WO2017049237A1 - Personalized approach to dosage of anti-fugetactic agent for treatment of cancer - Google Patents

Abstract

The invention described herein relates to methods and systems for determining an effective amount of an anti-fugetactic agent for treating cancer in a patient or a tumor cell.

Description

PERSONALIZED APPROACH TO DOSAGE OF ANTI-FUGETACTIC AGENT FOR TREATMENT OF CANCER

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application Nos. 62/220,911, filed September 18, 2015; and 62/220,916, filed September 18, 2015, each of which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] Cell movement in response to specific stimuli occurs in prokaryotes and eukaryotes. Cell movement seen in these organisms has been classified into three types: chemotaxis or the movement of cells along a gradient towards an increasing concentration of a chemical; negative chemotaxis which has been defined as the movement down a gradient of a chemical stimulus; and chemokinesis or the increased random movement of cells induced by a chemical agent.

[0003] Chemotaxis and chemokinesis have been observed in mammalian cells in response to a class of proteins called chemokines. Additionally, chemorepellent, or fugetactic, activity has been observed in mammalian cells. See, U.S. Patent No. 6,448,054 (incorporated herein by reference in its entirety). For example, some tumor cells secrete concentrations of chemokines that are sufficient to repel immune cells from the site of a tumor, thereby reducing the immune system's ability to target and eradicate the tumor. Metastasizing cancer cells may use a similar mechanism to evade the immune system.

[0004] Repulsion of tumor antigen-specific T-cells, e.g. from a tumor expressing high levels of CXCL12 or interleukin 8 (IL-8), allows the tumor cells to evade immune control. Anti-fugetactic agents have been described that inhibit the fugetactic activity of tumor cells and allow the patient's immune system to target the tumor (see US 2008/0300165, incorporated herein by reference in its entirety). However, treatment with such agents may not be sufficient to eradicate a tumor in all patients, depending on the type of tumor, size of tumor, number of metastases, site(s) of metastasis, patient's health, etc. [0005] There remains a need for treatments and compositions that target tumors to efficiently kill tumors and/or metastasizing cancer cells.

SUMMARY OF THE INVENTION

[0006] This invention relates to methods for determining an effective therapeutic amount of an anti-fugetactic agent for reducing a fugetactic effect of a tumor in a patient. Tumors differ in the amount of fugetactic chemokine that is produced by the tumor cells and/or present in the tumor microenvironment. The number of receptors for the fugetactic chemokine expressed on the surface of an immune cell also varies according to the individual, depending on factors including genetics, the amount of chemokine present (e.g., in the peripheral blood or in the microenvironment), type of immune cell, and other factors. Accordingly, and without being bound by theory, it is believed that the amount of anti-fugetactic agent that is required to counteract the fugetactic effect of a tumor in a patient may depend on the amount of fugetactic chemokine that is produced and/or the number of receptors expressed on immune cells.

[0007] As many as 85% of solid tumors and 1 eukemias express CXCL 12 at a level sufficient to have fugetactic effects, e.g. repulsion of immune cells from the tumor. Cancers that express CXCL12 at such levels include, but are not limited to, prostate cancer, lung cancer, breast cancer, pancreatic cancer, ovarian cancer, gastric cancer, esophageal cancer, melanoma, glioma and leukemia

[0008] In one aspect, the invention relates to a method for determining a therapeutic amount of an anti-fugetactic agent, the method comprising providing a sample having a fugetactic effect and using the sample to conduct a migration assay with immune cells. In one embodiment, an effective local amount of anti-fugetactic agent is determined.

[0009] In one aspect, the invention relates to a method for determining an effective local amount of an anti-fugetactic agent by determining a concentration of the anti-fugetactic agent that is sufficient to increase immune cell migration toward the tumor of a patient. In one embodiment, a migration assay is performed using immune cells, a sample comprising a fugetactic chemokine, and varying amounts of anti-fugetactic agent. The sample comprising the fugetactic chemokine may be, for example and without limitation, tumor cells (from the patient or from a different patient, e.g. a patient having a similar tumor), cancer cell line, conditioned medium from tumor cells or a cell line (including concentrated or diluted conditioned medium), a known amount of the chemokine, etc. Preferably, the sample comprises a similar or proportional amount of fugetactic chemokine compared to the tumor. More preferably, the sample is derived from a tumor in a patient to be treated with the anti- fugetactic agent.

[0010] In one embodiment, the method comprises: a) providing a plurality of tumor cells derived from a mammalian tumor; b) combining into an apparatus comprising a culture medium the plurality of tumor cells with a plurality of immune cells, wherein the apparatus permits the monitoring of the immune cells relative to the tumor cells; c) contacting the culture medium with known incremental amounts of an anti-fugetactic agent; and d) assessing the aggregate concentration at which the anti-fugetactic agent permits the immune cells or increasing numbers of immune cells to migrate towards the tumor cells, thereby determining the effective local amount of the anti-fugetactic agent mat reverses the fugetactic properties of the tumor cells. In one embodiment, the plurality of cells has a defined tumor cell volume. In one embodiment, the plurality of cells has fugetactic properties (i.e., a fugetactic effect), for example due to secretion of a chemokine. In one embodiment, the method includes monitoring the position of the immune cells relative to the tumor cells. In one embodiment, the plurality of immune cells is capable of attraction to the tumor cells, but the attraction is inhibited by the fugetactic properties of the tumor cells. In one embodiment, the immune cells express CXCR4. In one embodiment, the sample comprises CXCL12 and/or cells that express CXCL12.

[0011] In one aspect, the invention relates to a method for determining an effective local amount of an anti-fugetactic agent, the method comprising: a) providing a plurality of tumor cells derived from a mammalian tumor; b) combining into a plurality of apparatuses comprising a culture medium the plurality of tumor cells with a plurality of immune cells, wherein the apparatuses permit the monitoring of the immune cells relative to the tumor cells; c) contacting the culture medium with a plurality of known amounts of an anti-fugetactic agent; and d) assessing an amount of anti-fugetactic agent at which the immune cells or increasing numbers of immune cells migrate towards the tumor cells, thereby determining the effective local amount of the anti-fugetactic agent that reverses the fugetactic properties of the defined volume of tumor cells. In one embodiment, the immune cells are capable of attraction to the tumor cells but the attraction is inhibited by the fugetactic properties of the tumor cells. In one embodiment, the plurality of cells have a defined tumor cell volume and further have fugetactic properties. In one embodiment, the plurality of apparatuses are a plurality of wells in a multi-well tissue culture plate. In one embodiment, the immune cells express CXCR4. In one embodiment, the sample comprises CXCL12 and/or cells that express CXCL12.

[0012] In one aspect, the invention relates to a method for determining an effective local amount of an anti-fugetactic agent, the method comprising: a) providing a plurality of tumor cells derived from a mammalian tumor; b) placing the plurality of tumor cells into a culture medium for a period of time to provide a conditioned medium; c) combining into an apparatus (or plurality of apparatuses) the conditioned medium with a plurality' of immune cells, wherein the apparatus permits the monitoring of the immune cells relative to the conditioned medium; d) contacting the immune cells with known amounts (e.g., incremental amounts) of an anti-fugetactic agent; and e) assessing the concentration or aggregate concentration at which the anti-fugetactic agent permits the immune cells or increasing numbers of immune cells to migrate towards the conditioned medium, thereby determining the effective local amount of the anti-fugetactic agent that reverses the fugetactic properties of the conditioned medium. In one embodiment, the plurality of cells have a defined tumor cell volume and further have fugetactic properties. In one embodiment, the plurality of immune cells are capable of attraction to the tumor cells but such attraction is inhibited by the fugetactic properties of the conditioned medium. In one embodiment, the immune cells express CXCR4. In one embodiment, the culture medium comprises CXCL12.

[0013] In one aspect, the invention relates to a method for determining the therapeutic dose of an anti-fugetactic agent in a subject with cancer, the method comprising: a) obtaining from the subject a plurality of tumor cells which express SDF-1/CXCL12, and a plurality of immune cells which express CXCR4, each of the plurality of cells having a defined number and volume; b) combining into an apparatus comprising a culture medium the plurality of tumor cells with the plurality of immune cells, wherein the apparatus permits the monitoring of the immune cells relative to the tumor cells; c) contacting the culture medium with known incremental amounts of an anti-fugetactic agent; d) assessing the aggregate concentration at which the anti-fugetactic agent permits the immune cells or increasing numbers of immune cells to migrate towards the tumor cells, thereby determining the effective local amount of the anti-fugetactic agent that reverses the fugetactic properties of the known volume of tumor cells.

[0014] In one aspect, the invention relates to a method for determining the therapeutic dose of an anti-fugetactic agent in a subject with cancer, the method comprising: a) obtaining from the subject a plurality of tumor cells which express a fugetactic chemokine (or a tumor sample comprising a fugetactic chemokine), and a plurality of immune cells which express a receptor for the chemokine, each of the plurality of cells having a defined number and volume; b) dividing the immune cells into first, second and further samples, exposing a first immune cell sample to a known first amount of an anti-fugetactic agent, exposing a second immune cell sample to a known second amount of an anti-fugetactic agent, exposing further immune cell samples to known further amounts of an anti-fugetactic agent, wherein the first, second and further amounts are different; c) dividing the tumor cells into first, second and further tumor samples, and placing each tumor sample into an apparatus(es) comprising a culture medium, the apparatus permits the monitoring of the immune cells relative to the tumor cells; d) adding the first immune cell sample to the apparatus comprising the culture medium and the first tumor sample; and e) assessing the aggregate concentration at which the anti-fugetactic agent permits the immune cells or increasing numbers of immune cells to migrate towards the tumor cells, thereby determining the effective local amount of the anti- fugetactic agent that reverses the fugetactic properties of the known volume of tumor cells. In one embodiment, the method further comprises d') adding the second immune cell sample to the apparatus comprising culture medium and the second tumor sample, and optionally adding the further immune cell samples to the apparatuses comprising culture medium and the further tumor samples.

[0015] In one embodiment, the sample is a plurality of tumor cells derived from a mammalian tumor. In one embodiment, the sample is a conditioned medium from a plurality of tumor cells derived from a mammalian tumor. In one embodiment, the sample is a plurality of tumor cells, the tumor cells taken from a tumor and cultured in vitro for a period of time, e.g., to expand the number of the tumor cells. In one embodiment, the sample is a sample taken from a tumor microenvironment. In one embodiment, the sample is a known concentration of a fugetactic chemokine, preferably at a concentration that correlates with the amount of the chemokine that is produced by a tumor and/or present in a tumor

microenvironment. In one embodiment, the chemokine is CXCL12 or IL-8.

[0016] In one aspect, the invention relates to a system for determining an effective local amount of an anti-fugetactic agent. In one embodiment, the system comprises: a) an apparatus having an injection port and a chamber where the contents of the chamber are capable of being monitored; b) the chamber comprising a plurality of mammalian tumor cells derived from a selected tumor, the plurality of cells having a defined tumor cell volume (or a tumor sample comprising a fugetactic chemokine, said sample derived from tumor cells having a defined tumor cell volume), a culture medium and a plurality of immune cells; and c) an injection port configured to permit the addition of sequentially increasing known amounts of an anti-fugetactic agent to the medium, such that when an effective amount of the anti-fugetactic agent is included in the medium, the immune cells will migrate toward the tumor cells.

[0017] In one embodiment, the system comprises: a) an apparatus having an injection port and a chamber where the contents of the chamber are capable of being monitored; b) the chamber comprising a plurality of human tumor cells derived from a patient with a tumor, the plurality of cells having a defined tumor cell volume (or a tumor sample comprising a fugetactic chemokine, said sample derived from tumor cells having a defined tumor cell volume), a culture medium and a plurality of immune cells; and c) the injection port being configured to permit the addition of sequentially increasing known amounts of an anti- fugetactic agent to the medium such that when an effective amount of the anti-fugetactic agent is included in the medium, the immune cells will migrate toward the tumor cells.

[0018] In one aspect, the invention relates to a method for determining the therapeutic dose of an anti-fugetactic agent in a subject with cancer, the method comprising a) obtaining from the subject a plurality of tumor cells which express a fugetactic chemokine; b) combining into an apparatus comprising a culture medium the plurality of tumor cells with a plurality of immune cells which express a receptor for the chemokine, each of the plurality of cells having a defined number and volume, the apparatus permits the monitoring of the immune cells relative to the tumor cells; c) contacting the culture medium with known incremental amounts of an anti-fugetactic agent; d) assessing the aggregate concentration at which the anti-fugetactic agent permits the immune cells or increasing numbers of immune cells to migrate towards the tumor cells, thereby determining the effective local amount of the anti- fugetactic agent that reverses the fugetactic properties of the known volume of tumor cells.

[0019] In one aspect, the invention relates to a method for determining the therapeutic dose of an anti-fugetactic agent in a subject with a cancerous tumor, the method comprising a) obtaining from the subject a plurality of tumor cells which express a fugetactic chemokine; b) providing a plurality of immune cells which express a receptor for the chemokine, each of the plurality of cells having a defined number and volume, dividing the immune cells into a plurality of samples each of which have a defined number, and exposing each of the immune cell sample to a known different amount of an anti-fugetactic agent under conditions wherein the receptors of the immune cells bind the anti-fugetactic agent; c) dividing the tumor cells into separate samples of a defined cell number wherein the number of tumor samples correspond to the number of immune cell samples prepared in b); d) combining each of the immune cell samples with one of the tumor cell samples into a separate apparatus containing a culture medium such that each apparatus contains immune cells with a different amount of anti-fugetactic agent bound thereto; e) assessing which apparatus demonstrates that the immune cells or increasing numbers of immune cells migrate towards the tumor cells; and f) correlating that apparatus having the lowest concentration of anti-fugetactic agent per immune cell which exhibits anti-fugetactic properties to the effective concentration required for anti-fugetaxis for that tumor.

[0020] In some embodiments, the tumor cells are used to provide a sample to be used in the method, rather man using the tumor cells directly. Thus, where "tumor cells" are recited herein, it is contemplated that a sample derived from tumor cells may be used. In one embodiment, the plurality of tumor cells is used to provide a conditioned medium In one embodiment, the tumor cells are cultured in vitro for a period of time, e.g., to expand the number of the tumor cells. In one embodiment, a sample is taken from a tumor microenvironment. In one embodiment, the sample is a known concentration of a fugetactic chemokine, preferably at a concentration that correlates with the amount of the chemokine mat is produced by a tumor and/or present in a tumor microenvironment. In one embodiment, the chemokine is CXCL12 or IL-8. In a preferred embodiment, the chemokine is CXCL12.

[0021] In one embodiment, the sample is taken from or derived from a patient. In one embodiment, the sample is at least a portion of a biopsy taken from a patient. In one embodiment, tumor cells are taken from two or more sites within the tumor. In one embodiment, the plurality of tumor cells have a defined tumor cell volume and/or density.

[0022] In one embodiment, the immune cells include T cells and/or NK cells. In a preferred embodiment, the immune cells include T cells. In one embodiment, the immune cells include PBMCs or antigen-presenting cells. In one embodiment, the immune cells are heterologous (i.e., not derived from the patient having the tumor). In one embodiment, the immune cells are autologous (i.e., derived from the same patient that the sample is taken from). In one embodiment, the immune cells are cells that express a receptor for the chemokine. In one embodiment, the number (or average number) of chemokine receptors expressed by the immune cells is quantitated. In one embodiment, the chemokine receptor is CXCR4.

[0023] In one embodiment, the method further comprises determining the maximum effective concentration of the anti-fugetactic agent by further contacting the immune cells with known amounts (e.g. incremental amounts) of the agent to determine at which concentration the agent ceases to exhibit the anti-fugetactic property.

[0024] In a preferred embodiment, the cancer, tumor, or cell expresses an amount of a chemokine sufficient to produce a fugetactic effect. In one embodiment, the chemokine is secreted by the cell or tumor, such that the fugetactic effect is present in the tumor microenvironment. In one embodiment, the concentration of the chemokine in the tumor microenvironment is greater than about 100 nM. In one embodiment, the chemokine is CXCL12 or IL-8. In a preferred embodiment, the chemokine is CXCL12.

[0025] In one embodiment, the tumor is a solid tumor. In one embodiment, the tumor is a non-solid tumor. In one embodiment, the tumor is a leukemia [0026] The anti-fugetactic agent may be any such agent known in the art. In one embodiment, the anti-fugetactic agent is an anti-fugetactic agent as described in U.S. Patent Application Publication No. 2008/0300165, which is hereby incorporated by reference in its entirety. In a preferred embodiment, the anti-fugetactic agent is AMD3100

(mozobil/plerixafor) or derivative thereof, KRH-1636, T-20, T-22, T-140, TE-14011, T- 14012, TN14003, TAK-779, AK602, SCH-351125, Tannic acid, NSC 651016, thalidomide, GF 109230X, an antibody that interferes with dimerization of a fugetactic chemokine, or an antibody that interferes with dimerization of the receptor for a fugetactic chemokine. For example, the antibody may inhibit dimerization of CXCL12, IL-8, CXCR3, or CXCR4. In one embodiment, the anti-fugetactic agent is an antibody mat interferes with binding of the chemokine to its receptor. In an especially preferred embodiment, the anti-fugetactic agent is AMD3100 (l,l'-[l,4-phenylenebis(methylene)]bis [1,4,8,11-tetraazacyclotetradecane]).

[0027] In one embodiment, the amount of anti-fugetactic agent tested is between about 0.001 μΜ and about 100 mM. In one embodiment, the amount of anti-fugetactic agent tested is between about 0.01 μΜ and about 10 mM. In one embodiment, the amount of fugetactic agent tested is between about 0.1 μΜ and about 1 mM. Following testing, the therapeutic level of antifugetactic agent for that patient's cancer is known.

[0028] In one aspect, the method further comprises administering the therapeutic amount of anti-fugetactic agent to the patient. In one embodiment, the anti-fugetactic agent is administered locally to the tumor. In one embodiment, the anti-fugetactic agent is administered systemically. In one embodiment, the anti-fugetactic agent is administered systemically, followed by local administration.

[0029] In one aspect is provided a method for inhibiting angiogenesis within a tumor by locally delivering into or proximate the tumor an effective amount of a CXCR4 receptor antagonist, thereby inhibiting CXCL12 from binding to the receptor wherein the binding is an integral part of angiogenesis. In one embodiment, the CXCR4 antagonist is AMD3100 or derivative thereof, KRH-1636, T-20, T-22, T-140, TE-14011, T-14012, TN14003, TAK-779, AK602, SCH-351125, Tannic acid, NSC 651016, thalidomide, GF 109230X, an antibody that interferes with dimerization of a fugetactic chemokine, or an antibody' that interferes with dimerization of the receptor for a fugetactic chemokine. BRIEF DESCRIPTION OF THE FIGURES

[0030] FIGURE 1 represents the bimodal chemotactic effect of increasing amounts of AMD3100 on human T cells.

[0031] FIGURE 2 represents the bimodal fugetactic effect of increasing amounts of AMD3100 on human T cells.

DETAILED DESCRIPTION

[0032] After reading this description, it will become apparent to one skilled in the art how to implement the invention in various alternative embodiments and alternative applications. However, not all embodiments of the present invention are described herein. It will be understood that the embodiments presented here are presented by way of example only, and not limitation. As such, this detailed description of various alternative embodiments should not be construed to limit the scope or breadth of the present invention as set forth below.

[0033] Before the present invention is disclosed and described, it is to be understood that the aspects described below are not limited to specific compositions, methods of preparing such compositions, or uses thereof as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

Definitions

[0034] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0035] In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings:

[0036] The terminology' used herein is for the purpose of describing particular

embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0037] All numerical designations, e.g., pH, temperature, time, concentration, amounts, and molecular weight, including ranges, are approximations which are varied (+) or (-) by 10%, 1%, or 0.1%, as appropriate. It is to be understood, although not always explicitly stated, that all numerical designations may be preceded by the term "about." It is also to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0038] "Optional" or "optionally" means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0039] The term "comprising" or "comprises" is intended to mean mat the compositions and methods include the recited elements, but not excluding others. "Consisting essentially of when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination. For example, a composition consisting essentially of the elements as defined herein would not exclude other elements that do not materially affect the basic and novel characteristic(s) of the claimed invention. "Consisting of shall mean excluding more than trace amount of other ingredients and substantial method steps recited. Embodiments defined by each of these transition terms are within the scope of mis invention.

[0040] The terms "patient," "subject," "individual," and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In a preferred embodiment, the patient, subject, or individual is a mammal. In some embodiments, the mammal is a mouse, a rat, a guinea pig, a non-human primate, a dog, a cat, or a domesticated animal (e.g. horse, cow, pig, goat, sheep). In especially preferred embodiments, the patient, subject or individual is a human.

[0041] The term "cancer" refers to any disease caused by an uncontrolled division of abnormal cells in a part of the body. The term "tumor" refers to any cancerous growth. [0042] Examples of solid tumors include but are not limited to: Adrenal Cancer, Anal Cancer, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Brain/CNS Tumors, Breast Cancer (including inflammatory breast cancer), Cancer of Unknown Primary, Castleman Disease, Cervical Cancer, Colon/Rectum Cancer, Endometrial Cancer, Esophagus Cancer, Ewing Family Of Tumors, Eye Cancer, Gallbladder Cancer, Gastrointestinal Carcinoid Tumors, Gastrointestinal Stromal Tumor (GIST), Gestational Trophoblastic Disease, Hodgkin Disease, Kaposi Sarcoma, Kidney Cancer, Laryngeal and Hypopharyngeal Cancer, Liver Cancer, Lung Cancer, Lung Cancer - Non-Small Cell, Lung Cancer - Small Cell, Lung Carcinoid Tumor, Lymphoma of the Skin, Malignant Mesothelioma, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin Lymphoma, Oral Cavity and Oropharyngeal Cancer, Osteosarcoma, Ovarian Cancer, Pancreatic Cancer, Penile Cancer, Pituitary Tumors, Prostate Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoma - Adult Soft Tissue Cancer, Skin Cancer, Skin Cancer - Basal and Squamous Cell, Skin Cancer - Melanoma, Skin Cancer - Merkel Cell, Small Intestine Cancer, Stomach Cancer, Testicular Cancer, Thymus Cancer, Thyroid Cancer, Uterine Sarcoma, Vaginal Cancer, Vulvar Cancer, Waldenstrom Macroglobulinemia, and Wilms Tumor.

[0043] Examples of non-solid tumors include but are not limited to: Leukemia, Leukemia - Acute Lymphocytic (ALL) in Adults, Leukemia - Acute Myeloid (AML), Leukemia - Chronic Lymphocytic (CLL), Leukemia - Chronic Myeloid (CML), Leukemia - Chronic Myelomonocytic (CMML) Lymphoma, Multiple Myeloma, and Myelodysplastic Syndrome, Myelodysplastic Syndrome.

[0044] The term "treating" or "treatment" covers the treatment of a disease or disorder described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, i.e., arresting its development; (ii) relieving a disease or disorder, i.e., causing regression of the disorder; (iii) slowing progression of the disorder; and/or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder. For example, treatment of a cancer or tumor includes, but is not limited to, reduction in size of the tumor, elimination of the tumor and/or metastases thereof, remission of the cancer, inhibition of metastasis of the tumor, reduction or elimination of at least one symptom of the cancer, increase in progression-free survival, increase in survival, and the like.

[0045] The term "administering" or "administration" of an agent or drug to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or

subcutaneously), or topically. Administration includes self-administration and the administration by another.

[0046] The term "direct administration" or "local administration" refers to administration of the anti-fugetactic agent in a manner that targets the agent to the tumor and/or the tumor microenvironment. For example, direct injection into the tumor, tumor vasculature or tumor microenvironment; administration of the anti-fugetactic agent in a formulation that targets it to the tumor (e.g., bound to or otherwise associated with an anti-tumor antibody), and the like.

[0047] It is also to be appreciated that the various modes of treatment or prevention of medical diseases and conditions as described are intended to mean "substantial," which includes total but also less than total treatment or prevention, and wherein some biologically or medically relevant result is achieved.

[0048] The term "therapeutic" as used herein means a treatment and/or prophylaxis. A therapeutic effect is obtained by prevention, suppression, remission, or eradication of a disease state.

[0049] The term "therapeutically effective amount" or "effective amount" refers to an amount of the agent that, when administered, is sufficient to cause the desired effect. For example, an effective amount of an anti-fugetactic agent may be an amount sufficient to have an anti-fugetactic effect on a cancer cell or tumor (e.g. to attenuate a fugetactic effect from the tumor or cancer cell). The therapeutically effective amount of the agent will vary depending on the tumor being treated (type, size, severity, etc.) as well as the age, weight, etc., of the patient to be treated. The skilled artisan will be able to determine appropriate dosages depending on these and other factors using the methods and systems described herein. The compositions can also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic compounds may be administered to a subject having one or more signs or symptoms of a disease or disorder.

[0050] The term "kiU" with respect to a cell/cell population is directed to include any type of manipulation that will lead to the death of that cell/cell population.

[0051] "Cytokine" is a generic term for non-antibody, soluble proteins which are released from one cell subpopulation and which act as intercellular mediators, for example, in the generation or regulation of an immune response. See Human Cytokines: Handbook for Basic & Clinical Research (Aggrawal, et al. eds., Blackwell Scientific, Boston, Mass. 1991) (which is hereby incorporated by reference in its entirety for all purposes).

[0052] " CXCR4/CXCL 12 antagonist" refers to a compound mat antagonizes CXCL12 binding to CXCR4 or otherwise reduces the fugetactic activity of CXCL12.

[0053] By "fugetactic activity" or "fugetactic effect" it is meant the ability of an agent to repel (or chemorepel) a eukaryotic cell with migratory capacity (i.e., a cell that can move away from a repellant stimulus). The term also refers to the chemorepellant effect of a chemokine secreted by a cell, e.g. a tumor cell. Usually, the fugetactic effect is present in an area around the cell wherein the concentration of the chemokine is sufficient to provide the fugetactic effect. Some chemokines, including interleukin 8 and CXCL12, may exert fugetactic activity at high concentrations (e.g., over about 100 nM), whereas lower concentrations exhibit no fugetactic effect and may even be chemoattractant.

[0054] Accordingly, an agent with fugetactic activity is a "fugetactic agent." Such activity can be detected using any of a variety of systems well known in the art (see, e.g., U.S. Pat. No. 5,514,555 and U.S. Patent Application Pub. No. 2008/0300165, each of which is incorporated by reference herein in its entirety). A preferred system for use herein is described in U.S. Patent 6,448,054, which is incorporated herein by reference in its entirety.

[0055] The term "anti-fugetactic effect" refers to the effect of the anti-fugetactic agent to attenuate or eliminate the fugetactic effect of the chemokine. In some aspects, the anti- ftigetactic effect is indicated by the migration of immune cells to the tumor or tumor microenvironment, to the tumor cells, and/or infiltration of the tumor by the immune cells.

[0056] "Immune cells" as used herein are cells of hematopoietic origin that are involved in the specific recognition of antigens. Immune cells include antigen presenting cells (APCs), such as dendritic cells or macrophages, B cells, T cells, etc. Immune cells further include natural killer (NK) cells. Immune cells used in the methods described herein may be autologous, allogenic, or immortalized (e.g., cell lines).

[0057] The term "autologous" or "autologous cells" as used herein refers to immune cells obtained from, and then administered to the same patient.

[0058] The term "allogenic'^'' or "allogenic cells" as used herein refers to immune cells obtained from a subject other than the patient to whom they are administered. The terms "allogenic" and "allogeneic," as well as "heterologous," may be used interchangeably herein.

[0059] The term "effective local amount" refers to the amount of anti-fugetactic agent that is expected to be effective to counteract the ftigetactic effect of the tumor when such an amount is present locally at the tumor. That is, the effective local amount may, but does not necessarily, represent the amount of the anti-fugetactic agent that will be administered to the patient.

[0060] The term "plurality" refers to more than one. Thus, a "plurality of tumor cells" refers to at least two, and preferably more than two, tumor cells.

[0061] The term "conditioned medium" refers to a cell culture medium or other aqueous solution that is suitable for short- or long-term cell culture (e.g., saline) that has been incubated with the tumor cells for a period of time such that factors secreted by the tumor cells (e.g., chemokines) are present in the medium Preferably, the cells are removed from the medium prior to using the medium in the methods described herein. Conditioned medium also refers to, for example, conditioned medium that has been processed (e.g., concentrated, dialyzed, lyophilized, etc.) but retains the factors of interest, namely the ftigetactic chemokine(s) secreted by the tumor cells. Anti-fugetactic Agents

[0062] Many tumors have fugetactic effects, e.g. on immune cells, due to chemokines secreted by the tumor cells. High concentrations of chemokines can have fugetactic

(chemorepellant) effects on cells, whereas lower concentrations do not have such effects or even result in chemoattraction. For example, T-cells are repelled by CXCL12 (SDF-1) by a concentration-dependent and CXCR4 receptor-mediated mechanism.

[0063] The anti-fugetactic agent may be any such agent known in the art, for example an anti-fugetactic agent as described in U.S. Patent Application Publication No. 2008/0300165, which is hereby incorporated by reference in its entirety.

[0064] Anti-fugetactic agents include any agents that specifically inhibit chemokine and/or chemokine receptor dimerization, thereby blocking the chemorepellent response to a fugetactic agent. Certain chemokines, including IL-8 and CXCL12 can also serve as chemorepellents at high concentrations (e.g., above 100 nM) where much of the chemokine exists as a dimer. Without being bound by theory, it is believed that dimerization of the chemokine elicits a differential response in cells, causing dimerization of chemokine receptors, an activity which is interpreted as a chemorepellent signal. Blocking the chemorepellent effect of high concentrations of a chemokine secreted by a tumor can be accomplished, for example, by anti-fugetactic agents which inhibit chemokine dimer formation or chemokine receptor dimer formation. For example, antibodies that target and block chemokine receptor dimerization, for example, by interfering with the dimerization domains or ligand binding, can be anti-fugetactic agents. Anti-fugetactic agents that act via other mechanisms of action, e.g. that reduce the amount of fugetactic cy tokine secreted by the cells, inhibit dimerization, and/or inhibit binding of the chemokine to a target receptor, are also encompassed by the present invention. Where desired, this effect can be achieved without inhibiting the chemotactic action of monomelic chemokine.

[0065] In some embodiments, the anti-fugetactic agent is a CXCR4 antagonist, CXCR3 antagonist, CXCR4/CXCL12 antagonist or selective PKC inhibitor.

[0066] The CXCR4 antagonist can be but is not limited to AMD3100, KRH-1636, T-20, T- 22, T-140, TE-14011, T-14012, or TN14003, or an antibody that interferes with the dimerization of CXCR4. Additional CXCR4 antagonists are described, for example, in U.S. Patent Pub. No. 2014/0219952 and Debnath et al. Theranostics, 2013; 3(1): 47-75, each of which is incorporated herein by reference in its entirety, and include TG-0054 (burixafor), AMD3465, NIBR1816, AMD070, and derivatives thereof.

[0067] The CXCR3 antagonist can be but is not limited to TAK-779, AK602, or SCH- 351125, or an antibody that interferes with the dimerization of CXCR3.

[0068] The CXCR4/ CXCL 12 antagonist can be but is not limited to Tannic acid, NSC 651016, or an antibody that interferes with the dimerization of CXCR4 and/or CXCL 12.

[0069] The selective PKC inhibitor can be but is not limited to thalidomide or GF

109230X.

[0070] In a preferred embodiment, the anti-fugetactic agent is AMD3100 (plerixafor). AMD3100 is described in U.S. Patent No. 5,583,131, which is incorporated by reference herein in its entirety. Anti-fugetactic activity of AMD3100 is described in U.S. Patent Application Publication No. 2008/0300165, which is hereby incorporated by reference in its entirety.

[0071] In one embodiment, the anti-fugetactic agent is an AMD3100 derivative. AMD3100 derivatives include, but are not limited to, those found in U.S. Patent Nos. 7,935,692 and 5,583,131 (USRE42152), each of which is incorporated herein by reference in its entirety.

[0072] In one embodiment, the anti-fugetactic agent is coupled with a molecule that allows targeting of a tumor. In one embodiment, the anti-fugetactic agent is coupled with (e.g., bound to) an antibody specific for the tumor to be targeted. In one embodiment, the anti- fugetactic agent coupled to the molecule that allows targeting of the tumor is administered systemically.

[0073] CXCL12 expression by a tumor may also promote tumor growth, angiogenesis, and metastasis. Accordingly, methods for inhibiting tumor growth, angiogenesis, and metastasis are contemplated by this invention. [0074] In one embodiment, the anti-fugetactic agent is administered in combination with an additional compound that enhances the anti-fugetactic activity of the agent. In one embodiment, the additional compound is granulocyte colony stimulating factor (G-CSF). In one embodiment, G-CSF is not administered. When an additional agent is to be administered to the patient, the additional agent may or may not be present in the system as described herein when the effective dose is determined. That is, an additional agent may be added to the culture medium during the migration assay. Preferably, the additional agent is not added to the culture medium.

Methods of Assessing a Therapeutic Amount

[0075] This invention is predicated on the surprising discovery that the effective dose of an anti-fugetactic agent to reduce the fugetactic effects of a tumor is dependent upon factors including the amount of fugetactic chemokine expressed by the tumor (e.g., present in the tumor microenvironment), tumor volume, and/or on the chemokine receptor expression of immune cells.

[0076] In one aspect, this invention is directed to a method for determining an effective local amount of an anti-fugetactic agent for treating a tumor, based on the fugetactic activity of the tumor and/or the receptor expression of at least a subset of immune cells.

[0077] In one aspect, a sample having or suspected of having a fugetactic effect is provided for use in the method described herein. In one embodiment, the sample is a plurality of tumor cells. The tumor cells have or are expected to have a fugetactic activity. In one embodiment, the fugetactic activity is mediated by CXCL12. The tumor cells are preferably taken from a mammalian tumor. In one embodiment, the mammalian tumor has been removed from a subject, e.g. by surgical means. In one embodiment, a plurality of tumor cells is removed from the patient, for example by biopsy. In one embodiment, several samples of cells are removed from a single tumor (for example, from different areas of the tumor). Without being bound by theory, it is believed that different regions of a tumor may express different levels of fugetactic chemokine(s). In some embodiments, the cells taken from different areas of the tumor may be combined, or may be assayed separately. [0078] In one embodiment, the tumor cells are manipulated in vitro to provide the sample. In one embodiment, the tumor cells are expanded in culture to provide a larger number of tumor cells for the method. In one embodiment, the tumor cells are incubated in a cell culture medium or other aqueous solution that is suitable for short- or long-term cell culture (e.g., saline) for a period of time to provide conditioned medium, i.e., such that factors secreted by the tumor cells (e.g., chemokines) are present in the medium Preferably, the cells are removed from the conditioned medium prior to using the conditioned medium in the methods described herein.

[0079] In one embodiment, the sample is a biological sample taken from the tumor microenvironment. In one aspect, the sample is a peripheral blood sample.

[0080] In some embodiments, the sample has been concentrated (e.g., concentrated, lyophilized, dialyzed, or the like) to produce a sample with sufficient fugetactic activity for use in the methods described herein.

[0081] In one embodiment, the sample comprises a known amount of a fugetactic chemokine. In one embodiment, the sample does not comprise and/or is not derived from tumor cells.

[0082] In one aspect of the invention, a plurality of immune cells is provided for use in the methods described herein. Preferably, the immune cells are repelled by the fugetactic activity of the tumor. In one embodiment, the immune cells are capable of migrating towards (e.g., being attracted to) the sample. In one embodiment, the immune cells are heterologous (that is, not from the same patient as the tumor cells or other sample). In one embodiment, the immune cells are autologous (from the same patient as the tumor sample). In a preferred embodiment, the immune cells express a receptor for the fugetactic chemokine(s) expressed by the tumor cells. In another preferred embodiment, the immune cells are T cells and/or NK cells. In an especially preferred embodiment, the chemokine receptor expressed by the cells is CXCR4.

[0083] In one aspect, the number (or average number) of one or more receptors expressed by the immune cells is determined. Such determination may be performed by any means, such as fluorescent activated cell sorting (FACS) or reverse transcriptase polymerase chain reaction (RT-PCR). Preferably, the receptor(s) bind to at least one chemokine, e.g. fugetactic chemokine, expressed by the tumor cells. In one embodiment, the receptor is CXCR4.

[0084] In one aspect, the number (or average number) of one or more receptors expressed by the tumor cells is determined. Preferably, the receptor(s) are a target of the anti -fugetactic agent. In one embodiment, the receptor is CXCR4.

[0085] In one aspect, the method comprises combining the sample and the plurality of immune cells in an apparatus. In one embodiment, the sample is a plurality of tumor cells having a defined tumor volume. In a preferred embodiment, the apparatus further comprises a cell culture medium or other suitable medium In one embodiment, the apparatus comprises a plurality of chambers, and the sample and immune cells are combined in one or more of the plurality of chambers. In this way, multiple amounts of the anti-fugetactic agent can be assayed at one time.

[0086] In one aspect, the method comprises adding a known amount of the anti-fugetactic agent to the medium In one embodiment, increasing amounts of the anti-fugetactic agent are added to a series of chambers. In one embodiment, the sample and immune cells may be added to a chamber and known incremental amounts of the anti-fugetactic agent added over time, with the migration of the immune cells determined at a certain period of time after each incremental addition. In such an embodiment, the aggregate amount of anti-fugetactic agent that was added is used to determine the effective local amount. In a preferred embodiment, the anti-fugetactic agent is an antagonist of CXCR4. In an especially preferred embodiment, the anti-fugetactic agent is AMD3100.

[0087] In one aspect, the method comprises contacting the plurality of immune cells with a known amount of the anti-fugetactic agent prior to combining the immune cells with the sample in the apparatus. Without being bound by theory, it is believed that chemokine receptors on the tumor cells may compete with the immune cells for binding of AMD3100. Pre-incubating the immune cells with AMD3100 prior to combining with the sample allows AMD3100 to specifically bind to receptors on the immune cells.

[0088] In one aspect, the method comprises assessing the effective amount (e.g. , concentration) of the anti-fugetactic agent that reverses the fugetactic effect of the tumor. That is, the amount of the anti-fugetactic agent that allows the immune cells or increasing numbers of immune cells to migrate towards the sample. Where a plurality of amounts is assayed, the effective amount is any amount that allows the immune cells or increasing numbers of immune cells to migrate towards the sample, with the optimal amount being the amount that allows the largest number of immune cells to migrate towards the sample.

However, a skilled clinician would understand that one or several of the effective amounts or optimal amount may be undesirable or particularly desirable, depending on factors including the type and size of the tumor, the planned route of administration, the patient's age, weight, or other characteristics, and the like. Thus, the skilled clinician can choose the effective amount mat is suited to the particular patient.

[0089] In one aspect, the method comprises assessing a therapeutic amount of the anti- fugetactic agent that reverses the fugetactic effect of the tumor when administered to the subject. In one embodiment, the therapeutic amount is equivalent to the effective amount. In one embodiment, the method further comprises determining a therapeutic amount based on the effective amount. For example, the effective amount may be converted to an amount suitable for administration to a subject; adjusted based on tumor volume, patient weight and/or surface area; adjusted based on the expression (average expression) of the chemokine receptor in the subject's immune cells; and the like. In one embodiment, the therapeutic amount of the anti-fugetactic agent is determined based on tumor volume in the patient.

[0090] In one aspect, the method comprises correlating the number (or average number) of CXCR4 receptors expressed by the immune cells, the volume of the plurality of tumor cells and/or the amount of fugetactic chemokine produced by the tumor cells are correlated with the effective amount as determined by the assay to determine the amount of anti-fugetactic agent that should be administered to a patient.

[0091] In one aspect, the method comprises determining a maximum effective

concentration of the anti-fugetactic agent. That is, amounts of anti-fugetactic agent are added to the immune cells or to the culture medium that are higher than the effective amount and/or the optimal amount, such that an amount (or amounts) at which the agent ceases to exhibit the anti-fugetactic property is determined. In one embodiment, the amount at which the agent ceases to exhibit the anti-fugetactic property is an amount at which the anti-fugetactic agent exhibits fugetactic properties.

Correlation

[0092] In one aspect, this disclosure relates to methods of correlating the amount(s) of the anti-fugetactic agent that exhibits an anti-fugetactic effect with regard to a particular sample and/or immune cells with the amount to be administered to a patient.

[0093] In one embodiment, a locally effective amount of anti-fugetactic agent is determined. In one embodiment, the locally effective amount of anti-fugetactic agent is the amount of the anti-fugetactic agent that, when present in the tumor microenvironment, will have an anti-fugetactic effect (e.g., allow immune cells to infiltrate the tumor).

[0094] In one embodiment, a therapeutic amount of anti-fugetactic agent is determined. In one embodiment, the therapeutic amount is an amount of the anti-fugetactic agent that provided an anti-fugetactic effect in vitro in the methods described herein. In one

embodiment, the therapeutic amount is an amount of the anti-fugetactic agent that resulted in migration of immune cells, or increased migration of immune cells, to the tumor cells (or other sample having a fugetactic effect) in the methods described herein. In one embodiment, the therapeutic amount is the amount of the anti-fugetactic agent that resulted in the greatest migration of immune cells to the tumor cells (or other sample having a fugetactic effect) in the methods described herein.

[0095] In one embodiment, a therapeutic amount of anti-fugetactic agent is determined by further assessing the patient and/or tumor. For example, the number of chemokine receptors (e.g., CXCR4) expressed by immune cells, tumor type, tumor volume, amount of CXCL12 or other chemokine secreted by the tumor (e.g., the chemokine concentration of the tumor microenvironment), the weight and/or surface area of the patient, etc., may be analyzed to further refine the therapeutically effective amount.

[0096] In one embodiment, the methods as described herein are used to determine an amount of anti-fugetactic agent to be administered systemically to a patient. In one embodiment, the methods as described herein are used to determine an amount of anti- ftigetactic agent to be administered ex vivo to a known quantity of immune cells (e.g., T cells), the T cells to be administered to a patient. In one embodiment, the methods as described herein are used to determine an amount of anti-fugetactic agent to be administered locally to a tumor or tumor environment in a patient.

[0097] In some embodiments, the anti-fugetactic agent is localized to the tumor by conjugating the anti-fugetactic agent to an antibody specific for an antigen expressed by the tumor.

Systems

[0098] In one aspect, mis disclosure relates to systems for performing the methods as described herein.

[0099] In one aspect, a system is provided comprising an apparatus comprising at least one chamber. In one embodiment, the apparatus is a cell culture dish. In one embodiment, the apparatus is a multi-well cell culture plate. In one embodiment, the chamber comprises a Transwell® or similar permeable support insert.

[0100] In one embodiment, the apparatus comprises an injection port for applying anti- fugetactic agent to the chamber. The injection port can be any opening into the chamber that allows injection of reagents, cells, etc.

[0101] In one aspect, the system further comprises a plurality of immune cells and a sample in the at least one chamber. In one embodiment, the plurality of immune cells and the sample are separated by the permeable support insert. In one embodiment, the sample is a plurality of cells derived from a tumor. In one embodiment, the sample is derived from a tumor, e.g., conditioned medium from culturing of tumor cells. In a preferred embodiment, the sample comprises a fugetactic chemokine.

[0102] In one embodiment, the system further comprises instructions in a readable medium for determining the effective amount of the anti-fugetactic agent using the system.

[0103] The term "readable medium" as used herein refers to a representation of data that can be read, for example, by a human or by a machine. Non-limiting examples of human- readable formats include pamphlets, inserts, or other written forms. Non-limiting examples of machine-readable formats include any mechanism that provides (i.e., stores and/or transmits) information in a form readable by a machine (e.g., a computer, tablet, and/or smartphone). For example, a machine-readable medium includes read-only memory (ROM); random access memory (RAM); magnetic disk storage media; optical storage media; and flash memory devices. In one embodiment, the machine-readable medium is a CD-ROM. In one embodiment, the machine-readable medium is a USB drive. In one embodiment, the machine-readable medium is a Quick Response Code (QR Code) or other matrix barcode.

EXAMPLES

[0104] The following examples are for illustrative purposes only and should not be interpreted as limitations of the claimed invention. There are a variety of alternative techniques and procedures available to those of skill in the art which would similarly permit one to successfully perform the intended invention.

Example 1: Determination of the Anti-fugetactic versus Fugetactic Amount of

AMD3100

[0105] Freshly prepared and purified human CD3⁺ T cells were prepared from healthy donor peripheral blood. 20,000 T cells were loaded into the upper chamber of the Transwell in control, chemotactic or fugetactic settings with AMD3100 at concentrations between 0.1 μΜ and 10 μΜ. Migrated cells were counted in the lower chamber and migration quantitated as previously described. Vianello et al. The Journal of Immunology, 2006, 176: 2902-2914; Righi et al., Cancer Res. ; 71(16); 5522-34, each of which is incorporated herein in its entirety.

[0106] Clear evidence of binary or bimodal chemotactic (Figure 1; CI 2.3 at 1 μΜ) and fugetactic (Figure 2; CI = 1.6 at 0.1 μΜ) responses of human CD3+ T cells to AMD3100 (where a CI or chemotactic index of 1.0 is the control) was observed. All wells were run in triplicate. Example 2: Determination of a Local Anti-fugetactic Amount of AMD3100

[0107] For quantitative transmigration assays, purified human CD3¹ T cells (approximately 2 x 10⁴ cells) are added to the upper chamber of a Transwell® insert in each well, to a total volume of ISO μΐ of Iscove's modified medium. Tumor cells isolated from a mammalian tumor in DMEM containing 0.5% FCS, are added in the lower, upper, or both lower and upper chambers of the Transwell to generate a standard "checkerboard" analysis of cell migration, including measurements of chemotaxis, fugetaxis, and chemokinesis.

[0108] To determine the anti-fugetactic concentration of AMD3100, the T cells are incubated with 0.01 μΜ to 10 mM AMD3100 prior to addition to the chamber.

[0109] Cells are harvested from the lower chamber after 3 h, and cell counts are performed using a hemocytometer.

[0110] It is expected that T cells that are pre-incubated with a concentration of AMD3100 will exhibit a bimodal effect, with anti-fugetactic effects observed at lower concentrations and fugetactic effects at higher concentrations.

Example 3: Determination of a Local Anti-fugetactic Amount of AMD3100

[0111] For quantitative transmigration assays, purified human CD3⁺ T cells (approximately 2 x l0⁴ cells) are added to the upper chamber of a Transwell® insert in each well, to a total volume of 150 μΐ of Iscove's modified medium. Tumor cells isolated from a mammalian tumor in DMEM containing 0.5% FCS, are added in the lower, upper, or both lower and upper chambers of the Transwell to generate a standard "checkerboard" analysis of cell migration, including measurements of chemotaxis, fugetaxis, and chemokinesis.

[0112] To determine the anti-fugetactic concentration of AMD3100, 0.01 μΜ to 10 mM of AMD3100 is added to the upper or lower chamber of the insert.

[0113] Cells are harvested from the lower chamber after 3 h, and cell counts are performed using a hemocytometer. [0114] It is expected that T cells that are pre-incubated with a concentration of AMD3100 will exhibit a bimodal effect, with anti-fugetactic effects observed at lower concentrations and fugetactic effects at higher concentrations.

Example 4: Determination of a Therapeutic Amount of AMD3100 for Administration to a Patient

[0115] The method as described in Example 2 is performed, using tumor cells and CD3⁺ T cells taken from a human patient. In other embodiments, PBMCs are used.

[0116] To determine the anti-fugetactic concentration of AMD3100, the T cells are incubated with 0.01 μΜ to 10 mM AMD3100 prior to addition to the chamber.

[0117] Cells are harvested from the lower chamber after 3 h, and cell counts are performed using a hemocytometer.

[0118] It is expected that T cells that are pre-incubated with a concentration of AMD3100 will exhibit a bimodal effect, with anti-fugetactic effects observed at lower concentrations and fugetactic effects at higher concentrations.

Claims

WHAT IS CLAIMED IS:

1. A method for determining the therapeutic dose of an anti-fugetactic agent in a subj ect with cancer, said method comprising:

a) providing a tumor sample comprising a fugetactic chemokine;

b) combining into an apparatus comprising a culture medium said tumor sample with a plurality of immune cells which express a receptor for said chemokine, each of said plurality of cells having a defined number and volume, said apparatus permits the monitoring of said immune cells relative to said tumor sample;

c) contacting the culture medium with known incremental amounts of an anti- fugetactic agent;

d) assessing the aggregate concentration at which said anti-fugetactic agent permits said immune cells or increasing numbers of immune cells to migrate towards said tumor sample, thereby determining the effective local amount of said anti-fugetactic agent that reverses the fugetactic properties of said tumor sample.

2. A method for determining the therapeutic dose of an anti-fugetactic agent in a subject with a cancerous tumor, said method comprising:

a) providing a tumor sample comprising a fugetactic chemokine;

b) providing a plurality of immune cells which express a receptor for said chemokine, each of said plurality of cells having a defined number and volume, dividing the immune cells into a plurality' of samples each of which have a defined number, and exposing each of said immune cell sample to a known different amount of an anti-fugetactic agent under conditions wherein said receptors of said immune cells bind said anti-fugetactic agent; c) dividing the tumor sample into separate samples wherein the number of separate samples correspond to the number of immune cell samples prepared in b);

d) combining each of said immune cell samples with one of the separate samples into a separate apparatus containing a culture medium, such that each apparatus contains immune cells with a different amount of anti-fugetactic agent bound thereto;

e) assessing which apparatus demonstrates that said immune cells or increasing numbers of immune cells migrate towards said tumor sample; and f) correlating that apparatus having the lowest concentration of anti-fugetactic agent per immune cell which exhibits anti-fugetactic properties to the effective concentration required for anti-fugetaxis for that tumor.

3. The method of claims 1 or 2, wherein the tumor sample comprises tumor cells, tumor cell-conditioned media, a tumor cell line, a defined concentration of a fugetactic agent, expanded tumor cells, or a sample taken from a tumor microenvironment.

4. The method of claim 3, wherein the tumor sample comprises a plurality of tumor cells that express the fugetactic chemokine.

5. The method of any one of the above claims, wherein the tumor sample is or is derived from tumor cells from the subject.

6. The method of any one of the above claims, wherein said anti-fugetactic agent is selected from the group consisting of AMD3100 or derivative thereof, KRH-1636, T-20, T- 22, T-140, TE-14011, T-14012, TN14003, TAK-779, AK602, SCH-351125, Tannic acid, NSC 651016, thalidomide, and GF 109230X.

7. The method of any one of the above claims, wherein the fugetactic chemokine is CXCL12.

8. The method of any one of the above claims, wherein the chemokine receptor is CXCR4.

9. The method of any one of the above claims, wherein the subject is a human.

10. The method of any one of the above claims, wherein the immune cells are derived from the subject.

11. The method of any one of the above claims, wherein the immune cells are T cells.

12. The method of claim 6, wherein the anti-fugetactic agent is AMD3100.

13. The method of any one of the above claims, further comprising determining an average number of CXCR4 expressed by the immune cells.

14. The method of any one of the above claims, wherein the assessing step further comprises correlating the average number of CXCR4 expressed by the immune cells with the tumor cell volume to determine the effective local amount of said anti-fugetactic agent.

15. The method of any one of the above claims, further comprising:

determining a maximum effective concentration of said anti-fugetactic agent by further adding known incremental amounts of said agent into said chamber to determine at which concentration said agent ceases to permit said immune cells or increasing numbers of immune cells to migrate towards said tumor cells.

16. A method for determining an effective local amount of an anti-fugetactic agent, said method comprising:

a) providing a tumor sample having fugetactic properties;

b) combining into an apparatus comprising a culture medium said tumor sample with a plurality of immune cells, said plurality of immune cells being capable of attraction to said tumor sample but which attraction is inhibited by the fugetactic properties of said tumor sample, wherein said apparatus permits the monitoring of said immune cells relative to said tumor sample;

c) contacting the culture medium with known incremental amounts of an anti- fugetactic agent;

d) assessing the aggregate concentration at which said anti-fugetactic agent permits said immune cells or increasing numbers of immune cells to migrate towards said tumor sample, thereby determining the effective local amount of said anti-fugetactic agent that reverses the fugetactic properties of said tumor sample.

17. The method of claim 16, wherein the tumor sample comprises a plurality' of tumor cells derived from a mammalian tumor, said plurality of cells having a defined tumor cell volume.

18. A method for determining an effective local amount of an anti-fugetactic agent, said method comprising:

a) providing a plurality of tumor cells derived from a mammalian tumor, said plurality of cells having a defined tumor cell volume and further having fugetactic properties; b) combining into a plurality of apparatuses comprising a culture medium said plurality of tumor cells with a plurality of immune cells, said immune cells being capable of attraction to said tumor cells but which attraction is inhibited by the fugetactic properties of said tumor cells, wherein said apparatuses permit the monitoring of said immune cells relative to said tumor cells;

c) contacting the culture medium with a plurality of known amounts of an anti- fugetactic agent; and

d) assessing an amount of anti-fugetactic agent at which said immune cells or increasing numbers of immune cells migrate towards said tumor cells, thereby' determining the effective local amount of said anti-fugetactic agent that reverses the fugetactic properties of said known volume of tumor cells.

19. A method for determining an effective local amount of an anti-fugetactic agent, said method comprising:

a) contacting at least two pluralities of immune cells with known amounts of an anti-fugetactic agent such that each plurality is contacted with a different amount of the anti- fugetactic agent;

b) combining each plurality of tumor cells with a plurality of immune cells derived from a mammalian tumor, said plurality of cells having a defined tumor cell volume and further having fugetactic properties; and

c) assessing an amount of the anti-fugetactic agent at which said immune cells or increasing numbers of immune cells migrate towards said tumor cells, thereby determining the effective local amount of said anti-fugetactic agent that reverses the fugetactic properties of said volume of tumor cells.

20. A method for determining an effective local amount of an anti-fugetactic agent, said method comprising:

a) providing a plurality of tumor cells derived from a mammalian tumor, said plurality of cells having a defined tumor cell volume and further having fugetactic properties; b) placing said plurality of tumor cells into a culture medium for a period of time to provide a conditioned medium; c) combining into an apparatus said conditioned medium with a plurality of immune cells, said plurality of immune cells being capable of attraction to said tumor cells but which attraction is inhibited by the fugetactic properties of said conditioned medium, wherein said apparatus permits the monitoring of said immune cells relative to said conditioned medium;

d) contacting the immune cells with known incremental amounts of an anti- fugetactic agent; and

e) assessing the aggregate concentration at which said anti-fugetactic agent permits said immune cells or increasing numbers of immune cells to migrate towards said conditioned medium, thereby determining the effective local amount of said anti-fugetactic agent that reverses the fugetactic properties of said conditioned medium.

21. A method for determining the therapeutic dose of an anti-fugetactic agent in a subject with cancer, said method comprising:

a) obtaining from said subject a plurality of tumor cells which express a fugetactic chemokine, and a plurality of immune cells which express a receptor for the chemokine, each of said plurality of cells having a defined number and volume;

b) dividing the immune cells into first, second and further samples, and

(i) exposing a first immune cell sample to a known first amount of an anti- fugetactic agent,

(ii) exposing a second immune cell sample to a known second amount of an anti-fugetactic agent,

(ii) exposing further immune cell samples to known further amounts of an anti-fugetactic agent,

wherein the first, second and further amounts are different;

c) dividing the tumor cells into first, second and further tumor samples;

d) combining the first immune cell sample and the first tumor sample to an apparatus comprising a culture medium, said apparatus permits the monitoring of said immune cells relative to said tumor cells; and

e) assessing the aggregate concentration at which said anti-fugetactic agent permits said immune cells or increasing numbers of immune cells to migrate towards said tumor cells, thereby determining the effective local amount of said anti-fiigetactic agent that reverses the fugetactic properties of said known volume of tumor cells.

22. The method of any one of claims 16-21, wherein the tumor cells or tumor sample are derived from a tumor in a human patient.

23. The method of claim 22, wherein the immune cells are derived from the human patient.

24. The method of any one of claims 1, wherein the immune cells are T cells.

25. The method of any one of claims 16-24, wherein said anti-fugetactic agent is selected from the group consisting of AMD3100 or derivative thereof, KRH-1636, T-20, T-22, T-140, TE-14011, T-14012, TN14003, TAK-779, AK602, SCH-351125, Tannic acid, NSC 651016, thalidomide, and GF 109230X.

26. The method of claim 25, wherein the anti-fugetactic agent is AMD3100.

27. The method of any one of claims 16-26, further comprising determining an average number of CXCR4 expressed by the immune cells.

28. The method of any one of claims 16-27, wherein the assessing step further comprises correlating the average number of CXCR4 expressed by the immune cells with the tumor cell volume to determine the effective local amount of said anti-fugetactic agent.

29. The method of any one of claims 16-28, further comprising:

determining a maximum effective concentration of said anti-fugetactic agent by further adding known incremental amounts of said agent into said chamber to determine at which concentration said agent ceases to permit said immune cells or increasing numbers of immune cells to migrate towards said tumor cells.

30. A system for determining an effective local amount of an anti-fugetactic agent, said system comprising:

a) an apparatus having an injection port and a chamber where the contents of said chamber are capable of being monitored; b) said chamber comprising a plurality of mammalian tumor cells derived from a selected tumor, said plurality of cells having a defined tumor cell volume, a culture medium and a plurality of immune cells, said immune cells being capable of migrating to said tumor cells but, in the absence of an effective amount of an anti-fugetactic agent, are repelled by the fugetactic properties of said tumor cells; and

c) said injection port being configured to permit the addition of sequentially increasing known amounts of an anti-fugetactic agent to said medium such that when an effective amount of said anti-fugetactic agent is included in said medium, said immune cells will migrate toward said tumor cells.

31. A system for determining an effective amount of an anti-fugetactic agent, said system comprising:

a) an apparatus having an injection port and a chamber where the contents of said chamber are capable of being monitored;

b) said chamber comprising a plurality of human tumor cells derived from a patient with a tumor, said plurality of cells having a defined tumor cell volume, a culture medium and a plurality of immune cells said immune cells being capable of migrating to said tumor cells but, in the absence of an effective amount of an anti-fugetactic agent, are repelled by the fugetactic properties of said tumor cells; and

c) said injection port being configured to permit the addition of sequentially increasing known amounts of an anti-fugetactic agent to said medium such that when an effective amount of said anti-fugetactic agent is included in said medium, said immune cells will migrate toward said tumor cells.

32. The system of claim 30 or 31 , wherein the apparatus is a cell culture dish.

33. The system of claim 30 or 31, wherein the apparatus comprises a plurality of chambers comprising said tumor cells and said immune cells.