutes to about 120 minutes. In other embodiments, the one or more immune checkpoint inhibitors are administered over a period of about 30 minutes to about 90 minutes. In some embodiments, the one or more immune checkpoint inhibitors are administered over a period of about 30 minutes. In other embodiments, the one or more immune checkpoint inhibitors are administered over a period of about 60 minutes.

[0013] In some embodiments of the first aspect, the pravastatin is administered on day 1 of the treatment cycle. In some embodiments, the pravastatin is administered on day 4 of the treatment cycle. In some embodiments, the pravastatin is administered on days 1 and 4 of the treatment cycle.

[0014] In some embodiments of the first aspect, the dose of punabulin is approximately 10 mg / m². 2 Approximately 50 mg / m 2 In some embodiments, the dose of punabulin is approximately 20 mg / m². 2 In other embodiments, the dose of punabrine is approximately 30 mg / m². 2 .

[0015] In some embodiments of the first aspect, the pravastatin is administered over a period of about 10 minutes to about 128 minutes. In some embodiments, the pravastatin is administered over a period of about 10 minutes to about 120 minutes. In other embodiments, the pravastatin is administered over a period of about 20 minutes to about 90 minutes. In some embodiments, the pravastatin is administered over a period of about 30 minutes to about 60 minutes.

[0016] In some embodiments of the first aspect, when the one or more immune checkpoint inhibitors are administered on the same day as ponabulin, ponabulin is administered approximately 0.5 hours to approximately 3 hours after the administration of the one or more immune checkpoint inhibitors. In some embodiments, ponabulin is administered approximately 1 hour to approximately 2 hours after the administration of the one or more immune checkpoint inhibitors.

[0017] In some embodiments of the first aspect, the method further includes administering radiation therapy to the subject. In some embodiments, the radiation therapy is administered in 1 to 10 fractions. In other embodiments, the radiation therapy is administered in 3 to 5 fractions. In some embodiments, the radiation therapy is administered in 3 fractions. In other embodiments, the radiation therapy is administered in 4 fractions. In still other embodiments, the radiation therapy is administered in 5 fractions. In some embodiments, the total dose of radiation administered is from about 1 Gy to about 20 Gy. In other embodiments, the total dose of radiation administered is from about 2 Gy to about 15 Gy. In still other embodiments, the total dose of radiation administered is from about 4 Gy to about 15 Gy. In some embodiments, the total dose of radiation administered is about 4 Gy. In some embodiments, the total dose of radiation administered is about 8 Gy. In some embodiments, the total dose of radiation administered is about 12.5 Gy.

[0018] In some embodiments of the first aspect, radiotherapy is administered on days 1, 2, and 3 of the treatment cycle. In other embodiments, radiotherapy is administered on days 1, 2, 3, and 4 of the treatment cycle. In still other embodiments, radiotherapy is administered on days 1, 2, 3, 4, and 5 of the treatment cycle. In some embodiments, when radiotherapy and ponabulin are administered on the same day, ponabulin is administered approximately 3 to approximately 12 hours after the completion of radiotherapy administration.

[0019] In some embodiments of the first aspect, the biological sample is a blood sample. In some such embodiments, the blood sample is a peripheral blood sample. In other embodiments, the biological sample is tumor biopsy tissue.

[0020] In some embodiments of the first aspect, the one or more cells are dendritic cells (DCs) and their subsets (e.g., myeloid dendritic cells (mDCs), plasmacytoid dendritic cells (pDCs), and conventional dendritic cells (cDCs)); or the one or more cells are selected from the group consisting of CD3+ T cells, CD4+ T cells, CD8+ T cells, B cells, IgA+ plasma cells, IgG+ plasma cells, and IgM+ plasma cells; or the one or more cells are selected from the group consisting of CD16+ monocytes, CD14+ monocytes, natural killer (NK) cells, granzyme K+ T cells, CD8+ TRM cells, CD8+ TEM cells, CD4+ TCM cells, CD4+ TEM cells, CD4+ naive T cells, naive B cells, cDC1 cells, cDC2 cells, and DC3 cells. In some embodiments, the one or more cells are monocyte-derived macrophages (MoMacs) or mesenchymal macrophages (IMs). In other embodiments, the one or more cells are immune cells derived from peripheral blood mononuclear cells (PBMCs).

[0021] In some embodiments of the first aspect, the method includes determining the levels of one or more dendritic cells and their subsets expressing one or more biomarkers in a biological sample. In some embodiments, the dendritic cells are: plasmacytoid dendritic cells (pDCs), conventional dendritic cells (cDCs), or myeloid dendritic cells (mDCs). In some embodiments, the one or more biomarkers are: CCR7, CD40, CD80, CD83, CD86, GEF-H1, IL-2, IFNγ, IL-6, IL-12p70, IL-12p40, IL-13, IL-17A, IL-23, G-CSF, IL-8, IFN-β, or combinations thereof. In some embodiments, the one or more biomarkers are CCR7, CD40, CD80, CD83, CD86, or combinations thereof. In other embodiments, the one or more biomarkers are the GEF-H1 immune activation gene signature score (i.e., the GEF-H1 immune activation score).

[0022] In some embodiments of the first aspect, the biological sample is obtained on day 4 of the treatment cycle.

[0023] In some embodiments of the first aspect, the cancer is selected from: breast cancer, bladder cancer, glioma, glioblastoma, head and neck cancer, non-small cell lung cancer, small cell lung cancer, recurrent small cell lung cancer (SCLC), colorectal cancer, gastrointestinal stromal tumor, gastroesophageal cancer, renal cell carcinoma, prostate cancer, liver cancer, colon cancer, pancreatic cancer, ovarian cancer, lymphoma, cutaneous T-cell lymphoma, or melanoma.

[0024] In some embodiments of the first aspect, the method includes determining baseline levels of the one or more biomarkers or one or more cells in an initial biological sample. In some embodiments, the initial biological sample is acquired on day 1 of the treatment cycle, prior to administration of the one or more immune checkpoint inhibitors and ponabulin. In some embodiments, the method includes comparing the levels determined in step (iv) with baseline levels. In some embodiments, the comparison includes determining changes in the levels of the one or more biomarkers or one or more cells.

[0025] In some embodiments of the first aspect, treatment with the one or more immune checkpoint inhibitors and punabulin is discontinued if the changes in the levels of the one or more biomarkers or the one or more cell types are below a predetermined threshold. In other embodiments, treatment with the one or more immune checkpoint inhibitors and punabulin is discontinued if the changes in the levels of the one or more biomarkers or the one or more cell types are above a predetermined threshold.

[0026] In some embodiments, treatment with one or more immune checkpoint inhibitors and ponabulin is discontinued if the level of CCR7-expressing pDCs is lower than baseline by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% or more. In some embodiments, treatment with one or more immune checkpoint inhibitors and ponabulin is discontinued if the level of CCR7-expressing pDCs is higher than baseline by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% or more.

[0027] In some embodiments, treatment with one or more immune checkpoint inhibitors and ponabulin is discontinued if the level of CD80-expressing pDCs is lower than baseline by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% or more. In some embodiments, treatment with one or more immune checkpoint inhibitors and ponabulin is discontinued if the level of CD80-expressing pDCs is higher than baseline by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% or more.

[0028] In some embodiments, treatment with one or more immune checkpoint inhibitors and ponabulin is discontinued if the level of CD83-expressing pDCs is lower than baseline by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% or more. In some embodiments, treatment with one or more immune checkpoint inhibitors and ponabulin is discontinued if the level of CD83-expressing pDCs is higher than baseline by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% or more.

[0029] In a second aspect of this disclosure, a method for treating cancer in a subject is described, the method comprising a treatment cycle including the following steps: (i) Obtain biological samples from the subject; (ii) Determining the levels of one or more biomarkers or one or more cells in the biological sample; and (iii) If the level of one or more biomarkers or one or more cells in the biological sample is higher or lower than a threshold level, or if the score determined by mathematically combining the levels of two or more biomarkers or two or more cells is higher or lower than a predetermined threshold, then the subject shall be administered punabrine or a pharmaceutically acceptable salt thereof.

[0030] In some embodiments of the second aspect, the biological sample is tumor biopsy tissue. In other embodiments, the biological sample is peripheral blood.

[0031] In some embodiments of the second aspect, the one or more cells are dendritic cells (DCs) and their subsets (e.g., myeloid dendritic cells (mDCs), plasmacytoid dendritic cells (pDCs), and conventional dendritic cells (cDCs)); or the one or more cells are selected from the group consisting of CD3+ T cells, CD4+ T cells, CD8+ T cells, B cells, IgA+ plasma cells, IgG+ plasma cells, and IgM+ plasma cells; or the one or more cells are selected from the group consisting of CD16+ monocytes, CD14+ monocytes, natural killer (NK) cells, granzyme K+ T cells, CD8+ TRM cells, CD8+ TEM cells, CD4+ TCM cells, CD4+ TEM cells, CD4+ naive T cells, naive B cells, cDC1 cells, cDC2 cells, and DC3 cells. In some embodiments, the one or more cells are monocyte-derived macrophages (MoMacs) and interstitial macrophages (IMs). In other embodiments, the one or more cells are immune cells derived from peripheral blood mononuclear cells (PBMCs).

[0032] In some embodiments of the second aspect, the score is a GEF-H1 immune activation score. In some embodiments, the GEF-H1 immune activation score is determined using a combination of gene expression levels of one or more genes selected from the group consisting of: Mmp12, Csf1, Ccl6, Osm, Ccl9, Cxcl3, Clec7a, Hdc, Slc15a3, Dcstamp, Trem1, Cxcl2, Cd300a, Ncf2, Cd80, Lilr4b, Pdcd1lg2, Lilrb4a, Mxd 1. Sp140, Il1a, Ppbp, Gpr171, Ccl7, Il1b, Ccl4, Man1a, Aqp9, Creb5, Traf4, Nod2, Ptger2, Mefv, C d6, Reps2, Tlr7, Sirpb1b, Fcgr4, P2ry2, Apol8, Lag3, Pdcd1, Cxcr1, Rab27b, Ctla4, Cxcl9 and Fasl.

[0033] In some embodiments of the second aspect, the threshold for the GEF-H1 immune activation score is about 10 to about 40. In other embodiments, the threshold for the GEF-H1 immune activation score is about 20 to about 40. In some embodiments, the threshold for the GEF-H1 immune activation score is about 30. In other embodiments, the threshold for the GEF-H1 immune activation score is about 25.

[0034] In some embodiments of the second aspect, the cancer is selected from: breast cancer, bladder cancer, glioma, glioblastoma, head and neck cancer, non-small cell lung cancer, small cell lung cancer, recurrent small cell lung cancer (SCLC), colorectal cancer, gastrointestinal stromal tumor, gastroesophageal cancer, renal cell carcinoma, prostate cancer, liver cancer, colon cancer, pancreatic cancer, ovarian cancer, lymphoma, cutaneous T-cell lymphoma, or melanoma.

[0035] In some embodiments of the second aspect, the method further includes administering radiation therapy, an immune checkpoint inhibitor, a chemotherapeutic agent, or a combination thereof to the subject. In some embodiments, the method further includes administering radiation therapy and an immune checkpoint inhibitor to the subject. Attached Figure Description

[0036] Figure 1 The mean fluorescence intensity (MFI) levels of MHC-II, CD40, CD80 and CD86 expressed on the surface of XS106 DCs are shown by flow cytometry.

[0037] Figure 2A shows the change in the percentage of pDCs expressing CCR7, CD80, and CD83 in PD or PR+SD patients three days after the first administration of punabulin.

[0038] Figure 2B shows the changes in the percentage of peripheral blood mononuclear cells characterized by classic and inflammatory phenotypes in PD or PR+SD patients three days after the first administration of ponabulin.

[0039] Figures 3A and 3B show the proportion of various cell types in tumor biopsy samples in PR+SD subjects compared to PD subjects in scRNA-seq analysis.

[0040] Figure 4A The standardized baseline GEF-H1 immune activation score bars for cDC1, cDC2, DC3, and total DC cells are shown in patients with clinical benefit (PR+SD) and no clinical benefit (PD).

[0041] Figure 4B Box plots showing the GEF-H1 standardized immune scores of DC subsets and total DC cells before and after treatment in patients with clinical benefit (PR+SD) and no clinical benefit (PD).

[0042] Figure 5A Single-cell RNAseq analysis showed the GEF-H1-dependent immunogenicity of monocyte-derived macrophages in tumor biopsies before and after treatment in responding (PR+SD) and non-responding (PD) patients.

[0043] Figure 5B This study presents a single-cell RNAseq analysis of ARG1 (arginase 1) gene expression in monocyte-derived macrophages from tumor biopsies before and after treatment in responding (PR+SD) and non-responding (PD) patients.

[0044] Figure 6A A bubble heatmap of GEF-H1 standardized immune scores for 18 immune cell types associated with clinical response is shown at three time points, highlighting cell types with statistically significant differences between the PR+SD group and the PD group.

[0045] Figure 6B A bubble heatmap of the GEF-H1 standardized immune score at baseline (C1D1) is shown, displaying cell types relevant to clinical response.

[0046] Figure 6C Presented in tabular form Figure 6A Bubble heatmap data of GEF-H1 standardized immune scores.

[0047] Figure 7 A beehive diagram depicting single-cell RNA and T-cell receptor (TCR) sequencing analysis of PBMCs was created. Detailed Implementation

[0048] This disclosure provides methods and therapeutic compositions for reversing or enhancing the response to immune checkpoint inhibitor therapy, for use in treating, improving, or preventing cancer or tumors in a subject using pranabulin. In some embodiments, the methods and compositions provided herein can be used to treat, delay progression, prevent recurrence, or alleviate symptoms of cancer and other tumor conditions using pranabulin. Pranabulin, (3Z,6Z)-3-benzyl-6-{[5-(2-methyl-2-propyl)-1H-imidazol-4-yl]methylene}-2,5-piperazinedione, is a synthetic analog of the natural compound phenylahistin. Pranabulin can be prepared according to the methods and procedures detailed in U.S. Patent Nos. 7,064,201 and 7,919,497 (the entire contents of which are incorporated herein by reference). Punabulin effectively promotes the processing and presentation of antigens by dendritic cells to effector cells (such as T cells), as well as the migration of dendritic cells to lymph nodes, where they present tumor-specific antigens to naïve immune effector cells. Exposure of dendritic cells to punabulin induces dendritic cell maturation and significantly enhances their ability to activate T cells.

[0049] Some embodiments disclosed herein involve administering prazolam or a pharmaceutically acceptable salt thereof to a cancer subject in combination with one or more immune checkpoint inhibitors. In some embodiments, prazolam and one or more immune checkpoint inhibitors may be administered in combination with radiation therapy.

[0050] Some embodiments disclosed herein include measuring the levels of one or more biomarkers or one or more cells in a subject's biological sample after administration of ponabulin and one or more immune checkpoint inhibitors, and determining whether to discontinue treatment. In some embodiments, the level of a specific cell expressing one or more biomarkers may be used to determine whether to discontinue treatment. In some embodiments, the cells are dendritic cells (DCs). In such embodiments, the dendritic cells are myeloid dendritic cells (mDCs). In other embodiments, the cells are plasmacytoid dendritic cells (pDCs). In still other embodiments, the cells are conventional dendritic cells (cDCs). In some embodiments, changes in the levels of one or more biomarkers or one or more cells in a subject's biological sample may be assessed relative to a reference level of one or more biomarkers or one or more cells in the subject's biological sample (e.g., relative to the level of one or more biomarkers or one or more cells in the subject's biological sample before the start of the treatment methods described herein).

[0051] Other embodiments disclosed herein include determining the level of one or more biomarkers or one or more cells in a subject's biological sample prior to administration of ponabulin and one or more immune checkpoint inhibitors, and administering ponabulin treatment if the level of the one or more biomarkers or one or more cells is above or below a predetermined threshold.

[0052] Before further describing this disclosure, it should be understood that the invention is not limited to the specific embodiments described, as variations are certainly possible. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the invention is defined only by the appended claims.

[0053] If a numerical range is provided, it should be understood that, unless the context explicitly specifies otherwise, every intermediate value between the upper and lower limits of the range (to one-tenth of the lower limit unit), and any other stated or intermediate value within the range, is included within this invention. The upper and lower limits of these smaller ranges may be independently included in the smaller range and also within this invention, but are subject to any explicitly excluded limits within the range. If the range contains one or two limits, the range excluding one or both of those limits is also included within this invention.

[0054] The method described herein can be implemented in any logically feasible order of the events, or executed in the order of the events.

[0055] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention, the methods and materials described herein are preferred.

[0056] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials associated with the cited publications.

[0057] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly specifies otherwise. It should also be noted that claims may be drafted to exclude any preferred elements. Therefore, this statement is intended as a precondition for the use of exclusive terms such as “only,” “only,” or “negative” restrictions when stating elements of a claim.

[0058] The publications discussed herein are provided because they were published prior to the filing date of this application. Nothing herein shall be construed as an admission that the invention is not entitled to prior publication due to prior invention. Furthermore, the publication dates provided may differ from the actual publication dates and may require independent verification.

[0059] the term Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains. All patents, applications, published applications, and other publications are incorporated herein by reference in their entirety. Where multiple definitions exist for terms herein, the definitions in this section shall prevail unless otherwise stated.

[0060] As used in this article, the term "substance" is used to refer to compounds, mixtures of compounds, biological macromolecules, or extracts made from biological materials.

[0061] As used herein, the term "antagonist" refers to a compound that can bind to a receptor (e.g., an immune checkpoint receptor) to block cellular activity. Antagonists can be ligands that directly bind to the receptor. Alternatively, antagonists can bind to the receptor indirectly, by forming a complex with another molecule that directly binds to the receptor, such as (a) or (b) otherwise modifying another compound to allow that compound to directly bind to the receptor.

[0062] As used in this article, the term “improvement” refers to any reduction in the degree, severity, frequency and / or likelihood of symptoms or clinical signs of a particular condition.

[0063] The term "antibody" or "antibody fragment" is intended to encompass a polypeptide chain containing a molecular structure of a specific shape capable of fitting and recognizing an epitope, wherein one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope. Antibodies used in this disclosure may be polyclonal or monoclonal antibodies. Antibodies also include free antibodies and their derived antigen-binding fragments, as well as conjugates, such as pegylated antibodies, drug conjugates, radioisotope conjugates, or toxin conjugates. Monoclonal antibodies targeting a specific epitope or combination of epitopes can be used to target and / or eliminate cell populations expressing that marker. Various techniques can be used to screen cell populations expressing that marker using monoclonal antibodies, including: magnetic sorting using antibody-coated magnetic beads, "screening" with antibodies attached to a solid matrix (such as a plate), and flow cytometry (see, for example, U.S. Patent No. 5,985,660; and Morrison et al., Cell, Vol. 96, pp. 737-749 (1999)). These technologies can be used to screen specific cell populations, perform immunohistochemical analysis of biopsy samples, and detect markers released by cancer cells into the blood and other bodily fluids. Humanized versions of these antibodies are also within the scope of this disclosure. Humanized antibodies, due to their low antigenicity, are particularly suitable for in vivo application.

[0064] The terms “cancer,” “tumor,” and “carcinoma” are used interchangeably herein to refer to cells exhibiting relatively autonomous growth, that is, cells displaying an abnormal growth phenotype characterized by a significant loss of control over cell proliferation. Typically, the target cells to be detected or treated in this application include: precancerous cells (e.g., benign), malignant cells, pre-metastatic cells, metastatic cells, and non-metastatic cells. Detection of cancer cells is particularly important.

[0065] As used herein, the term "immune checkpoint inhibitor" refers to molecules (e.g., small molecules, peptides, polypeptides, proteins, antibodies, antibody fragments, etc.) that act as inhibitors (antagonists) of the immune checkpoint pathway. Inhibition of the pathway may include blocking the pathway by binding to receptors or signaling molecules that are components of the immune checkpoint pathway.

[0066] As used herein, the term "peptide" is a general term referring to a natural protein, a fragment of a polypeptide sequence, or an analogue. Therefore, natural proteins, fragments, and analogues are all species of the genus *Polypeptide*.

[0067] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes all solvents, dispersion media, coating materials, antibacterial and antifungal agents, isotonic agents, and absorption delayers. The use of these media and formulations in pharmaceutically active substances is well known in the art. Unless any conventional media or formulation is incompatible with the active ingredient, its use in therapeutic compositions is considered. In addition, various adjuvants commonly used in the art may be included. For considerations regarding the addition of various components to pharmaceutical compositions, see, for example, Gilman et al. (ed.) (1990) 20, *Goodman and Gilman's: The Pharmacological Basis of Therapeutics*, 8th ed., Pergamon, the entire contents of which are incorporated herein by reference. Pharmaceutically acceptable excipients can be monosaccharides or monosaccharide derivatives.

[0068] As used herein, the term “subject” refers to human or non-human mammals such as dogs, cats, mice, rats, cattle, sheep, pigs, goats, non-human primates or birds (such as chickens), and any other vertebrates or invertebrates.

[0069] The term "mammal" is used in its usual biological sense. Therefore, it specifically includes, but is not limited to: primates (including apes (chimpanzees, apes, monkeys) and humans), cattle, horses, sheep, goats, pigs, rabbits, dogs, cats, rodents, rats, mice, guinea pigs, etc.

[0070] As used herein, the term "effective dose" or "therapeutic effective dose" refers to the likelihood that one or more symptoms of a disease or condition will be effectively relieved or reduced to some extent, and may include a therapeutic dose that cures a disease or condition.

[0071] As used herein, the terms “treat,” “treatment,” or “treating” refer to the administration of a compound or pharmaceutical composition to a subject for preventive and / or therapeutic purposes. “Preventive treatment” refers to treatment of a subject who has not yet exhibited symptoms of a disease or condition but is susceptible to or at risk of developing a specific disease or condition, thereby reducing the likelihood of that patient developing the disease or condition. The term “therapeutic treatment” refers to treatment of a subject who already has a disease or condition.

[0072] As used herein, the term "biomarker" refers to a molecular marker based on DNA, RNA, protein, carbohydrate, or glycolipid that is expressed or present in a subject or patient sample and can be detected by standard methods (or the methods disclosed herein) and helps monitor a subject's responsiveness or sensitivity to the combination therapies described herein. Such biomarkers include, but are not limited to: CCR7, CD40, CD80, CD83, CD86, GEF-H1, IL-2, IFNγ, IL-6, IL-12p70, IL-12p40, IL-13, IL-17A, IL-23, G-CSF, IL-8, and IFN-β, or combinations thereof. In some embodiments, the biomarkers may be: Mmp12, Csf1, Ccl6, Osm, Ccl9, Cxcl3, Clec7a, Hdc, Slc15a3, Dcstamp, Trem1, Cxcl2, Cd300a, Ncf2, Cd80, Lilr4b, Pdcd1lg2, Lilrb4a, Mxd1, Sp140, Il1a, Ppbp, Gpr171, Ccl7, Il1b, Ccl4, Man1a, Aqp9, Creb5, Traf4, Nod2, Ptger2, Mefv, Cd6, Reps2, Tlr7, Sirpb1b, Fcgr4, P2ry2, Apol8, Lag3, Pdcd1, Cxcr1, Rab27b, Ctla4, Cxcl9, and Fasl, or combinations thereof. In some embodiments, the levels of multiple biomarkers may be measured and combined mathematically to determine a comprehensive score. In some embodiments, the comprehensive score may be a GEF-H1 immune activation score. For subjects who are sensitive to or respond to the combination therapy described herein, the expression of biomarkers in their samples may be determined to be above or below a predetermined threshold or reference level (e.g., the level in samples obtained from previous time points of the individual).

[0073] As used herein, the term "chemotherapeutic agent" refers to an agent that can reduce, prevent, alleviate, limit, and / or delay the growth of metastatic tumors or tumors, or directly kill tumor cells through tumor necrosis, apoptosis, or any other mechanism, or can be used in a pharmaceutically effective dose or other manner to reduce, prevent, alleviate, limit, and / or delay the growth of metastatic tumors or tumors in a subject with cancer. Chemotherapy agents include, but are not limited to, the following: fluoropyrimidines; pyrimidine nucleosides; purine nucleosides; antifolate agents; platinum-based preparations; anthracyclines / anthraquinones; epipodophyllotoxins; camptothecins; hormones; hormone complexes; anti-hormonal agents; enzymes, proteins, peptides, and polyclonal and / or monoclonal antibodies; vinca alkaloids; taxanes; epothilones; antimicrotubule agents; alkylating agents; antimetabolites; topoisomerase inhibitors; antiviral agents; and various other cytotoxic and cell growth-inhibiting drugs.

[0074] Immune checkpoint inhibitors In some embodiments, one or more immune checkpoint inhibitors may be administered in combination with punabulin. Pardoll's review in *Nature Reviews Cancer* (April 2012): pp. 252-264 (the full text of which is incorporated herein by reference) describes the immune checkpoint pathway and the blocking of these pathways with immune checkpoint inhibitor compounds. Immune checkpoint inhibitor compounds exert their antitumor activity by blocking one or more endogenous immune checkpoint pathways that downregulate antitumor immune responses. Inhibition or blocking of immune checkpoint pathways typically involves using immune checkpoint inhibitor compounds to inhibit the interaction between checkpoint receptors and ligands, thereby reducing or eliminating downregulated signaling and resulting in a weakened antitumor response.

[0075] In some embodiments of this disclosure, immune checkpoint inhibitor compounds inhibit signaling interactions between immune checkpoint receptors and their corresponding ligands. Immune checkpoint inhibitor compounds can block activation of immune checkpoint pathways by inhibiting (antagonizing) immune checkpoint receptors (examples of which include CTLA-4, PD-1, LAG-3, TIM-3, BTLA, and KIR) or by inhibiting ligands of immune checkpoint receptors (examples of which include PD-L1 and PD-L2). In such embodiments, the immune checkpoint inhibitor compounds function to reduce or eliminate the downregulation of certain aspects of the immune system's antitumor response in the tumor microenvironment.

[0076] Programmed death 1 (PD-1) is a repressive member of the extended family of T cell regulatory factors CD28 / CTLA-4 (Okazaki et al., *Curr Opin Immunol*, Vol. 14, pp. 779-782 (2002); Bennett et al., *Journal of Immunology*, Vol. 170, pp. 711-718 (2003), both cited in full). Other members of the CD28 family include CD28, CTLA-4, ICOS, and BTLA. PD-1 is thought to exist in monomeric form, lacking the unpaired cysteine ​​residues characteristic of other CD28 family members. PD-1 is expressed on the surface of activated B cells, T cells, and monocytes.

[0077] The PD-1 gene encodes a 55 kDa type I transmembrane protein (Agata et al., (1996) *Int Immunol*, Vol. 8, pp. 765-772, the full text of which is incorporated herein by reference). Although structurally similar to CTLA-4, PD-1 lacks the MYPPY motif, which is crucial for the binding of B7-1 and B7-2. Two ligands for PD-1 have been identified—PD-L1 (B7-H1) and PD-L2 (B7-DC)—which have been shown to downregulate T cell activity upon binding to PD-1 (Freeman et al., (2000) *Journal of Experimental Medicine*, Vol. 192, pp. 1027-1034; Carter et al., (2002) *Eur. J. Immunol*, Vol. 32, pp. 634-643, the full text of which is incorporated herein by reference). Both PD-L1 and PD-L2 are B7 homologs that bind to PD-1 but not to other CD28 family members. PD-L1 is highly expressed in a variety of human cancers (Dong et al., (2002) Nature Medicine, Vol. 8, pp. 787-789, the full text of which is incorporated herein by reference).

[0078] PD-1 is considered an immunosuppressive protein that negatively regulates TCR signaling (Ishida, Y. et al. (1992), Journal of the European Society for Molecular Biology (EMBO J), Vol. 11, pp. 3887-3895; Blank, C. et al. (first published online on December 29, 2006), Immunology and Immunotherapy (Immunol. Immunother), Vol. 56, No. 5, pp. 739-745, both of which are incorporated herein by reference). The interaction between PD-1 and PD-L1 can act as an immune checkpoint, leading to, for example, a reduction in tumor-infiltrating lymphocytes, a decrease in T-cell receptor-mediated proliferation, and / or immune escape from cancer cells (Dong et al., (2003) J. Mol. Med, Vol. 81, pp. 281-287; Blank et al., (2005) Cancer Immunol. Immunother, Vol. 54, pp. 307-314; Konishi et al., (2004) Clin. Cancer Res, Vol. 10, pp. 5094-5100, all of which are incorporated herein by reference in their entirety). Immunosuppression can be reversed by inhibiting the local interaction between PD-1 and PD-L1 or PD-L2; when the interaction between PD-1 and PD-L2 is blocked simultaneously, the effects are additive (Iwai et al., (2002) Proc. Nat'l. Acad. Sci. USA, Vol. 99, pp. 12293-12297; Brown et al., (2003) Journal of Immunology, Vol. 170, pp. 1257-1266, both of which are incorporated herein by reference in their entirety).

[0079] Immune checkpoint receptor cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) is expressed on T cells and participates in signaling pathways that reduce T cell activation levels. CTLA-4 is believed to downregulate T cell activation by competitively binding to and isolating CD80 from CD86. Furthermore, CTLA-4 has been shown to enhance T cell activation. Reg Cellular immunosuppressive activity.

[0080] The immune checkpoint receptor programmed death 1 (PD-1) is expressed by activated T cells that have been exposed to antigens for extended periods. The binding of PD-1 to its known binding ligands PD-L1 and PD-L2 occurs primarily in the tumor microenvironment, leading to a downregulation of antitumor-specific T cell responses. Both PD-L1 and PD-L2 are known to be expressed on tumor cells. The expression of PD-L1 and PD-L2 on tumors is associated with decreased patient survival.

[0081] Immune checkpoint receptor T cell membrane protein 3 (TIM-3) is expressed on Th1 and Tc1 cells, but not on other T cells. The interaction between TIM-3 and its ligand galactolectin-9 generates Th1 cell death signals. TIM-3 has been reported to play a role in maintaining T cell exhaustion, and blocking TIM-3 can restore the activity of exhausted T cells.

[0082] Immune checkpoint receptors B-cell and T-cell attenuating factor (BTLA) receptors are expressed on both resting and activated B cells and T cells. BTLA is activated upon binding to its ligand HVEM (herpesvirus invasion mediator), leading to downregulation of T cell activation and proliferation. HVEM is expressed in certain tumors (such as melanoma) and tumor-associated endothelial cells.

[0083] Killer cell immunoglobulin-like receptors (KIRs) are a polymorphic family of immune checkpoint receptors expressed on NK cells and some T cells, and act as regulators of immune tolerance associated with natural killer (NK) cells. Blocking specific KIR receptors with inhibitory compounds can enhance NK cell activity, thereby promoting tumor destruction.

[0084] In some embodiments of this disclosure, the immune checkpoint inhibitor compound is a small molecule organic compound (molecular weight less than 1000 Daltons), peptide, polypeptide, protein, antibody, antibody fragment, or antibody derivative. In some embodiments, the immune checkpoint inhibitor compound is an antibody. In some embodiments, the antibody is a monoclonal antibody, specifically a human or humanized monoclonal antibody.

[0085] Monoclonal antibodies, antibody fragments, and antibody derivatives that block immune checkpoint pathways can be prepared using a variety of methods well known to those skilled in the art, including but not limited to somatic cell hybridization techniques and hybridoma methods. For details on hybridoma preparation methods, see *Antibodies, A Laboratory Manual*, Harlow and Lane, 1988, Cold Spring Harbor Publishing, New York, the full text of which is incorporated herein by reference. Human monoclonal antibodies can be identified and isolated by screening phage display libraries of human immunoglobulin genes, as described in U.S. Patents 5,223,409, 5,403,484, 5,571,698, 6,582,915, and 6,593,081, all of which are incorporated herein by reference. Monoclonal antibody preparation can be performed using the general method described in U.S. Patent No. 6,331,415 (Cabilly), the full text of which is incorporated herein by reference.

[0086] For example, XenoMouse can be used. TMHuman monoclonal antibodies were prepared from B-cell hybridomas derived from (Abgenix, Freemont, Calif.) or from XenoMouse. XenoMouse is a mouse host with functional human immunoglobulin genes, described in U.S. Patent No. 6,162,963 (Kucherlapati), the full text of which is incorporated herein by reference.

[0087] The following illustrative documents describe methods for the preparation and use of immune checkpoint antibodies. The preparation and therapeutic use of anti-CTLA-4 antibodies are described in U.S. Patent Nos. 7,229,628 (Allison), 7,311,910 (Linsley), and 8,017,144 (Korman), all of which are incorporated herein by reference in their entirety. The preparation and therapeutic use of anti-PD-1 antibodies are described in U.S. Patent No. 8,008,449 (Korman) and U.S. Patent Application No. 2011 / 0271358 (Freeman), all of which are incorporated herein by reference in their entirety. The preparation and therapeutic use of anti-PD-L1 antibodies are described in U.S. Patent No. 7,943,743 (Korman), all of which are incorporated herein by reference in their entirety. The preparation and therapeutic use of anti-TIM-3 antibodies are described in U.S. Patent Nos. 8,101,176 (Kuchroo) and 8,552,156 (Tagayanagi), the entire contents of which are incorporated herein by reference. The preparation and therapeutic use of anti-LAG-3 antibodies are described in U.S. Patent Application No. 2011 / 0150892 (Thudium) and International Publication No. WO2014 / 008218 (Lonberg), the entire contents of which are incorporated herein by reference. The preparation and therapeutic use of anti-KIR antibodies are described in U.S. Patent No. 8,119,775 (Moretta), the entire contents of which are incorporated herein by reference. The preparation of antibodies that block the BTLA-regulated inhibitory pathway (anti-BTLA antibodies) is described in U.S. Patent No. 8,563,694 (Mataraza), the entire contents of which are incorporated herein by reference.

[0088] In some embodiments, the one or more immune checkpoint inhibitors are PD-1, PD-L1, or CTLA-4 inhibitors. In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor. In some embodiments, the immune checkpoint inhibitor is a PD-L1 binding ligand. In some embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor. In some embodiments, the immune checkpoint inhibitor is a CTLA-4 inhibitor.

[0089] In some embodiments, the one or more immune checkpoint inhibitors described herein include a first immune checkpoint inhibitor and a second immune checkpoint inhibitor, wherein the first immune checkpoint inhibitor and the second immune checkpoint inhibitor are different. In some embodiments, the first and second immune checkpoint inhibitors are each independently a PD-1, PD-L1, or CTLA-4 inhibitor. In some embodiments, the first immune checkpoint inhibitor is a PD-1 inhibitor and the second immune checkpoint inhibitor is a CTLA-4 inhibitor.

[0090] In some embodiments, the immune checkpoint inhibitor is selected from: pembrolizumab (SEQ ID NO:1), nivolumab (SEQ ID NO:2), cimiprimab (SEQ ID NO:3), atezolizumab (SEQ ID NO:4), avelumab (SEQ ID NO:5), pembrolizumab (SEQ ID NO:6), pildizumab (SEQ ID NO:7), ipilimumab (SEQ ID NO:8), BMS 936559 (SEQ ID NO:9), durvalumab (SEQ ID NO:10), camrelizumab (SEQ ID NO:10), dotalimab (SEQ ID NO:11), tislelizumab (SEQ ID NO:12), sintilimab (SEQ ID NO:13), toripalimab (SEQ ID NO:14), or any combination thereof. In some embodiments, the one or more immune checkpoint inhibitors may comprise anti-PD-1 HuMAbs, selected from: 17D8, 2D3, 4H1, 5C4 (also referred to herein as nivolumab), 4A11, 7D3, and 5F4, all of which are described in U.S. Patent No. 8,008,449 (the entire contents of which are incorporated herein by reference). In some embodiments, anti-PD-1 HuMAbs may be selected from: 3G10, 12A4 (also referred to herein as BMS-936559), 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4, all of which are described in U.S. Patent No. 7,943,743 (the entire contents of which are incorporated herein by reference).

[0091] In some embodiments, the one or more immune checkpoint inhibitors may be contained in a pharmaceutically acceptable formulation. In some embodiments, the one or more immune checkpoint inhibitors may be contained in a pharmaceutically acceptable aqueous formulation. Examples of acceptable aqueous formulations include isotonic buffers and physiological saline solutions with a pH adjusted to 4.5-8, such as Ringer's lactate solution.

[0092] In some embodiments, the immune checkpoint inhibitor compound may be included in a pharmaceutically acceptable liposomal formulation, wherein the formulation may be a passive or targeted liposomal formulation. Methods for preparing suitable antibody liposomal formulations are described in U.S. Patent Nos. 5,399,331 (Loughrey), 8,304,565 (Wu), and 7,780,882 (Chang), the entire contents of which are incorporated herein by reference.

[0093] In some embodiments, the one or more immune checkpoint inhibitors may be antibodies. In some embodiments, the antibody is a dry lyophilized solid that needs to be reconstituted with an aqueous rehydration solvent before use. In some embodiments, the antibody is contained in a pharmaceutically acceptable formulation that can be directly injected into the tumor. In some embodiments, the immune checkpoint inhibitor antibody is contained in a pharmaceutically acceptable formulation that can be injected into the peritumoral region surrounding the tumor. The peritumoral region may contain anti-tumor immune cells. In some embodiments, the antibody is contained in a pharmaceutically acceptable formulation that is administered via intravenous injection or infusion. In some embodiments, the immune checkpoint inhibitor antibody is contained in a pharmaceutically acceptable formulation that is administered via subcutaneous or intradermal injection. In some embodiments, the antibody is contained in a pharmaceutically acceptable formulation that is administered via intraperitoneal injection or irrigation.

[0094] The precise dosage of the immune checkpoint inhibitor compound included in the specific methods or treatment combinations disclosed herein may vary depending on factors known in the art, such as the physiological and clinical condition of the subject, the route of administration, the formulation components, the physical and chemical properties of the immune checkpoint inhibitor compound, the intended dosing regimen or sequence, etc. However, those skilled in the art can readily determine the appropriate dosage after appropriately taking these factors into consideration.

[0095] Chemotherapy In some embodiments, the punabulin and one or more immune checkpoint inhibitors may be administered in combination with additional chemotherapeutic agents. In some embodiments, the additional chemotherapeutic agents may be selected from the following group: abiraterone, acetate, abitrexate, abraxane, ABVD, ABVE, ABVE-PC, AC, AC-T, ADE, trastuzumab-metazidine conjugate, adriamycin, afatinib dimaleate, afinitor, everolimus, Akynzeo, netupitant and palonosetron hydrochloride, Aldara, imiquimod, interleukin, alecensa, alectinib, alecenumab, alimta, pemetrexed disodium, and aloxi (pemetrexed disodium). Lonosetron, Ambochlorin, Amboclorin, Amylone, Anastrozole, Aprepitant, Aredia, Arimidex, Aromasin, Arranon, Arsenic trioxide, Arzerra, Erwinia chrysanthemi asparaginase, Avastin, Azacitidine, Beacoppin, Becenum, Beleodaq, Bendamustine hydrochloride, BEP, Bevacizumab, Bexarotin, Bexxar (tosimomab and iodine I) 131Tosimomumab, Bicalutamide, BiCNU (carmustine), Bleomycin, Bonatumab, Blincyto (bonatumab), Bortezomib, Bosulif (bosutinib), Bosutinib, Brentuximab velituximab, Busulfan, Cabazitaxel, Cabozantinib malate, CAF, Camppath (alemumab), Camptosar (irinotecan hydrochloride), Capecitabine, CAPOX, Carac (topical fluorouracil), Carboplatin, CARBOPLATIN-TAXOL, Carfilzomib, Carmubris (carmustine), Carmustine implant, Casodex (bicalutamide), CeeNU (lomustine), Ceritinib, Ce Rubidine (daunorubicin hydrochloride), Cervarix (recombinant bivalent HPV vaccine), Cetuximab, Chlorambucil-Prednisone, CHOP, Cisplatin, Clafen (cyclophosphamide), Clofarex (clopraline), Clolar (clopraline), CMF, Cobitinib, Cometriq (cazozidinib malate), COPDA, COPP formula, COPP-ABV, Cosmegen (actinomycin D), Cotellic (cobitinib), Crizotinib, CVP, Cyclophosphamide, Cyfos (ifosfamide), Cyramza (ramucirumab), A Cytosine, Liposome Cytarabine, Cytosar-U (Cytarabine), Cytoxan (Cyclophosphamide), Darafenib, Dacarbazine, Dacogen (Decitabine), Actinomycin D, Darazalex, Darazalex, Dasatinib, Daunoromycin Hydrochloride, Decitabine, Degarelix, Denisulin, Denorubin, DepoCyt (Liposome Cytarabine), Dexamethasone, Dexrazoxane Hydrochloride, Ditoximab, Docetaxel, Doxil (Liposome Doxorubicin Hydrochloride), Doxorubicin Hydrochloride, Dox-SL (Liposome Doxorubicin Hydrochloride), Efudex (Topical Fluorouracil), Elitek (Raburicase) Ellence (eporarubicin hydrochloride), erlotinumab, Eloxatin (oxaliplatin), eltrombopag ethanolamine, Emend (aprepitant), Empliciti (erlotinumab), enzalutamide, epirubicin hydrochloride, EPOCH, Erbitux (cetuximab), eribulin mesylate, Erivedge (vemodiger), erlotinib hydrochloride, Erwinaze (Erwinia chrysanthum asparaginase), Etopophos (etoposide phosphate), etoposide, etoposide phosphate, Evacet (doxorubicin hydrochloride liposome), everolimus, Evista (raloxifene hydrochloride), exemestane, fluorouracil injection, topical fluorouracil.Fareston (Toremifene), Farydak (Pabistat), Faslodex (Flushexant), FEC, Femara (Letrozole), Figex, Fludara (Fludarabine Phosphate), Fludarabine Phosphate, Fluoroplex (Topical Fluorouracil), Fluorouracil Injection, Topical Fluorouracil, Flutamide, Foles (Methotrexate), Foles PFS (Methotrexate), FOLFIRI (Fluorouracil, Leucovorin, and Irinotecan), FOLFIRI-BEVACIZUMAB, FOLFIRI-CETUXIMAB, FOLFIRINOX, FOLFOX, Folotyn (Platrexate), FU-LV, Fluvestrant, Gardasil (Recombinant Quadrivalent HPV Vaccine). 9 (recombinant HPV 9-valent vaccine), Gazyva (Oxaliplatin), Gefitinib, Gemcitabine Hydrochloride, Gemcitabine-Cisplatin, Gemcitabine-Oxaliplatin, Gemcitabine-Ozomicin, Gemzar (Gemcitabine Hydrochloride), Gilotrif (Afatinib Dimaleate), Gleevec (Imatinib Mesylate), Gliadel (Carmustine Implant), Gliadel Wafer (carmustine implant), glucuronidase, goserelin acetate, Halaven (eribulin mesylate), Herceptin (trastuzumab), recombinant bivalent HPV vaccine, recombinant nine-valent HPV vaccine, recombinant quadrivalent HPV vaccine, Hycamtin (topotecan hydrochloride), Hyper-CVAD, Ibrance (pebocilib), tiimomab, ibrutinib, ICE, Iclusig (ponatinib hydrochloride), Idamycin (idarubicin hydrochloride), edalaris, Ifex (ifosfamide), ifosfamide, IL-2 (aldeleukin), imatinib mesylate, Imbruvica (ibrutinib), imiquimod, Imlygic (talimox), Inlyta (axitinib), recombinant interferon α-2b, interleukin-2 (aldeleukin), Intron A (recombinant interferon α-2b), tosimomab-iodine I. 131Tosimomab combination therapy, Iressa (gefitinib), irinotecan hydrochloride, liposomal irinotecan hydrochloride, Istodax (romidesin), ixaprone, ixazomib citrate, Ixempra (ixaprone), Jakafi (ruxotinib phosphate), Jevtana (cabazitaxel), Kadcyla (trastuzumab-mettansine conjugate), Keoxifene (raloxifene hydrochloride), Kepivance (palivmin), Kyprolis (carbohydrate) Zomi), Lanreitide Acetate, Lapatinib Tosylate, Lenalidomide, Lenvatinib Mesylate, Lenvima (Lenvatinib Mesylate), Letrozole, Calcium Leucovorin, Leukeran (Chloramic Acid), Leuprolide Acetate, Levulan (Aminolevulinic Acid), Linfolizin (Chloramic Acid), LipoDox (Doxorubicin Hydrochloride Liposome), Lonsurf (Trifluuridine-Tippirimidine Combination), Lupron (Leuprolide Acetate). Depot (Leuprolide Acetate), Lupron Depot-Ped (Leuprolide Acetate), Lupron Depot-3 Month (Leuprolide Acetate), Lupron Depot-4 Month (Leuprolide Acetate), Lynparza (Olaparib), Marqibo (Liposome Vincristine Sulfate), Matulane (Procarbazine Hydrochloride), Nitrogen Mustard Hydrochloride, Megace (Megestrol Acetate), Mekinist (Trametinib), Mercaptopurine, Sodium Mercaptosulfonate, Mesnex (Sodium Mercaptosulfonate), Methazolastone (Temozolomide), MethotrexateLPF (Methotrexate), Mexate (Methotrexate), Mexate-AQ (Methotrexate), Mitomycin C, Mitoxantrone Hydrochloride, Mitozytrex (Mitomycin C), MOPP, Mozobil (Prexafo), Mustargen (Nitrogen Mustard Hydrochloride), Mustamycin (Mitomycin C), Myleran (Busulfan), Mylosar (Azacitidine), Mylotarg (Gemtuzumab / Ozomicin), Nanoparticle Paclitaxel (Albumin-bound Paclitaxel Nanoparticles), Navelbine (Vinorelbine Tartrate), Nexituzumab, Nerabin, Neosar (Cyclophosphamide), Netopitant and Palonosetron Hydrochloride, Neupogen (Figrex), Nexavar (Sorafenib Tosylate), Nilotinib, Ninlaro (Ixazomib Citrate), N olvadex (tamoxifen citrate), Nplate (romilastine), obbitulumab, Odomzo (sonidiazole), OEPA, olvadex, OFF, olaparib, homoharringtonine methanesulfonate, Oncaspar (pegaspargase), ondansetron hydrochloride, Onivyde (liposomal irinotecan hydrochloride), Ontak (denis interleukin), OPPA, osimertinib, oxaliplatin, paclitaxel, albumin-bound Paclitaxel nanoparticle formulations, PAD, pebocillin, parivmin, palonosetron hydrochloride, palonosetron hydrochloride and netotipant, pamidronate disodium, panitumumab, pabistat, paraplatin (carboplatin), paraplatin (carboplatin), pazopanib hydrochloride, PVC, pegaspargase, pegylated interferon α-2b, PEG-Intron (pegylated interferon α-2b), Pemetrexed DisodiumPerjeta (pertuzumab), Pertuzumab, Platinol (cisplatin), Platinol-AQ (cisplatin), Plexafo, Pomalidomide, Pomalyst (pomalidomide), Ponatinib Hydrochloride, Portrazza (nexituzumab), Prandtolexa, Prednisone, Procarbazine Hydrochloride, Proleukin (aldeleukin), Prolia (denomab), Promacta (eltrombopag ethanolamine), Provenge (Cyproxetine-T), Purinethol (mercaptopurine), Purixan (Mercaptopurine), R-CHOP, R-CVP, R-EPOCH, recombinant human papillomavirus (HPV) bivalent vaccine, recombinant human papillomavirus (HPV) nine-valent vaccine, recombinant human papillomavirus (HPV) quadrivalent vaccine, recombinant interferon alpha-2b, regorafenib, R-EPOCH, Revlimid (lenalidomide), Rheumatrex (methotrexate), rituximab, rorapipitan hydrochloride, romidixin, romistachytin, Rubidomycin (daunorubicin hydrochloride), ruxotetinib phosphate, sterile talc aerosol (Sclerosol) Intrapleural Aerosol (Talc), Steximab, Ciproxetine-T, Somatuline Depot (Lanrexate Acetate), Sonidergi, Sorafenib Tosylate, Sprycel (Dasatinib), STANFORD V, Sterile Talc Powder (Talc), Sterile Talc (Talc), Stivarga (Regorafenib), Sunitinib Malate, Sutent (Sunitinib Malate), Sylatron (Pegylated Interferon Alpha-2b), Sylvant (Steuximab), Synovir (Thalidomide), Synribo (Homoharringtonine), Tabloid (Thioguanine), TAC, Tafinlar (Dabrafenib), Tagrisso (Osimertinib), Talc, Talimogenes Lahparevivic, Tamoxifen Citrate, Tarabine PFS (cytarabine), Tarceva (erlotinib hydrochloride), Targretin (bexarotin), Tasigna (nilotinib), Taxol (paclitaxel), Taxotere (docetaxel), Temodar (temozolomide), Temozolomide, tesimolimus, thalidomide, thioguanine, thiotepa, Tolak (topical fluorouracil), Toposar (etoposide), topotecan hydrochloride, toremifene, Torisel (tesimolimus), tosimomab and iodine I 131Tosimomumab, Totect (dexrazoxane hydrochloride), TPF, Trabectin, Trametinib, Trastuzumab, Treanda (bendamustine hydrochloride), Trifluuridine and Tipiridine hydrochloride, Trisenox (arsenic trioxide), Tykerb (lapatinib tosylate), Unituxin (ditoximab), Triacetyluridine, VAC, Vandetanib, VAMP, Varubi (lorapidan hydrochloride), Vectibix (panitumumab), VeIP, Velban (vincaline sulfate), Velcade (bortezomib), Velsar (vincaline sulfate), Vemurafenib, VePesid (etoposide), Viadur (leuprolide acetate), Vidaza (azacitidine), Vincaline sulfate, Vincasar PFS (Vincristine Sulfate), Vincristine Sulfate, Liposome Vincristine Sulfate, Vinorelbine Tartrate, VIP, Vimodegiline, Visogard (Uric acid triacetate), Voraxaze (Glucuronidase), Vorinostat, Votrient (Pazopanib Hydrochloride), Wellcovorin (Calcium Leucovorin), Xalkori (Crizotinib), Xeloda (Capecitabine), XELIRI, XELOX, Xgeva (Denoxin), Xofigo (Radium-223 Chloride), Xtandi (Enzalutamide), Yervoy (Ivory) Pilmumab), Yondelis (trabectedin), Zaltrap (aflibercept), Zarxio (filgrastim), Zelboraf (vemurafenib), Zevalin (teimomab), Zinecard (dexrazoxan hydrochloride), aflibercept, Zofran (ondansetron hydrochloride), Zoladex (goserelin acetate), zoledronic acid, Zolinza (vorinostat), Zometa (zoledronic acid), Zydelig (aderalis), Zykadia (ceritinib), and Zytiga (abiraterone acetate).

[0096] In some embodiments, the additional chemotherapy agent is docetaxel.

[0097] The precise dosage of additional chemotherapeutic agents included in the specific methods of this disclosure may vary depending on factors known in the art, such as the physiological and clinical condition of the subject, the route of administration, the formulation composition, the physical and chemical properties of the additional chemotherapeutic agents, the intended dosing regimen or sequence, etc. However, those skilled in the art can readily determine the appropriate dosage after appropriately taking these factors into consideration.

[0098] Radiation therapy In some embodiments, a combination of punabulin and one or more immune checkpoint inhibitors may be administered in conjunction with radiotherapy. In some embodiments, the radiotherapy may be selected from external beam radiotherapy or internal beam radiotherapy. In some embodiments, the external beam radiotherapy may be selected from three-dimensional conformal radiotherapy (3D-CRT), intensity-modulated radiotherapy (IMRT), proton beam therapy, image-guided radiotherapy (IGRT), stereotactic radiotherapy (SRT), or combinations thereof. In some embodiments, the radiotherapy may be selected from intraoperative radiotherapy (IORT), whole-body radiotherapy, radioimmunotherapy, radiosensitizers, radioprotective agents, or combinations thereof.

[0099] Therapeutic uses and methods In some respects, this article provides a method for treating cancer in a subject, the method comprising a treatment cycle including the following steps: (i) administering one or more immune checkpoint inhibitors to the subject; (ii) administering punabulin or a pharmaceutically acceptable salt thereof to the subject; (iii) obtaining a biological sample from the subject; and (iv) determining the level of one or more biomarkers or the level of one or more cells in the biological sample.

[0100] The treatment cycle disclosed in this article begins on day 1 and ends on the last day of the cycle. For example, a three-week treatment cycle begins on day 1 and ends on day 21, while a four-week treatment cycle begins on day 1 and ends on day 28.

[0101] In some embodiments, the biological sample may be a blood sample. In other embodiments, the biological sample may be tumor biopsy tissue. In some embodiments, the biological sample may be obtained from the subject before the start of a treatment cycle (e.g., on day 1 of the treatment cycle or before day 1 of the treatment cycle). In some embodiments, the biological sample may be obtained during the treatment cycle. For example, in some embodiments, the biological sample may be obtained from the subject on days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 and / or 28 of the treatment cycle.

[0102] In some embodiments, the treatment cycle may be 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days, or longer. In some embodiments, the treatment cycle may be 14 days (two weeks or "Q2W"). In some embodiments, the treatment cycle may be 21 days (three weeks or "Q3W"). In some embodiments, the treatment cycle may be 28 days (four weeks or "Q4W"). In some embodiments, the treatment cycle may be repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. In some embodiments, the steps of obtaining biological samples from the subject and measuring the levels of one or more biomarkers or one or more cells in the biological samples may be repeated in each treatment cycle.

[0103] In some embodiments, the treatment cycle includes administering the one or more immune checkpoint inhibitors and punabulin to the subject on day 1 of the treatment cycle and obtaining a biological sample on day 4 of the treatment cycle. In such embodiments, the biological sample may be obtained from the subject prior to administering the one or more immune checkpoint inhibitors and punabulin on day 1 of the treatment cycle. In other such embodiments, the biological sample may be obtained from the subject prior to day 1 of the treatment cycle.

[0104] In some embodiments, the one or more biomarkers are: CCR7, CD40, CD80, CD83, CD86, GEF-H1, IL-2, IFNγ, IL-6, IL-12p70, IL-12p40, IL-13, IL-17A, IL-23, G-CSF, PD-L1, IL-8, and IFN-β, or combinations thereof. In some embodiments, the one or more biomarkers are: CCR7, CD40, CD80, CD83, CD86, or combinations thereof. In some embodiments, the biomarkers are: Mmp12, Csf1, Ccl6, Osm, Ccl9, Cxcl3, Clec7a, Hdc, Slc15a3, Dcstamp, Trem1, Cxcl2, Cd300a, Ncf2, Cd80, Lilr4b, Pdcd1lg2, Lilrb4a, Mxd1, Sp140, Il1a, Ppbp, Gpr171, Ccl7, Il1b, Ccl4, Man1a, Aqp9, Creb5, Traf4, Nod2, Ptger2, Mefv, Cd6, Reps2, Tlr7, Sirpb1b, Fcgr4, P2ry2, Apol8, Lag3, Pdcd1, Cxcr1, Rab27b, Ctla4, Cxcl9, and Fasl, or combinations thereof. In some embodiments, the levels of multiple biomarkers can be measured and combined using mathematical methods to determine a combined score. In some embodiments, the combined score is a GEF-H1 immune activation score. In some embodiments, the GEF-H1 immune activation score is determined using a combination of gene expression levels of one or more genes selected from the group consisting of: Mmp12, Csf1, Ccl6, Osm, Ccl9, Cxcl3, Clec7a, Hdc, Slc15a3, Dcstamp, Trem1, Cxcl2, Cd300a, Ncf2, Cd80, Lilr4b, Pdcd1lg2, Lilrb4a, Mxd 1. Sp140, Il1a, Ppbp, Gpr171, Ccl7, Il1b, Ccl4, Man1a, Aqp9, Creb5, Traf4, Nod2, Ptger2, Mefv, Cd6, Reps2, Tlr7, Sirpb1b, Fcgr4, P2ry2, Apol8, Lag3, Pdcd1, Cxcr1, Rab27b, Ctla4, Cxcl9, and Fasl. In some embodiments, the one or more biomarkers include CCR7. In some embodiments, the one or more biomarkers include CD40. In some embodiments, the one or more biomarkers include CD80. In some embodiments, the one or more biomarkers include CD83.In some embodiments, the one or more biomarkers include CD86.

[0105] In some embodiments, the levels of one or more cells in a biological sample can be determined using techniques known in the art. In some embodiments, the one or more cells may be dendritic cells. In such embodiments, the dendritic cells are myeloid dendritic cells (mDC). In other embodiments, the cells are plasmacytoid dendritic cells (pDC). In still other embodiments, the cells are conventional dendritic cells (cDC). In some embodiments, the levels of one or more dendritic cells expressing one or more biomarkers are determined in a biological sample. In some embodiments, the one or more biomarkers are: CCR7, CD40, CD80, CD83, CD86, GEF-H1, IL-2, IFNγ, IL-6, IL-12p70, IL-12p40, IL-13, IL-17A, IL-23, G-CSF, IL-8, IFN-β, or combinations thereof. In some embodiments, the one or more biomarkers are: CCR7, CD40, CD80, CD83, CD86, or combinations thereof. In some embodiments, the biomarkers are: Mmp12, Csf1, Ccl6, Osm, Ccl9, Cxcl3, Clec7a, Hdc, Slc15a3, Dcstamp, Trem1, Cxcl2, Cd300a, Ncf2, Cd80, Lilr4b, Pdcd1lg2, Lilrb4a, Mxd1, Sp140, Il1a, Ppbp, Gpr171, Ccl7, Il1b, Ccl4, Man1a, Aqp9, Creb5, Traf4, Nod2, Ptger2, Mefv, Cd6, Reps2, Tlr7, Sirpb1b, Fcgr4, P2ry2, Apol8, Lag3, Pdcd1, Cxcr1, Rab27b, Ctla4, Cxcl9, and Fasl, or combinations thereof. In some embodiments, the levels of one or more cells (e.g., dendritic cells) having a GEF-H1 immune activation score above a predetermined threshold are measured. In some embodiments, the one or more biomarkers include CCR7. In some embodiments, the one or more biomarkers include CD40. In some embodiments, the one or more biomarkers include CD80. In some embodiments, the one or more biomarkers include CD83. In some embodiments, the one or more biomarkers include CD86. In some embodiments, when the biological sample is tumor biopsy tissue, the one or more biomarkers include PD-L1.

[0106] The level of a specific biomarker or cell in a biological sample can be assessed based on a specific threshold level, or based on the level of that specific biomarker or cell in a biological sample obtained from the subject before the start of the treatment cycle or at an earlier time point during treatment. In some embodiments, the degree of change of the biomarker or cell relative to a previously measured reference level can be measured.

[0107] The levels of specific biomarkers or cells in a biological sample can indicate the likelihood of a subject responding to treatment (i.e., being a responder or non-responder). Therefore, in some embodiments, treatment is terminated if the level of a specific biomarker or cell falls below a predetermined threshold, or if the change in the level of a specific biomarker or cell relative to a previously measured reference level is less than a predetermined level. In some embodiments, treatment is terminated if the level of a specific biomarker or cell is above a predetermined threshold, or if the change in the level of a specific biomarker or cell relative to a previously measured reference level is greater than a predetermined level. In some embodiments, termination of treatment includes ceasing the administration of immune checkpoint inhibitors or punabulin. In some embodiments, termination of treatment includes ceasing treatment cycles involving the administration of immune checkpoint inhibitors and punabulin. In some embodiments, the biological sample may be tumor biopsy tissue. In other embodiments, the biological sample may be a blood sample. In such embodiments, the blood sample is a peripheral blood sample. In some embodiments, the peripheral blood sample contains peripheral blood mononuclear cells (PBMCs).

[0108] In some embodiments, treatment is terminated if the level of a specific biomarker in the biological sample is below a specific threshold. In some embodiments, treatment is terminated if the level of a specific biomarker in the biological sample is below approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of a specific threshold, or falls within a range of any two of these values. In some embodiments, treatment is terminated if the level of a specific biomarker in the biological sample is above a specific threshold. In some embodiments, treatment is terminated if the level of a specific biomarker in the biological sample is above approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of a specific threshold, or falls within a range of any two of these values.

[0109] In some embodiments, treatment is terminated if the percentage of a specific biomarker level in the biological sample is below a reference level and decreases by approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%, or falls within any two of these values. In some embodiments, treatment is terminated if the percentage of a specific biomarker level in the biological sample is above a reference level and increases by approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%, or falls within any two of these values. In such embodiments, the reference level is the level of biomarkers in biological samples obtained from the subject before the start of the treatment cycle (e.g., day 1 of the treatment cycle before the administration of any therapeutic agent and / or radiation therapy).

[0110] In some embodiments, the decision to discontinue treatment can be made by comparing the reduction in the level of CCR7 expressed by dendritic cells or their subsets in biological samples obtained from the subject after administration of one or more checkpoint inhibitors and / or punabulin and / or radiation therapy, compared to the level of CCR7 expressed by dendritic cells or their subsets in biological samples obtained from the subject before administration of one or more checkpoint inhibitors and / or punabulin and / or radiation therapy. In some patients (responders), the reduction in the level of CCR7 expressed by dendritic cells or their subsets in biological samples collected on day 4 of the treatment cycle is less than in other patients (non-responders). In some embodiments, treatment is terminated if the level of CCR7 expressed in dendritic cells or a subset thereof in a biological sample obtained from the subject on day 4 of the treatment cycle is lower than approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the level of CCR7 expressed in dendritic cells or a subset thereof in a biological sample obtained from the subject before day 1 of the treatment cycle, or within a range of any two of these values. In some embodiments, the cells are plasmacytoid dendritic cells (pDCs). In other embodiments, the cells are conventional dendritic cells (cDCs).

[0111] In some embodiments, the decision to discontinue treatment can be made by comparing the reduction in the level of CD80 expressed by dendritic cells or their subsets in biological samples obtained from a subject after administration of one or more checkpoint inhibitors and / or punabulin and / or radiation therapy, compared to the level of CD80 expressed by dendritic cells or their subsets in biological samples obtained from the subject before administration of one or more checkpoint inhibitors and / or punabulin and / or radiation therapy. In some patients (responders), the reduction in the level of CD80 expressed by dendritic cells or their subsets in biological samples collected on day 4 of the treatment cycle is less than in other patients (non-responders). In some embodiments, treatment is terminated if the level of CD80 expressed by dendritic cells or a subset thereof in a biological sample obtained from the subject on day 4 of the treatment cycle is lower than approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the level of CD80 expressed by dendritic cells or a subset thereof in a biological sample obtained from the subject before administration of one or more checkpoint inhibitors and ponabulin on day 1 of the treatment cycle, or within a range of any two of these values. In some embodiments, the cells are plasmacytoid dendritic cells (pDCs). In other embodiments, the cells are conventional dendritic cells (cDCs).

[0112] In some embodiments, the decision to discontinue treatment can be made by comparing the reduction in the level of CD83 expressed by dendritic cells or their subsets in biological samples obtained from the subject after administration of one or more checkpoint inhibitors and / or punabulin and / or radiation therapy, compared to the level of CD83 expressed by dendritic cells or their subsets in biological samples obtained from the subject before administration of one or more checkpoint inhibitors and / or punabulin and / or radiation therapy. In some patients (responders), the reduction in the level of CD83 expressed by dendritic cells or their subsets in biological samples collected on day 4 of the treatment cycle is less than in other patients (non-responders). In some embodiments, treatment is terminated if the level of CD83 expressed in dendritic cells or a subset thereof in a biological sample obtained from the subject on day 4 of the treatment cycle is lower than approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the level of CD83 expressed in dendritic cells or a subset thereof in a biological sample obtained from the subject before administration of one or more checkpoint inhibitors and punabulin on day 1 of the treatment cycle, or within a range of any two of these values. In some embodiments, the cells are plasmacytoid dendritic cells (pDCs). In other embodiments, the cells are conventional dendritic cells (cDCs).

[0113] In some embodiments, the decision to discontinue treatment can be made by comparing the reduction in the level of CD86 expressed by dendritic cells or their subsets in biological samples obtained from the subject after administration of one or more checkpoint inhibitors and / or punabulin and / or radiation therapy, compared to the level of CD86 expressed by dendritic cells or their subsets in biological samples obtained from the subject before administration of one or more checkpoint inhibitors and / or punabulin and / or radiation therapy. In some patients (responders), the reduction in the level of CD86 expressed by dendritic cells or their subsets in biological samples collected on day 4 of the treatment cycle is less than in other patients (non-responders). In some embodiments, treatment is terminated if the level of CD86 expressed by dendritic cells or a subset thereof in a biological sample obtained from the subject on day 4 of the treatment cycle is lower than approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the level of CD86 expressed by dendritic cells or a subset thereof in a biological sample obtained from the subject before day 1 of the treatment cycle, or within a range of any two of these values. In some embodiments, the cells are plasmacytoid dendritic cells (pDCs). In other embodiments, the cells are conventional dendritic cells (cDCs).

[0114] In some aspects, this document provides a method for treating cancer in a subject, the method comprising: (i) obtaining a biological sample from the subject; (ii) determining the level of one or more biomarkers or one or more cells in the biological sample; and (iii) administering prazolam or a pharmaceutically acceptable salt thereof to the subject if the level of one or more biomarkers or one or more cells in the biological sample is above or below a threshold level. In some embodiments, the method may further comprise administering radiotherapy, an immune checkpoint inhibitor, a chemotherapeutic agent, or a combination thereof to the subject. In such embodiments, the method may include administering radiotherapy and an immune checkpoint inhibitor to the subject.

[0115] In some embodiments, the subject may be resistant to immuno-oncology therapy. In other embodiments, the subject may not have received immuno-oncology therapy.

[0116] In some embodiments, the biological sample is tumor biopsy tissue. In some embodiments, the biological sample may be a blood sample. In some embodiments, the biological sample is peripheral blood. In some embodiments, the one or more cells are dendritic cells. In some specific embodiments, the dendritic cells are: DC3 cells, mDC cells, pDC cells, cDC cells (cDC1 and cDC2 cells). In other embodiments, the cells are monocyte-derived macrophages (MoMac). In such embodiments, the cells may be MoMac-III cells. In some embodiments, the one or more biomarkers are guanylate exchange factor H1 (GEF-H1) immune activation scores.

[0117] The GEF-H1 immune pathway comprises 47 genes that drive different cellular signaling programs in dendritic cells (DCs). These programs are dominated by the c-Jun N-terminal kinase (JNK) pathway and the AP-1 / ATF transcriptional response to regulate innate and adaptive immune responses. In some embodiments, the GEF-H1 immune activation score can be determined based on the expression levels of one or more of these 47 genes. In one embodiment, obtaining the GEF-H1 immune activation score first involves normalizing gene expression counts using log2 fragments per thousand bases per million mapped reads (FPKM), and then summing the expression levels of these genes to establish a standardized GEF-H1 immune activation score. For a discussion of GEF-H1, see Kashyap A. et al., *Cell Reports*, September 24, 2019, Vol. 28, No. 13, pp. 3367-3380, the entire contents of which are incorporated herein by reference. The genes included in the GEF-H1 immune pathway are shown in the table below. In some embodiments, the GEF-H1 immune activation score can be calculated using all 47 genes in the GEF-H1 immune pathway. In other embodiments, the GEF-H1 immune activation score can be calculated using the gene expression levels of a subset of genes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or 46 in the GEF-H1 immune pathway.

[0118] In some embodiments, GEF-H1 immune activation scores for multiple cell types can be measured, including but not limited to: T cells (including CD4+ TEM, CD4+ TCM, CD8+ TEM, CD8+ TCM, gdT, naive CD8, dnT, MAIT, naive CD4, proliferating CD4, and Treg), NK cells (including NK cells, proliferating NK, and NK_CD56bright), B cells (including naive, intermediate, memory, and plasmablasts), monocytes (including CD16+ and CD14+ monocytes), and dendritic cells (including cDC1, cDC2, DC3, mregDC, and pDC). In some embodiments, GEF-H1 immune activation scores for CD16+ and CD14+ monocytes, NK cells, CD8+ TEM, CD4+ TCM, CD4+ naive T cells, naive B cells, cDC1 cells, and cDC2 cells can be measured. In some embodiments, the cell samples may be derived from tumor biopsy tissue or peripheral blood samples. In some embodiments, the GEF-H1 immune activation score for a specific cell type may be higher in responding subjects than in non-responding subjects. Therefore, in some embodiments, treatment is administered to subjects with a GEF-H1 immune activation score above a predetermined threshold. For each of the aforementioned cell types, the threshold level for the GEF-H1 immune activation score may be approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, or higher, or within the range defined by any two of the foregoing scores. For example, the threshold levels for the GEF-H1 score can be approximately 1 to approximately 50, approximately 10 to approximately 20, approximately 10 to approximately 30, approximately 20 to approximately 40, or approximately 5 to approximately 30.

[0119] In some embodiments, if the cells are dendritic cells, the threshold level of the GEF-H1 immune activation score can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, or higher, or within the range defined by any two of the foregoing scores. For example, the threshold level of the GEF-H1 immune activation score can be about 1 to about 50, about 10 to about 20, about 10 to about 30, about 20 to about 40, or about 5 to about 30. In some embodiments, if the cells are monocyte-derived macrophages, the threshold level of GEF-H1 can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. For example, the threshold level of the GEF-H1 immune activation score can be about 1 to about 50, about 10 to about 20, about 10 to about 30, about 20 to about 40, or about 5 to about 30.

[0120] In some embodiments, a GEF-H1 score below a threshold level indicates that the patient may not respond to treatment. Therefore, if the GEF-H1 immune activation score measured from tumor biopsy tissue or peripheral blood samples is below a threshold level, treatment may not be initiated.

[0121] As used herein, the terms "co-administer," "co-administering," or "co-administration" refer to two or more formulations or therapies that simultaneously have biological efficacy on a subject, regardless of when or how they are actually administered. In one embodiment, the formulations or therapies are administered simultaneously. In one such embodiment, co-administration can be achieved by combining the formulations in a single dosage form. In another embodiment, the formulations or therapies are administered sequentially. In some embodiments, administration may be spaced at intervals, such as 30 minutes, 1 hour, 2 hours, 1 day, 2 days, 3 days, or 1 week. In one embodiment, the formulations are administered via the same route, such as orally. In another embodiment, the formulations are administered via different routes, such as one orally and another intravenously.

[0122] In some embodiments, a method of treating a subject's cancer may include the combined administration of a therapeutically effective amount of ponabulin or a pharmaceutically acceptable salt thereof and one or more immune checkpoint inhibitors. In some embodiments, ponabulin is administered after administration of one or more immune checkpoint inhibitors. In some embodiments, when the one or more immune checkpoint inhibitors are administered on the same day as ponabulin, ponabulin is administered approximately 0.5 hours to approximately 3 hours after the administration of the one or more immune checkpoint inhibitors. For example, when the one or more immune checkpoint inhibitors are administered on the same day as ponabulin, ponabulin is administered 0.5 hours, 1 hour, 1.5 hours, 2 hours, 2.5 hours, or 3 hours after the administration of the one or more immune checkpoint inhibitors, or within any two of these times.

[0123] In some embodiments, the methods of treating cancer as described herein may further include the combined administration of additional chemotherapy agents.

[0124] In some embodiments, the method of treating cancer as described herein may include combined administration of radiation therapy. In such embodiments, radiation therapy may be administered on days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, and / or 28 of a treatment cycle. For example, in some embodiments, radiation therapy may be administered on days 1, 2, and 3 of a treatment cycle. In other embodiments, radiation therapy may be administered on days 1, 2, 3, and 4 of a treatment cycle. In still other embodiments, radiation therapy may be administered on days 1, 2, 3, 4, and 5 of a treatment cycle. In yet another embodiment, radiation therapy may be administered on days 2, 3, and 4 of a treatment cycle.

[0125] The total radiation dose administered to the subject during the treatment period may be about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 Gy, or higher, or within a range defined by any two of the foregoing values. For example, the total radiation dose administered to the subject may be about 1 Gy to about 20 Gy, about 2 Gy to about 15 Gy, or about 4 Gy to about 15 Gy. In some embodiments, the total radiation dose administered to the subject is about 4 Gy. In other embodiments, the total radiation dose administered to the subject is about 8 Gy. The total radiation dose administered to the subject is approximately 12.5 Gy. In some embodiments, the radiation therapy may be administered in 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 fractions, or more. In some embodiments, the radiation therapy may be administered in 3 to 5 fractions. In some embodiments, the radiation therapy may be administered in 3 fractions. In some embodiments, the radiation therapy may be administered in 4 fractions. In some embodiments, the radiation therapy may be administered in 5 fractions.

[0126] In some embodiments, when radiotherapy and pranabulin are administered on the same day, pranabulin is administered approximately 3 to approximately 12 hours after the completion of radiotherapy. For example, in such embodiments, pranabulin may be administered approximately 4 to approximately 10 hours, approximately 4 to approximately 8 hours, or approximately 5 to approximately 8 hours after the completion of radiotherapy.

[0127] In some embodiments, the one or more immune checkpoint inhibitors are administered on day 1 of the treatment cycle, and pravastatin is administered on day 1 of the treatment cycle. In other embodiments, the one or more immune checkpoint inhibitors are administered on day 1 of the treatment cycle, and pravastatin is administered on days 1 and 4 of the treatment cycle. In some embodiments, the one or more immune checkpoint inhibitors are administered on day 1 of the treatment cycle, pravastatin is administered on days 1 and 4 of the treatment cycle, and radiotherapy is administered on days 1, 2, and 3 of the treatment cycle. In some embodiments, the one or more immune checkpoint inhibitors are administered on day 1 of the treatment cycle, pravastatin is administered on days 1 and 4 of the treatment cycle, and radiotherapy is administered on days 1, 2, 3, and 4 of the treatment cycle. In some embodiments, the one or more immune checkpoint inhibitors are administered on day 1 of the treatment cycle, pravastatin is administered on days 1 and 4 of the treatment cycle, and radiotherapy is administered on days 1, 2, 3, 4, and 5 of the treatment cycle. In some embodiments, the one or more immune checkpoint inhibitors are administered on days 1 and 15 of the treatment cycle, ponabulin is administered on days 1 and 4 of the treatment cycle, and radiotherapy is administered on days 1, 2, and 3 of the treatment cycle. In some embodiments, the one or more immune checkpoint inhibitors are administered on days 1 and 15 of the treatment cycle, ponabulin is administered on days 1 and 4 of the treatment cycle, and radiotherapy is administered on days 1, 2, 3, 4, and 5 of the treatment cycle.

[0128] In some embodiments, radiation therapy may be administered in each treatment cycle. In other embodiments, radiation therapy may be administered only in certain treatment cycles. For example, in some embodiments, radiation therapy may be administered only in the first treatment cycle. In other embodiments, radiation therapy may be administered in the first and second treatment cycles. In other embodiments, radiation therapy may be administered in the first, second, and third treatment cycles.

[0129] In some embodiments, pranabulin is administered on days 1 and 4 of the first treatment cycle, and on day 1 of each subsequent (i.e., the second, third, fourth, and fifth) treatment cycle. In some embodiments, pranabulin is administered on days 1 and 4 of the first and second treatment cycles, and on day 1 of each subsequent treatment cycle.

[0130] In some embodiments, the one or more checkpoint inhibitors are administered on day 1 of the first treatment cycle and on day 1 of each subsequent (i.e., the second, third, fourth, and fifth) treatment cycle. In some embodiments, the one or more checkpoint inhibitors are administered on days 1 and 15 of the first treatment cycle and on day 1 of each subsequent treatment cycle. In some embodiments, the one or more checkpoint inhibitors are administered on days 1 and 15 of the first treatment cycle and on days 1 and 15 of each subsequent treatment cycle.

[0131] In some embodiments, this disclosure provides a method for treating breast cancer, bladder cancer, glioma, glioblastoma, head and neck cancer, non-small cell lung cancer, small cell lung cancer, recurrent small cell lung cancer (SCLC), colorectal cancer, gastrointestinal stromal tumor, gastric and esophageal cancer, renal cell carcinoma, prostate cancer, liver cancer, colon cancer, pancreatic cancer, ovarian cancer, lymphoma, cutaneous T-cell lymphoma, or melanoma.

[0132] In some embodiments, this disclosure provides a method for treating fibrolamellar hepatocellular carcinoma, any histologically classified MSI-H cancer (including but not limited to: colorectal cancer, endometrial cancer, adrenocortical carcinoma, anal cancer, appendix cancer, biliary tract cancer, bladder cancer, brain cancer, breast cancer, cervical cancer, gastric cancer or gastroesophageal junction cancer, head and neck squamous cell carcinoma, liver cancer, mesothelioma, nasopharyngeal carcinoma, neuroendocrine tumors, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, retroperitoneal cancer, salivary gland cancer, sarcoma, small cell lung cancer, small intestinal cancer, testicular cancer, thyroid cancer, vaginal cancer, and vulvar cancer). In some embodiments, this disclosure provides a method for treating tumor types that have been approved as checkpoint inhibitors.

[0133] In some embodiments, the subject may be an animal, such as a mammal or a human. In some embodiments, the subject is a human.

[0134] In some embodiments, punabrine or a pharmaceutically acceptable salt thereof is contained in a pharmaceutically acceptable solution. In some embodiments, punabrine or a pharmaceutically acceptable salt thereof is contained in an injectable formulation. In some embodiments, punabrine or a pharmaceutically acceptable salt thereof is contained in an injectable formulation that allows punabrine or a pharmaceutically acceptable salt thereof to remain substantially stable at or near the injection site.

[0135] The precise dosage of punabulin or its pharmaceutically acceptable salts included in specific methods or treatment combinations disclosed herein can vary depending on factors known in the art, such as the physiological and clinical condition of the subject, the route of administration, the formulation composition, and the intended dosing regimen or sequence. Therefore, it is impractical to specify a universal dose of punabulin or its pharmaceutically acceptable salts that is therapeutically effective for all possible applications. However, those skilled in the art can readily determine an appropriate dose after properly considering these factors.

[0136] Dosage The pharmaceutical compositions described herein can be administered via any acceptable route of administration for similar purposes, including but not limited to: oral, sublingual, oral, subcutaneous, intravenous, intranasal, intratumoral, topical, transdermal, intradermal, intraperitoneal, intramuscular, intrapulmonary, vaginal, rectal, or intraocular administration. Oral and parenteral administration are routine methods of treatment for the indications of the subjects in the preferred embodiments.

[0137] The compositions described herein may be provided in a single dosage form. As used herein, a “single dosage form” means a composition comprising a certain amount of compound or composition suitable for administration in a single dose to animals (especially mammalian subjects) according to good medical practice. However, the preparation of a single dosage form or a single dosage form does not imply that the dosage form is administered once daily or once per treatment course. Such dosage forms may include once, twice, three or more times daily, or infusion over a period of time (e.g., about 30 minutes to about 2-6 hours), or continuous infusion, and may be administered multiple times within a treatment course, but single administration is not excluded. Those skilled in the art will understand that this formulation does not specifically define the entire treatment course, and such decisions are left to professionals in the therapeutic field.

[0138] The compositions described above can be in any suitable form suitable for various routes of administration (e.g., oral, sublingual, oral, nasal, rectal, topical (including transdermal and intradermal), ocular, intracranial, intrathecal, intrathecal, intra-arterial, intravenous, intramuscular, or other parenteral routes). Those skilled in the art will understand that oral and nasal compositions include those administered by inhalation and can be prepared using available methods, employing a variety of pharmaceutically acceptable carriers well known in the art, depending on the specific route of administration desired. Pharmaceutically acceptable carriers include, for example, solid or liquid fillers, diluents, solubilizers, surfactants, and encapsulating materials. Optional pharmaceutically active materials may be included that do not materially interfere with the inhibitory activity of the compound or composition. The amount of carrier used in conjunction with the compound or composition should be sufficient to provide the actual amount of material required to administer each unit dose of the compound. The techniques and compositions used to prepare the dosage forms described herein are documented in the following references, all of which are incorporated herein by reference: Modern Pharmaceutics, 4th Edition, Chapters 9 and 10 (Banker and Rhodes, eds., 2002); Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1989); and Ansel, Introduction to Pharmaceutical Dosage Forms, 8th Edition (2004).

[0139] Various oral dosage forms are available, including solid forms such as tablets, capsules (e.g., solid gel capsules and liquid gel capsules), granules, and powders. Tablets may comprise compressed tablets, molded tablets, enteric-coated tablets, sugar-coated tablets, film-coated tablets, or multilayer compressed tablets, and may contain suitable binders, lubricants, diluents, disintegrants, colorants, flavoring agents, gliding agents, and solubilizers. Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and / or suspensions reconstituted from non-effervescent particles, and effervescent formulations reconstituted from effervescent particles, and contain suitable solvents, preservatives, emulsifiers, suspending agents, diluents, sweeteners, solubilizers, colorants, and flavoring agents.

[0140] Pharmaceutically acceptable carriers suitable for preparing oral dosage forms are well known in the art. Tablets typically contain conventional pharmaceutically compatible excipients as inert diluents, such as calcium carbonate, sodium carbonate, mannitol, lactose, and cellulose; binders such as starch, gelatin, and sucrose; disintegrants such as starch, alginate, and croscarmellose sodium; and lubricants such as magnesium stearate, stearic acid, and talc. Flow aids (such as silica) may be used to improve the flowability of powder mixtures. Colorants (such as FD&C dyes) may be added to improve appearance. Sweeteners and flavoring agents (such as aspartame, saccharin, menthol, peppermint, and fruit flavorings) are useful excipients for chewable tablets. Capsules typically contain one or more of the solid diluents disclosed above. The selection of carrier components depends on secondary considerations (such as taste, cost, and storage stability), which are not critical and can be readily selected by those skilled in the art.

[0141] Oral compositions also include liquid solutions, emulsions, suspensions, etc. Pharmaceutically acceptable carriers suitable for preparing such compositions are well known in the art. Typical carrier components for syrups, elixirs, emulsions, and suspensions include ethanol, glycerin, propylene glycol, polyethylene glycol, liquid sucrose, sorbitol, and water. For suspensions, typical suspending agents include methylcellulose, sodium carboxymethylcellulose, AVICEL RC591, tragacanth gum, and sodium alginate; typical wetting agents include lecithin and polysorbate 80; typical preservatives include methylparaben and sodium benzoate. Oral liquid compositions may also contain one or more components, such as the sweeteners, flavoring agents, and coloring agents disclosed above.

[0142] Such compositions can also be coated using conventional methods, typically employing pH-dependent or time-dependent coating materials, to allow the target composition to be released in the gastrointestinal tract near the intended site of action, or released at different times to prolong the intended duration of action. These dosage forms typically include, but are not limited to, one or more of the following: cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate, ethyl cellulose, Eudragit coating materials, waxes, and shellac.

[0143] The compositions described herein may optionally contain additional pharmaceutically active ingredients.

[0144] Other compositions for achieving systemic delivery of the target compound include sublingual, oral, and nasal dosage forms. Such compositions typically contain one or more soluble fillers (such as sucrose, sorbitol, and mannitol) and binders (such as gum arabic, microcrystalline cellulose, carboxymethyl cellulose, and hydroxypropyl methylcellulose). They may also include the flow aids, lubricants, sweeteners, colorants, antioxidants, and flavoring agents disclosed above.

[0145] Liquid compositions for topical ophthalmic use should be formulated to be suitable for topical application to the eye. Usage comfort should be maximized as much as possible, although sometimes optimal comfort may need to be appropriately reduced due to formulation considerations (such as drug stability). If maximizing comfort cannot be achieved, the liquid should be formulated to a level tolerable for topical ophthalmic use by the patient. Furthermore, ophthalmally acceptable liquids may be packaged for single use or contain preservatives to prevent contamination during repeated use.

[0146] For ophthalmic applications, physiological saline is often used as the primary solvent to prepare solutions or medications. Preferably, the ophthalmic solution is maintained at a comfortable pH using a suitable buffer system. The formulation may also contain conventional, pharmaceutically acceptable preservatives, stabilizers, and surfactants.

[0147] Preservatives that may be used in the pharmaceutical compositions disclosed herein include, but are not limited to, benzalkonium chloride, PHMB, chlorobutanol, thimerosal, phenylmercuric acid, acetate, and phenylmercuric nitrate. Surfactants that may be used include, for example, Tween 80. Similarly, a variety of suitable solvents that may be used in the ophthalmic formulations disclosed herein include, but are not limited to, polyvinyl alcohol, povidone, hydroxypropyl methylcellulose, poloxamer, carboxymethyl cellulose, hydroxyethyl cellulose, and purified water.

[0148] Osmoregulators may be added as needed or for convenience, including but not limited to: salts (especially sodium chloride and potassium chloride), mannitol and glycerin, or any other suitable ophthalmic acceptable osmoregulators.

[0149] A variety of buffers and pH adjustment methods can be used, provided the final formulation is ocularly acceptable. For many compositions, the pH is between 4 and 9. Accordingly, buffers include acetate buffer, citrate buffer, phosphate buffer, and borate buffer. The pH of these formulations can be adjusted using acids or bases as needed.

[0150] Ophthalmic acceptable antioxidants include, but are not limited to: sodium metabisulfite, sodium thiosulfate, acetylcysteine, butylated hydroxyanisole, and butylated hydroxytoluene.

[0151] Other excipients that can be included in ophthalmic formulations are chelating agents. One useful chelating agent is disodium ethylenediaminetetraacetate, but other chelating agents can be used in place of or in combination with it.

[0152] For topical application, creams, ointments, gels, solutions, or suspensions containing the compositions disclosed herein may be used. Topical formulations typically consist of a pharmaceutical carrier, cosolvent, emulsifier, penetration enhancer, preservative system, and emollient.

[0153] For intravenous administration, the compositions described herein may be dissolved or dispersed in a pharmaceutically acceptable diluent, such as physiological saline or glucose solution. Suitable excipients may be included to achieve the desired pH, including but not limited to: sodium hydroxide, sodium carbonate, sodium acetate, hydrochloric acid, and citric acid. In various different embodiments, the pH range of the final composition is 2 to 8, or preferably 4 to 7. Antioxidant excipients may include: sodium bisulfite, sodium acetone bisulfite, sodium formaldehyde sulfite, sodium bisulfite, thiourea, and EDTA. In some embodiments, excipients for intravenous delivery may include Kolliphor HS 15 (polyoxyhydroxy 15 stearate or dissolving alcohol HS-15), propylene glycol, and a 5% aqueous glucose solution (D5W). Other non-limiting examples of excipients suitable for the final intravenous formulation may include: sodium or potassium phosphate, citric acid, tartaric acid, gelatin, and carbohydrates such as glucose, mannitol, and dextran. More acceptable excipients are described in Powell et al., *Compendium of Excipients for Parenteral Formulations*, PDA Pharmaceutical Science & Technology Journal, 1998, Vol. 52, pp. 238-311, and Nema et al., *Excipients and Their Role in Approved Injectable Products: Current Usage and Future Directions*, PDA Pharmaceutical Science & Technology Journal, 2011, Vol. 65, pp. 287-332, both of which are incorporated herein by reference in their entirety. Antimicrobial agents may also be included to obtain antibacterial or antifungal solutions, including but not limited to: phenylmercuric nitrate, thimerosal, benzyl chloride, benzalkonium chloride, phenol, cresol, and chlorobutanol.

[0154] Compositions for intravenous administration may be provided to caregivers in one or more solid forms, which are reconstituted shortly before administration with a suitable diluent, such as sterile water, saline, or a glucose solution. In other embodiments, the composition is provided as a ready-to-use solution. In still other embodiments, the composition is provided as a solution that requires further dilution before administration. In embodiments involving the administration of a combination of the compound described herein with another pharmaceutical agent, the combination may be provided to caregivers as a mixture, or the two agents may be mixed by caregivers before administration, or the two agents may be administered separately.

[0155] The actual dosage of the active compounds described herein depends on the specific compound and the condition being treated; the selection of an appropriate dosage is entirely within the knowledge of those skilled in the art. In some embodiments, the dosage range for punabulin is approximately 1 mg / m². 2 Approximately 50 mg / m 2 In some embodiments, the dosage range of punabrine is about 1-50 mg / m². 2Body surface area. In some embodiments, the dosage range of punabulin is approximately 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-13.75, 1-14, 1-15, 1-16, 1-17, 1-18, 1-19, 1-20, 1-22.5, 1-25, 1-27.5, 1-30, 1.5-2, 1.5-3, 1.5-4, 1.5-5, 1.5-6, 1.5-7, 1.5-8, 1.5-9, 1.5-10, 1.5-11, 1.5-12, 1.5-13, 1.5-13.75, 1.5-14, 1.5-15, 1. 5-16, 1.5-17, 1.5-18, 1.5-19, 1.5-20, 1.5-22.5, 1.5-25, 1.5-27.5, 1.5-30, 2.5-2, 2.5-3, 2.5-4, 2.5-5, 2.5-6, 2.5-7, 2.5-8, 2.5-9, 2.5-10, 2.5-11, 2.5-12, 2.5-13, 2.5-13.75, 2.5-14, 2.5-15, 2.5-16, 2.5-17, 2.5-18, 2.5-19, 2.5-20, 2.5-22.5, 2.5-25, 2.5-27.5, 2.5-30, 2.5-7.5, 3- 4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-11, 3-12, 3-13, 3-13.75, 3-14, 3-15, 3-16, 3-17, 3-18, 3-19, 3-20, 3-22.5, 3-25, 3-27.5, 3-30, 3.5-6.5, 3.5-13.75, 3.5-15, 2.5-17.5, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 4-11, 4-12, 4-13, 4-13.75, 4-14, 4-15, 4-16, 4-17, 4-18, 4-19, 4-20, 4-22.5, 4-25, 4-27 .5, 4-30, 5-6, 5-7, 5-8, 5-9, 5-10, 5-11, 5-12, 5-13, 5-13.75, 5-14, 5-15, 5-16, 5-17, 5-18, 5-19, 5-20, 5-22.5, 5-25, 5-27.5, 5-30, 6-7, 6-8, 6-9, 6-10, 6-11, 6-12, 6-13, 6-13.75, 6-14, 6-15, 6-16, 6-17, 6-18, 6-19, 6-20, 6-22.5, 6-25, 6-27.5, 6-30, 7-8, 7-9, 7-10, 7-11, 7-12, 7-13, 7-13.75, 7-14, 7-15, 7-16, 7-17, 7-18, 7-19, 7-20, 7-22.5, 7-25, 7-27.5, 7-30, 7.5-12.5, 7.5-13.5, 7.5-15, 8-9, 8-10, 8-11, 8-12, 8-13, 8-13.75, 8-14, 8-15, 8-16, 8-17, 8-18, 8-19, 8-20, 8-22.5, 8-25, 8-27.5, 8-30, 9-10, 9-11, 9-12, 9-13, 9-13.75, 9-14, 9-15, 9-16, 9-17, 9-18, 9-19, 9-20, 9-22.5, 9 -25, 9-27.5, 9-30, 10-11, 10-12, 10-13, 10-13.75, 10-14, 10-15, 10-16, 10-17, 10-18, 10-19, 10-20, 10-22.5, 10-25, 10-27.5, 10-30, 11.5-15.5, 1 2.5-14.5, 7.5-22.5, 8.5-32.5, 9.5-15.5, 15.5-24.5, 5-35, 17.5-22.5, 22.5-32.5, 25-35, 25.5-34.5, 27.5-32.5, 2-20, 2.5-22.5, or 9.5-21.5 mg / m². 2 Body surface area. In some embodiments, the dosage of punabulin is approximately 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17. 5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40mg / m 2Body surface area. In some embodiments, the dose of punabulin is less than about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17. .5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40mg / m 2 Body surface area. In some embodiments, the dose of punabulin is greater than about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 1 9.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 2 9, 29.5, 30, 30.5, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50mg / m 2 Body surface area.

[0156] In some embodiments, the dosage of punabulin is about 5 mg-300 mg, 5 mg-200 mg, 7.5 mg-200 mg, 10 mg-100 mg, 15 mg-100 mg, 20 mg-100 mg, 30 mg-100 mg, 40 mg-100 mg, 10 mg-80 mg, 15 mg-80 mg, 20 mg-80 mg, 30 mg-80 mg, 40 mg-80 mg, 10 mg-60 mg, 15 mg-60 mg, 20 mg-60 mg, 30 mg-60 mg, or about 40 mg-60 mg. In some embodiments, the dosage of punabulin is about 20 mg-60 mg, 27 mg-60 mg, 20 mg-45 mg, or 27 mg-45 mg. In some embodiments, the dosage of punabulin is approximately 5 mg-7.5 mg, 5 mg-9 mg, 5 mg-10 mg, 5 mg-12 mg, 5 mg-14 mg, 5 mg-15 mg, 5 mg-16 mg, 5 mg-18 mg, 5 mg-20 mg, 5 mg-22 mg, 5 mg-24 mg, 5 mg-26 mg, 5 mg-28 mg, 5 mg-30 mg, 5 mg-32 mg, 5 mg-34 mg, 5 mg-36 mg, 5 mg-38 mg, 5 mg-30 mg, 5 mg-32 mg, 5 mg-34 mg, 5 mg-36 mg, 5 mg-38 mg, 5 mg-30 mg, 5 mg-32 mg, 5 mg-34 mg, 5 mg-36 mg, 5 mg-38 mg, 5 mg-30 mg, 5 mg-32 mg, 5 mg-34 mg, 5 mg-36 mg, 5 mg-38 mg, 5 mg-35 ... mg-40mg, 5mg-42mg, 5mg-44mg, 5mg-46mg, 5mg-48mg, 5mg-50mg, 5mg-52mg, 5mg-54mg, 5mg-56mg, 5mg-58mg, 5m g-60mg, 7mg-7.7mg, 7mg-9mg, 7mg-10mg, 7mg-12mg, 7mg-14mg, 7mg-15mg, 7mg-16mg, 7mg-18mg, 7mg-20mg, 7mg- 22mg, 7mg-24mg, 7mg-26mg, 7mg-28mg, 7mg-30mg, 7mg-32mg, 7mg-34mg, 7mg-36mg, 7mg-38mg, 7mg-40mg, 7mg-4 2mg, 7mg-44mg, 7mg-46mg, 7mg-48mg, 7mg-50mg, 7mg-52mg, 7mg-54mg, 7mg-56mg, 7mg-58mg, 7mg-60mg, 9mg-10 mg, 9mg-12mg, 9mg-14mg, 9mg-15mg, 9mg-16mg, 9mg-18mg, 9mg-20mg, 9mg-22mg, 9mg-24mg, 9mg-26mg, 9mg-28m g, 9mg-30mg, 9mg-32mg, 9mg-34mg, 9mg-36mg, 9mg-38mg, 9mg-40mg, 9mg-42mg, 9mg-44mg, 9mg-46mg, 9mg-48mg,9mg-50mg、9mg-52mg、9mg-54mg、9mg-56mg、9mg-58mg、9mg-60mg、10mg-12mg、10mg-14mg、10mg-15mg、10mg-16mg、10mg-18mg、10mg-20mg、10mg-22mg、10mg-24mg、10mg-26mg、10mg-28mg、10mg-30mg、10mg-32mg、10mg-34mg、10mg-36mg、10mg-38mg、10mg-40mg、10mg-42mg、10mg-44mg、10mg-46mg、10mg-48mg、10mg-50mg、10mg-52mg、10mg-54mg、10mg-56mg、10mg-58mg、10mg-60mg、12mg-14mg、12mg-15mg、12mg-16mg、12mg-18mg、12mg-20mg、12mg-22mg、12mg-24mg、12mg-26mg、12mg-28mg、12mg-30mg、12mg-32mg、12mg-34mg、12mg-36mg、12mg-38mg、12mg-40mg、12mg-42mg、12mg-44mg、12mg-46mg、12mg-48mg、12mg-50mg、12mg-52mg、12mg-54mg、12mg-56mg、12mg-58mg、12mg-60mg、15mg-16mg、15mg-18mg、15mg-20mg、15mg-22mg、15mg-24mg、15mg-26mg、15mg-28mg、15mg-30mg、15mg-32mg、15mg-34mg、15mg-36mg、15mg-38mg、15mg-40mg、15mg-42mg、15mg-44mg、15mg-46mg、15mg-48mg、15mg-50mg、15mg-52mg、15mg-54mg、15mg-56mg、15mg-58mg、15mg-60mg、17mg-18mg、17mg-20mg、17mg-22mg、17mg-24mg、17mg-26mg、17mg-28mg、17mg-30mg、17mg-32mg、17mg-34mg、17mg-36mg、17mg-38mg、17mg-40mg、17mg-42mg、17mg-44mg、17mg-46mg、17mg-48mg、17mg-50mg、17mg-52mg、17mg-54mg、17mg-56mg、17mg-58mg、17mg-60mg、20mg-22mg、20mg-24mg、20mg-26mg、20mg-28mg、20mg-30mg、20mg-32mg、20mg-34mg、20mg-36mg、20mg-38mg、20mg-40mg、20mg-42mg、20mg-44mg、20mg-46mg、20mg-48mg、20mg-50mg、20mg-52mg、20mg-54mg、20mg-56mg、20mg-58mg、20mg-60mg、22mg-24mg、22mg-26mg、22mg-28mg、22mg-30mg、22mg-32mg、22mg-34mg、22mg-36mg、22mg-38mg、22mg-40mg、22mg-42mg、22mg-44mg、22mg-46mg、22mg-48mg、22mg-50mg、22mg-52mg、22mg-54mg、22mg-56mg、22mg-58mg、22mg-60mg、25mg-26mg、25mg-28mg、25mg-30mg、25mg-32mg、25mg-34mg、25mg-36mg、25mg-38mg、25mg-40mg、25mg-42mg、25mg-44mg、25mg-46mg、25mg-48mg、25mg-50mg、25mg-52mg、25mg-54mg、25mg-56mg、25mg-58mg、25mg-60mg、27mg-28mg、27mg-30mg、27mg-32mg、27mg-34mg、27mg-36mg、27mg-38mg、27mg-40mg、27mg-42mg、27mg-44mg、27mg-46mg、27mg-48mg、27mg-50mg、27mg-52mg、27mg-54mg、27mg-56mg、27mg-58mg、27mg-60mg、30mg-32mg、30mg-34mg、30mg-36mg、30mg-38mg、30mg-40mg、30mg-42mg、30mg-44mg、30mg-46mg、30mg-48mg、30mg-50mg、30mg-52mg、30mg-54mg、30mg-56mg、30mg-58mg、30mg-60mg、33mg-34mg、33mg-36mg、33mg-38mg、33mg-40mg、33mg-42mg、33mg-44mg、33mg-46mg、33mg-48mg、33mg-50mg、33mg-52mg, 33mg-54mg, 33mg-56mg, 33mg-58mg, 33mg-60mg, 36mg-38mg, 36mg-40mg, 36mg-42mg, 36mg-44mg, 36mg-46mg, 36mg-48mg, 36mg-50mg, 36mg-52mg, 36mg-54mg, 36 mg-56mg, 36mg-58mg, 36mg-60mg, 40mg-42mg, 40mg-44mg, 40mg-46mg, 40mg-48mg, 40mg-50mg, 40mg-52mg, 40mg-54mg, 40mg-56mg, 40mg-58mg, 40mg-60mg, 43mg-46mg, 43mg- 48mg, 43mg-50mg, 43mg-52mg, 43mg-54mg, 43mg-56mg, 43mg-58mg, 42mg-60mg, 45mg-48mg, 45mg-50mg, 45mg-52mg, 45mg-54mg, 45mg-56mg, 45mg-58mg, 45mg-60mg, 48mg-50m g, 48mg-52mg, 48mg-54mg, 48mg-56mg, 48mg-58mg, 48mg-60mg, 50mg-52mg, 50mg-54mg, 50mg-56mg, 50mg-58mg, 50mg-60mg, 52mg-54mg, 52mg-56mg, 52mg-58mg, or 52mg-60mg. In some embodiments, the dose of punabulin is greater than about 5 mg, about 10 mg, about 12.5 mg, about 13.5 mg, about 15 mg, about 17.5 mg, about 20 mg, about 22.5 mg, about 25 mg, about 27 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 125 mg, about 150 mg, or about 200 mg. In some embodiments, the dose of punabulin is less than about 5 mg, about 10 mg, about 12.5 mg, about 13.5 mg, about 15 mg, about 17.5 mg, about 20 mg, about 22.5 mg, about 25 mg, about 27 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 125 mg, about 150 mg, or about 200 mg.

[0157] In some embodiments, the dose of one or more immune checkpoint inhibitors may be from about 100 µg to about 5000 mg, from about 500 µg or less to about 800 mg, from about 1.0 mg to about 600 mg, from about 100 mg to about 600 mg, or from about 200 mg to 500 mg. In some embodiments, the dose of one or more immune checkpoint inhibitors may be from about 240 mg to about 480 mg per dose. In some embodiments, the dose of the one or more immune checkpoint inhibitors is about 240 mg. In some embodiments, the dose of the one or more immune checkpoint inhibitors is about 480 mg.

[0158] In some embodiments, the dosage range of one or more immune checkpoint inhibitors is from about 100 mg / kg to about 5000 mg / kg. In some embodiments, the dosage range of one or more immune checkpoint inhibitors is from about 100 to 1000 mg / kg. In some embodiments, the dosage range of one or more immune checkpoint inhibitors is approximately 100-200, 100-300, 100-400, 100-500, 100-600, 100-700, 100-800, 100-900, 100-1000, 100-1100, 100-1200, 100-1300, 100-1375, 100-1400, 100-1500, 100-1600, 100-1700, 100-1800, 100-1900, 100-2000, 100-2250, 100- 2500, 100-2750, 100-3000, 150-200, 150-300, 150-400, 150-500, 150-600, 150-700, 150-800, 150-900, 150-1000, 150-1100, 150-1200, 150-1300, 150-1375, 150-1400, 150-1500, 150-1600, 150-1700, 150-1800, 150-1900, 150-2000, 150-2250, 150-2500, 150 -2750, 150-3000, 250-2000, 250-3000, 250-4000, 250-5000, 250-600, 250-700, 250-800, 250-900, 250-1000, 250-1100, 250-1200, 250-1300, 250-1375, 250-1400, 250-1500, 250-1600, 250-1700, 250-1800, 250-1900, 250-2000, 250-2250, 250-2500, 250-275 0, 250-3000, 250-750, 300-400, 300-500, 300-600, 300-700, 300-800, 300-900, 300-1000, 300-1100, 300-1200, 300-1300, 300-1375, 300-1400, 300-1500, 300-1600, 300-1700, 300-1800, 300-1900, 300-2000, 300-2250, 300-2500, 300-2750, or 300-300 mg / kg.In some embodiments, the dosage of one or more immune checkpoint inhibitors is approximately 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 1 9.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100mg.

[0159] In some embodiments, the dosage of one or more immune checkpoint inhibitors is about 0.5 mg-3000 mg, 0.5 mg-2500 mg, 0.5 mg-2000 mg, 0.5 mg-1500 mg, 0.5 mg-1000 mg, 0.5 mg-500 mg, 0.5 mg-200 mg, 0.75 mg-200 mg, 1.0 mg-100 mg, 1.5 mg-100 mg, 2.0 mg-100 mg, 3.0 mg-100 mg, 4.0 mg-100 mg, 1.0 mg-80 mg, 1.5 mg-80 mg, 2.0 mg-80 mg, 3.0 mg-80 mg, 4.0 mg-80 mg, 1.0 mg-60 mg, 1.5 mg-60 mg, 2.0 mg-60 mg, 3.0 mg-60 mg, or about 4.0 mg-60 mg. In some embodiments, one or more immune checkpoint inhibitors are administered at about 20 mg-60 mg, 27 mg-60 mg, 20 mg-45 mg, or 27 mg-45 mg. In some embodiments, one or more immune checkpoint inhibitors are administered at doses of approximately 5 mg-7.5 mg, 5 mg-9 mg, 5 mg-10 mg, 5 mg-12 mg, 5 mg-14 mg, 5 mg-15 mg, 5 mg-16 mg, 5 mg-18 mg, 5 mg-20 mg, 5 mg-22 mg, 5 mg-24 mg, 5 mg-26 mg, 5 mg-28 mg, 5 mg-30 mg, 5 mg-32 mg, 5 mg-34 mg, 5 mg-36 mg, 5 mg-38 mg, 5 mg-40 mg, 5 mg-42 mg, 5 mg-44 mg, 5 mg-46 mg, 5 mg-48 mg, 5 mg-50 mg, 5 mg-52 mg, 5 mg-54 mg, 5 mg-56 mg, 5 mg-58 mg, 5 mg-60 mg, 7 mg-7.7 mg, 7 mg-9 mg, 7 mg-10 mg, 7 mg-12 mg, etc. mg, 7mg-14mg, 7mg-15mg, 7mg-16mg, 7mg-18mg, 7mg-20mg, 7mg-22mg, 7mg-24mg, 7mg-26mg, 7mg- 28mg, 7mg-30mg, 7mg-32mg, 7mg-34mg, 7mg-36mg, 7mg-38mg, 7mg-40mg, 7mg-42mg, 7mg-44mg, 7mg -46mg, 7mg-48mg, 7mg-50mg, 7mg-52mg, 7mg-54mg, 7mg-56mg, 7mg-58mg, 7mg-60mg, 9mg-10mg, 9 mg-12mg, 9mg-14mg, 9mg-15mg, 9mg-16mg, 9mg-18mg, 9mg-20mg, 9mg-22mg, 9mg-24mg, 9mg-26mg,9mg-28mg、9mg-30mg、9mg-32mg、9mg-34mg、9mg-36mg、9mg-38mg、9mg-40mg、9mg-42mg、9mg-44mg、9mg-46mg、9mg-48mg、9mg-50mg、9mg-52mg、9mg-54mg、9mg-56mg、9mg-58mg、9mg-60mg、10mg-12mg、10mg-14mg、10mg-15mg、10mg-16mg、10mg-18mg、10mg-20mg、10mg-22mg、10mg-24mg、10mg-26mg、10mg-28mg、10mg-30mg、10mg-32mg、10mg-34mg、10mg-36mg、10mg-38mg、10mg-40mg、10mg-42mg、10mg-44mg、10mg-46mg、10mg-48mg、10mg-50mg、10mg-52mg、10mg-54mg、10mg-56mg、10mg-58mg、10mg-60mg、12mg-14mg、12mg-15mg、12mg-16mg、12mg-18mg、12mg-20mg、12mg-22mg、12mg-24mg、12mg-26mg、12mg-28mg、12mg-30mg、12mg-32mg、12mg-34mg、12mg-36mg、12mg-38mg、12mg-40mg、12mg-42mg、12mg-44mg、12mg-46mg、12mg-48mg、12mg-50mg、12mg-52mg、12mg-54mg、12mg-56mg、12mg-58mg、12mg-60mg、15mg-16mg、15mg-18mg、15mg-20mg、15mg-22mg、15mg-24mg、15mg-26mg、15mg-28mg、15mg-30mg、15mg-32mg、15mg-34mg、15mg-36mg、15mg-38mg、15mg-40mg、15mg-42mg、15mg-44mg、15mg-46mg、15mg-48mg、15mg-50mg、15mg-52mg、15mg-54mg、15mg-56mg、15mg-58mg、15mg-60mg、17mg-18mg、17mg-20mg、17mg-22mg、17mg-24mg、17mg-26mg、17mg-28mg、17mg-30mg、17mg-32mg、17mg-34mg、17mg-36mg、17mg-38mg、17mg-40mg、17mg-42mg、17mg-44mg、17mg-46mg、17mg-48mg、17mg-50mg、17mg-52mg、17mg-54mg、17mg-56mg、17mg-58mg、17mg-60mg、20mg-22mg、20mg-24mg、20mg-26mg、20mg-28mg、20mg-30mg、20mg-32mg、20mg-34mg、20mg-36mg、20mg-38mg、20mg-40mg、20mg-42mg、20mg-44mg、20mg-46mg、20mg-48mg、20mg-50mg、20mg-52mg、20mg-54mg、20mg-56mg、20mg-58mg、20mg-60mg、22mg-24mg、22mg-26mg、22mg-28mg、22mg-30mg、22mg-32mg、22mg-34mg、22mg-36mg、22mg-38mg、22mg-40mg、22mg-42mg、22mg-44mg、22mg-46mg、22mg-48mg、22mg-50mg、22mg-52mg、22mg-54mg、22mg-56mg、22mg-58mg、22mg-60mg、25mg-26mg、25mg-28mg、25mg-30mg、25mg-32mg、25mg-34mg、25mg-36mg、25mg-38mg、25mg-40mg、25mg-42mg、25mg-44mg、25mg-46mg、25mg-48mg、25mg-50mg、25mg-52mg、25mg-54mg、25mg-56mg、25mg-58mg、25mg-60mg、27mg-28mg、27mg-30mg、27mg-32mg、27mg-34mg、27mg-36mg、27mg-38mg、27mg-40mg、27mg-42mg、27mg-44mg、27mg-46mg、27mg-48mg、27mg-50mg、27mg-52mg、27mg-54mg、27mg-56mg、27mg-58mg、27mg-60mg、30mg-32mg、30mg-34mg、30mg-36mg、30mg-38mg、30mg-40mg、30mg-42mg、30mg-44mg、30mg-46mg、30mg-48mg、30mg-50mg、30mg-52mg、30mg-54mg、30mg-56mg、30mg-58mg、30mg-60mg, 33mg-34mg, 33mg-36mg, 33mg-38mg, 33mg-40mg, 33mg-42mg, 33mg-44mg, 33mg-46mg, 33mg-48mg, 33mg-5 0mg, 33mg-52mg, 33mg-54mg, 33mg-56mg, 33mg-58mg, 33mg-60mg, 36mg-38mg, 36mg-40mg, 36mg-42mg, 36mg-44mg, 36m g-46mg, 36mg-48mg, 36mg-50mg, 36mg-52mg, 36mg-54mg, 36mg-56mg, 36mg-58mg, 36mg-60mg, 40mg-42mg, 40mg-44mg , 40mg-46mg, 40mg-48mg, 40mg-50mg, 40mg-52mg, 40mg-54mg, 40mg-56mg, 40mg-58mg, 40mg-60mg, 43mg-46mg, 43mg-4 8mg, 43mg-50mg, 43mg-52mg, 43mg-54mg, 43mg-56mg, 43mg-58mg, 42mg-60mg, 45mg-48mg, 45mg-50mg, 45mg-52mg, 45 mg-54mg, 45mg-56mg, 45mg-58mg, 45mg-60mg, 48mg-50mg, 48mg-52mg, 48mg-54mg, 48mg-56mg, 48mg-58mg, 48mg-60mg 50mg-52mg, 50mg-54mg, 50mg-56mg, 50mg-58mg, 50mg-60mg, 52mg-54mg, 52mg-56mg, 52mg-58mg, 52mg-60mg, 100mg-200mg, 200mg-300mg, 300mg-400mg, 400mg-500mg, 50mg-2000mg, 500mg-1000mg, 1000mg-2000mg, or 1000mg-3000mg. In some embodiments, the dose of one or more immune checkpoint inhibitors is greater than about 1 mg, 5 mg, about 10 mg, about 12.5 mg, about 13.5 mg, about 15 mg, about 17.5 mg, about 20 mg, about 22.5 mg, about 25 mg, about 27 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 125 mg, about 150 mg, or about 200 mg. In some embodiments, the dose of one or more immune checkpoint inhibitors is less than about 5 mg, about 10 mg, about 12.5 mg, about 13.5 mg, about 15 mg, or about 200 mg.Approximately 17.5 mg, approximately 20 mg, approximately 22.5 mg, approximately 25 mg, approximately 27 mg, approximately 30 mg, approximately 40 mg, approximately 50 mg, approximately 60 mg, approximately 70 mg, approximately 80 mg, approximately 90 mg, approximately 100 mg, approximately 125 mg, approximately 150 mg, or approximately 200 mg, approximately 300 mg, approximately 400 mg, approximately 500 mg, approximately 1000 mg, approximately 2000 mg, or approximately 3000 mg.

[0160] In some embodiments, the one or more immune checkpoint inhibitors are administered intravenously. In such embodiments, the one or more immune checkpoint inhibitors are administered over a time period of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, or 240 minutes, or longer, or within any two of the aforementioned defined ranges. In some embodiments, the one or more immune checkpoint inhibitors are administered over a time period of about 10 minutes to about 180 minutes, about 10 minutes to about 120 minutes, about 20 minutes to about 100 minutes, about 30 minutes to about 90 minutes, or about 30 minutes to about 60 minutes. In some embodiments, the one or more immune checkpoint inhibitors are administered over a time period of about 30 minutes. In other embodiments, the one or more immune checkpoint inhibitors are administered over a time period of about 60 minutes.

[0161] In some embodiments, the one or more immune checkpoint inhibitors are administered 1, 2, 3, 4, or 5 times per treatment cycle. In some embodiments, the one or more immune checkpoint inhibitors are administered once per cycle. In other embodiments, the one or more immune checkpoint inhibitors are administered twice per cycle. In some embodiments, if the treatment cycle is three weeks (Q3W), the one or more immune checkpoint inhibitors are administered once per cycle. In such embodiments, the one or more immune checkpoint inhibitors are administered on day 1 of the cycle. In some embodiments, if the treatment cycle is four weeks (Q4W), the one or more immune checkpoint inhibitors are administered twice per cycle. In such embodiments, the one or more immune checkpoint inhibitors are administered on days 1 and 15 of the cycle. In some embodiments, the one or more immune checkpoint inhibitors are: pembrolizumab, nivolumab, cimiprimab, atezolizumab, avelumab, pildilizumab, ipilimumab, BMS 936559, durvalumab, camrelizumab, dotalimab, tislelizumab, sintilimab, toripalimab, or combinations thereof. In some embodiments, the one or more immune checkpoint inhibitors are avelumab. In other embodiments, the one or more immune checkpoint inhibitors are atezolizumab. In still other embodiments, the one or more immune checkpoint inhibitors are durvalumab. In yet another embodiment, the one or more immune checkpoint inhibitors are nivolumab. In some embodiments, the one or more immune checkpoint inhibitors are pembrolizumab.

[0162] In some embodiments, punabulin is administered before the administration of one or more immune checkpoint inhibitors. In some embodiments, punabulin and one or more immune checkpoint inhibitors are administered simultaneously. In some embodiments, punabulin is administered after the administration of one or more immune checkpoint inhibitors.

[0163] In some embodiments, punabulin is administered at approximately 1 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 24 h, 30 h, 36 h, 40 h, or 48 h, or within any two of these values, after administration of one or more immune checkpoint inhibitors. In some embodiments, punabulin is administered approximately 1 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 24 h, 30 h, 36 h, 40 h, or 48 h, or within any two of these values, before administration of one or more immune checkpoint inhibitors. In some embodiments, punabulin is administered less than about 1 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, 30 h, 36 h, 40 h, or 48 h, or within any two of these values, after administration of one or more immune checkpoint inhibitors. In some embodiments, punabulin is administered more than about 1 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, 30 h, 36 h, 40 h, or 48 h, or within any two of these values, after administration of one or more immune checkpoint inhibitors. In some embodiments, punabulin is administered less than about 1 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, 30 h, 36 h, 40 h, or 48 h, or between any two of these values, before administration of one or more immune checkpoint inhibitors.In some embodiments, punabulin is administered more than about 1 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, 30 h, 36 h, 40 h, or 48 h, or within any two of these values, before administration of one or more immune checkpoint inhibitors. In some embodiments, punabulin is administered approximately 1 min–5 min, 1 min–10 min, 1 min–15 min, 1 min–20 min, 1 min–25 min, 1 min–30 min, 0.25 h–0.5 h, 0.25 h–0.75 h, 0.25 h–1 h, 0.5 h–1 h, 0.5 h–2 h, 0.5 h–2.5 h, 1 h–2 h, 1 h–3 h, 1 h–5 h, 1 h–24 h, 1 min–24 h, or 1 min–2 h, 1 day–2 days, 1 day–3 days, 1 day–4 days, 1 day–5 days, or 1 day–6 days after administration of one or more immune checkpoint inhibitors. In some embodiments, punabulin is administered approximately 1 min–5 min, 1 min–10 min, 1 min–15 min, 1 min–20 min, 1 min–25 min, 1 min–30 min, 0.25 h–0.5 h, 0.25 h–0.75 h, 0.25 h–1 h, 0.5 h–1 h, 0.5 h–2 h, 0.5 h–2.5 h, 1 h–2 h, 1 h–3 h, 1 h–5 h, 1 h–24 h, 1 min–24 h, or 1 min–2 h, 1 day–2 days, 1 day–3 days, 1 day–4 days, 1 day–5 days, or 1 day–6 days before administration of one or more immune checkpoint inhibitors.

[0164] In some embodiments, the treatment regimen includes the combined administration of one or more immune checkpoint inhibitors and punabulin. In some embodiments, the treatment regimen includes the combined administration of one or more immune checkpoint inhibitors and punabulin once every 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In some embodiments, the treatment regimen includes the combined administration of one or more immune checkpoint inhibitors and punabulin twice every 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In some embodiments, the treatment regimen includes the combined administration of one or more immune checkpoint inhibitors and punabulin once weekly during a treatment cycle of 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In some embodiments, the treatment regimen includes the combined administration of one or more immune checkpoint inhibitors and punabulin twice weekly during a treatment cycle of 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In some embodiments, the treatment regimen includes the combined administration of one or more immune checkpoint inhibitors and punabulin on days 1, 8, and 15 of a 21-day treatment cycle. In some embodiments, the treatment regimen includes the combined administration of one or more immune checkpoint inhibitors and punabulin on day 1 of a 21-day treatment cycle. In some embodiments, the treatment regimen includes the combined administration of one or more immune checkpoint inhibitors and punabulin on days 1 and 4 of a 28-day treatment cycle. In some embodiments, the combined administration of one or more immune checkpoint inhibitors and punabulin includes administering one or more immune checkpoint inhibitors before administering punabulin. In some embodiments, the combined administration of one or more immune checkpoint inhibitors and punabulin includes administering one or more immune checkpoint inhibitors after administering punabulin. In some embodiments, the combined administration of one or more immune checkpoint inhibitors and punabulin includes administering one or more immune checkpoint inhibitors and punabulin simultaneously. In some embodiments, the one or more immune checkpoint inhibitors mentioned in this paragraph may independently be the first, second, third, fourth, fifth, sixth, seventh, or eighth immune checkpoint inhibitor. In some embodiments, the treatment regimen includes the combined administration of one or more immune checkpoint inhibitors and punabulin every day of the week for one week. In some embodiments, the treatment regimen includes the combined administration of one or more immune checkpoint inhibitors and punabulin every day of the week for two, three, or four weeks. In some embodiments, the treatment regimen includes the combined administration of one or more immune checkpoint inhibitors and punabulin on day 1 of weekly treatment. In some embodiments, the treatment regimen includes the combined administration of one or more immune checkpoint inhibitors and punabulin on days 1 and 2 of weekly treatment. In some embodiments, the treatment regimen includes the combined administration of one or more immune checkpoint inhibitors and punabulin on days 1, 2, and 3 of weekly treatment.In some embodiments, the treatment regimen includes the combined administration of one or more immune checkpoint inhibitors and punabulin on days 1, 2, and 3 of each week of treatment. In some embodiments, the treatment regimen includes the combined administration of one or more immune checkpoint inhibitors and punabulin on days 1, 2, 3, and 4 of each week of treatment. In some embodiments, the treatment regimen includes the combined administration of one or more immune checkpoint inhibitors and punabulin on days 1, 2, 3, 4, 5, and 6 of each week of treatment. In some embodiments, the treatment regimen includes the combined administration of one or more immune checkpoint inhibitor compositions and punabulin on days 1, 3, and 5 of each week of treatment. In some embodiments, the treatment cycle for punabulin and the one or more immune checkpoint inhibitors may be the same. In some embodiments, the treatment cycle for punabulin and the one or more immune checkpoint inhibitors may be different. For example, in some embodiments, the treatment cycle for ponabulin is 21 days, while the treatment cycle for one or more immune checkpoint inhibitors is 14 days. In some embodiments, the immune checkpoint inhibitor used on each dosing day may be the same or different. In some embodiments, the immune checkpoint inhibitor used on the first dosing day may be different from the immune checkpoint inhibitor used on subsequent dosing days. In some embodiments, the immune checkpoint inhibitor used on the first dosing day may be the same or different from the immune checkpoint inhibitor used on the second dosing day. In some embodiments, the immune checkpoint inhibitor used on the first dosing day may be the same or different from the immune checkpoint inhibitor used on the third dosing day. In some embodiments, the immune checkpoint inhibitor used on the first dosing day may be the same or different from the immune checkpoint inhibitor used on the fourth dosing day. In some embodiments, the immune checkpoint inhibitor used on the first dosing day may be the same or different from the immune checkpoint inhibitor used on the fifth dosing day. In some embodiments, the immune checkpoint inhibitor used on the first dosing day may be the same or different from the immune checkpoint inhibitor used on the sixth dosing day. In some embodiments, the immune checkpoint inhibitor used on the first dosing day may be the same or different from the immune checkpoint inhibitor used on the seventh dosing day.

[0165] In some embodiments, the treatment regimen includes administering one or more immune checkpoint inhibitors (e.g., the first, second, third, fourth, fifth, sixth, seventh, or eighth) once every 3 weeks. In some embodiments, the treatment regimen includes administering one or more immune checkpoint inhibitors once every 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In some embodiments, the treatment regimen includes administering one or more immune checkpoint inhibitors twice every 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In some embodiments, the treatment regimen includes administering one or more immune checkpoint inhibitors once weekly during a treatment cycle of 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In some embodiments, the treatment regimen includes administering one or more immune checkpoint inhibitors twice weekly during a treatment cycle of 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In some embodiments, the treatment regimen includes administering one or more immune checkpoint inhibitors three times a week (e.g., days 1, 2, and 3, or days 1, 3, and 5) during a treatment cycle of 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In some embodiments, the treatment regimen includes administering one or more immune checkpoint inhibitors on days 1, 8, and 15 during a 21-day treatment cycle. The one or more immune checkpoint inhibitors mentioned in this paragraph may independently be the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, or 8th or more immune checkpoint inhibitors. In some embodiments, the treatment regimen includes administering one or more immune checkpoint inhibitors every day of the week for one week. In some embodiments, the treatment regimen includes administering one or more immune checkpoint inhibitors every day of the week for 2, 3, or 4 weeks. In some embodiments, the treatment regimen includes administering one or more immune checkpoint inhibitors on day 1 of each week of treatment. In some embodiments, the treatment regimen includes administering one or more immune checkpoint inhibitors on days 1 and 2 of each week of treatment. In some embodiments, the treatment regimen includes administering one or more immune checkpoint inhibitors on days 1, 2, and 3 of weekly treatment. In some embodiments, the treatment regimen includes administering one or more immune checkpoint inhibitor compositions on days 1, 3, and 5 of weekly treatment. In some embodiments, the treatment regimen includes administering one or more immune checkpoint inhibitors on days 1, 2, 3, and 4 of weekly treatment. In some embodiments, the treatment regimen includes administering one or more immune checkpoint inhibitors on days 1, 2, 3, 4, and 5 of weekly treatment. In some embodiments, the treatment regimen includes administering one or more immune checkpoint inhibitors on days 1, 2, 3, 4, 5, and 6 of weekly treatment.

[0166] In some embodiments, the treatment regimen includes administering pranabulin once every 3 weeks. In some embodiments, the treatment regimen includes administering pranabulin once every 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In some embodiments, the treatment regimen includes administering pranabulin twice every 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In some embodiments, the treatment regimen includes administering pranabulin once weekly during a treatment cycle of 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In some embodiments, the treatment regimen includes administering pranabulin twice weekly during a treatment cycle of 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In some embodiments, the treatment regimen includes administering pranabulin three times weekly (e.g., days 1, 2, and 3, or days 1, 3, and 5) during a treatment cycle of 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In some embodiments, the treatment regimen includes administering pranabulin on day 1 of a 21-day treatment cycle. In some embodiments, the treatment regimen includes administering pranabulin on days 1 and 8 of a 21-day treatment cycle. In some embodiments, the treatment regimen includes administering pranabulin on days 1, 8, and 15 of a 21-day treatment cycle. In some embodiments, the treatment regimen includes administering pranabulin every day of the week for one week. In some embodiments, the treatment regimen includes administering pranabulin every day of the week for two, three, or four weeks. In some embodiments, the treatment regimen includes administering pranabulin on day 1 of each week of treatment. In some embodiments, the treatment regimen includes administering pranabulin on days 1 and 2 of each week of treatment. In some embodiments, the treatment regimen includes administering pranabulin on days 1, 2, and 3 of each week of treatment. In some embodiments, the treatment regimen includes administering pranabulin on days 1, 3, and 5 of each week of treatment. In some embodiments, the treatment regimen includes administering pranabulin on days 1, 2, 3, and 4 of each week of treatment. In some embodiments, the treatment regimen includes administering punabrine on days 1, 2, 3, 4, and 5 of each week of treatment. In some embodiments, the treatment regimen includes administering punabrine on days 1, 2, 3, 4, 5, and 6 of each week of treatment.

[0167] Treatment cycles can be repeated provided the treatment is clinically tolerable. In some embodiments, the treatment cycle of one or more immune checkpoint inhibitors and ponabulin is repeated n times, where n is an integer ranging from 2 to 30. In some embodiments, n is 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, a new treatment cycle may begin immediately after the end of the previous treatment cycle. In some embodiments, a new treatment cycle may begin some time after the end of the previous treatment cycle. In some embodiments, a new treatment cycle may begin 1, 2, 3, 4, 5, 6, or 7 weeks after the end of the previous treatment cycle.

[0168] The compositions disclosed herein can be administered via any acceptable route of administration for pharmaceutical purposes, including but not limited to: oral, subcutaneous, oral, intravenous, intranasal, intratumoral, topical, transdermal, intraperitoneal, intramuscular, intrapulmonary, vaginal, rectal, gastric, or intraocular administration. Oral and parenteral administration are routine methods for treating the indications of the subjects in the preferred embodiments. In some embodiments, the compositions described herein may be used in combination with other therapeutic agents. In some embodiments, the compositions described herein may be administered or used in combination with treatments such as chemotherapy, radiotherapy, and biotherapy.

[0169] To further illustrate the present invention, the following embodiments are included. Of course, these embodiments should not be construed as constituting specific limitations on the present invention. Variations of these embodiments within the scope of the claims are all within the understanding of those skilled in the art and should be considered to fall within the scope of the invention described herein and claimed. Readers should recognize that those skilled in the art, possessing the knowledge of this disclosure, can prepare and use the present invention without detailed embodiments.

[0170] Example Example 1: In vitro experiment of ponabulin combined with radiotherapy Radiation therapy can release local antigens, which, when combined with mature dendritic cells (DCs), can enhance systemic immunity against immune checkpoint inhibitors (ICIs), even in environments of ICI resistance. Here, we validated this hypothesis preclinically and clinically in a phase I basket study.

[0171] In in vitro experiments, purnabulin and radiotherapy were combined at different time points before and after radiotherapy. Flow cytometry was used to assess the expression of CD80, CD86, and major histocompatibility complex class II (MHC-II) molecules to detect DC activation levels. Animals implanted with the TS / A mouse breast cancer cell line (TSA) were irradiated with 8 Gy × 3 fractions, with or without purnabulin and with or without anti-PD-1 monoclonal antibody.

[0172] SP37A3 and XS106 DC lines were irradiated with 10 Gy, and ponabulin was added to these cells. Radiotherapy combined with ponabulin promoted DC maturation, especially when radiotherapy was performed 3-6 hours before the addition of ponabulin, while adding ponabulin before radiotherapy had no such effect. Figure 1 The results of treating the XS106 cell line with punabulin 3 hours before irradiation or 1, 3, and 6 hours after irradiation were presented. The results showed that punabulin treatment 3 hours after irradiation resulted in the highest DC activation. BALB / c mouse TSA-derived breast cancer cells were irradiated with 24 Gy / 3fr, combined with αPD1 and punabulin treatment. The αPD1 monotherapy group, the punabulin monotherapy group, and the αPD1 + punabulin group showed only weak antitumor effects. However, triple therapy showed a stronger tumor regression effect than irradiation and the αPD1 group. In the triple therapy group, the percentage of CD8+ T cells and CD86+ DCs in the tumor was significantly increased, a phenomenon not observed in the monotherapy or dual therapy groups (p < 0.05, Dunnett test).

[0173] Punabulin combined with radiotherapy and immune checkpoint inhibitors can induce a systemic immune response in immunotherapy-resistant tumors.

[0174] Example 2: This is an open-label, single-center study designed to evaluate the safety and tolerability of ponabulin in combination with radiotherapy / immunotherapy in patients with one of multiple metastatic or locally advanced cancers who have experienced disease progression following standard-of-care anti-PD-1 / PD-L1 mAb therapy, and to assess the objective response rate of the study regimen.

[0175] The study groups were divided according to tumor type specificity. This study comprised eight groups: (1) bladder cancer; (2) melanoma; (3) Merkel cell carcinoma; (4) MSI-H cancer (any histological type); (5) non-small cell lung cancer; (6) renal cell carcinoma; (7) small cell lung cancer; and (8) any tumor type for which an immune checkpoint inhibitor has been approved. The treatment cycle for subjects in the study was based on the treatment cycle of anti-PD-1 / PD-L1 mAb.

[0176] All subjects received triple therapy (RT) plus prazolam and anti-PD-1 / PD-L1 mAb in Cycle 1, followed by a combination of anti-PD-1 / PD-L1 mAb and prazolam in Cycle 2 and subsequent cycles until disease progression, unacceptable toxicity, withdrawal from study treatment, or termination of the study. In Cycle 1, short-term local consolidation RT was administered starting on day 1. In Cycle 2 of any regimen, RT of any optional order was administered to other untreated lesions at the discretion of the treating physician. In any anti-PD-1 / PD-L1 regimen, prazolam was administered on days 1 and 4 of Cycle 1; if optional RT was administered in Cycle 2, prazolam was also administered on day 4 of Cycle 2. In Cycle 3 and subsequent cycles, prazolam was administered on day 1. Anti-PD-1 / PD-L1 mAb is administered on day 1 of each treatment cycle (or day 15 for a four-week (Q4W) regimen containing avelumab, durvalumab, or nivolumab as the anti-PD-1 / PD-L1 mAb). Subjects must receive an alloanti-PD-1 / PD-L1 mAb that has been unresponsive to prior treatment.

[0177] Radiation therapy: Radiation therapy is performed using external beam radiation therapy, including 2D / conventional techniques, three-dimensional conformal radiotherapy, intensity-modulated radiotherapy (IMRT), stereotactic radiosurgery (SRS), or proton beam therapy (PBT), as determined by the treating radiation oncologist. Radiation therapy (RT) is administered according to one of the following three regimens: 8 Gy × 3 fractions, 12.5 Gy × 4 fractions, and / or 4 Gy × 5 fractions on days 1-3, 1-4, or 1-5 of cycle 1. The choice of RT regimen targeting the tumor and lesions is determined by the treating radiation oncologist. RT can target up to 5 tumor lesions, and the radiation therapy regimens can be administered concurrently or sequentially. If it is necessary to target other untreated lesions with the same regimens (cycle 2 Q4W; cycle 2 Q3W), the treating radiation oncologist may choose any sequential RT. The treatment area includes any lesions in lymph nodes and organs (including the brain and bones). At least one measurable lesion was left untreated for disease assessment during the study. Treatment response was independent of RT-treated tumor and bone lesions. Intracranial metastases were used for irRECIST response assessment.

[0178] If a patient experiences a radiation-induced toxicity after receiving at least one dose of radiation therapy, and the treating radiation oncologist determines it is in the best interest of the patient, subsequent radiation therapy may be discontinued and the adverse event (AE) recorded. Since the number of treatments is between 3 and 5, the patient receives a minimum of 1 and a maximum of 5 treatments. The initially prescribed single dose may change, but the total dose administered may be reduced. Patients are required to undergo a visit from their treating radiation oncologist at the end of each radiation therapy cycle.

[0179] Punabulin administration: Punabulin was administered intravenously on days 1 and 4 of cycle 1, and if optional RT was performed in cycle 2, then punchabulin was also administered on days 1 and 4 of cycle 2. Optional RT was always performed on day 1 of cycle 2. Punabulin was always administered on day 1 of cycle 3 and subsequent cycles. Two punchabulin dose levels were explored: initially 30 mg / m² was tested. 2 Dosage level. If deemed intolerable, explore 20 mg / m². 2 Dosage level.

[0180] For 30 mg / m 2 For dose levels of ponabulin, a 60-minute intravenous infusion with a ±10-minute window period is recommended; for 20 mg / m²... 2 For dosage levels, a 30-minute intravenous infusion window of ±5 minutes is recommended. This is especially important for individuals with a body surface area (BSA) greater than 2.4 m². 2 The patient, with a height of 2.4 m 2 Calculate the dose of pranabulin for maximum BSA. Pranabulin is administered 1–2 hours (if applicable) after the completion of anti-PD-1 or PD-L1 mAb infusion, or at least 3 hours (but no more than 12 hours) after radiotherapy. Grouping for this study is shown in Table 1.

[0181] Table 1. Study Groups C = cycle, D = day; Q3W = 3-week cycle; Q4W = 4-week cycle Peripheral blood collection: Where feasible, peripheral blood is collected from the patient before administration of pranabulin on day 1 of cycle 1 (C1D1), day 4 of cycle 1 (C1D4), day 1 of cycle 3 (C3D1), day 1 of cycle 5 (C5D1), and day 1 of cycle 7 (C7D1). The first sample is collected before administration of pranabulin, the second sample is collected 3 days after the pranabulin infusion, and subsequent samples are usually collected several weeks after the most recent infusion. Blood is usually processed within 4 hours of collection.

[0182] Peripheral blood phenotypic analysis: Immunophenotyping was performed using fresh heparinized peripheral blood, with 200 µL of whole blood per test plate. After incubation with a suitable fluorescein-conjugated antibody mixture for 30 minutes, whole blood was lysed using BD FACS lysis buffer according to the production protocol and washed with PBS. Countbright absolute counting microspheres (Invitrogen) were added for absolute quantification. Data were acquired using BDLSR II and analyzed using FlowJo version 9. Dendritic cells were identified using a four-color gating strategy. First, all peripheral blood dendritic cells (DCs) were gated to be LIN-FITC negative and HLA-DR-APC-H7 positive. Plasma-like dendritic cells (pDCs) were defined as CD11c-APC positive and CD123-PerCP negative; while conventional dendritic cells (cDCs) were defined as CD11c-APC-10 and CD123-PerCP positive. After establishing isotype controls from parental gating, separate flow cytometry was used for different DC maturation marker pairs. PE-conjugated antibodies were used to quantify CD86, CD40, and CCR5, while PE-Cy7-conjugated antibodies were used to quantify CD80, CD83, and CCR7. A separate assay panel was also provided for lymphocyte and monocyte immunophenotypic analysis.

[0183] Patients with clinical benefit (PR+SD) had significantly higher percentages of CCR7+, CD80+, and CD83+ pDCs than patients without clinical benefit (PD). On day 3 after the first administration of ponabulin (C1D4), the triple therapy of radiotherapy / ponabulin / immune checkpoint inhibitor induced pDC maturation, as evidenced by the expression of CCR7 (a chemokine receptor that guides mature DCs to lymph nodes), along with enhanced expression of the co-stimulatory molecules CD80 and CD83 (Figure 2A). Patients with clinical benefit (PR+SD) had a significantly higher percentage of inflammatory monocytes (weakly CD14-positive, strongly CD16-positive). The percentage of CD14-weakly CD16-positive inflammatory monocytes was dramatically increased in PR+SD patients, accompanied by a shift towards the classic CD14+CD16- monocyte phenotype, while the monocyte phenotype change was minimal in PD patients (Figure 2B). Overall, the analysis showed that, compared with PD patients, PR+SD patients exhibited a significant or positive trend of increased expression of pDC maturation markers (CCR7 / CD80 / CD83). Increased CCR7 expression in pDCs and the shift of monocytes from the classical phenotype to the inflammatory phenotype were only observed in PR+SD patients.

[0184] Example 3: Computational Analysis of scRNAseq Localization and gene counting: Raw sequencing data were processed using Cell Ranger software from 10x Genomics and the hg38 human reference genome (GRCm38 v86 and the corresponding GENCODE annotation file). Frankish, Adam et al., “GENCODE reference annotation for the human and mouse genomes,” Nucleic acids research, Vol. 47, No. 1 (2019): 766-773. During localization preprocessing, PCR repetitive sequences were removed. Gene expression levels were measured by statistically analyzing the UMI (Ultra-Specific Molecular Tag) of each gene. Cellular duplex status was inferred using DoubletFinder software. McGinnis, Christopher S., Lyndsay M. Murrow, and Zev J. Gartner, “DoubletFinder: doublet detection in single-cell RNA sequencing data using artificial nearest neighbors,” *Cell Systems*, Vol. 8, No. 4 (2019): 329-337. Data quality was controlled by evaluating several quality control metrics, including: total UMI count, total number of detected genes, mitochondrial gene percentage, log10 (number of genes) per UMI, and inferred cell duplex status. For subsequent analysis, low-quality cells with a total UMI count below 500, a total number of genes below 200, and a mitochondrial gene percentage exceeding 50% were removed. Kang, Yunhee, et al., “A human forebrain organoid model of fragile X syndrome exhibits altered neurogenesis and highlights new treatment strategies,” Nature Neuroscience, Vol. 24, No. 10 (2021): 1377-1391.

[0185] Dimensionality Reduction and Cluster Analysis: The main computational analysis of the scRNA-seq readout matrix was performed using the Seurat package (v 4.1.1) in R (v 4.2.1). (Satija, Rahul et al., "Spatial reconstruction of single-cell gene expression data," *Nature Biotechnology*, Vol. 33, No. 5 (2015): 495-502). After loading the count matrix and metadata into Seurat, highly variable genes were identified based on gene expression changes, and dimensionality reduction was performed using principal component analysis (PCA). Principal components were screened based on the interpreted total gene expression variation, typically using a carefully selected scree plot method to capture at least 70% of the total variation. The selected principal components were converted into t-SNE (t-distributed random neighborhood embedding) components or UMAP for visualization. McInnes, Leland, John Healy, and James Melville, “Umap: Uniformmanifold approximation and projection for dimension reduction,” arXiv preprint, arXiv:1802.03426, (2018); Cieslak, Matthew C. et al., “t-Distributed Stochastic Neighbor Embedding (t-SNE): A tool for eco-physiological transcriptomic analysis,” Marinegenomics, Vol. 51 (2020): p. 100723. Subsequently, canonical correlation analysis (CCA) and the IntegrateData() function were used to integrate the created objects into a merged object. Stuart, Tim et al., "Comprehensive integration of single-cell data," *Cell*, Vol. 177, No. 7 (2019): 1888-1902. The integrated data were normalized, and the sample identity effect was eliminated through regression using the SCTransform function in Seurat.Hafemeister, Christoph, and Rahul Satija, “Analyzing scRNA-seq data with the sctransform and offset models” (2020). Subsequently, the list of CCA objects was analyzed using the FindNeighbors() and FindClusters() functions and a graph-based clustering algorithm.

[0186] Cell type annotation: A semi-supervised approach was used to annotate cell type information. First, inferCNV (a widely used algorithm for identifying aneuploid cells based on large-scale copy number variation) was applied to distinguish malignant and non-malignant cell subpopulations. Second, for cells not labeled as tumor cells, single-cell data annotated in Leader et al., *Cancer Cell*, Vol. 39, No. 12 (2021): pp. 1594-1609, was used as a reference panel, and the scPred algorithm was used to assign cell type labels in a supervised manner. (See Alquicira-Hernandez, Jose et al., "scPred: accurate supervised method for cell-type classification from single-cell RNA-seq data", *Genome Biology*, Vol. 20, No. 1 (2019): pp. 1-17). Supervised cell type annotation generally yields more accurate annotation results than unsupervised methods. All cell type annotation analyses were performed using data from each individual to correct for locus and disease differences.

[0187] Differential expression and pathway analysis: Differentially expressed genes were detected by integrating all sample data within each cell type. The MAST algorithm based on a generalized linear model (with cell detection rate as a covariate) was used to detect differentially expressed genes. (Finak, Greg et al., "MAST: a flexible statistical framework for assessing transcriptional changes and characterizing heterogeneity in single-cell RNAsequencing data," *Genome Biology*, Vol. 16, No. 1 (2015): pp. 1-13). Genes with a corrected p-value less than 0.05 were identified as differentially expressed signals. Gene enrichment analysis was performed using the clusterProfiler software package in conjunction with the GeneOntology biological processes and KEGG pathway databases, as seen in Yu, Guangchuang et al., “clusterProfiler: an R package for comparing biological themes among gene clusters,” *Omics: a journal of integrative biology*, Vol. 16, No. 5 (2012): 284-287; Ashburner, Michael et al., “Gene ontology: tool for the unification of biology,” *Nature Genetics*, Vol. 25, No. 1 (2000): 25-29; Kanehisa, Minoru, and Susumu Goto, “KEGG: kyoto encyclopedia of genes and genomes,” *Nucleic Acid Research*. Acids Research, Vol. 28, No. 1 (2000): pp. 27-30.

[0188] Single-cell RNAseq analysis of unirradiated tumor biopsies from unirradiated targeted lesions before treatment (C1D1) and after treatment (C3D1). In PR+SD subjects, granzyme K+ T cells (T_GZMK, P=0.038) were significantly increased, while resident memory CD8+ T cells (CD8 Trm, P=0.0095), MoMac-III (monocyte-derived macrophage subset 3, P=0.038), IgA+ plasma cells (P=0.023), IgG+ plasma cells (P=0.042), and IgM+ plasma cells (P=0.037) were all significantly reduced (Figures 3A and 3B).

[0189] The results of the GEF-H1-dependent immunophenotypic analysis of tumor-infiltrating dendritic cells are as follows: Figure 4A and 4B As shown, the GEF-H1 immune activation scores of cDC1, cDC2, and total DCs in baseline PR+SD subjects were significantly higher than those in PD subjects. Figure 4A However, despite increased GEF-H1 immune activation scores for cDC2, DC3, and total DCs in PR+SD subjects after treatment on C3D1, PD subjects showed the opposite trend. Figure 4B ).

[0190] Single-cell RNAseq analysis of tumor biopsy tissues before and after triple therapy showed that GEF-H1-dependent immune signatures were activated in monocyte-derived macrophages (MoMac III) in responding patients. Figure 5A The study found lower levels of GEF-H1-dependent immunomarkers in the MoMac cells of responders. For a description of GEF-H1, see Kashyap et al., *Cell Reports*, September 24, 2019, Vol. 28, No. 13: pp. 3367-3380 (the entire contents of which are incorporated herein by reference). Further analysis showed that ARG1 (arginase 1) expression in MoMac-III was higher in PD subjects than in PR+SD subjects, suggesting an M2-biased phenotype in PD patients. Figure 5B ).

[0191] Single-cell RNA sequencing (scRNA-seq) of PBMCs was primarily performed using the Seurat package (version 4.3.0) in R (version 4.3.1). Seurat objects were initialized by loading the counting matrix and related metadata. The methods for identifying hypervariable genes and performing principal component analysis (PCA) were consistent with those used in the scRNA-seq analysis of tumor biopsy tissues, and normalization was performed using the SCTransform function in Seurat. Subsequently, the harmony algorithm from the Harmony package was used for data integration to remove batch effects. UMAP visualization was stratified by patient response category (partial response PR, stable disease SD, and progressive disease PD) and different time points. Finally, cluster analysis was performed on the integrated dataset using the FindNeighbors and FindClusters functions in Seurat.

[0192] The mean standardized baseline GEF-H1-dependent immunophenotypic score of peripheral blood mononuclear cells (PBMCs) was assessed. Figure 6A Response patients are defined as those demonstrating partial response (PR) or stable disease (SD). Responders (PR+SD) had significantly higher baseline PBMC GEF-H1 scores across multiple cell types (including T cells, NK cells, B cells, monocytes, and dendritic cells) than non-responders. In the PR+SD group, a steady upward trend in GEF-H1-related gene expression was observed; conversely, in the PD group, the GEF-H1 immune activation score first decreased from C1D1 to C1D4, then rebounded from C1D4 to C3D1. Figure 6B From Figure 6A The same chart extracted shows baseline data related to clinical response (at C1D1). Figure 6C Presented in tabular form Figure 6A The data.

[0193] Single-cell and T-cell receptor sequencing analyses of PBMCs revealed log-fold distributions of small clonal family cell abundance changes between C1D1 and C3D1 in the PR+SD and PD groups, respectively. Figure 7Each point represents a cell. MiloR collected neighboring cells for each point and calculated differential abundance. To compare differential cell abundance across different conditions (e.g., time points or response types), a beehive plot was generated using the MiloR package. This package randomly collected cells from the UMAP. For selected cells, neighboring cells were collected using a K-NN plot, and a generalized linear model was used to test for differential cell abundance across different conditions. Cells with a fraction below 70% were defined as mixed cells. Cells with a fraction above 70% retained their original cell type. For newly defined cell types from MiloR, significantly enriched cells were extracted. The p-value for cell abundance differences was <0.01. In downstream analyses, all plots were drawn using the ggplot2 package (v3.4.4) and the ggpubr package (v0.6.0). Significant compositional differences were observed over time only in PR+SD patients and small clonal family cells. Specifically, compared to C1D1, the PR+SD group showed a significant increase in CD4+ naive cells, CD4+ central memory T cells (TCM), CD4+ CTLs, regulatory T cells (Tregs), CD8+ naive cells, CD8+ TCM, and mucosa-associated inertial T cells (MAIT) at C3D1. These changes may reflect shifts in cell proportions over time with immune activation, potentially related to the migration patterns of T lymphocyte subsets in and out of the bloodstream during treatment.

Claims

1. A method of treating cancer in a subject, the method comprising a treatment cycle comprising the steps of: (i) administering one or more immune checkpoint inhibitors to the subject; (ii) administering plinabulin or a pharmaceutically acceptable salt thereof to the subject; (iii) obtaining a biological sample from the subject; and (iv) determining the level of one or more biomarkers or the level of one or more cells in the biological sample.

2. The method of claim 1, wherein, If the level of one or more biomarkers or one or more cells in the biological sample is above or below a predetermined threshold level, or a score determined by mathematically combining the levels of two or more biomarkers or two or more cells is above or below a predetermined threshold, then treatment with the one or more immune checkpoint inhibitors and plinabulin is discontinued.

3. The method of claim 1 or 2, wherein the one or more biomarkers are: CCR7, CD40, CD80, CD83, CD86, GEF-H1, IL-2, IFNy, IL-6, IL-12p70, IL-12p40, IL-13, IL-17A, IL-23, G-CSF, PD-L1, IL-8, and IFN-β, or a combination thereof.

4. The method of claim 3, wherein the one or more biomarkers comprise CCR7.

5. The method of claim 3 or 4, wherein the one or more biomarkers comprise CD40.

6. The method of any one of claims 3-5, wherein the one or more biomarkers comprise CD80.

7. The method of any one of claims 3-6, wherein the one or more biomarkers comprise CD83.

8. The method of any one of claims 3-7, wherein the one or more biomarkers comprise CD86.

9. The method of any one of claims 3-7, wherein the one or more biomarkers comprise GEF-H1.

10. The method of any one of claims 1-9, wherein the one or more immune checkpoint inhibitors are: pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab, pidilizumab, ipilimumab, BMS 936559, durvalumab, camrelizumab, domvanalimab, tislelizumab, sintyrozumab, and tremelimumab, or a combination thereof.

11. The method of claim 10, wherein the one or more immune checkpoint inhibitors are avelumab.

12. The method of claim 10, wherein the one or more immune checkpoint inhibitors are atezolizumab.

13. The method of claim 10, wherein the one or more immune checkpoint inhibitors are durvalumab.

14. The method of claim 10, wherein the one or more immune checkpoint inhibitors are nivolumab.

15. The method of claim 10, wherein the one or more immune checkpoint inhibitors are pembrolizumab.

16. The method of any one of claims 1-15, wherein the treatment cycle is 21 days.

17. The method of any one of claims 1-15, wherein the treatment cycle is 28 days.

18. The method of any one of claims 1-17, wherein the one or more immune checkpoint inhibitors are administered by intravenous injection.

19. The method of any one of claims 1-18, wherein the one or more immune checkpoint inhibitors are administered on day 1 of the treatment cycle.

20. The method of any one of claims 1-19, wherein the one or more immune checkpoint inhibitors are administered on day 15 of the treatment cycle.

21. The method of any one of claims 1-20, wherein the one or more immune checkpoint inhibitors are administered to the subject at a dose of 50 mg to 2000 mg.

22. The method of any one of claims 1-21, wherein the one or more immune checkpoint inhibitors are administered over a period of about 10 minutes to about 180 minutes.

23. The method of claim 22, wherein the one or more immune checkpoint inhibitors are administered over a period of about 30 minutes to about 90 minutes.

24. The method of claim 22, wherein the one or more immune checkpoint inhibitors are administered over a period of about 30 minutes.

25. The method of claim 22, wherein the one or more immune checkpoint inhibitors are administered over a period of about 60 minutes.

26. The method of any one of claims 1-24, wherein the plinabulin is administered on day 1 of the treatment cycle.

27. The method of any one of claims 1-26, wherein the plinabulin is administered on day 4 of the treatment cycle.

28. The method of any one of claims 1-27, wherein the dose of plinabulin is about 10 mg / m 2 to about 50 mg / m 2 .

29. The method of claim 28, wherein the dose of plinabulin is about 20 mg / m 2 .

30. The method of claim 28, wherein the about 30 mg / m 2 .

31. The method of any one of claims 1-30, wherein the plinabulin is administered over a period of about 10 minutes to about 180 minutes.

32. The method of claim 31, wherein the plinabulin is administered over a period of about 20 minutes to about 90 minutes.

33. The method of claim 31, wherein the plinabulin is administered over a period of about 30 minutes to about 60 minutes.

34. The method of any one of claims 1-33, wherein when the one or more immune checkpoint inhibitors are administered on the same day as plinabulin, plinabulin is administered about 0.5 hours to about 3 hours after completion of administration of the one or more immune checkpoint inhibitors.

35. The method of claim 34, wherein plinabulin is administered about 1 hour to about 2 hours after completion of administration of the one or more immune checkpoint inhibitors.

36. The method of any one of claims 1-35, further comprising administering radiation therapy to the subject.

37. The method of claim 36, wherein the radiation therapy is administered in 1 to 10 fractions.

38. The method of claim 36, wherein the radiation therapy is administered in 3 to 5 fractions.

39. The method of claim 36, wherein the radiation therapy is administered in 3 fractions.

40. The method of claim 36, wherein the radiation therapy is administered in 4 fractions.

41. The method of claim 36, wherein the radiation therapy is administered in 5 fractions.

42. The method of any one of claims 36-41, wherein the total dose of radiation administered is about 1 Gy to about 20 Gy.

43. The method of claim 42, wherein the total dose of radiation administered is about 2 Gy to about 15 Gy.

44. The method of claim 42, wherein the total dose of radiation administered is about 4 Gy to about 15 Gy.

45. The method of claim 42, wherein the total dose of radiation administered is about 4 Gy.

46. The method of claim 42, wherein the total dose of radiation administered is about 8 Gy.

47. The method of claim 42, wherein the total dose of radiation administered is about 12.5 Gy.

48. The method of any one of claims 36-47, wherein the radiation therapy is performed on days 1, 2, and 3 of the treatment cycle.

49. The method of any one of claims 36-47, wherein the radiation therapy is performed on days 1, 2, 3, and 4 of the treatment cycle.

50. The method of any one of claims 36-47, wherein the radiation therapy is performed on days 1, 2, 3, 4, and 5 of the treatment cycle.

51. The method of any one of claims 36-50, wherein when the radiation therapy and plinabulin are administered on the same day, the plinabulin is administered about 3 hours to about 12 hours after the radiation therapy administration is completed.

52. The method of any one of claims 1-51, wherein the biological sample is a blood sample.

53. The method of any one of claims 1-51, wherein the biological sample is a tumor biopsy.

54. The method of any one of claims 1-53, wherein the one or more cells are dendritic cells or a subset thereof; or wherein the one or more cells are selected from the group consisting of CD4+ T cells, CD8+ T cells, B cells, IgA+ plasma cells, IgG+ plasma cells, and IgM+ plasma cells; or wherein the one or more cells are selected from the group consisting of CD16+ monocytes, CD14+ monocytes, natural killer (NK) cells, granzyme K+ T cells, CD8+ TRM cells, CD8+ TEM cells, CD4+ TCM cells, CD4+ TEM cells, CD4+ naive T cells, naive B cells, cDC1 cells, cDC2 cells, and DC3 cells; or wherein the one or more cells are monocyte-derived macrophages (MoMac) or interstitial macrophages (IM); or wherein the one or more cells are immune cells from peripheral blood mononuclear cells (PBMCs).

55. The method of claim 54, wherein the method comprises determining the level of one or more dendritic cells expressing one or more biomarkers in the biological sample.

56. The method of claim 54 or 55, wherein the one or more biomarkers is: CCR7, CD40, CD80, CD83, CD86, GEF-Hl, IL-2, IFNy, IL-6, IL-12p70, IL-12p40, IL-13, IL-17A, IL-23, G-CSF, IL-8, IFN-β, or a combination thereof.

57. The method of claim 56, wherein the one or more biomarkers is: CCR7, CD40, CD80, CD83, CD86, or a combination thereof.

58. The method of claim 56, wherein the one or more biomarkers is GEF-Hl.

59. The method of any one of claims 54-58, wherein the dendritic cells are myeloid dendritic cells.

60. The method of any one of claims 1-59, wherein the biological sample is obtained on day 4 of the treatment cycle.

61. The method of any one of claims 1-60, wherein the cancer is selected from the group consisting of: breast cancer, bladder cancer, glioma, glioblastoma, head and neck cancer, non-small cell lung cancer, small cell lung cancer, recurrent small cell lung cancer (SCLC), colorectal cancer, gastrointestinal stromal tumor, gastroesophageal cancer, renal cell carcinoma, prostate cancer, liver cancer, colon cancer, pancreatic cancer, ovarian cancer, lymphoma, cutaneous T-cell lymphoma, or melanoma.

62. The method of any one of claims 1-61, comprising determining a baseline level of the one or more biomarkers or one or more cells in an initial biological sample.

63. The method of claim 62, wherein the initial biological sample is obtained prior to day 1 of the treatment cycle, administration of the one or more immune checkpoint inhibitors and plinabulin.

64. The method of claim 62 or 63, comprising comparing the level determined in step (iv) to the baseline level.

65. The method of claim 64, wherein the comparison comprises determining a change in the level of the one or more biomarkers or one or more cells.

66. The method of claim 65, wherein if the change in the level of the one or more biomarkers or one or more cells is less than a predetermined threshold, then treatment with the one or more immune checkpoint inhibitors and plinabulin is discontinued.

67. The method of claim 66, wherein if the level of CCR7-expressing pDCs is greater than about 10% above the baseline level, then treatment with the one or more immune checkpoint inhibitors and plinabulin is discontinued.

68. The method of claim 66, wherein if the level of CCR7-expressing pDCs is greater than about 20% above the baseline level, then treatment with the one or more immune checkpoint inhibitors and plinabulin is discontinued.

69. The method of claim 66, wherein if the level of CCR7-expressing pDCs is less than about 20% below the baseline level, then treatment with the one or more immune checkpoint inhibitors and plinabulin is discontinued.

70. A method of treating cancer in a subject, comprising, the method comprises a treatment cycle comprising the steps of: (i) obtaining a biological sample from the subject; (ii) determining the level of one or more biomarkers or the level of one or more cells in the biological sample; and (iii) administering plinabulin or a pharmaceutically acceptable salt thereof to the subject if the level of one or more biomarkers or one or more cells in the biological sample is higher or lower than a threshold level, or a score determined by mathematically combining the levels of two or more biomarkers or two or more cells is higher or lower than a predetermined threshold.

71. The method of claim 70, wherein the biological sample is a tumor biopsy.

72. The method of claim 70, wherein the biological sample is peripheral blood.

73. The method of any one of claims 70-72, wherein the one or more cells are dendritic cells or a subset thereof; or wherein the one or more cells are selected from the group consisting of T cells, CD4+ T cells, CD8+ T cells, B cells, IgA+ plasma cells, IgG+ plasma cells, and IgM+ plasma cells; or wherein the one or more cells are selected from the group consisting of CD 16+ monocytes, CD 14+ monocytes, natural killer (NK) cells, granzyme K+ T cells, CD8+ TRM cells, CD8+ TEM cells, CD4+ TCM cells, CD4+ TEM cells, CD4+ naive T cells, naive B cells, cDC1 cells, cDC2 cells, and DC3 cells; or wherein the one or more cells are monocyte-derived macrophages (MoMac) or interstitial macrophages (IM); or wherein the one or more cells are immune cells from peripheral blood mononuclear cells (PBMCs).

74. The method of any one of claims 70-72, wherein the one or more cells are monocyte-derived macrophages (MoMac).

75. The method of any one of claims 70-72, wherein the one or more cells are peripheral blood mononuclear cells.

76. The method of any one of claims 70-75, wherein the score is a GEF-H1 immune activation score.

77. The method of claim 76, wherein the GEF-Hl immune activation score is determined using a combination of gene expression levels of one or more genes selected from the group consisting of: Mmp12, Csf1, Ccl6, Osm, Ccl9, Cxcl3, Clec7a, Hdc, Slc15a3, Dcstamp, Trem1, Cxcl2, Cd300a, Ncf2, Cd80, Lilr4b, Pdcd1lg2, Lilrb4a, Mxd1, Sp140, Il1a, Ppbp, Gpr171, Ccl7, Il1b, Ccl4, Man1a, Aqp9, Creb5, Traf4, Nod2, Ptger2, Mefv, Cd6, Reps2, Tlr7, Sirpb1b, Fcgr4, P2ry2, Apol8, Lag3, Pdcd1, Cxcr1, Rab27b, Ctla4, Cxcl9, and Fasl.

78. The method of claim 76 or 77, wherein the threshold for the GEF-Hl immune activation score is about 10 to about 40.

79. The method of claim 76 or 77, wherein the threshold for the GEF-Hl immune activation score is about 20 to about 40.

80. The method of claim 76 or 77, wherein the threshold for the GEF-Hl immune activation score is about 30.

81. The method of claim 76 or 77, wherein the threshold for the GEF-Hl immune activation score is about 25.

82. The method of any one of claims 70-81, wherein the cancer is selected from the group consisting of: breast cancer, bladder cancer, glioma, glioblastoma, head and neck cancer, non-small cell lung cancer, small cell lung cancer, recurrent small cell lung cancer (SCLC), colorectal cancer, gastrointestinal stromal tumor, gastroesophageal cancer, renal cell carcinoma, prostate cancer, liver cancer, colon cancer, pancreatic cancer, ovarian cancer, lymphoma, cutaneous T-cell lymphoma, or melanoma.

83. The method of any one of claims 70-82, further comprising administering to the subject a radiation therapy, an immune checkpoint inhibitor, a chemotherapeutic agent, or a combination thereof.

84. The method of any one of claims 70-83, further comprising administering to the subject a radiation therapy and an immune checkpoint inhibitor.

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