Cultivation of tumor-infiltrating lymphocytes from tumor digesta

Abstract

Tumor infiltrating lymphocytes (TILs) are immune cells that have left the bloodstream and migrated to tumors. TILs have been used in autologous adoptive transfer therapy for the treatment of cancer. A method for rapidly expanding tumor infiltrating lymphocytes using digested tumor cells is disclosed.

Description

[Technical field]

[0001] Background technology Tumor infiltrating lymphocytes (TILs) are immune cells that have left the bloodstream and migrated into tumors. TILs have been used in autologous adoptive transfer therapy for the treatment of cancer. Typically, freshly surgically resected tumors are used as starting material for successful initiation and expansion of tumor-specific TIL cultures to produce clinically relevant doses of TIL therapy. Thus, candidate patients for TIL therapy must be eligible for surgery. If a patient is eligible for surgery, the tumor must be resectable. In the presence of multiple tumor anatomical sites, a skilled selection of resection of the appropriate tumor metastasis(s) with potential T cell infiltration must be performed for each patient.

[0002] In the generation of TILs, once a surgically resectable tumor is obtained, the tumor is typically cut into small pieces and individual pieces are placed in separate wells of a culture plate where the first TIL expansion (termed "Pre-REP") occurs. The first expanded TIL population is then selected for tumor reactivity. Tumor-reactive clones are subjected to a second expansion round (termed "REP"). A total of 5-7 weeks of culture is required, and this culture condition requires the use of a clean room, checking for confluence by dissociating the culture, and a significant amount of time to maintain the cells. Any method of rapidly expanding TILs that is less invasive, faster, or requires fewer resources would be beneficial. Summary of the Invention

[0003] Methods and compositions are disclosed for the rapid production of expanded tumor infiltrating lymphocyte (TIL) populations from bulk, unpurified tumor digesta.

[0004] In one aspect, disclosed herein is a method for rapidly producing an expanded population of tumor infiltrating lymphocytes (TILs) for use in adoptive cell therapy in human patients, comprising culturing bulk non-purified tumor digesta from a human subject in a culture medium comprising an effective amount of IL-2 to expand highly tumor-reactive and / or specific tumor infiltrating lymphocytes.

[0005] Also disclosed herein is a method for rapidly generating an expanded tumor infiltrating lymphocyte (TIL) population of any of the aforementioned embodiments, further comprising obtaining one or more tissue samples (including but not limited to, one or more biopsies (e.g., core biopsies, etc.) and / or one or more surgical resections) from which a bulk crude tumor digest is derived or obtained, the one or more tissue samples comprising TILs from a subject; and digesting the one or more tissue samples (including but not limited to, one or more biopsies (e.g., core biopsies, etc.) and / or one or more surgical resections) with one or more human or humanized enzymes, including but not limited to, collagenase (e.g., XIAFLEX®), hyaluronidase (e.g., HYLENEX®), and / or DNAse (e.g., PULMOZYME®). In some embodiments, the method may further comprise harvesting the expanded TIL population.

[0006] In one embodiment, the TILs are obtained from one or more core biopsy tissue samples. Also disclosed herein is a method of any of the above embodiments, wherein one or more core biopsies are digested directly from the patient without disaggregation of the specimen. Digestion may include the use of one or more human or humanized enzymes, including but not limited to collagenase (e.g., XIAFLEX®), hyaluronidase (e.g., HYLENEX®), and / or DNAse (e.g., PULMOZYME®).

[0007] Also disclosed are methods of any of the above embodiments, further comprising performing one or more biopsies (e.g., core biopsy and / or surgical resection, etc.) prior to plating the TILs.In one embodiment, disclosed is a method of rapidly producing an expanded TIL population, further comprising harvesting the expanded TIL population.

[0008] In one aspect, a method for rapidly producing a TIL population of any preceding aspect is disclosed, wherein digestion further comprises mechanical disruption of a tissue sample (including but not limited to, one or more biopsies (e.g., core biopsies, etc.) and / or one or more surgical resections).

[0009] The disclosed expanded TIL populations may be used to treat human cancers (e.g., soft tissue sarcomas (e.g., but not limited to, fibrous sarcomas, including, but not limited to, atypical lipomatous tumors, well-differentiated liposarcoma, myxofibrosarcoma, leiomyosarcoma, solitary fibrous tumor, and leiomyosarcoma), any connective tissue neoplasm, or osteosarcoma. In one aspect, disclosed herein is a method of treating cancer in a human subject, comprising administering to the subject the rapidly expanded TILs of any of the preceding aspects. In other words, in one aspect disclosed herein, a method of treating cancer in a subject, comprising culturing bulk crude tumor digests from the subject in a culture medium comprising an amount of IL-2 effective to expand tumor-infiltrating lymphocytes with high tumor reactivity and specificity; harvesting the expanded TIL cells; adoptively transferring the expanded TILs into the subject. In some embodiments, the method of treating cancer may further include obtaining one or more tissue samples (including but not limited to, one or more biopsies (e.g., core biopsies, etc.) and / or one or more surgical resections) from which a bulk crude tumor digest is derived or obtained, the one or more tissue samples comprising TILs from a subject; and digesting the one or more tissue samples (including but not limited to, one or more biopsies (e.g., core biopsies, etc.) and / or one or more surgical resections) with one or more human or humanized enzymes, including but not limited to, collagenase (e.g., XIAFLEX®), hyaluronidase (e.g., HYLENEX®), and / or DNAse (e.g., PULMOZYME®). [Brief description of the drawings]

[0010] [Figure 1] The total yield from the digestion method for TIL proliferation with IL-2 (6000 IU / mL) alone is shown. [Diagram 2] 1 shows the digestion method and the phenotype of preREP TILs grown from soft tissue sarcoma tumors using IL-2 (6000 IU / mL) as the only supplement. [Diagram 3]Figure 1 shows a comparison of total cell yields between enzyme formulations. The FDA approved enzyme formulation utilizing xeno-free enzymes showed superior yields in total viable cell counts after processing fresh tumors. [Figure 4] Comparison of viability and phenotype: FDA approved enzyme formulation using xeno-free enzymes showed superior viability of digesta samples from fresh tumors and more CD8+ lymphocytes in preREP cultures. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] 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 to specific recombinant biotechnology methods, unless otherwise specified, or to particular reagents, which, unless otherwise specified, can, 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.

[0012] A.Definition As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to a "pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

[0013] 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, it will be understood that by using the antecedent "about," the particular value forms another embodiment. It will be further understood that each of the endpoints of a range is significant in relation to the other endpoint, and independently of the other endpoint. It is also understood that certain numerical values ​​are disclosed herein, and that each numerical value is also disclosed herein as "about" that particular value in addition to the numerical value itself. For example, when a numerical value is disclosed, it is also understood that "less than or equal to," "greater than or equal to," and possible ranges between the numerical values ​​are also disclosed, as appropriately understood by one of ordinary skill in the art. For example, when a numerical value is disclosed, "less than or equal to 10" as well as "greater than or equal to 10" are also disclosed. It is also understood that throughout this application, data is provided in several different formats, and that the data represents endpoints and starting points, as well as ranges for any combination of the data points. For example, if a specific data point "10" and a specific data point 15 are disclosed, it is understood that 10 and greater than, greater than, less than, less than, and equal to 15, as well as 10 to 15, are considered to be disclosed. It is also understood that each unit between two specific units is also disclosed. For example, if 10 and 15 are disclosed, 11, 12, 13, and 14 are also disclosed.

[0014] In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings.

[0015] "Optional" or "optionally, optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes cases where the event or circumstance occurs and cases where it does not occur.

[0016] "Reduction" can refer to any change that reduces the amount of a symptom, disease, composition, condition, or activity. A substance is also understood to reduce the genetic production of a gene when the genetic production of the gene product in the presence of the substance is less than the production of the gene product in the absence of the substance. For example, a reduction can also be a change in the symptoms of a disorder such that the symptoms are less than those previously observed. A reduction can be any individual, median, or average reduction in a condition, symptom, activity, or composition by a statistically significant amount. Thus, a reduction 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% reduction, so long as the reduction is statistically significant.

[0017] "Inhibit", "inhibiting", and "inhibition" refer to a decrease in an activity, response, condition, disease, or other biological parameter. This can include, but is not limited to, the complete elimination of the activity, response, condition, or disease. It can also include, for example, a 10% decrease in the activity, response, condition, or disease compared to native or control levels. Thus, the decrease can be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of decrease in between, compared to native or control levels.

[0018] "Reduce" or other forms of the word, such as "reducing" or "reduction," refers to a decrease in an event or characteristic (e.g., tumor growth). It is understood that this is usually relative to some standard or expected value, in other words, relative, but not necessarily with reference to a standard or relative value. For example, "reducing tumor growth" means reducing the rate of tumor growth compared to a standard or control.

[0019] As used herein, "treat", "treating", "treatment" and grammatical variations thereof include administering a composition with the intent or purpose of partially or completely preventing, delaying, curing, curing, attenuating, mitigating, altering, repairing, restoring, improving, stabilizing, alleviating, and / or reducing the intensity or frequency of one or more diseases or conditions, symptoms of a disease or condition, or the underlying causes of a disease or condition. Treatment according to the present invention may be applied preventatively, prophylactically, palliatively, or therapeutically. Prophylactic treatment is administered to a subject prior to the onset of cancer (e.g., before overt signs of cancer), at early onset (e.g., at early signs and symptoms of cancer), or after established onset. Prophylactic administration may occur several days to several years before symptoms of infection appear.

[0020] "Prevent" or other forms of the word, such as "preventing" or "prevention," means to stop a particular event or characteristic, to stabilize or delay the occurrence or progression of a particular event or characteristic, or to minimize the likelihood of a particular event or characteristic occurring. Prevent is usually absolute, as compared to, for example, reduce, and does not require a comparison to a control. As used herein, something may be reduced but not prevented, or something may be reduced and both prevented. Similarly, something may be prevented but not reduced, or something may be prevented and both reduced. When reduce or prevent is used, it is understood that the use of the other word is also expressly disclosed unless specifically indicated otherwise.

[0021] "Biocompatible" generally refers to a material and any metabolic or decomposition products thereof that are generally non-toxic to the recipient and do not pose significant adverse effects in the subject.

[0022] The term "comprising" shall mean that the composition, method, etc. includes the recited elements, but does not exclude other elements. When used to define compositions and methods, "consisting essentially of" shall mean including the recited elements, but excluding any other elements of essential importance to the combination. Thus, a composition consisting essentially of the elements defined herein does not exclude trace contaminants from the isolation and purification methods, as well as pharma- ceutically acceptable carriers, e.g., phosphate buffered saline, preservatives, and the like. "Consisting of" shall mean excluding more than trace elements of other components 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.

[0023] A "control" is a substitute control or sample used in an experiment for comparison purposes. Controls may be "positive" or "negative."

[0024] The term "subject" refers to any individual that is the target of administration or treatment. The subject may be a vertebrate, e.g., a mammal. In one embodiment, the subject may be a human, a non-human primate, a cow, a horse, a pig, a dog, or a cat. The subject may also be a guinea pig, a rat, a hamster, a rabbit, a mouse, or a mole. Thus, the subject may be a human or animal patient. The term "patient" refers to a subject receiving treatment from a clinician, e.g., a physician.

[0025] An "effective amount" of a drug refers to an amount of the drug sufficient to provide a desired effect. The amount of a drug that is "effective" will vary from subject to subject, depending on many factors, such as the age and general condition of the subject, the specific drug(s), and the like. Thus, a quantified "effective amount" cannot always be specified. However, an appropriate "effective amount" in any subject's case can be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, unless otherwise specified, an "effective amount" of a drug can refer to an amount that encompasses both a therapeutically effective amount and a prophylactically effective amount. The "effective amount" of a drug required to obtain a therapeutic effect can vary depending on factors such as the age, sex, and weight of the subject. Dosage regimens can also be adjusted to provide an optimal therapeutic response. For example, several divided doses can be administered daily, or the dose can be relatively reduced as indicated by the exigencies of the therapeutic situation.

[0026] A "pharmaceutical acceptable" ingredient may refer to an ingredient that is not biologically or otherwise undesirable, i.e., the ingredient may be incorporated into a pharmaceutical formulation provided in this 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 ingredients of the formulation containing the ingredient. When used in relation to human administration, the term generally means that the ingredient has met the necessary standards of toxicological and manufacturing testing or that the ingredient is included in the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.

[0027] A "pharmaceutically acceptable carrier" (sometimes referred to as a "carrier") generally refers to a carrier or excipient useful in preparing a safe and non-toxic pharmaceutical or therapeutic composition, including carriers that are acceptable for veterinary and / or human pharmaceutical or therapeutic use. The term "carrier" or "pharmaceutically acceptable carrier" can include, but is not limited to, phosphate buffered saline, water, emulsions (such as oil / water or water / oil emulsions), and / or various types of wetting agents. As used herein, the term "carrier" includes, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material known in the art for use in pharmaceutical formulations, and as further described herein.

[0028] "Pharmacologically active" (or simply "active"), as used herein as in "pharmacologically active" derivative or analog, can refer to a derivative or analog (e.g., a salt, ester, amide, conjugate, metabolite, isomer, fragment, etc.) that has the same type and approximately the same degree of pharmacological activity as the parent compound.

[0029] "Therapeutic agent" refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, such as, for example, the treatment of a disorder or other undesirable physiological condition, and prophylactic effects, such as, for example, the prevention of a disorder or other undesirable physiological condition (e.g., non-immunogenic cancer). These terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of the beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, etc. When the term "therapeutic agent" is used or a particular agent is specifically identified, the term includes the agent itself, as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.

[0030] A "therapeutically effective amount" or "therapeutically effective dose" of a composition (e.g., a composition containing an agent) refers to an amount effective to achieve a desired therapeutic outcome. In some embodiments, the desired therapeutic outcome is control of type I diabetes. In some embodiments, the desired therapeutic outcome is control of obesity. The therapeutically effective amount of a given therapeutic agent will typically vary depending on factors such as the type and severity of the disorder or disease being treated, the age, sex, and weight of the subject. The term may also refer to the amount of therapeutic agent effective to promote a desired therapeutic effect, such as pain relief, or the rate of delivery of the therapeutic agent (e.g., amount over time). The exact desired therapeutic effect will vary depending on the condition being treated, the tolerance of the subject, the agent and / or agent formulation being administered (e.g., potency of the therapeutic agent, concentration of the agent in the formulation, etc.), as well as various other factors understood by those skilled in the art. In some cases, the desired biological or medical response is achieved over a period of days, weeks, or years following multiple administrations of the composition.

[0031] 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. The term includes active treatment, i.e., treatment specifically aimed at ameliorating a disease, condition, or disorder, and also includes causal treatment, i.e., treatment aimed at eliminating the cause of the associated disease, condition, or disorder. In addition, the term includes palliative treatment, i.e., treatment designed to relieve symptoms, but not cure, of a disease, pathological condition, or disorder; preventive treatment, i.e., treatment aimed at minimizing or partially or completely inhibiting the onset of the associated disease, pathological condition, or disorder; and supportive treatment, i.e., treatment used to complement another specific therapy aimed at ameliorating the associated disease, pathological condition, or disorder.

[0032] 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 into this application for the material contained in them that is discussed in the sentence in which the reference is relied upon.

[0033] B. Compositions and Methods Tumor infiltrating lymphocytes (TILs) are immune cells that have left the bloodstream and migrated into tumors. TILs have been used in autologous adoptive transfer therapy (ACT) for the treatment of cancer. Typically, freshly surgically resected tumors are used as starting material for successful initiation and expansion of tumor-specific TIL cultures to produce clinically relevant doses of TIL therapy. Thus, candidate patients for TIL therapy must be eligible for surgery. If a patient is eligible for surgery, the tumor must be resectable. When multiple tumor anatomical sites are present, a skilled selection of the appropriate tumor site with potential T cell infiltration for resection must be performed for each patient.

[0034] Before TIL production can begin with the prior art methods, a surgically resectable tumor must be obtained. Acquisition of tumor for TIL culture (the first step in TIL therapy, called preREP) requires a surgical procedure. Although there are risks to the patient, the invasive acquisition of tissue samples is not the most technically demanding part of the protocols used prior to this disclosure. After acquisition, tissue samples from prior art methods must undergo intensive laboratory preparation of the tumor for culture, including further sectioning of the surgical resection (i.e., fragmentation of the resection). In practice, 5-7 weeks of culture are required before TIL numbers are expanded sufficiently for use in ACT. To achieve this, culture conditions require the use of clean rooms, isolation of cultures to check for confluence, and significant personnel time to maintain the cells. Despite the extensive range of culture methods, TILs do not grow from every surgical resection fragment, and TILs may not migrate from tissue fragments subjected to culture.

[0035] To overcome the obstacles of the methods employed in the art, applicants have developed a reliable method for initiating TIL cultures from tumor samples by directly digesting tissue samples to create bulk, unpurified digests. Furthermore, the methods disclosed herein recognize that individual fragments of tumors will yield widely different TIL cultures with varying efficacy against tumors. This method eliminates the need to maintain multiple cultures and allows for faster TIL expansion. These advantages increase the eligibility of TIL treatment (enabling an increase in unresectable patients). Furthermore, by using core biopsies rather than surgically resected tumors, this method allows for image-guided sampling of high-yield areas in heterogeneous tumors (i.e., viable areas rather than necrotic areas).

[0036] Furthermore, the prior art purified fragmentation method leaves cells in the tissue microenvironment, and the digestion of bulk unpurified tumor fragments removes cellular structures in situ. This removal of cellular structures in situ allows co-culture interactions that are beneficial for the formation of tumor-reactive TILs. Furthermore, the exact starting number of TILs may be determined, which is not possible with the prior art purified fragmentation method.

[0037] Thus, in one aspect, disclosed herein is a method of rapidly generating an expanded tumor infiltrating lymphocyte (TIL) population, comprising obtaining one or more tissue samples (including but not limited to, a biopsy (e.g., a core biopsy) and / or one or more surgical resections) containing TILs from a subject; digesting the one or more tissue samples (including but not limited to, a biopsy (e.g., a core biopsy) and / or one or more surgical resections) with one or more human or humanized enzymes, including but not limited to, collagenase (e.g., XIAFLEX®), hyaluronidase (e.g., HYLENEX®), and / or DNAse (e.g., PULMOZYME®); and culturing cells from the biopsy in complete medium containing IL-2. In one aspect, the method may further comprise harvesting the expanded TIL cells. In one aspect, the digestion may be performed using Miltyeni's digestion kit.

[0038] The concentration of bulk, unpurified digested cells used in the pre-REP of the disclosed method can affect the yield and efficacy of the disclosed method. In one embodiment, the method uses 5x10 6 For example, this method utilizes less than 4x10 cells per tissue culture well. 6 , 3x10 6 , 2x10 6 , 1x10 6 , 9x10 5 , 8x10 5 , 7x10 5 , 6x10 5 , 5x10 5 , 4x10 5 , 3x10 5 , 2x10 5 , or 1x10 5 The following bulk unpurified digested cells may be used:

[0039] It is understood and contemplated herein that the concentration of IL-2 can be adjusted to maximize the proliferation of TILs. For example, the IL-2 concentration used in the culture of TILs can be 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000 IU / mL or more.

[0040] In one embodiment, the disclosed method of producing an expanded TIL population comprises obtaining one or more biopsies (e.g., percutaneous tumor samples) from a subject. As used herein, "biopsy" can include any partial removal of tissue, such as excision biopsy, incision biopsy, core biopsy, or fine needle aspiration biopsy. It is understood and contemplated herein that the use of TILs obtained from biopsies (e.g., core biopsies, including core needle biopsies, etc.) can enable TIL treatment for patients who are not eligible for surgery and for patients with unresectable tumors. In addition, core biopsies (e.g., core needle biopsies, etc.) allow image-guided sampling from multiple anatomical sites.

[0041] When a biopsy, particularly a core biopsy, is used as the source of the tissue sample, it is understood and contemplated herein that the core biopsy (e.g., core needle biopsy, etc.) may be obtained using any device capable of obtaining a core biopsy (see, e.g., Bard Core Biopsy Instruments such as BARD MAGNUM®, BARD MAX-CORE®, BARD BIOPTY-CUT®, BARD MARQUEE®, BARD MISSION®, and BARD MONOPTY® (CR Bard, Inc.) and Carefusion's Temno Biopsy Systems). The needle for obtaining the biopsy may be a 6, 8, 10, 12, 14, 16, 18, or 20 gauge needle, with a needle length of about 2 cm to about 30 cm, preferably about 10 cm to about 25 cm, and more preferably about 16 cm to about 20 cm. For example, the needle length for obtaining a core biopsy may be 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, 10 cm, 11 cm, 12 cm, 13 cm, 14 cm, 15 cm, 16 cm, 17 cm, 18 cm, 19 cm, 20 cm, 21 cm, 22 cm, 23 cm, 24 cm, 25 cm, 26 cm, 27 cm, 28 cm, 29 cm, or 30 cm long. The needle penetration depth may be about 15 mm to 30 mm, preferably about 20 mm to 25 mm. For example, the penetration depth may be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 mm.

[0042] In one embodiment, the use of core biopsies allows the ability to target specific and possibly multiple regions of a tumor. In one embodiment, the method disclosed herein is a method for rapidly producing an expanded TIL population, the method further comprising using imaging techniques such as radiomics to guide TIL acquisition.

[0043] Once a tissue sample has been obtained, including but not limited to a biopsy (e.g., a core biopsy, including a core needle biopsy) and / or a surgical resection, the additional advantage is that it does not require further sectioning (i.e., creating fragments) but may be directly digested. In one aspect, the disclosed methods may include placing the tissue sample directly into a digestion solution (e.g., one or more human or humanized enzymes, including but not limited to collagenase (e.g., XIAFLEX®), hyaluronidase (e.g., HYLENEX®), and / or DNAse (e.g., PULMOZYME®), etc.).

[0044] The culture process used by methods understood in the art requires 5-7 weeks to grow TILs from bulk non-purified tumor digest. This is a significant issue in the art since the additional time to initiate adoptive transfer therapy of TILs represents an increased risk to the patient due to progression of malignancy while the cell product is being prepared. Furthermore, the additional time required for culture requires additional resources of the hospital in additional personnel for the requirements of culture maintenance and media costs as well as clean room maintenance. The present method reduces the growth time to less than 5 weeks, thereby reducing patient attrition from secondary treatments for disease progression. For example, culturing to obtain an expanded population of TILs may be performed for any time between 1 day and 5 weeks (35 days), preferably 21 days (3 weeks) and 5 weeks (35 days), more preferably 4 weeks (28 days) and 5 weeks (35 days). For example, the culture time may 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. According to some embodiments, the pre-REP expansion is harvested when the desired expansion is reached, but not later than 4 weeks. Thus, disclosed herein are TIL expansion methods in which the pre-REP culture is 1, 2, 3, 4, 5, 6, 7 (1 week), 8, 9, 10, 11, 12, 13, 14 (2 weeks), 15, 16, 17, 18, 19, 20, 21 (3 weeks), 22, 23, 24, 25, 26, 27, or 28 (4 weeks) days. Following pre-REP, the pre-REP TILs may be frozen and used at a later time. Finally, the fresh or thawed pre-REP TILs are subjected to a rapid expansion protocol (REP) that may last for less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days. If frozen TILs are used, the TILs may be thawed for 1-3 days. In some embodiments, thawed TILs are used and the recovery of thawed TILs is 40×10 6 If less than 100, a second culture of thawed TILs can be used to expand the number of TILs.

[0045] It is understood and contemplated herein that all or a portion of the medium in the reservoir may be replaced to maintain the quality of nutrients and remove any waste products during the culture. The medium replacement may include removal and replacement of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% of the medium. This medium replacement may be accomplished using any acceptable method for proper tissue culture maintenance known in the art. In one aspect, the medium replacement may be performed at least once during the culture of the TILs. For example, the medium in the reservoir may be changed 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, i.e., once every 15 days, once every 10 days, once every 7 days, once every 5 days, once every other day, or about 2-3 times a week during the culture period.

[0046] The culture methods used herein may utilize any complete medium containing IL-2 suitable for the growth and proliferation of TILs, including, but not limited to, Minimum Essential Medium (MEM), Eagle'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 Hemocyte Medium.

[0047] TILs may be cultured in any gas permeable reservoir suitable for cell culture and growth of TILs. In one aspect, it is understood and contemplated herein that large tissue culture flasks may slow the growth of TILs as it takes longer for the cells to reach confluency. In one aspect, the gas permeable reservoir is a 6 (approximately 10 cm 2 Surface area / well and 60cm 2 Total surface area), 12 (approx. 4 cm 2 Surface area / well: approx. 48 cm 2 Total surface area), 24 (approx. 2 cm 2Surface area / well: approx. 48 cm 2 Total surface area), 48 (approx. 1 cm 2 Surface area / well: approx. 48 cm 2 total surface area), or 96 (approx. 0.32 cm 2 Surface area / well and 31cm 2 The total surface area of ​​the wells may be a tissue culture plate containing 100 μm of wells (e.g., a G-Rex 24-well plate or a G-Rex 6-well plate (Wilson Wolf). In some embodiments, the plate may be silicone coated.

[0048] The intention of the method for rapidly producing expanded TIL populations is to use TILs in adoptive transfer therapy for cancer. The new method derives several advantages from previous processes. First, it is more successful in expanding TILs from previously poorly performing tumor subtypes. Furthermore, this method successfully produces TILs for ACT with lower risk and reduced cost for patients who would not have been able to access it before with current methods. This new method is performed with significantly less technical intervention time and increases the efficiency of TIL production.

[0049] Also disclosed herein is a method for rapidly producing an expanded TIL population, the method further comprising harvesting the expanded TIL population.

[0050] It is understood and contemplated herein that the TILs generated by the disclosed methods are both tumor specific and function as preREPs. In one aspect, the method may further include verifying the tumor specificity and activity of the preREP TILs by IFN-γ release assay, intracellular IFN-γ staining, ELISA, and / or ELIspot.

[0051] Once expanded, the disclosed expanded TIL populations may be used for the treatment of cancer. In one aspect, a method disclosed herein is a method of treating, reducing, inhibiting and / or preventing cancer and / or metastasis in a subject comprising administering to a subject any of the rapidly expanded TILs disclosed herein, including any TILs produced and / or expanded by the disclosed methods. In other words, disclosed herein, in one aspect, is a method of treating, reducing, inhibiting and / or preventing cancer and / or metastasis in a subject, the method comprising obtaining one or more tissue samples (including but not limited to, a biopsy (e.g., a core biopsy, etc.) and / or one or more surgical sections) from which a bulk, unpurified tumor digest is derived or obtained, the one or more tissue samples comprising TILs from the subject; digesting the one or more tissue samples (including but not limited to, a biopsy (e.g., a core biopsy, etc.) and / or one or more surgical sections) with one or more human or humanized enzymes, including but not limited to, collagenase (e.g., XIAFLEX®), hyaluronidase (e.g., HYLENEX®), and / or DNAse (e.g., PULMOZYME®); culturing cells from the biopsy in complete medium containing IL-2; harvesting the expanded TIL cells; and adoptively transferring the expanded TILs into a subject.

[0052] The TILs rapidly expanded by the disclosed methods may be used to treat, inhibit, reduce, and / or prevent any disease in which uncontrolled cell proliferation occurs, such as cancer. A non-limiting list of various types of cancer is as follows: carcinoma, cancer of solid tissue, squamous cell carcinoma, adenocarcinoma, sarcoma (including but not limited to soft tissue sarcoma (including but not limited to atypical lipomatous tumor, well-differentiated liposarcoma, myxofibrosarcoma, leiomyosarcoma, solitary fibrous tumor, or leiomyosarcoma) and bone tissue sarcoma, glioma, high-grade glioma, blastoma, neuroblastoma, plasmacytoma, histiocytoma, melanoma, adenoma, hypoxic tumor, myeloma, AIDS-related lymphoma or sarcoma, metastatic cancer, or cancer in general.

[0053] A representative, but non-limiting list of cancers that the disclosed compositions can be used to treat are: sarcoma, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of the head and neck, kidney cancer, lung cancer, including small cell lung cancer and non-small cell lung cancer, neuroblastoma / glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, melanoma, squamous cell carcinoma of the mouth, throat, larynx, and lung, colon cancer, cervical cancer, cervical carcinoma, breast cancer, and epithelial cancer, renal cancer, genitourinary cancer, lung cancer, esophageal cancer, head and neck carcinoma, colon cancer, hematopoietic cancer; testicular cancer; colorectal cancer, prostate cancer, or pancreatic cancer.

[0054] 1. Pharmaceutical Carriers / Drug Delivery As mentioned above, TILs may be administered in a pharma- ceutically acceptable carrier in vivo. By "pharma-ceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject together with a nucleic acid or vector without causing any undesirable biological effects or interacting in a detrimental manner with any of the other components of the pharmaceutical composition in which it is contained. Naturally, the carrier will be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as is well known to those skilled in the art.

[0055] The compositions may be administered parenterally (e.g., intravenously), by intramuscular injection, intraperitoneal injection, transdermally, externally, topically, etc., including topical intranasal administration or administration by inhalation. As used herein, "topical intranasal administration" means delivery of the composition to the nose and nasal cavity via one or both of the nostrils, and may include delivery by spray or droplet mechanism, or by aerosolization of the nucleic acid or vector. Administration of the composition by inhalation may be through the nose or mouth by delivery by spray or droplet mechanism. It may also be delivered directly to any area of ​​the respiratory system (e.g., lungs) by intubation. The exact amount of the composition required will vary from subject to subject, depending on the species, age, weight, and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, the mode of administration, etc. Thus, it is not possible to specify exact amounts for all compositions. However, appropriate amounts can be determined by one of ordinary skill in the art using only routine experimentation with the teachings herein.

[0056] Parenteral administration of compositions, when used, is generally characterized by injection.Injectables can be prepared in conventional form, either as liquid solution or suspension, solid form suitable for solution of suspension in liquid before injection, or as emulsion.The more recent revised approach for parenteral administration includes the use of sustained release or sustained release system, so that a constant dosage is maintained.See, for example, U.S. Patent No. 3,610,795 (incorporated herein by reference).

[0057] The materials may be in solution, suspension (e.g., incorporated into microparticles, liposomes, or cells). They may be targeted to specific cell types via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, KD, Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al. al., Biochem. Pharmacol, 42:2062-2065, (1991)). Vehicles such as "stealth" and other antibody-conjugated liposomes (including lipid-mediated drug targeting to colon cancer), receptor-mediated targeting of DNA via cell-specific ligands, lymphocyte-tropic tumor targeting, and highly specific therapeutic retroviral targeting of mouse glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214-6220, (1989), and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general, receptors are involved in pathways of endocytosis, either constitutive or ligand-induced. These receptor clusters in clathrin-coated pits enter the cell via clathrin-coated vesicles and pass through acidified endosomes, where the receptors are sorted and then either recycled to the cell surface, stored intracellularly, or degraded in lysosomes.Internalization pathways serve a variety of functions, including nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, ligand dissociation and degradation, and regulation of receptor levels. Many receptors follow multiple intracellular pathways, depending on the cell type, receptor concentration, ligand type, ligand valency, and ligand concentration.

[0058] a) Pharmaceutically acceptable carrier The compositions containing the antibodies can be used therapeutically in combination with a pharma- ceutically acceptable carrier.

[0059] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. AR Gennaro, Mack Publishing Company, Easton, PA 1995. Usually, an appropriate amount of a pharma- ceutically acceptable salt is used in the formulation to make the formulation isotonic. Examples of pharma- ceutically acceptable carriers include, but are not limited to, saline, Ringer's solution, and dextrose solution. The pH of the solution is preferably about 5 to about 8, more preferably about 7 to about 7.5. Further carriers include sustained release preparations, such as semipermeable matrices of solid hydrophobic polymers containing the antibody, the matrices being in the form of shaped articles, such as films, liposomes, or microparticles. It will be apparent to one skilled in the art that certain carriers may be more preferable depending, for example, on the route of administration and the concentration of the composition to be administered.

[0060] Pharmaceutical carriers are known to those skilled in the art. These are most commonly standard carriers for administering drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

[0061] Pharmaceutical compositions may include, in addition to the molecule of choice, carriers, thickeners, diluents, buffers, preservatives, surfactants, etc. Pharmaceutical compositions may also include one or more active ingredients such as antibacterial agents, anti-inflammatory agents, anesthetics, etc.

[0062] The pharmaceutical compositions may be administered in a number of ways, depending on whether local or systemic treatment is desired and on the area to be treated. Administration may be topical (including ophthalmological, vaginal, rectal, intranasal), oral, by inhalation, or parenteral, for example, intravenous drip, subcutaneous, intraperitoneal, or intramuscular injection. The disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.

[0063] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as, for example, antibacterial agents, antioxidants, chelating agents, and inert gases.

[0064] Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0065] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets.Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders may be desirable.

[0066] Some of the compositions may optionally be administered as pharma- ceutically acceptable acid or base addition salts formed by reaction with inorganic acids, such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids, such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with inorganic bases, such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases, such as mono-, di-, tri-alkyl and aryl amines, and substituted ethanol amines.

[0067] b) Therapeutic uses Effective dosages and schedules for administering the composition may be empirically determined, and making such determinations is within the skill of the art. The dosage range for administration of the composition is sufficient to produce the desired effect in which the symptoms of the disorder are affected. The dosage should not be so high as to cause adverse side effects, such as undesirable cross-reactions, anaphylactic reactions, and the like. In general, dosage will vary according to the age, condition, sex, and extent of the disease of the patient, the route of administration, or whether other drugs are included in the dosing regimen, and can be determined by one of skill in the art. Dosage can be adjusted by the individual physician in the event of any indications to the contrary. Dosage can vary and may be administered in one or more doses per day for one or several days. Guidance regarding appropriate dosages for a given class of pharmaceuticals can be found in the literature. For example, guidance in selecting an appropriate dose of an antibody can be found in the literature on therapeutic use of antibodies (e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, NJ, (1985) ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp. 365-389). Typical daily dosages of antibodies used alone can range from about 1 μg / kg body weight up to 100 mg / kg body weight or more per day, depending on the factors mentioned above.

[0068] 2. Kit Disclosed herein are kits relating to reagents that can be used in carrying out the methods disclosed herein. The kits may include any reagent or combination of reagents discussed herein or that would be understood to be necessary or beneficial in carrying out the disclosed methods. For example, the kits may include needles for taking core biopsies (such as core needle biopsies), core biopsy instruments, media for culturing core biopsy tissue samples, IL-2, and required buffers and enzymes. EXAMPLES

[0069] 3. Working Example Tumor infiltrating lymphocytes (TILs) reside within tumors and are subject to a series of tumor suppressive mechanisms within the tumor microenvironment (TME). Adoptive cell therapy (ACT) is a strategy used to overcome TME suppression and involves culturing TILs from tumors, expanding the TIL cell product, and then injecting the expanded TILs with IL-2. The classical method of TIL culture requires tumor fragments as the starting tissue source. Historically, TILs have been cultured using tumor fragments (1 mm ) based on their success in melanoma patients. 3 As efforts with ACT have been expanded to other solid tumors, culturing TILs with tumor-specific reactivity has had less success, especially in sarcoma specimens.

[0070] In this study, we explore an alternative strategy of utilizing tumor digests (e.g., bulk crude tumor digests) as the initial TIL culture source, since some TILs may not be able to migrate out of the tumor using the fragmentation method. Additionally, shown herein are differences between the growth, phenotype, and reactivity of TILs grown from fragments compared to bulk crude tumor digests. Here, we report the successful growth, lymphocyte subpopulation phenotype, and tumor-specific reactivity of TILs generated from digests compared to fragments derived from soft tissue sarcomas.

[0071] method Patients with soft tissue sarcomas consented to an IRB-approved protocol, and primary tumor specimens were obtained fresh from the operating room. Tumor fragments (1 mm 3 ) were crushed and placed into a single well of a 24-well plate containing 2 mL of medium supplemented with 6000 IU / mL IL-2. Excess tumor tissue was digested using collagenase enzyme and mechanical disruption. Digested tumor cells (5 × 10 5 Viable cells) were placed into a single well of a 48-well plate with medium containing IL-2 (6000 IU / ml). All TILs were cultured for 30 days and then harvested.

[0072] Lymphocyte phenotypes (CD3, CD4, and CD8 T cells and CD56 NK cells) from cultured TILs based on digestion or fragments were measured using flow cytometry after 4 weeks of culture.

[0073] result Seven sarcoma specimens were obtained. TILs were expanded from each specimen, with no significant difference in median total TIL numbers between digestion and fragmentation methods (4.3 x 106 vs. 2.7 x 106, p=0.6250). The expansion of TILs from tumor digests for each specimen is shown in Figure 1. Median yield 3.4 x 10 7 (Range 1.4×10 7 -9.2×10 7 ) and median survival rate of 97% (93%-99%).

[0074] To determine the percentage or phenotype of TILs using the digestion methodology, lymphocytes were stained with anti-CD3, anti-CD4, anti-CD8, and anti-CD56 antibodies and measured using flow cytometry to determine the numbers of total T cells, CD4+ T cells, CD8+ T cells, and NK cells, respectively (Figure 2). Digests were prepared for use in humans, and the collagenase, hyaluronidase, and / or DNAse enzymes used in the digestion were switched to human or humanized enzymes (e.g., HYLENEX®, PULMOZYME®, and / or XIAFLEX®). In doing so, the human enzymes (non-xeno) showed a surprising and unexpected significant improvement in the total number of viable cells following digestion (Figure 3). Furthermore, digestion survival was surprisingly improved and total CD8 T cells were increased when human / humanized (non-xeno) digestion was used.

[0075] conclusion TILs can be grown directly from the digesta of soft tissue sarcoma tumors, as these are highly fibrotic sets of primary malignancies, and this method may also work well for TIL cultures derived from sources other than metastatic lymph nodes.

[0076] TIL cultures generated from tumor digests were comparable in overall numbers and phenotypic presentation and did not differ across the various sarcoma subtypes, although individual differences exist.

[0077] TILs cultured from digestion have a higher probability of tumor-specific reactivity compared to TILs cultured from fragments.

Claims

1. 1. A method for rapidly producing an expanded population of tumor-reactive tumor-infiltrating lymphocytes (TILs) for use in adoptive cell therapy, comprising culturing bulk non-purified tumor digesta from a human subject in a culture medium containing IL-2 in an amount effective to expand highly tumor-reactive tumor-infiltrating lymphocytes.

2. The method of claim 1 , wherein the expanded TIL population also has high tumor specificity.

3. 10. The method of claim 1, further comprising obtaining one or more tissue samples from a subject and digesting the one or more tissue samples with one or more human or humanized enzymes.

4. 4. The method of claim 3, wherein the one or more human or humanized enzymes comprise collagenase, hyaluronidase, and / or DNAse.

5. 5. The method of claim 4, wherein the one or more human or humanized enzymes comprise HYLENEX®, PULMOZYME®, and / or XIAFLEX®.

6. The method of claim 3 , wherein the one or more tissue samples comprise one or more core biopsy tissue samples.

7. 7. The method of claim 6, further comprising performing one or more core biopsies prior to digesting the tissue sample.

8. The method of claim 3 , wherein the one or more tissue samples comprise one or more surgical resections.

9. 9. The method of claim 8, further comprising performing one or more surgical resections prior to digesting the tissue sample.

10. 4. The method of claim 3, wherein the one or more core biopsies or one or more surgical resections are digested without disaggregating the specimen.

11. The method of claim 1 , wherein the culture medium is a complete medium.

12. The method of claim 1 , further comprising harvesting the expanded TIL population.

13. The method of claim 1, wherein the TILs are cultured in medium containing IL-2 for up to 5 weeks.

14. A pharmaceutical composition for use in a method for treating cancer in a subject, the method comprising administering to the subject a rapidly expanded TIL population produced by the method of any one of claims 1 to 13.

15. A pharmaceutical composition for use in a method for treating cancer in a human subject, the method comprising: culturing bulk crude tumor digest from the subject in a culture medium containing IL-2 in an amount effective to expand highly tumor-reactive and / or specific tumor-infiltrating lymphocytes; harvesting the expanded TIL cells; and adoptively transferring the expanded TILs into the subject.

16. The pharmaceutical composition of claim 15, wherein the method further comprises obtaining one or more tissue samples from a subject and digesting the one or more tissue samples with one or more human or humanized enzymes.

17. 17. The pharmaceutical composition of claim 16, wherein the one or more human or humanized enzymes comprise collagenase, hyaluronidase, and / or DNAse.

18. 18. The pharmaceutical composition of claim 17, wherein the one or more human or humanized enzymes comprise HYLENEX®, PULMOZYME®, and / or XIAFLEX®.

19. 17. The pharmaceutical composition of claim 16, wherein the one or more tissue samples comprise one or more core biopsy tissue samples.

20. The pharmaceutical composition of claim 19, wherein the method further comprises performing one or more core biopsies prior to digesting the tissue sample.

21. 20. The pharmaceutical composition of claim 19, wherein the one or more tissue samples comprise one or more surgical resections.

22. The pharmaceutical composition of claim 21, wherein the method further comprises performing one or more surgical resections prior to digesting the tissue sample.

23. 20. The pharmaceutical composition of claim 19, wherein the one or more core biopsies or one or more surgical resections are digested without disaggregating the specimen.

24. The pharmaceutical composition of claim 14, wherein the cancer is a solid tumor.

25. The pharmaceutical composition of claim 24, wherein the cancer is a sarcoma.

26. 26. The pharmaceutical composition of claim 25, wherein the sarcoma is a soft tissue sarcoma.

27. 27. The pharmaceutical composition of claim 26, wherein the soft tissue sarcoma is a fibrous sarcoma.

28. 28. The pharmaceutical composition of claim 27, wherein the fibrous sarcoma is selected from the group consisting of atypical lipomatous tumor, well-differentiated liposarcoma, myxofibrosarcoma, leiomyosarcoma, solitary fibrous tumor, and leiomyosarcoma.

29. The pharmaceutical composition of claim 15, wherein the cancer is a solid tumor.

30. 30. The pharmaceutical composition of claim 29, wherein the cancer is a sarcoma.

31. 31. The pharmaceutical composition of claim 30, wherein the sarcoma is a soft tissue sarcoma.

32. 32. The pharmaceutical composition of claim 31, wherein the soft tissue sarcoma is a fibrous sarcoma.

33. 33. The pharmaceutical composition of claim 32, wherein the fibrous sarcoma is selected from the group consisting of atypical lipomatous tumor, well-differentiated liposarcoma, myxofibrosarcoma, leiomyosarcoma, solitary fibrous tumor, and leiomyosarcoma.

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