Method of generating TILS from cerebro spinal fluid for leptomeningeal disease

Abstract

Disclosed are methods of isolating and expanding tumor infiltrating lymphocytes (TILs) from cerebrospinal fluid and the use of said TILs in the treatment of Leptomeningeal disease.

Description

[0001] METHOD OF GENERATING TILS FROM CEREBRO SPINAL FLUID FOR LEPTOMENINGEAL DISEASE

[0002] I. BACKGROUND

[0003] Leptomeningeal disease (LMD) has a grave prognosis with a median survival of 6-8 weeks. Melanoma has the greatest likelihood of metastasizing to the central nervous system (CNS), and the incidence of LMD is highest in melanoma (5-25%), which carries the worst prognosis. M- LMD is becoming increasingly prevalent as patients survive longer with improved therapies. Nevertheless, the pathophysiology of LMD is poorly understood and there are no effective therapies for LMD. The advent of immunocellular therapies revolutionized the treatment for metastatic melanoma and may be a promising approach for LMD. Immune checkpoint inhibitors (ICIs) in melanoma BM showed intracranial response, but 50% of asymptomatic and 80% of symptomatic patients do not respond to treatment. In LMD, ICIs showed a modest improved survival both via and intrathecal (IT) delivery, with up to ~616to 7.5-month survival. What are needed are new methods of treating LMD.

[0004] II. SUMMARY

[0005] Disclosed are methods related to the isolation, expansion and use of tumor infiltrating lymphocyte (TILs) from cerebrospinal fluid.

[0006] In one aspect, disclosed herein are methods of treating, reducing, decreasing, inhibiting, ameliorating, and / or preventing leptomeningeal disease (LMD)(including, but not limited to melanoma LMD (M-LMD)) in a subject comprising administering (such as, intrathecal administration including, but not limited to use of an Ommaya reservoir) to the subject tumor infiltrating lymphocytes (TILs) obtained from cerebrospinal fluid and expanded ex vivo. In some aspects TILs are autologous to the subject.

[0007] Also disclosed are methods of treating, reducing, decreasing, inhibiting, ameliorating, and / or preventing LMD of any preceding aspect, further comprising culturing the TILs in media comprising IL-2, IL-2 + an anti-CD3 antibody, IL-2 + an anti-4- IBB agonistic antibody, IL-2 + an anti-CD28 antibody, and / or IL-2+ any combination of an anti-CD3 antibody, anti-CD28 antibody, and / or an anti-4- IBB agonistic antibody to expand the TILs and harvesting the expanded TILs. In some aspects, the culture is maintained at normoxic or hypoxic conditions.

[0008] In one aspect, disclosed herein are methods of treating, reducing, decreasing, inhibiting, ameliorating, and / or preventing LMD of any preceding aspect wherein the TILs are cultured for at least 4 weeks. III. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and together with the description illustrate the disclosed compositions and methods.

[0010] Figure 1 shows the proposed schema for T cell expansion from CSF for ACT. (1 ) CSF obtained either from an Ommaya Tap (top) or lumbar puncture (bottom). (2a) After centrifugation, a total cell count is achieved and plated in an appropriate plate for culture, making its way from a 96-well U-bottom plate to a 96-well flat-bottom plate to a (2b) 48-well plate to a (2c) 24 well plate. (3) After 4-6 weeks in culture, the T cells are collected and counted to be placed in either a rapid expansion protocol or (4) used in a reactivity assay with HLA- matched melanoma cell lines (after HL A matching). (5) After identifying reactive clones, the product would be introduced back into the patient following a clinical-scale REP.

[0011] Figures 2A, 2B, 2C, 2D, 2E, 2F, and 2G show flow cytometry phenotypes as part of a screening tool after initial culture. Figure 2A shows CD3+ and CD3-CD56+ frequency of T cells collected after 4-6 weeks. Figure 2B shows CD4+ and CD8+ frequency of T cells collected after 4-6 weeks in culture. Figure 2C shows representative gating strategy for a preREP CSF T cell culture (CSF001 Day 133 0KT3). Figure 2D shows phenotype of CSF001 samples cultured in IL-2 only (6000IU / mL). Figure 2E shows phenotype of CSF001 samples cultured in IL-2+ agonistic 4-1BB antibody (lOug / mL). Figure 2F shows phenotype of CSF001 samples cultured in IL-2+OKT3 (lOng / mL). Figure 2G shows phenotype of CSF001 samples cultured in IL-2 +CD3 / CD28 human dynabeads (5beads: ITcell).

[0012] Figure 3A shows preREP fold expansion of CSF T cell samples cultured in media with IL-2 only (6000IU / mL).

[0013] Figure 3B shows REP fold expansion from 21 unique patient samples.

[0014] Figure 3C shows preREP fold expansion for CSF001 across 4 unique culture conditions (left) and the associated growth kinetics curve (right; normalized to the surface area of a traditional 24 well plate). Two separate unpaired t-tests were performed, comparing the means of the preREP fold expansion in IL-2 + 0KT3 (*,p<0.05) and IL-2+ anti-CD3 / CD28 human dynabeads (**,p<0.01)

[0015] Figure 4 shows preliminary reactivity data for patient 6.Sample was selected for coculture with HLA-matched melanoma cell lines as having more than 25% CD8+ T cells (in a postREP sample). For the 4 culture conditions outlined, 1) T cells + HLA-matched cell lines, 2) T cells + w6 / 32 MHC Class I blocked cell lines, 3) T cells alone, 4) T cells + CD3 / CD28 Human Dynabeads (1:1), cells were to be plated at IxlO5cells per condition and incubated for approximately 18-24 hours at which point supernatants were collected and analyzed for IFN-y release. An additional HLA-mismatched cell line (WM2032) was plated for this co-culture to serve as an extra control.

[0016] Figure 5 shows preliminary reactivity data for patient 1 across 3 unique preREP culture conditions. Indicated patient 1 samples were screened across 3 plating conditions for more than 25% CD8+ T cells and 3 samples were selected for co-culture with HLA-matched melanoma cell lines. For the 4 culture conditions outlined, 1) T cells + HLA-matched cell lines, 2) T cells + w6 / 32 MHC Class I blocked cell lines, 3) T cells alone, 4) T cells + CD3 / CD28 Human Dynabeads (1:1), cells were to be plated at IxlO5cells precondition and incubated for approximately 18-24 hours at which point supernatants were collected and analyzed for IFN-y release. Assay was conducted with postREP T cell cultures following standard rapid expansion protocol.

[0017] Figure 6A, 6B, 6C, and 6D show phenotype of T cells expanded from CSF. Figure 6A shows CD4 and CDS ratio of T cells expanded after 4-6 weeks. Figure 6B shows phenotype cultured in IL-2 + 0KT3 (10 ng / ml). Figure 6C shows phenotype cultured in IL-2 + 4-1 BB (10 ug / ml). Figure 6D shows phenotype following 2 weeks in rapid expansion.

[0018] Figure 7 shows Post-REP reactivity in samples of >25% CDS+ T cells, co-cultured with HL.A-matched melanoma cells lines, and HLA unmatched tumor cell line WM2032. T cells alone, -i-tumor cells, +turnor cells+W6 / 32. Cell lines were plated at 1 x 105cells per condition and incubated overnight for 18-24 hours. Supernatant was analyzed for cytokines IFN-y, granzyme B, and TNF-a.

[0019] Figure 8. BM TILs are highly reactive. TILs were expanded from 2 fragments from melanoma BM. TIL.s were collected after 4 weeks of culture and co-cultured overnight with autologous tumor. MHC class I was blocked by incubating autologous tumor with W6 / 32. antibody. Supernatants were collected and IFN-y was measured by ELISA.

[0020] Figure 9 shows Tumor burden decrease with TILs. NOG-IL2 mice were injected subcutaneously with autologous tumor. When tumors reached 25-50 mm2, mice were injected with BM TIL. Control mice received no TIL.

[0021] IV. DETAILED DESCRIPTION

[0022] Before the present compounds, compositions, articles, devices, and / or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods or specific recombinant biotechnology methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0023] A. Definitions

[0024] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like.

[0025] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed the “less than or equal to 10”as well as “greater than or equal to 10” is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0026] In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:

[0027] “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0028] An "increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition or activity. An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, the increase can be a 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, 90, 95, or 100% increase so long as the increase is statistically significant.

[0029] A "decrease" can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease can be a 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, 90, 95, or 100% decrease so long as the decrease is statistically significant.

[0030] "Inhibit," "inhibiting," and "inhibition" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

[0031] By “reduce” or other forms of the word, such as “reducing” or “reduction,” is meant lowering of an event or characteristic (e.g., tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, “reduces tumor growth” means reducing the rate of growth of a tumor relative to a standard or a control.

[0032] By “prevent” or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed.

[0033] The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. In one aspect, the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline. The subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.

[0034] The term “therapeutically effective” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.

[0035] The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

[0036] "Biocompatible" generally refers to a material and any metabolites or degradation products thereof that are generally non-toxic to the recipient and do not cause significant adverse effects to the subject.

[0037] "Comprising" is intended to mean that the compositions, methods, etc. include the recited elements, but do not exclude others. "Consisting essentially of' when used to define compositions and methods, shall mean including the recited elements, but excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. "Consisting of' shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions provided and / or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0038] A “control” is an alternative subject or sample used in an experiment for comparison purposes. A control can be "positive" or "negative." “Effective amount” of an agent refers to a sufficient amount of an agent to provide a desired effect. The amount of agent that is “effective” will vary from subject to subject, depending on many factors such as the age and general condition of the subject, the particular agent or agents, and the like. Thus, it is not always possible to specify a quantified “effective amount.” However, an appropriate “effective amount” in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an “effective amount” of an agent can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts. An “effective amount” of an agent necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0039] A "pharmaceutically acceptable" component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation provided by the disclosure and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained. When used in reference to administration to a human, the term generally implies the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.

[0040] "Pharmaceutically acceptable carrier" (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic and includes a carrier that is acceptable for veterinary and / or human pharmaceutical or therapeutic use. The terms "carrier" or "pharmaceutically acceptable carrier" can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil / water or water / oil emulsion) and / or various types of wetting agents. As used herein, the term "carrier" encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and as described further herein.

[0041] “Pharmacologically active” (or simply “active”), as in a “pharmacologically active” derivative or analog, can refer to a derivative or analog (e.g., a salt, ester, amide, conjugate, metabolite, isomer, fragment, etc.) having the same type of pharmacological activity as the parent compound and approximately equivalent in degree. “Therapeutic agent” refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition (e.g., a non-immunogenic cancer). The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the terms “therapeutic agent” is used, then, or when a particular agent is specifically identified, it is to be understood that the term includes the agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.

[0042] “Therapeutically effective amount” or “therapeutically effective dose” of a composition (e.g. a composition comprising an agent) refers to an amount that is effective to achieve a desired therapeutic result. In some embodiments, a desired therapeutic result is the control of type I diabetes. In some embodiments, a desired therapeutic result is the control of obesity. Therapeutically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject. The term can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect, such as pain relief. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the agent and / or agent formulation to be administered (e.g., the potency of the therapeutic agent, the concentration of agent in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art. In some instances, a desired biological or medical response is achieved following administration of multiple dosages of the composition to the subject over a period of days, weeks, or years.

[0043] Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.

[0044] B. Method of treating Leptomeningeal disease (LMD)

[0045] Adoptive T Cell Therapy (ACT) using tumor infiltrating lymphocytes (TILs) is a novel, successful therapeutic approach demonstrating complete and durable responses in patients with advanced metastatic melanoma, including regression of brain metastases (BM). The phenotype and function of T cells in CSF was previously unknown, and no one was able to expand tumor reactive T cells from CSF and study its therapeutic effect in LMD. Data provided herein shows successful expansion of tumor-reactive T cells (i.e., CD8+ T cells) ex vivo from the cerebrospinal fluid (CSF) of patients with melanoma LMD (M-LMD). We also have strong data supporting that the tumor microenvironment (TME) in the cerebrospinal fluid (CSF) of patients with M-LMD is significantly different at the molecular and cellular level from the TME of BM and extracranial metastases (EM). Using single cell RNA sequencing (sc RNAseq), the studies show that T cell populations shift to more exhausted phenotypes in the CSF of patients with LMD compared to EM and BM. Although the leptomeningeal space are suppressive environments, ex vivo expansion of tumor-reactive T cells with large expansion in the sum of billions can be generated and infused back into a patient to target the tumor cells and treat LMD. T cells in the CSF can be expanded to clinically relevant numbers for ACT and have significant therapeutic anti-tumor responses in LMD. The basis for the approach described herein of using T cells derived from the CSF is based on the idea that 1) T cells recognize antigens in LMD tumor cells that were not identical to antigens in the systemic tumors, and 2) that T cells in the CSF space have adapted to the unique environment of the CSF and better able to survive in that environment.

[0046] The disclosed compositions can be used to treat any disease where uncontrolled cellular proliferation occurs such as cancers. A representative but non-limiting list of cancers that the disclosed compositions can be used to treat is the following: lymphomas such as B cell lymphoma and T cell lymphoma; mycosis fungoides; Hodgkin’s Disease; myeloid leukemia (including, but not limited to acute myeloid leukemia (AML) and / or chronic myeloid leukemia (CML)); bladder cancer; brain cancer; nervous system cancer; head and neck cancer; squamous cell carcinoma of head and neck; renal cancer; lung cancers such as small cell lung cancer, nonsmall cell lung carcinoma (NSCLC), lung squamous cell carcinoma (LUSC), and Lung Adenocarcinomas (LUAD); neuroblastoma / glioblastoma; ovarian cancer; pancreatic cancer; prostate cancer; skin cancer; hepatic cancer; melanoma; squamous cell carcinomas of the mouth, throat, larynx, and lung; cervical cancer; cervical carcinoma; breast cancer including, but not limited to triple negative breast cancer; genitourinary cancer; pulmonary cancer; esophageal carcinoma; head and neck carcinoma; large bowel cancer; hematopoietic cancers; testicular cancer; and colon and rectal cancers.

[0047] The way melanoma tumor cells interact within the tumor microenvironment (TME) in distal sites plays a key role in metastasis and tumor survival. The mechanisms by which cancer cells invade, adapt, and survive in the leptomeninges is poorly understood. We have strong data supporting that the TME in the cerebrospinal fluid (CSF) of patients with M-LMD is significantly different at the molecular and cellular level from the TME of BM and extracranial metastases (EM), and that TME of BM differs from that of the EM. Normal CSF composition is nearly acellular and contains mostly CD4+ T cells. 19 Once, cancer cells access into the leptomeningeal space they trigger an inflammatory response with innate immune responses. TILs activate a strong adaptive immune response. Immunomodulatory mechanisms seem to be key players with inflammatory cells like lymphocytes, macrophages, and dendritic cells. Tumor cells must learn to evade the immune system and adapt to the harsh microenvironment of the leptomeningeal space (devoid of oxygen and micronutrients) to survive. In LMD, tumor cells survive under hypoxic and low iron conditions by relying on lipocalin-2 (LCN2), induced by macrophage-derived cytokines.

[0048] We have strong data from comparing T cell populations from CSF in patients with LMD using single cell RNA sequencing (sc RNAseq) that show the T cell populations shifts to more exhausted phenotype, with highest numbers of exhausted and apoptotic T cells, that we believe reflects metabolic adaptation to the harsh, poor nutrient CSF TME. Increased numbers of T cells and dendritic cells (DCs) in CSF were associated with prolonged survival in M-LMD, whereas poor T cell responses were associated with poor prognosis. Interferon gamma (IFN-y) is a classical cytokine with tumor suppressive capability derived mostly by Thl CD4+ T cells and CD8+ T cells, natural killer (NK) cells, NKT cells, and few other immune cells. The proportion of immune cells expressing IFN-y in CSF is inadequately low. Increased levels of IFN-y in CSF plays a key role in recruitment and activation of peripheral myeloid cells, causing a variety of DC subtypes. In addition, a rare sub-population of DCs was associated with increased survival and via modulatory T-cell mediated mechanisms and histocompatibility complex (MHC) expression. This led us to hypothesize that CSF T cells that survive in CSF have adapted to this different environment to be more efficient for tumor killing, disclosed are methods related to the isolation, expansion and use of tumor infiltrating lymphocyte (TILs) from cerebrospinal fluid.

[0049] In one aspect, the treatment of the LMD can include adoptive cell therapy employing administration of TILs obtained from CSF and expanded ex vivo. For example, disclosed herein are methods of treating, reducing, decreasing, inhibiting, ameliorating, and / or preventing leptomeningeal disease (LMD)(including, but not limited to melanoma LMD (M-LMD)) in a subject comprising administering to the subject tumor infiltrating lymphocytes (TILs) obtained from cerebrospinal fluid (CSF) and expanded ex vivo. In some aspects TILs are autologous to the subject. It is understood and herein contemplated that the isolation of TILs from any sample including CSF does note produce sufficient numbers to provide a therapeutic effect in an adoptive cell therapy comprising administering the isolated TILs to the subject. Accordingly, the TILs can be cultured to expand the TIL numbers.

[0050] The culture process employed by the art understood methods takes 5-7 weeks to expand TILs. This is a significant problem in the art as additional time to initiating adoptive transfer therapy of TILs represents an increased risk to the patient due to progression of malignancy while the cell product is being prepared. Moreover, the added time needed for culturing requires additional resources of the hospital in additional personnel to requirements to maintain the culture and costs for media and maintaining a cleanroom. The present method decreases the expansion time to less than 5 weeks resulting in decreased attrition patients from therapy secondary to disease progression. For example, culturing to obtain an expanded population of TILs can occur for any time between 1 day and 5 weeks (35 days), preferably between 21 days (3 weeks) and 5 weeks (35 days), more preferably between 4 weeks (28 days) and 5 weeks (35 days). For example, the culture time can be less than 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, or 35 days. Accordingly, disclosed herein are methods of treating, reducing, decreasing, inhibiting, ameliorating, and / or preventing LMD of any preceding aspect wherein the TILs are cultured for at least 4 weeks.

[0051] As shown herein, culturing conditions are important to the expansion of the TIL population. Such conditions can include the addition of cytokines, chemokines, and or antibodies that will stimulate the TIL population, including, but not limited to IL-2, anti-CD3, anti-CD28, anti-4- IBB agonistic antibody or other agonist, and any combination thereof. Thus, in one aspect, disclosed herein are methods of treating, reducing, decreasing, inhibiting, ameliorating, and / or preventing LMD, further comprising culturing the TILs in media comprising IL-2, IL-2 + an anti-CD3 antibody, IL-2 + an anti-4- IBB agonistic antibody, IL-2 + an anti-CD28 antibody, and / or IL-2+ any combination of an anti-CD3 antibody, anti-CD28 antibody, and / or an anti-4- IBB agonistic antibody to expand the TILs and harvesting the expanded TILs. In some aspects, the culture is maintained at normoxic or hypoxic conditions.

[0052] To maintain the quality of the nutrients in culture and remove any waste, it is understood and herein contemplated that the all or a portion of the media in the reservoir may be exchanged. The exchange of media can comprise 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% removal and replacement of media. This media exchange can be accomplished employing any acceptable method for proper tissue culture maintenance known in the art. In one aspect, the media exchange can occur at least one time during the culture of the TILs. For example, the media in the reservoir can be exchanged 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, or 35 times during the culture period. That is, the media exchange can occur once during the culture period, once every 15 days, once every 10 days, once every 7 days, once every 5 days, once every other day, or about 2 to 3 times per week.

[0053] The culture methods employed herein can utilize any complete media comprising IL-2 appropriate for the growth and propagation of the TILs, including, but not limited to Minimum Essential Medium (MEM), Eagles’s Minimum Essential Medium (EMEM), Dulbecco’s Minimum Essential Medium (DMEM) Medium 199, RPMI 1640, CMRL-1066, BGJb Medium, Iscove’s Modified Dulbecco’s Medium (IMDM), and Blood Cell Media.

[0054] TILs can be administered by any route that is therapeutically efficacious including, but not limited to intrathecal administration such as, for example, through the use of an Ommaya reservoir.

[0055] It is understood and herein contemplated that the disclosed treatment regimens can used alone or in combination with any anti -cancer therapy known in the art including, but not limited to Abemaciclib, Abiraterone Acetate, ABITREXATE® (Methotrexate), ABRAXANE® (Paclitaxel Albumin-stabilized Nanoparticle Formulation), ABVD, ABVE, ABVE-PC, AC, AC- T, ADCETRIS® (Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine, ADRIAMYCIN® (Doxorubicin Hydrochloride), Afatinib Dimaleate, AFINITOR® (Everolimus), AKYNZEO® (Netupitant and Palonosetron Hydrochloride), ALDARA® (Imiquimod), Aldesleukin, ALECENSA® (Alectinib), Alectinib, Alemtuzumab, ALIMTA® (Pemetrexed Disodium), ALIQOPA® (Copanlisib Hydrochloride), ALKERAN™ for Injection (Melphalan Hydrochloride), ALKERAN™ Tablets (Melphalan), ALOXI® (Palonosetron Hydrochloride), ALUNBRIG® (Brigatinib), AMBOCHLORIN® (Chlorambucil), AMBOCLORIN® (Chlorambucil), Amifostine, Aminolevulinic Acid, Anastrozole, Aprepitant, AREDIA® (Pamidronate Disodium), ARIMIDEX® (Anastrozole), AROMASIN® (Exemestane),ARRANON® (Nelarabine), Arsenic Trioxide, ARZERRA® (Ofatumumab), Asparaginase Erwinia chrysanthemi, Atezolizumab, AVASTIN® (Bevacizumab), Avelumab, Axitinib, Azacitidine, BAVENCIO® (Avelumab), BEACOPP, BECENUM® (Carmustine), BELEODAQ® (Belinostat), Belinostat, Bendamustine Hydrochloride, BEP, BESPONSA® (Inotuzumab Ozogamicin) , Bevacizumab, Bexarotene, BEXXAR® (Tositumomab and Iodine I 131 Tositumomab), Bicalutamide, BICNU® (Carmustine), Bleomycin, Blinatumomab, BLINCYTO® (Blinatumomab), Bortezomib, BOSULIF® (Bosutinib), Bosutinib, Brentuximab V edotin, Brigatinib, BuMel, Busulfan, BUSULFEX® (Busulfan), Cabazitaxel, CABOMETYX® (Cabozantinib-S-Malate), Cabozantinib-S-Malate, CAF, CAMPATH® (Alemtuzumab), CAMPTOSAR® (Irinotecan Hydrochloride), Capecitabine, CAPOX, CARAC® (Fluorouracil— Topical), Carboplatin, CARBOPLATIN-TAXOL, Carfilzomib, CARMUBRIS® (Carmustine), Carmustine, Carmustine Implant, CASODEX® (Bicalutamide), CEM, Ceritinib, CERUBIDINE® (Daunorubicin Hydrochloride), CERVARIX® (Recombinant HPV Bivalent Vaccine), Cetuximab, CEV, Chlorambucil, CHLORAMBUCIL-PREDNISONE, CHOP, Cisplatin, Cladribine, CLAFEN® (Cyclophosphamide), Clofarabine, CLOFAREX® (Clof arabine), CLOLAR® (Clofarabine), CMF, Cobimetinib, COMETRIQ® (Cabozantinib-S- Malate), Copanlisib Hydrochloride, COPDAC, COPP, COPP-AB V, COSMEGEN® (Dactinomycin), COTELLIC® (Cobimetinib), Crizotinib, CVP, Cyclophosphamide, CYFOS® (Ifosfamide), CYRAMZA® (Ramucirumab), Cytarabine, Cytarabine Liposome, CYTOSAR- U® (Cytarabine), CYTOXAN® (Cyclophosphamide), Dabrafenib, Dacarbazine, DACOGEN® (Decitabine), Dactinomycin, Daratumumab, DARZALEX® (Daratumumab), Dasatinib, Daunorubicin Hydrochloride, Daunorubicin Hydrochloride and Cytarabine Liposome, Decitabine, Defibrotide Sodium, DEFITELIO® (Defibrotide Sodium), Degarelix, Denileukin Diftitox, Denosumab, DEPOCYT® (Cytarabine Liposome), Dexamethasone, Dexrazoxane Hydrochloride, Dinutuximab, Docetaxel, DOXIL® (Doxorubicin Hydrochloride Liposome), Doxorubicin Hydrochloride, Doxorubicin Hydrochloride Liposome, DOX-SL® (Doxorubicin Hydrochloride Liposome), DTIC-DOME® (Dacarbazine), Durvalumab, EFUDEX® (Fluorouracil— Topical), ELITEK® (Rasburicase), ELLENCE® (Epirubicin Hydrochloride), Elotuzumab, ELOXATIN® (Oxaliplatin), Eltrombopag Olamine, EMEND® (Aprepitant), EMPLICITI® (Elotuzumab), Enasidenib Mesylate, Enzalutamide, Epirubicin Hydrochloride , EPOCH, ERBITUX® (Cetuximab), Eribulin Mesylate, ERIVEDGE® (Vismodegib), Erlotinib Hydrochloride, ERWINAZE® (Asparaginase Erwinia chrysanthemi), ETHYOL® (Amifostine), Etopophos ETOPOPHOS® (Etoposide Phosphate), Etoposide, Etoposide Phosphate, EV ACET® (Doxorubicin Hydrochloride Liposome), Everolimus, EVISTA® (Raloxifene Hydrochloride), EVOMELA® (Melphalan Hydrochloride), Exemestane, 5-FU® (Fluorouracil Injection), 5-FU® (Fluorouracil— Topical), FARESTON® (Toremifene), FARYDAK® (Panobinostat), FASLODEX® (Fulvestrant), FEC, FEMARA® (Letrozole), Filgrastim, FLUDARA® (Fludarabine Phosphate), Fludarabine Phosphate, FLUOROPLEX® (Fluorouracil- -Topical), Fluorouracil Injection, Fluorouracil— Topical, Flutamide, FOLEX® (Methotrexate), FOLEX PFS® (Methotrexate), FOLFIRI, FOLFIRI-BEVACIZUMAB, FOLFIRL CETUXIMAB, FOLFIRINOX, FOLFOX, FOLOTYN® (Pralatrexate), FU-LV, Fulvestrant, GARDAS1L® (Recombinant HPV Quadrivalent Vaccine), GARDAS1L 9® (Recombinant HPV Nonavalent Vaccine), GAZYVA® (Obinutuzumab), Gefitinib, Gemcitabine Hydrochloride, GEMCITABINE-CISPLATIN, GEMCITABINE-OXALIPLATIN, Gemtuzumab Ozogamicin, GEMZAR® (Gemcitabine Hydrochloride), GILOTRIF® (Afatinib Dimaleate), GLEEVEC® (Imatinib Mesylate), GLIADEL® (Carmustine Implant), GLIADEL WAFER® (Carmustine Implant), Glucarpidase, Goserelin Acetate, HALAVEN® (Eribulin Mesylate), HEMANGEOL® (Propranolol Hydrochloride), HERCEPTIN® (Trastuzumab), HPV Bivalent Vaccine, Recombinant, HPV Nonavalent Vaccine, Recombinant, HPV Quadrivalent Vaccine, Recombinant, HYCAMTIN® (Topotecan Hydrochloride), HYDREA® (Hydroxyurea), Hydroxyurea, Hyper-CVAD, IBRANCE® (Palbociclib), Ibritumomab Tiuxetan, Ibrutinib, ICE, ICLUSIG® (Ponatinib Hydrochloride), IDAMYCIN® (Idarubicin Hydrochloride), Idarubicin Hydrochloride, Idelalisib, IDHIFA® (Enasidenib Mesylate), IFEX® (Ifosfamide), Ifosfamide, IFOSFAMIDUM® (Ifosfamide), IL-2 (Aldesleukin), Imatinib Mesylate, IMBRUVICA® (Ibrutinib), IMFINZI® (Durvalumab), Imiquimod, IMLYGIC® (Talimogene Laherparepvec), INLYTA® (Axitinib), Inotuzumab Ozogamicin, Interferon Alfa-2b, Recombinant, Interleukin-2 (Aldesleukin), INTRON A® (Recombinant Interferon Alfa-2b), Iodine I 131 Tositumomab and Tositumomab, Ipilimumab, IRESSA® (Gefitinib), Irinotecan Hydrochloride, Irinotecan Hydrochloride Liposome, ISTODAX® (Romidepsin), Ixabepilone, Ixazomib Citrate, IXEMPRA® (Ixabepilone), JAKAFI® (Ruxolitinib Phosphate), JEB, JEVTANA® (Cabazitaxel), KADCYLA® (Ado-Trastuzumab Emtansine), KEOXIFENE® (Raloxifene Hydrochloride), KEPIVANCE® (Palifermin), KEYTRUDA® (Pembrolizumab), KISQALI® (Ribociclib), KYMRIAH® (Tisagenlecleucel), KYPROLIS® (Carfilzomib), Lanreotide Acetate, Lapatinib Ditosylate, LARTRUVO® (Olaratumab), Lenalidomide, Lenvatinib Mesylate, LENVIMA® (Lenvatinib Mesylate), Letrozole, Leucovorin Calcium, LEUKERAN® (Chlorambucil), Leuprolide Acetate, LEUSTATIN® (Cladribine), LEVULAN® (Aminolevulinic Acid), LINFOLIZIN® (Chlorambucil), LIPODOX® (Doxorubicin Hydrochloride Liposome), Lomustine, LONSURF® (Trifluridine and Tipiracil Hydrochloride), LUPRON® (Leuprolide Acetate), LUPRON DEPOT® (Leuprolide Acetate), LUPRON DEPOT-PED® (Leuprolide Acetate), LYNPARZA® (Olaparib), MARQIBO® (Vincristine Sulfate Liposome), MATULANE® (Procarbazine Hydrochloride), Mechlorethamine Hydrochloride, Megestrol Acetate, MEKINIST® (Trametinib), Melphalan, Melphalan Hydrochloride, Mercaptopurine, Mesna, MESNEX® (Mesna), METHAZOLASTONE® (Temozolomide), Methotrexate, METHOTREXATE LPF® (Methotrexate), Methylnaltrexone Bromide, MEXATE® (Methotrexate), MEXATE-AQ® (Methotrexate), Midostaurin, Mitomycin C, Mitoxantrone Hydrochloride, M1T0ZYTREX® (Mitomycin C), MOPP, MOZOBIL® (Plerixafor), MUSTARGEN® (Mechlorethamine Hydrochloride) , MUTAMYCIN® (Mitomycin C), MYLERAN® (Busulfan), MYLOSAR® (Azacitidine), MYLOTARG® (Gemtuzumab Ozogamicin), NANOPARTICLE PACLITAXEL® (Paclitaxel Albumin-stabilized Nanoparticle Formulation), NAVELBINE® (Vinorelbine Tartrate), Necitumumab, Nelarabine, NEOSAR® (Cyclophosphamide), Neratinib Maleate, NERLYNX® (Neratinib Maleate), Netupitant and Palonosetron Hydrochloride, NEULASTA® (Pegfilgrastim), NEUPOGEN® (Filgrastim), NEXAVAR® (Sorafenib Tosylate), NILANDRON® (Nilutamide), Nilotinib, Nilutamide, NINLARO® (Ixazomib Citrate), Niraparib Tosylate Monohydrate, Nivolumab, NOLVADEX® (Tamoxifen Citrate), NPLATE® (Romiplostim), Obinutuzumab, ODOMZO® (Sonidegib), OEPA, Ofatumumab, OFF, Olaparib, Olaratumab, Omacetaxine Mepesuccinate, ONCASPAR® (Pegaspargase), Ondansetron Hydrochloride, ONIVYDE® (Irinotecan Hydrochloride Liposome), ONTAK® (Denileukin Diftitox), OPDIVO® (Nivolumab), OPPA, Osimertinib, Oxaliplatin, Paclitaxel, Paclitaxel Albumin-stabilized Nanoparticle Formulation, PAD, Palbociclib, Palifermin, Palonosetron Hydrochloride, Palonosetron Hydrochloride and Netupitant, Pamidronate Disodium, Panitumumab, Panobinostat, PARAPLAT® (Carboplatin), PARAPLATIN® (Carboplatin), Pazopanib Hydrochloride, PCV, PEB, Pegaspargase, Pegfilgrastim, Peginterferon Alfa-2b, PEG-INTRON® (Peginterferon Alfa-2b), Pembrolizumab, Pemetrexed Disodium, PERJETA® (Pertuzumab), Pertuzumab, PLATINOL® (Cisplatin), PLATINOL-AQ® (Cisplatin), Plerixafor, Pomalidomide, POMALYST® (Pomalidomide), Ponatinib Hydrochloride, PORTRAZZA® (Necitumumab), Pralatrexate, Prednisone, Procarbazine Hydrochloride, PROLEUKIN® (Aldesleukin), PROLIA® (Denosumab), PROMACTA® (Eltrombopag Olamine), Propranolol Hydrochloride, PROVENGE® (Sipuleucel-T), PURINETHOL® (Mercaptopurine), PURIXAN® (Mercaptopurine), Radium 223 Dichloride, Raloxifene Hydrochloride, Ramucirumab, Rasburicase, R-CHOP, R-CVP, Recombinant Human Papillomavirus (HPV) Bivalent Vaccine, Recombinant Human Papillomavirus (HPV) Nonavalent Vaccine, Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine, Recombinant Interferon Alfa- 2b, Regorafenib, RELISTOR® (Methylnaltrexone Bromide), R- EPOCH, REVLIMID® (Lenalidomide), RHEUMATREX® (Methotrexate), Ribociclib, R-ICE, RITUXAN® (Rituximab), RITUXAN HYCELA® (Rituximab and Hyaluronidase Human), Rituximab, Rituximab and , Hyaluronidase Human, ,Rolapitant Hydrochloride, Romidepsin, Romiplostim, RUBIDOMYCIN® (Daunorubicin Hydrochloride), RUBRACA® (Rucaparib Camsylate), Rucaparib Camsylate, Ruxolitinib Phosphate, RYDAPT® (Midostaurin), Sclerosol Intrapleural Aerosol (Talc), Siltuximab, Sipuleucel-T, S0MATUL1NE DEPOT® (Lanreotide Acetate), Sonidegib, Sorafenib Tosylate, SPRYCEL® (Dasatinib), STANFORD V, Sterile Talc Powder (Talc), STERITALC® (Talc), STIVARGA® (Regorafenib), Sunitinib Malate, SUTENT® (Sunitinib Malate), SYLATRON® (Peginterferon Alfa-2b), SYLVANT® (Siltuximab), Synribo SYNRIBO® (Omacetaxine Mepesuccinate), TABLOID® (Thioguanine), TAC, TAFINLAR® (Dabrafenib), TAGRISSO® (Osimertinib), Talc, Talimogene Laherparepvec, Tamoxifen Citrate, TARABINE PFS® (Cytarabine), TARCEVA® (Erlotinib Hydrochloride), TARGRETIN® (Bexarotene), TASIGNA® (Nilotinib), TAXOL® (Paclitaxel), TAXOTERE® (Docetaxel), TECENTRIQ® (Atezolizumab), TEMODAR® (Temozolomide), Temozolomide, Temsirolimus, Thalidomide, THALOMID® (Thalidomide), Thioguanine, Thiotepa, Tisagenlecleucel, TOLAK® (Fluorouracil-Topical), Topotecan Hydrochloride, Toremifene, TORISEL® (Temsirolimus), Tositumomab and Iodine 1 131 Tositumomab, TOTECT® (Dexrazoxane Hydrochloride), TPF, Trabectedin, Trametinib, Trastuzumab, TREANDA® (Bendamustine Hydrochloride), Trifluridine and Tipiracil Hydrochloride, TRISENOX® (Arsenic Trioxide), TYKERB® (Lapatinib Ditosylate) , UNITUXIN® (Dinutuximab), Uridine Triacetate, VAC, Vandetanib, VAMP, VARUBI® (Rolapitant Hydrochloride), VECTIBIX® (Panitumumab), VelP, VELBAN® (Vinblastine Sulfate), VELCADE® (Bortezomib), VELSAR® (Vinblastine Sulfate), Vemurafenib, VENCLEXTA® (Venetoclax), Venetoclax, VERZENIO® (Abemaciclib), VIADUR® (Leuprolide Acetate), VIDAZA® (Azacitidine), Vinblastine Sulfate, VINCASAR PFS® (Vincristine Sulfate), Vincristine Sulfate, Vincristine Sulfate Liposome, Vinorelbine Tartrate, VIP, Vismodegib, VISTOGARD® (Uridine Triacetate), VORAXAZE® (Glucarpidase), Vorinostat, VOTRIENT® (Pazopanib Hydrochloride), VYXEOS® (Daunorubicin Hydrochloride and Cytarabine Liposome), WELLCOVORIN® (Leucovorin Calcium), XALKORI® (Crizotinib), XELODA® (Capecitabine), XELIRI, XELOX, XGEVA® (Denosumab), XOFIGO® (Radium 223 Dichloride), XT ANDI® (Enzalutamide), YERVOY® (Ipilimumab), YONDELIS® (Trabectedin), ZALTRAP® (Ziv-Aflibercept), ZARXIO® (Filgrastim), ZEJULA® (Niraparib Tosylate Monohydrate), ZELBORAF® (Vemurafenib), ZEVALIN® (Ibritumomab Tiuxetan), ZINECARD® (Dexrazoxane Hydrochloride), Ziv-Aflibercept, ZOFRAN® (Ondansetron Hydrochloride), ZOLADEX® (Goserelin Acetate), Zoledronic Acid, ZOLINZA® (Vorinostat), ZOMETA® (Zoledronic Acid), ZYDELIG® (Idelalisib), ZYKADIA® (Ceritinib), and / or ZYTIGA® (Abiraterone Acetate). The treatment methods can include or further include checkpoint inhibitors including, but are not limited to antibodies that block PD-1 (such as, for example, Nivolumab (BMS-936558 or MDX1106), pembrolizumab, cemiplimab , CT-011, MK- 3475), PD-L1 (such as, for example, atezolizumab, avelumab, durvalumab, MDX-1105 (BMS- 936559), MPDL3280A, or MSB0010718C), PD-L2 (such as, for example, rHIgM12B7), CTLA- 4 (such as, for example, Ipilimumab (MDX-010), Tremelimumab (CP-675,206)), IDO, B7-H3 (such as, for example, MGA271, MGD009, omburtamab), B7-H4, B7-H3, T cell immunoreceplor with Ig and ITIM domains (TIGIT)(such as, for example BMS-986207, OMP- 313M32, MK-7684, AB- 154, ASP-8374, MTIG7192A, or PVSRIPO), CD96, B- and T- lymphocyte attenuator (BTLA), V-domain Ig suppressor of T cell activation (VISTA)(such as, for example, INJ-61610588, CA-170), TIM3 (such as, for example, TSR-022, MBG453, Sym023, INCAGN2390, LY3321367, BMS-986258, SHR-1702, RO7121661), LAG-3 (such as, for example, BMS-986016, LAG525, MK-4280, REGN3767, TSR-033, BI754111, Sym022, FS118, MGD013, and Immutep).

[0056] C. Examples

[0057] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and / or methods claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.

[0058] 1. Example 1: Expansion of T cells from the Cerebrospinal Fluid of Melanoma Patients with Leptomeningeal Disease a) Method

[0059] CSF was collected from 6 melanoma patients with LMD via lumbar puncture or Ommaya tap as shown in Figure 1. The initial volume was measured, and the isolated cells were plated following established TIL culture protocols to determine optimal expansion conditions, including: 1) 6000IU / mL of IL-2, 2) 0KT3, and 3) anti-4- IBB agonistic antibody. CSF cultures were evaluated after the initial 4-6 weeks and T cells were further propagated via the rapid expansion protocol (REP). The final T cell product was phenotyped for CD4+ and CD8+ T cells using flow cytometry. b) Results

[0060] An average of 6.31mL of CSF was collected from patients with LMD, resulting in an average yield of 2.74xl04viable cells available for expansion (range: 1 103-2.43 106). Isolated cells were analyzed by flow cytometry to determine the percent cells that were T cells and then the percentage of cells that were CD4+ vs CD8+ cells (see Figure 2). Prior to expansion, the isolated cells were predominantly T cells and specifically CD4+ T cells. Cells were then cultured in IL-2 alone, IL-2 + an anti-4- IBB agonistic antibody, IL-2 + 0KT3 (anti-CD3 antibody), or IL-2 + anti-CD3 / CD28 dynabeads. After culture, all samples were predominantly T cells (Figure 2A and 2B). The percentage of live cells grown in IL-2 alone that were CD8+ T cells or CD4+ T cells varied between samples. However, TILs grown in IL-2+ an anti-4- IBB agonistic antibody were mainly CD4+ T cells (approximately 67%). By contrast, culturing in IL-2 + 0KT3 or IL-2 + anti CD3 / CD28 dynabeads resulted in predominantly CD8+ T cells. After initial culture in IL-2, 75% of the samples tested demonstrated an increased cell yield, with an average of 271.79-fold expansion (range3.85-1054) (Figure 3A). Four samples subsequently underwent a REP, with a 100% success rate and an average of 231.99-fold expansion (Rangell.4-710.2)(Figure 3B). Comparing the expansion for each culture condition, the largest expansion was obtained in the IL-2+OKT3 (10,000-fold expansion) and IL-2+CD3 / CD38 Dynabeads (30,000-fold expansion) (Figure 3C). Although not as significant, TILs cultured in IL-2 alone still expanded 271.79-fold as indicated above and TILs cultured in IL-2+an anti-4- 1BB agonistic antibody expanded approximately 375-fold. Post-REP products were analyzed by flow cytometry for the frequency of CD4+ and CD8+ T cells. Data from the postREP analysis revealed similar results, with further expansion of CD4+ T cells.

[0061] We next determined the reactivity of TILs expanded from a patient with HLA-matched tumor cell lines (Figures 4 and 5). Assays showed that cells were reactive producing IFN-g when exposed to HLA-matched cancer cells.

[0062] 2. Example 2: The Use of ImmunoCellular Therapies in CNS Metastases:

[0063] The mechanisms of treatment response or T cell exhaustion (i.e., TME and role of cellular immunity) in CNS are unknown. T cells specifically recognize melanoma antigens and are essential to this immune-mediated response. ACT using TILs is a novel, successful therapy demonstrating complete and durable responses in patients with advanced / refractory metastatic melanoma, 1-4 with up to 51% (18 / 35 treated patients) clinical response, with a duration of 11.5 + / - 2.2 months. Sites of regression i.e., metastases in brain.4 ACT with TILs involves tumor acquisition, ex vivo expansion of T-cells, and re-infusion of the expanded T cells back into the patient. Trials using ACT with TILs have shown this is feasible, safe, and efficacious. The use of IT administration of autologous TILs obtained from EM, was shown by the MDACC group to be safe in a single case report of a patient with M-LMD.

[0064] The complexity and dynamics of the T-cell repertoire are poorly understood, especially in the CNS. TCRs are exceptionally variable T lymphocyte membrane proteins that identify antigens displayed on nonhomologous cells by the MHC. TCR genomic loci go through somatic recombination to generate structurally diverse T cells needed for antigen recognition. TCR(3 subunits can be recognized by their complement determining region 3 (CDR3) sequence, which is the location of V(D)J recombination encoding the main position of antigen interaction. The resultant CDR3 hypervariable sequences allow the recognition of diverse peptide MHC complexes. This TCR repertoire is continuously changing and adapting in response to immune challenges. Therefore, there is a wide variety of TCRP clone types, detailed measurements of the variety of lengths of CDR3P, use of non-template bases, sequence convergence, predilections for selection and pairing of TCRP variable and joining genes can provide valuable information to better understand the T cell anti-tumor response in the CSF, and in reaction to immune- modulatory challenges.

[0065] While systemic tumor-reactive T cells are effective, no one has determined if tumor- reactive T cells in the CSF environment of LMD are effective and their phenotype and function are unknown. Whereas the leptomeninges can be a suppressive microenvironment, ex vivo expansion of tumor-reactive T cells into the billions can be generated and infused back into a patient to target the tumor cells in patients with LMD with TILs adapted to the CSF with LMD specific antigen recognition. a) Data

[0066] MCC is one of the few cancer centers to offer ACT with TILs for the treatment of solid malignancies. Multiple studies have shown success with collecting and expanding TILs from resected melanoma samples. We also have data showing it is feasible to expand tumor-reactive T cells (i.e., CD8+ T cells) from the CSF of patients with M-LMD.

[0067] Expansion of CD4+ and CD8+ T cells from CSF in M-LMD (Fig. 6). We isolated and expanded CD4+ and CD8+ T cells from CSF from six patients with M-LMD. An average of 2.74 x 104viable cells were available for expansion (range 1 x 103to 2.43 x 106). We followed established T cell culture protocols with different expansion conditions (i.e., 6000 IU of interleukin-2 [IL-2], OKT3, anti-4- IBB agonistic antibody), and rapid expansion protocol (REP). After initial culture in IL-2 alone, 75% showed an increased cell yield average of 271.79- fold expansion (range 3.85- to 1054-fold). Phenotypic analysis of preREP TILs revealed approximately 67% of the samples were predominantly CD4+ » CD8+ T cells via flow cytometry. REP was done on 4 samples, with a 100% success rate and average 231.99-fold expansion (range 11.4-to 710.2-fold). Data from expansion of these T cells with OKT3 medium and 4- IBB showed similar results.

[0068] T cells are reactive to HLA matched tumor cells (Fig. 7). T cells produce IFN-yin response to HLA matched tumor cells (i.e., SKMEL 28 and WM88) with more than 2-fold increase compared to blockade of MHC class I with W6 / 32 antibody. These results were similar when T cells were cultured on OKT-3 medium. T cells from BM are highly reactive to autologous tumor cells and tumor burden decreases after TIL ACT. We showed that TILs from BM produce IFN-yin response to autologous tumor with more than 4-fold increase compared to blockade of MHC class I with W6 / 32 antibody (Fig. 8). Tumor rejection measured after ACT with BM TIL in N0G-IL2 mice shows decrease in tumor burden compared to control with no BM TIL (Fig. 9). N0G-IL2 mice were injected subcutaneously with autologous tumor. When tumors reached 25-50 mm2, mice were injected with BM TIL. Control mice received no TIL. b) Experimental Design and Methods

[0069] (1) Tumor-reactive CD8+ T cells from the CSF of patients with M-LMD can be expanded to clinically relevant numbers for ACT.

[0070] We have data with two samples showing that this is successful and feasible. We can also conduct the rapid expansion protocols with tumor reactive CD8+ T cells isolated from CSF. We can identify patients (n=8-10) with M-LMD. CSF (10-30 mL, via lumbar puncture or Ommaya reservoir) and peripheral blood (10 mL) can be collected and sent to the laboratory for processing i.e., isolation and expansion of T cells. CSF and blood samples can be spun down for T cell and tumor cell collection. T cells can be isolated from CSF and plated in media containing 6000 lU / mL of IL-2. The number of expanded T cells can be measured at 4 weeks. Cells can be grown on different mediums i.e., IL-2 only, OKT-3, CD33CD28, etc., and under different metabolic conditions (i.e., hypoxic versus normoxic with different pH levels). We estimate about 200 tumor cells that can be collected from the CSF in LMD. We can culture the autologous tumor cells from CSF following established protocols. We can evaluate T cells’ reactivity to autologous tumor or HLA-matched melanoma target cells. T cells expanded from CSF can be co-cultured with autologous tumor cells alone or in the presence of an HLA blocking antibody for 24 hours. Supernatants can be collected. IFN-y, Granzyme B, and TNF-a in supernatants can be measured by ELLA. Alternatively, if autologous tumor cells are not available, T cells can be co-cultured with HLA-matched melanoma tumor cell lines. Expanded T cells can also be phenotyped for immune cell markers (i.e., CD3+, CD4+, and CD8+ T cells, CD56+ NK cells, CD4+ foxp3+ regulatory T cells, a / TCR, y / 6 TCR, PD-1, and CD 16) and analyzed by flow cytometry on a Celesta flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NI). Data can be acquired using FACSDiva (Becton-Dickinson and Company) analyzed using Flowlo software (Becton, Dickinson and Company). The phenotype of the T cell product can be reported as a percentage of the live, single cell population. (2) Evaluation of the clonality, diversity, and convergence of TCR repertoires of tumorreactive T cells from CSF using TCR sequencing.

[0071] We were able to expand tumor-reactive T cells and have data on TCR sequencing on two samples and need to expand this to larger number of samples for analyses. T cells can be expanded from CSF as described herein. Autologous tissue from melanoma BM (>1 cm3) and EM biopsy (>1 cm3, e.g., tumor draining lymph nodes [TDLNs] or skin) can also be collected if accessible and feasible. When available, solid tumor specimens (i.e., BM and EM) can be minced into small fragments and plated as one fragment per well in media containing 6000 lU / mL of IL-2. T cells from the samples and mononuclear cells from peripheral blood can be counted. CD4+ and CD8+ T cells can be isolated by magnetic bead separation on MACS columns from bulk TIL products. DNA can be extracted using the Qiagen DNeasy Blood and Tissue Kit according to the kit’s protocol. DNA can be quantified via Nanodrop and deep level TCR sequencing can be performed by the MCC Molecular Genomics Core using the ImmunoSEQ TCRP Kit v3. Data can be uploaded to the Adaptive Biotechnologies server for analysis on the Adaptive ImmunoSEQ Analyzer 3.0. In addition, the lymphocyte landscape profile of T cells’ activation, exhaustion, and proliferation can be evaluated with scRNA seq. and immunohistochemistry using standard protocols. This can provide valuable data about the differences in competency of the T cells from different sites to migrate, proliferate, and kill tumor cells in CNS disease.

[0072] (3) Tumor reactive T cells delivered intrathecally provide a survival benefit in a melanoma model of LMD.

[0073] We can evaluate whether tumor reactive T cells via intrathecal (IT) delivery provides a survival advantage in the murine model of LMD, with a microsurgically implanted murine Ommaya reservoir designed for IT drug delivery. M05 melanoma-bearing mice can be used for therapeutic experiments. We can inject M05 (250 cells in 10 pL suspension in PBS) melanoma cells into the CSF space via cistema magna of 6- to 8-week old C57B1 / 6 mice. We have extensive experience with tumor animal models in LMD, therapeutic development in LMD, CSF LMD, and tumor collection and IITs in LMD. Mice displaying neurologic symptoms upon awakening after injecting M05 cells can be immediately euthanized. Bioluminescence signals can be detected by intraperitoneally injections 0.1 cc of a 1:40 dilution of NanoLuc-reporter substrate in sterile PBS. Bioluminescence images (BLI) can be captured using the Xenogen IVIS 200 system, and BLI analyses cam be performed using Living Image Software. Treatment with tumor reactive T cells can begin 7-14 days postinjection of cancer cells. T cells can be expanded as above, and can be infused via the Ommaya reservoir. A maximum tolerated dose (MTL) can be established by measuring weight loss, signs of neurotoxicity, and other extracranial adverse events (n=10). Since the volume and rate of production of CSF is significantly reduced in mice compared to human (i.e., 35 g L versus >100 mL; 0.32 g L / min vs. 350 g L / min), an equivalent of tumor reactive T cells in mice is not be feasible (i.e., 35x109 IT administration in a human, equivalent to -1.23x107 T cells in mice), and amount of T cells per infusion can be limited based on concentration (i.e., tumor reactive T cells per uL) and we estimate treating with -320,000-400,000 T cells in 8- 10 uL per infusion (40,000 cells / uL). Three different concentrations and a vehicle control group can be used for IT injection, initially in a once a week schedule for 3-4 weeks (n=10 mice per treatment group) or continue until disease progression or unacceptable toxicity. Another group of mice can be treated via systemic injections. Tumor reactive T cells can be given in combination with IL-2 IT. Response to treatment can be evaluated by measuring body weights and survival rates. Levels of IFN-y, IL-10, IL- 12 p70, IL- 13, IL-ip, IL-2, IL-4, IL-6, IL-8, TNF-a and TGF-P can be measured in CSF and blood at baseline and post-every IT treatment. Post-mortem, CSF, blood, meninges, brain, and lymph nodes can be collected for analyses. Moreover, the reactive tumor T cells from the CSF obtained from mouse and human can be evaluated via scRNA seq, including data we already have on human CSF samples of M-LMD.

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Claims

V. CLAIMSWhat is claimed is:

1. A method of treating leptomeningeal disease (LMD) in a subject comprising administering to the subject tumor infiltrating lymphocytes (TILs) obtained from cerebrospinal fluid and expanded ex vivo.

2. The method of claim 1 , wherein the LMD is melanoma LMD.

3. The method of claim 1, wherein the TILs are autologous to the subject.

4. The method of claim 1, further comprising culturing the TILs in media comprising IL-2 to expand the TILs and harvesting the expanded TILs.

5. The method of claim 4, wherein the culture media further comprises an anti-CD3 antibody, anti- CD28 antibody, and / or an anti-4-lBB agonistic antibody.

6. The method of claim 4 or 5, wherein the TILs are cultured in hypoxic conditions.

7. The method of any of claims 4-6, wherein the TILs are cultured for at least 4 weeks.

8. The method of any of claims 1-7, wherein the expanded TILs are administered to the subject intrathecally.

9. The method of claim 8, wherein the intrathecal administration is achieved via use of an Ommaya reservoir.

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