Methods for treating cancer using antisense

Abstract

To provide compositions and methods for treating cancer using antisense nucleic acid (AS ODN).SOLUTION: The disclosure relates to compositions and methods for treating cancer using antisense (AS) nucleic acids directed against Insulin-like Growth Factor 1 Receptor (IGF-1R). The AS may be administered to the patients systemically, or may be used to produce an autologous cancer cell vaccine. In embodiments, the AS are provided in an implantable irradiated biodiffusion chamber comprising tumor cells and an effective amount of the AS. The chambers are irradiated and implanted in the abdomen of subjects and stimulate an immune response that attacks tumors distally. The compositions and methods disclosed herein may be used to treat many different kinds of breast cancer, including glioblastoma. In embodiments, a method for predicting efficacy of an antisense nucleic acid targeting the insulin-like growth factor 1 receptor in a subject.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present disclosure relates to compositions and methods for treating cancer using antisense nucleic acids that target the insulin-like growth factor 1 receptor (IGF-1R). The present disclosure also relates to compositions and methods for treating cancer by treating a subject using at least one implantable diffusion chamber for irradiated living organisms (see U.S. Patent No. 6,541,036 and International Application No. PCT / US2016 / 026970, which are hereby incorporated by reference in their entireties) that includes tumor cells and an antisense nucleic acid that targets IGF-1R.

Background Art

[0002] Patent Document 1 (which is hereby incorporated by reference in its entirety) discloses "compositions and methods for treating cancer using antisense (AS) nucleic acids that target the insulin-like growth factor 1 receptor (IGF-1R)". The AS can be administered systemically to a patient or used to produce an autologous cancer cell vaccine. Since the patient's response to IGF-1R can vary, there is a need for a method to predict the prognosis of a subject with cancer who responds to treatment with IGF-1R AS ODN.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

[0004] The present disclosure relates, at least in part, to the use of antisense oligodeoxynucleotides (AS-ODNs) that target insulin-like growth factor receptor-1 (IGF-1R) (「IGF-1R AS ODN」) for treating a subject having cancer. In certain embodiments, the present disclosure relates to methods and compositions (including diagnostic and companion diagnostics) for identifying a subject likely to respond to treatment with IGF-1R AS ODN and administering IGF-1R AS ODN to such a subject. In certain embodiments, a subject likely to respond to treatment with IGF-1R AS ODN is identified by determining MGMT methylation and / or determining T cell function in the subject. In certain embodiments, a subject likely to respond to treatment with IGF-1R AS ODN is identified by determining MGMT methylation and / or determining T cell function in the subject, wherein a patient having methylated MGMT and / or having good T cell function in the subject has been shown to be likely to respond to treatment with IGF-1R AS ODN.

[0005] In some embodiments, a method is provided for predicting the prognosis of a subject having cancer (e.g., glioma or glioblastoma) who has responded to treatment with IGF-1R AS ODN, wherein the method comprises determining MGMT methylation and / or determining T cell function in the subject.

[0006] In one aspect, a method is provided that comprises determining MGMT methylation and / or determining T cell function in a subject diagnosed with cancer, and subsequently administering IGF-1R AS ODN to the subject. In related embodiments, a method is provided that comprises determining MGMT methylation and / or determining T cell function in a subject diagnosed with cancer, and subsequently administering IGF-1R AS ODN to the subject only if the subject is confirmed to have methylated MGMT and / or the subject has good T cell function.

[0007] As used herein, MGMT refers to O6-methylguanine-DNA methyltransferase (e.g., Uniprot accession number Q6LDD1). Methylation of the O6-methylguanine-DNA methyltransferase (MGMT) promoter stops the cell's ability to demethylate the methyl group of O6 guanine and enhances the therapeutic effect of temozolomide (TMZ) compared to patients with an unmethylated MGMT promoter. DNA methylation is the covalent addition of a methyl group, usually at the 5' position, to cytosine or guanine nucleotides. Evaluation of MGMT methylation can be performed and determined using methods well known in the art. In some embodiments, the methylation status of eight CpG islands within the MGMT gene promoter is evaluated. Methylated MGMT in various embodiments is an indicator of a favorable prognosis and / or prediction of a positive response to IGF-1R AS ODN treatment. On the other hand, unmethylated MGMT is an indicator of a favorable prognosis and / or prediction of a positive response to IGF-1R AS ODN treatment in various embodiments.

[0008] Determination of T cell function can also be determined using methods and criteria well known in the art. In some embodiments, T cell function is determined by evaluating the number of T cells that express IFN-γ in response to non-specific stimulation. In certain embodiments, the term "good T cell function" as used herein refers to a subject in which the number of T cells that express IFN-γ in response to non-specific stimulation is above the median. The term "insufficient T cell function" refers to a subject in which the number of T cells that express IFN-γ in response to non-specific stimulation is below the median.

[0009] In some embodiments, the IGF-1R AS ODN is administered to the subject before temozolomide is administered to the subject. In certain embodiments, the IGF-1R The AS ODN is administered to the subject at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; or at least 8 weeks before temozolomide is administered to the subject.

[0010] In some embodiments of any of the aspects and embodiments provided herein, the IGF-1R AS ODN is administered to the subject as an autologous cancer cell vaccine. In some embodiments of any of the aspects and embodiments provided herein, the IGF-1R AS ODN is administered to the subject as a fully formulated in vivo diffusion chamber.

[0011] The present disclosure demonstrates that an antisense oligodeoxynucleotide (AS-ODN) targeting the insulin-like growth factor 1 receptor (IGF-1R) effectively stimulates the response in subjects being treated for cancer when used in the treatment methods described herein. In certain aspects, the method is effective to treat cancer in a patient, alone or optionally in combination with systemic administration, as part of an autologous cancer cell vaccine. In a preferred method, the methods disclosed herein provide an effective cancer therapy as monotherapy, i.e., in the absence of chemotherapy and in the absence of radiation therapy.

[0012] In an embodiment, the present disclosure provides an in vivo diffusion chamber for implantation into a subject having a tumor. The in vivo diffusion chamber contains irradiated tumor cells and irradiated insulin-like growth factor 1 receptor antisense oligodeoxynucleotide (IGF-1R AS ODN). In an embodiment, the tumor cells are removed from the resection site of the subject.

[0013] In an embodiment, the present disclosure provides a diffusion chamber containing irradiated IGF-1R AS ODN and irradiated, adhesively enhanced fragmented tumor cells. The in vivo diffusion chamber contains a membrane that is impermeable to cells and permeable to IGF-1R AS ODN.

[0014] In embodiments, tumor cells are removed from the resection site using an endoscopic device. In further embodiments, tumor cells are removed from the resection site using a tissue morcellator. In some embodiments, tumor cells are viable when removed from the resection site using a tissue morcellator. In other embodiments, the tissue morcellator includes a high-speed reciprocating inner cannula within a stationary outer cannula. The outer cannula may include a side aperture, and further, tumor cells are aspirated into the side aperture by electronically controlled variable suction. In embodiments, the tissue morcellator does not generate heat at the resection site. In still further embodiments, tumor cells are enriched for nestin expression prior to placement in a biocompatible diffusion chamber. In some embodiments, implantation of the chamber inhibits tumor regrowth in a subject. In some embodiments, implantation of the chamber inhibits tumor regrowth for at least 3 months, at least 6 months, at least 12 months, or at least 36 months.

[0015] In other embodiments, the present disclosure provides a method of making a biocompatible diffusion chamber for implantation in a subject having a tumor. The method includes placing tumor cells in the biocompatible diffusion chamber in the presence of IGF-1R AS ODN and irradiating the biocompatible diffusion chamber with radiation, wherein the tumor cells are removed from the resection site using a tissue morcellator that does not generate heat at the resection site in the subject. Typically, a plurality of chambers are used. For example, about 10 chambers or about 20 chambers. Advantageously, an optimal anti-tumor response is achieved when the number of cells in the chamber is from about 750,000 to about 1,250,000. For example, when 20 chambers are implanted, it is about 1,000,000 / chamber.

[0016] In some embodiments, the tissue morcellator is an endoscopic device. In further embodiments, the tissue morcellator includes a high-speed reciprocating inner cannula within a stationary outer cannula. In other embodiments, the outer cannula includes a side aperture and tumor cells are aspirated into the side aperture by electronically controlled variable suction.

[0017] In an embodiment, the present disclosure provides a method for treating a subject suffering from a tumor. The method includes implanting one or more biocompatible diffusion chambers into the subject, the one or more biocompatible diffusion chambers containing irradiated tumor cells and irradiated insulin-like growth factor 1 receptor antisense oligodeoxynucleotide (IGF-1R AS ODN), and the tumor cells being removed from the subject at an excision site using a tissue morcellator that does not generate heat at the excision site.

Brief Description of the Drawings

[0018]

Figure 1

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Modes for Carrying Out the Invention

[0019] Detailed Description Definitions All terms not defined in this specification have their ordinary meanings as recognized in the art.

[0020] As used herein, terms such as "a", "an", and "the" include singular and plural referents unless the context clearly dictates otherwise. As used herein, the term "about" preceding a numerical value represents a range within ±10% of that value. For example, "about 100" encompasses 90 and 110. To avoid misunderstanding, it is understood that the term "about" includes the 10% range in addition to the indicated value itself. For example, "about 100" includes exactly 100 and the range from 90 to 100.

[0021] As used herein, the terms "self-" and "autologous-" mean cells or tissues obtained from the same individual. As used herein, the term "autologous cancer cell vaccine" in part means a therapeutic agent generated by isolating tumor cells from an individual and treating these tumor cells ex vivo. The cells are then re-administered to the individual from whom the tumor cells were isolated. In embodiments, the autologous cancer cell vaccine may include, in addition to the tumor cells, additional components such as buffers and / or antisense nucleic acids (e.g., IGF-1R AS ODN, etc.). In embodiments, the "autologous cancer cell vaccine" may mean a biocompatible diffusion chamber containing tumor cells and one or more additional components. In certain aspects, the "autologous cancer cell vaccine" may be what is herein also referred to as a "fully formulated biocompatible diffusion chamber" or a "fully formulated chamber".

[0022] As used herein, the term "fully formulated chamber" or "fully formulated biocompatible diffusion chamber" is a biocompatible diffusion chamber containing autologous tumor cells and other cells contained in the tumor microenvironment (TME), which may or may not be treated prior to encapsulation in the chamber with a first amount of IGF-1R AS ODN. An external addition of a second amount, for example at least 2 μg of IGF-1R AS ODN, is made to the cells for encapsulation, and then the chamber is irradiated with 5 Gy of gamma rays.

[0023] As used herein, the term "low molecule" includes nucleic acids, peptides, proteins, and other chemical substances (e.g., cytokines, growth hormones, etc. produced by cells), but does not include cells, exosomes, and microvesicles.

[0024] As used herein, the term "targeting IGF-1R expression" means administering an antisense nucleic acid having a sequence designed to bind to IGF-1R. As used herein, the term "systemic administration" means achieving delivery of a substance throughout the body of a subject. Typical systemic administration routes include parenteral administration, transdermal administration, intraperitoneal administration, intravenous administration, subcutaneous administration, and intramuscular administration.

[0025] Other administration routes include oral administration, nasal administration, topical administration, intraocular administration, intraoral administration, sublingual administration, vaginal administration, intrahepatic administration, intracardiac administration, intrapancreatic administration, inhalation administration, and implantable pump administration.

[0026] Antisense molecule An antisense molecule is a nucleic acid that functions by binding to a complementary target sequence of mRNA according to the Watson-Crick base pairing rule. Translation of the target mRNA is inhibited by an active mechanism and / or a passive mechanism when hybridization occurs between complementary helices. In the passive mechanism, hybridization of mRNA with an exogenous nucleotide sequence results in the formation of a double strand that prevents the ribosome complex from reading the message. In the active mechanism, hybridization promotes the binding of RnaseH, which destroys the RNA while leaving the antisense intact to hybridize to the other complementary mRNA target. One or both mechanisms inhibit the translation of proteins that contribute to or sustain a malignant phenotype. As a therapeutic agent, antisense molecules are much more selective, and thus more effective and less toxic than conventional drugs.

[0027] The methods and compositions disclosed herein include the use of antisense molecules for treating cancer. Typically, the antisense molecules are antisense oligodeoxynucleotides (AS-ODNs). In some embodiments, the antisense molecules include a modified phosphate backbone. In certain aspects, the phosphate backbone modification increases the resistance of the antisense to nuclease degradation. In certain embodiments, the modification is a locked antisense. In other embodiments, the modification is a phosphorothioate bond. In certain aspects, the antisense contains one or more phosphorothioate bonds. In certain embodiments, the phosphorothioate bond stabilizes the antisense molecule by conferring nuclease resistance, thereby increasing its half-life. In some embodiments, the antisense can be partially phosphorothioated. For example, up to about 1%, up to about 3%, up to about 5%, up to about 10%, up to about 20%, up to about 30%, up to about 40%, up to about 50%, up to about 60%, up to about 70%, up to about 80%, up to about 90%, up to about 95%, or up to about 99% of the antisense can be phosphorothioated. In some embodiments, the antisense is fully phosphorothioated. In other embodiments, the phosphorothioate bonds can alternate with phosphodiester bonds. In certain embodiments, the antisense has at least one terminal phosphorothioate monophosphate.

[0028] In some embodiments, the antisense molecule includes one or more CpG motifs. In other embodiments, the antisense molecule does not include a CpG motif. In certain aspects, one or more of the CpG motifs are methylated. In other aspects, one or more of the CpG motifs are not methylated. In certain embodiments, when the antisense molecule is administered to a subject, one or more unmethylated CpG motifs induce an innate immune response. In some aspects, the innate immune response is mediated by the binding of the unmethylated CpG-containing antisense molecule to Toll-like receptors (TLRs).

[0029] In certain embodiments, the antisense molecule includes at least one terminal modification or “cap”. The cap can be a 5’ and / or 3’ cap structure. The term “cap” or “end cap” includes chemical modifications at either end of the oligonucleotide (with respect to the terminal ribonucleotide), and also includes modifications of the bonds between the last two nucleotides at the 5’ end and between the last two nucleotides at the 3’ end. The cap structure can increase the resistance of the antisense molecule to exonucleases without impairing molecular interactions with the target sequence or cellular mechanisms. Such modifications can be selected based on an increase in its efficacy in vitro or in vivo. The cap can be present at the 5’ end (5’ cap) or the 3’ end (3’ cap), or at both ends. In certain embodiments, the 5’ and / or 3’ cap is independently selected from phosphorothioate monophosphate, abasic residue (moiety), phosphorothioate bond, 4’-thionucleotide, carbocyclic nucleotide, phosphorodithioate bond, inverted nucleotide or inverted abasic moiety (2’-3’ or 3’-3’), phosphorodithioate monophosphate, and methylphosphonate moiety. When a phosphorothioate bond or phosphorodithioate bond is part of the cap structure, it is generally disposed between the two terminal nucleotides at the 5’ end and between the two terminal nucleotides at the 3’ end.

[0030] In a preferred embodiment, the antisense molecule targets the expression of insulin-like growth factor 1 receptor (IGF-1R). IGF-1R is a tyrosine kinase cell surface receptor that shares 70% homology with the insulin receptor. Upon activation by its ligands (IGF-I, IGF-II, and insulin), it regulates a wide range of cellular functions including growth, transformation, and cell survival. IGF-1R is not an absolute requirement for normal growth but is essential for growth under anchorage-independent conditions that can occur in malignant tissues. A review of the role of IGF-1R in tumors is provided by Baserga et al., "Vitamins and Hormones," Volume 53, pp. 65-98, 1997 (which is hereby incorporated by reference in its entirety).

[0031] In certain embodiments, the antisense molecule is an oligonucleotide that targets DNA or RNA of a growth factor or growth factor receptor, such as IGF-1R. In certain embodiments, the antisense is a deoxynucleotide that targets IGF-1R (IGF-1R AS ODN). The full-length coding sequence of IGF-1R is provided as SEQ ID NO: 19 (see, for example, PCT / US2016 / 26970 pamphlet, which is hereby incorporated by reference in its entirety).

[0032] In certain embodiments, the antisense molecule comprises a nucleotide sequence complementary to the IGF-1R signal sequence, comprising either RNA or DNA. The signal sequence of IGF-1R is a 30-amino acid sequence. In other embodiments, the antisense molecule comprises a nucleotide sequence complementary to a portion of the IGF-1R signal sequence, comprising either RNA or DNA. In some embodiments, the antisense molecule comprises a nucleotide sequence complementary to codons 1-309 of IGF-1R, comprising either RNA or DNA. In other embodiments, the antisense molecule comprises a nucleotide sequence complementary to a portion of codons 1-309 of IGF-1R, comprising either RNA or DNA.

[0033] In certain embodiments, the IGF-1R AS ODN is at least about 5 nucleotides, at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, at least about 40 nucleotides, at least about 45 nucleotides, or at least about 50 nucleotides in length. In some embodiments, the IGF-1R AS ODN is from about 15 nucleotides to about 22 nucleotides in length. In certain aspects, the IGF-1R AS ODN is about 18 nucleotides in length.

[0034] In certain embodiments, the IGF-1R AS ODN forms a secondary structure at 18°C but does not form a secondary structure at about 37°C. In other embodiments, the IGF-1R AS ODN does not form a secondary structure at either about 18°C or about 37°C. In yet other embodiments, the IGF-1R AS ODN does not form a secondary structure at any temperature. In other embodiments, the IGF-1R AS ODN does not form a secondary structure at 37°C. In certain embodiments, the secondary structure is a hairpin loop structure.

[0035] In some aspects, the IGF-1R AS ODN comprises the nucleotide sequence of SEQ ID NO: 1 or a fragment thereof. In certain embodiments, the IGF-1R AS ODN can have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 98%, or 100% identity to SEQ ID NO: 1 or a fragment thereof. In some embodiments, the IGF-1R AS ODN comprises one or more phosphorothioate linkages.

[0036] In certain embodiments, the IGF-1R AS ODN consists of SEQ ID NO: 1. NOBEL is an 18-mer oligodeoxynucleotide having a phosphorothioate backbone and a sequence complementary to codons 2-7 of the IGF-1R gene. Thus, NOBEL is an antisense oligonucleotide (IGF-1R AS ODN) that targets IGF-1R. The NOBEL sequence derived as the complementary sequence of the IGF-1R gene at the 5' end is 5'-TCCTCCGGAGCCAGACTT-3' (SEQ ID NO: 1) is.

[0037] NOBEL has a stable shelf life due to its phosphorothioate backbone and is resistant to nuclease degradation. Administration of NOBEL can be provided by any of the standard methods associated with the introduction of oligodeoxynucleotides known to those of skill in the art. Advantageously, the AS ODNs disclosed herein, including NOBEL, can be administered with little to no toxicity. Even at a level of approximately 2 g / kg (scale adjusted) based on mouse studies (40 μg in the tail vein), no toxicity issues were shown. NOBEL can be manufactured according to the usual procedures known to those of skill in the art.

[0038] Antisense molecules, for example, the NOBEL sequence of SEQ ID NO: 1 may also contain one or more p-ethoxy backbone modifications as disclosed in U.S. Patent No. 9,744,187, which is incorporated herein by reference in its entirety. In some embodiments, the nucleic acid backbone of the antisense molecule contains at least one p-ethoxy backbone linkage. For example, up to about 1%, up to about 3%, up to about 5%, up to about 10%, up to about 20%, up to about 30%, up to about 40%, up to about 50%, up to about 60%, up to about 70%, up to about 80%, up to about 90%, up to about 95%, or up to about 99% of the antisense molecule may be p-ethoxy linked. The remainder of the linkages may be phosphodiester linkages or phosphorothioate linkages or combinations thereof. In a preferred embodiment, 50% to 80% of the phosphate backbone linkages in each oligonucleotide are p-ethoxy backbone linkages, and 20% to 50% of the phosphate backbone linkages in each oligonucleotide are phosphodiester backbone linkages.

[0039] The various IGF-1R antisense sequences are bioactive in some or all of the multimodality actions of the NOBEL sequence. The 18-mer NOBEL sequence has both downregulation activity of the IGF-1R receptor and even TLR agonist activity, and further experiments in mice suggest that both activities are required for in vivo antitumor immune activity. The AS ODN molecule has antitumor activity, while the complementary sense sequence does not have such activity despite also having CpG motifs.

[0040] In certain embodiments, the antisense sequence is selected from the group consisting of SEQ ID NOs: 1 to 14 shown in Table 1. In some embodiments, the antisense has 90% sequence identity to one or more of SEQ ID NOs: 1 to 14. In some embodiments, the antisense has 80% sequence identity to one or more of SEQ ID NOs: 1 to 14. In some embodiments, the antisense has 70% sequence identity to one or more of SEQ ID NOs: 1 to 14.

[0041]

Table 1

[0042] In certain embodiments, the IGF-1R AS ODN comprises any one of the nucleotide sequences of SEQ ID NOs: 1-14 or a fragment thereof. In certain embodiments, the IGF-1R AS ODN may have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 98%, or 100% identity to any one of SEQ ID NOs: 1-14 or a fragment thereof.

[0043] In some embodiments, the antisense molecule downregulates the expression of a gene downstream of the IGF-1R pathway intracellularly. In certain aspects, the downstream gene is hexokinase (HexII). In some embodiments, the antisense molecule downregulates the expression of a housekeeping gene intracellularly. In some aspects, the housekeeping gene is L13.

[0044] In certain aspects, the IGF-1R AS ODN is chemically synthesized. In certain embodiments, the IGF-1R AS ODN is produced by solid-phase organic synthesis. In some aspects, the synthesis of the IGF-1R AS ODN is performed in a synthesizer equipped with a flow-through technology and a sealed chemical column reactor. In some embodiments, each synthetic cycle sequence on the solid support consists of a plurality of steps that are sequentially performed until the full-length IGF-1R AS ODN is obtained. In certain embodiments, the IGF-1R AS The ODN is stored in liquid form. In other embodiments, the IGF-1R AS ODN is lyophilized prior to storage. In some embodiments, the lyophilized IGF-1R AS ODN is dissolved in water prior to use. In other embodiments, the lyophilized IGF-1R AS ODN is dissolved in an organic solvent prior to use. In yet other embodiments, the lyophilized IGF-1R AS ODN is formulated as a pharmaceutical composition. In some aspects, the pharmaceutical composition is a liquid pharmaceutical composition. In other aspects, the pharmaceutical composition is a solid pharmaceutical composition. Additional antisense nucleic acids are also described in U.S. Patent Application Publication No. 2017 / 0056430, which is incorporated herein by reference in its entirety.

[0045] Autologous cancer cell vaccine Introduction Immunotherapy is currently used to target hematological malignancies using one common cellular antigen. Unfortunately, solid tumors are far more complex, have an unidentifiable number of tumor-specific targets and exhibit epigenetic progression of genetic changes towards malignancy. Even more troublesome, there are significant variations in tumor phenotypes within the diagnostic cancer groups of the WHO. Autologous cell vaccines would be an ideal tumor-specific immunotherapy for solid tumor cancers as they would encompass all such variations and targets. However, autologous cancer cell vaccines cannot be derived from primary cell cultures because the tumor phenotype changes with serial passage and the array of tumor-specific antigens decreases. For this reason, lot release certification, which is difficult to achieve, would be required at each passage. The present disclosure eliminates such concerns by plating newly excised fragmented tumor cells and re-implanting them as depot antigens within 24 hours. In certain aspects, the excellent results achieved herein are obtained, among other things in the specific examples described herein, by ensuring that an appropriate number of cells are present within the chamber.

[0046] In previous studies, autologous cell vaccines have been designed using antigen-presenting cells instead of autologous tumor cells. In this paradigm, monocytes from the subject are collected from pre-treatment plasma leukopheresis and differentiated ex vivo into autologous dendritic cells (DCs). The dendritic cells are then presented with the subject's tumor crude lysate to induce DC activation / maturation, and at a later time point, the mature dendritic cells, now cross-primed with tumor antigens, are injected into the subject as a DC vaccine. However, ex vivo differentiation lacks some of the major stimulatory components that occur only in vivo. In addition, the differentiation of DCs from hematopoietic precursors requires large-scale in vitro manipulation involving expensive equipment and labor-intensive cell processing. The present disclosure circumvents such concerns by providing an endogenous DC maturation process and an immunomodulatory and immunostimulatory antisense oligodeoxynucleotide (AS-ODN) that promotes the generation of an appropriate immune response. More specifically, the present disclosure provides a biocompatible diffusion chamber containing disseminated tumor cells derived from a patient and irradiated antisense molecules. This is implanted into the patient over the course of the treatment efficacy period. Without being bound by any theory, the combination of irradiated tumor cells, antisense, and the biocompatible diffusion chamber is thought to act concertedly to simulate a local immune response and enhance the response by reducing or eliminating M2 cells and preventing attenuation of the immune system.

[0047] Therefore, the present disclosure demonstrates that an irradiated implantable biocompatible diffusion chamber containing newly resected tumor cells and IGF-1R AS ODN functions safely as a target-specific autologous cell vaccine effective in cancer immunotherapy. Accordingly, the claimed use of the implantable biocompatible diffusion chamber to initiate an immune response that selectively targets tumor cells in a subject provides a new and important approach for treating cancer, particularly GBM.

[0048] Biocompatible diffusion chamber A representative diffusion chamber includes a chamber barrel having two ends, a first end and a second end. In an embodiment, the diffusion chamber for a living body is a small ring with one side capped by a porous cell-impermeable membrane such as a Duropore membrane manufactured by Millipore Corporation. Optionally, only one end can be left open to seal with a porous membrane, and one of the ends can be sealed as part of the chamber body. The membrane can be made of a plastic, Teflon®, polyester, or any inert material that can withstand strong and flexible chemical treatments. The chamber can be made of any substance, for example, but not limited to, plastic, Teflon®, Lucite, titanium, Plexiglas, or any inert material that is non-toxic and well-tolerated to humans. Additionally, the chamber should be able to withstand sterilization treatment. In some aspects, the diffusion chamber is sterilized with ethylene oxide before use. Other suitable chambers are described in U.S. Provisional Patent Application No. 62 / 621,295 filed on January 24, 2018, U.S. Patent No. 6,541,036, PCT / US16 / 26970 pamphlet, and U.S. Patent No. 5,714,170 (each of which is incorporated herein by reference in its entirety).

[0049] In certain embodiments, the membrane allows the passage of small molecules but not cells (i.e., cells cannot enter or exit the chamber). In some aspects, the pore diameter of the membrane allows the diffusion of nucleic acids and other chemical substances (e.g., cytokines produced by cells) from the chamber, but prevents the passage of cells between the chamber and the implanted subject. The biocompatible diffusion chambers useful in the present disclosure include any chamber that prevents the passage of cells between the chamber and the implanted subject, provided that the chamber allows the exchange and passage of factors between the chamber and the subject. Thus, in certain aspects, the pore size has a cut-off that prevents the passage of substances with a volume greater than 100 μm 3 from entering or leaving the chamber. In some embodiments, the pores of the membrane have a diameter of about 0.25 μm or less. For example, the pores can have a diameter of about 0.1 μm. In certain aspects, the pores are in the range of 0.1 μm to 0.25 μm in diameter. See also Lange et al., Journal of Immunology (J. Immunol.), 1994, Vol. 153, pp. 205-211, and Lanza et al., Transplantation, 1994, Vol. 57, pp. 1371-1375 (each incorporated herein by reference in its entirety). This pore diameter prevents the passage of cells entering or leaving the chamber. In certain embodiments, the diffusion chamber is constructed from a 14 mm Lucite ring having a hydrophilic Durapore membrane (Millipore, Bedford, Massachusetts) with a pore size of 0.1 μm.

[0050] In certain embodiments, the biocompatible diffusion chamber includes a membrane that allows the diffusion of IGF-1R AS ODN from the chamber. In some embodiments, about 50% of the IGF-1R AS ODN diffuses from the chamber in about 12 hours, about 60% of the IGF-1R AS ODN diffuses from the chamber in about 24 hours, and IGF-1R AS Approximately 80% of the ODN diffuses from the chamber in about 48 hours and / or approximately 100% of the IGF-1R AS ODN diffuses from the chamber in about 50 hours.

[0051] In an exemplary method, to assemble a diffusion chamber for a living body, a first porous membrane is attached to one side of a first diffusion chamber using an adhesive and pressure to form a hermetic seal. A second porous membrane is similarly attached to a second diffusion chamber ring. The membranes can be fixed in place using a rubber gasket that can similarly provide a more hermetic seal. The diffusion chamber rings are left overnight (at least 8 hours) to dry. Next, the first diffusion chamber ring and the second diffusion chamber ring are attached to each other using an adhesive and left overnight (at least 8 hours) to dry. In a preferred embodiment, the joining process between the first chamber ring and the second chamber ring includes using 1,2-dichloroethane as a solvent to facilitate adhesion between the two rings. As an alternative, the chamber can have only one side containing the porous membrane.

[0052] One or more openings (e.g., ports) are provided in the barrel portion of the chamber that can be covered by a cap that allows access from the outside of the subject's body to replenish the diffusion chamber after the chamber has been implanted. The openings allow multiple sequential samplings of the contents without contamination and without harming the subject, thus significantly reducing the number of implantation procedures performed on the subject. Prior to implantation into the patient, one or more openings can be sealed using a cap made of bone wax, port plug, or PMMA. The cap can be a screw-on self-sealing rubber that can be attached to the opening. In some configurations, the diffusion chamber can contain two or more injection openings or ports. Sampling of the chamber contents can be performed by accessing the opening by removing the cap outside the subject's body and inserting a normal needle and syringe. In some embodiments, the chamber can further include an extraction device. Such a device facilitates removal of the chamber from the patient.

[0053] In an embodiment, the chamber functions as an antigen depot designed such that tumor antigens diffuse from the chamber for the purpose of promoting a therapeutic host immune response. Exogenous IGF-1R AS ODN and ex vivo irradiation promote an inflammatory response. This formulation is a novel autologous cell vaccine that is associated with clinical and radiographic improvement and long-term survival according to the protocol and includes an exogenous active pharmaceutical ingredient (API) and irradiation that is interpreted to induce or enhance tumor immune effects. Furthermore, the addition of low concentrations of IGF-1R AS ODN is extremely important for the inflammatory response.

[0054] In certain embodiments, the present disclosure provides a biocompatible diffusion chamber for implantation into a subject having cancer, comprising (a) tumor cells and (b) an effective amount of an antisense molecule. In other embodiments, a method of treating cancer in a subject is provided, comprising: (a) obtaining a biocompatible diffusion chamber comprising tumor cells and an effective amount of an antisense nucleic acid; (b) irradiating the biocompatible diffusion chamber and its contents; and (c) implanting the irradiated biocompatible diffusion chamber into the subject over a therapeutically effective period.

[0055] In certain embodiments, the IGF-1R AS ODN is present in the biocompatible diffusion chamber in an amount in the range of about 0.5 μg to about 10 μg. In certain aspects, IGF-1R The IGF-1R AS ODN is present in an amount in the range of about 1 μg to about 5 μg / chamber or about 2 μg to 4 μg / chamber. In some embodiments, the IGF-1R AS ODN is present in an amount of about 2 μg / chamber. In some embodiments, the IGF-1R AS ODN is present in an amount of about 4 μg / chamber. In some embodiments, it is present in an amount of about 1.0 microgram (μg) to about 5.0 μg. For example, the IGF-1R AS ODN is present in an amount of about 1.0 μg, about 2.0 μg, about 3.0 μg, about 4.0 μg, about 5.0 μg, about 6.0 μg, about 7.0 μg, about 8.0 μg, about 9.0 μg, or about 10.0 μg per chamber. In some embodiments, the IGF-1R AS ODN is present in an amount of about 5.0 μg to about 50.0 μg per chamber. In some embodiments, the IGF-1R AS ODN is present in an amount of about 50.0 μg to about 100.0 μg per chamber. In some embodiments, the IGF-1R AS ODN is present in an amount of about 10.0 μg to about 500.0 μg per chamber. In some embodiments, the IGF-1R AS ODN is present in an amount of about 100.0 μg to about 500.0 μg per chamber. In some embodiments, the IGF-1R AS ODN is present in an amount of about 500.0 μg to about 1.0 milligram (mg) per chamber. In some embodiments, the IGF-1R AS ODN is present in an amount of about 1.0 mg to about 3.0 mg per chamber. In some embodiments, the IGF-1R AS ODN is present in an amount of about 3.0 mg to about 5.0 mg per chamber. In some embodiments, the IGF-1R AS ODN is present in an amount of about 5.0 mg to about 10.0 mg per chamber. In some embodiments, the IGF-1R AS ODN is present in an amount of about 1.0 μg to about 10.0 mg per chamber. Without being bound by theory, these levels are thought to promote Th1 response enhancement in the subject while avoiding M2 immune stimulation responses in the subject.

[0056] In certain embodiments, the tumor cells are not treated with IGF-1R AS ODN prior to encapsulation. However, typically, the tumor cells are treated with IGF-1R AS ODN prior to encapsulation. The time for treating the cells prior to encapsulation can vary. For example, the tumor cells can be treated ex vivo with IGF-1R AS ODN immediately prior to encapsulation for up to about 4 hours, up to about 6 hours, up to about 8 hours, up to about 12 hours, or up to about 18 hours. Typically, the tumor tissue can be treated ex vivo prior to encapsulation for about 12 hours to about 18 hours. For convenience, the cells can be encapsulated after continuing the pretreatment overnight. Without being bound by theory, the pretreatment prior to encapsulation is thought to play a desirable role in the stimulatory generation of tumor antigens.

[0057] The amount of IGF-1R AS ODN used for the pretreatment prior to encapsulation can be in the range of about 1 mg to 8 mg per million cells, for example, in the range of about 2 mg to about 6 mg per million cells, about 3 mg to about 5 mg per million cells. Typically, the amount of IGF-1R AS ODN used for the pretreatment prior to encapsulation is about 4 mg per million cells.

[0058] In some embodiments, the IGF-1R AS ODN for ex vivo treatment of the tumor cells is used at a concentration in the range of at least about 2 mg / ml to at least about 5 mg / ml. In certain aspects, the IGF-1R AS ODN is used at a concentration of at least 4 mg / ml. In a specific embodiment, the IGF-1R AS ODN is used at a concentration of 4 mg / ml.

[0059] In certain embodiments, the IGF-1R AS ODN used to treat tumor cells ex vivo and the IGF-1R AS ODN present in the chamber are the same. In other embodiments, the IGF-1R AS ODN used to treat tumor cells ex vivo and the IGF-1R AS ODN present in the chamber are different. In certain embodiments, the IGF-1R AS ODN used to treat tumor cells ex vivo is at least about 5 nucleotides, at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, at least about 40 nucleotides, at least about 45 nucleotides, or at least about 50 nucleotides in length. In some embodiments, the IGF-1R AS ODN used to treat tumor cells ex vivo is about 15 nucleotides to about 22 nucleotides in length. In certain aspects, the IGF-1R AS ODN used to treat tumor cells is about 18 nucleotides in length.

[0060] In certain embodiments, the IGF-1R AS ODN used to treat tumor cells ex vivo forms a secondary structure at 18°C but does not form a secondary structure at about 37°C. In other embodiments, the IGF-1R AS ODN used to treat tumor cells does not form a secondary structure at either about 18°C or about 37°C. In yet other embodiments, the IGF-1R AS ODN used to treat tumor cells ex vivo does not form a secondary structure at any temperature. In other embodiments, the IGF-1R AS ODN used to treat tumor cells does not form a secondary structure at 37°C. In certain embodiments, the secondary structure is a hairpin loop structure.

[0061] In some embodiments, the IGF-1R AS ODN used to treat tumor cells comprises the nucleotide sequence of SEQ ID NO:1 or a fragment thereof. In certain embodiments, the IGF-1R AS ODN used to treat tumor cells can have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 98%, or 100% identity to SEQ ID NO:1 or a fragment thereof. In certain embodiments, the IGF-1R AS ODN used to treat tumor cells is SEQ ID NO:1.

[0062] After treating the tumor cells with the AS-ODN for a period of time, the AS-ODN is removed and fresh AS-ODN is added to the chamber, which is then irradiated prior to implantation into the subject. In certain embodiments, the in vivo diffusion chamber is treated by gamma irradiation in an amount of about 1 Gy, about 2 Gy, about 4 Gy, about 5 Gy, about 6 Gy, about 10 Gy, or up to about 15 Gy. In certain embodiments, the radiation dose is about 5 Gy or less. In other embodiments, the radiation dose is at least about 5 Gy. In some embodiments, the radiation dose is 5 Gy. In certain embodiments, the in vivo diffusion chamber can be irradiated at least once, at least twice, at least three times, at least four times, or at least five times. In some embodiments, the chamber is irradiated for less than about 24 hours prior to implantation into the subject. In other embodiments, the chamber is irradiated for about 24 hours prior to implantation into the subject. In still other embodiments, the chamber is irradiated for at least about 24 hours prior to implantation into the subject. In still other embodiments, the chamber is irradiated for about 48 hours or less prior to implantation into the subject. In still other embodiments, the chamber is irradiated for at least about 48 hours prior to implantation into the subject.

[0063] Tumor cells are typically killed before implantation, for example, by irradiation. However, it is not necessary to kill the cells, and in fact, it may be advantageous to keep the cells alive to promote the release of antigens. Therefore, in certain embodiments, the cells may not be irradiated before implantation. However, for safety purposes, it is desirable to prevent the release of viable tumor cells to the subject.

[0064] Tumor cells can be placed in the diffusion chambers in various numbers. In certain embodiments, about 1×10 4 ~ about 5×10 6 tumor cells are placed in each diffusion chamber. In other embodiments, about 1×10 5 ~ about 1.5×10 6 tumor cells are placed in the diffusion chamber. In yet other embodiments, about 5×10 5 ~ about 1×10 6 tumor cells are placed in the chamber. We have found that the number of tumor cells can affect the anti-tumor response of the subject and that an appropriate range should be selected to increase the chance of obtaining the desired result. The anti-tumor immune response of patients implanted with 20 chambers is optimal within the range of about 750,000 to about 1,250,000 cells per chamber and peaks at about 1 million cells / chamber. A plurality of chambers containing irradiated tumor cells are administered, and to maintain the optimal immune response, the cell number / chamber is preferably maintained within that range. Preferably, the tumor cells are intact and do not autolyze or become damaged as described herein.

[0065] In certain embodiments, it may be preferable to maintain the ratio of the cells in the chamber to the AS ODN. Therefore, in certain aspects, the chamber may contain about 2 μg of AS ODN and 750,000 - 1,250,000 cells, for example, 1,000,000 cells. Therefore, the ratio of cells to AS ODN is within the range of about 3.75×10 5 ~ about 6.25×10 5 / μg AS ODN, for example, about 5.0×10 5It can be in cells / μg. Therefore, in a typical patient accommodating 20 chambers, the total dosage of AS ODN is about 40 μg.

[0066] Typically, administration will be carried out in the chambers as described herein. However, in certain embodiments, the irradiated cells and IGF-1R AS ODN can be co-administered to a subject without being physically confined together in a chamber or other container. Thus, in certain methods using this approach, the irradiated cells and IGF-1R AS ODN are dispersed, diffused, or metabolized in the body, which is limited by the physiological functions of the subject. Therefore, in certain embodiments, for example, the tumor cells used can be prepared as described herein for use in the chambers and co-administered with IGF-1R AS ODN, but the administration need not be confined within a physical container. Such administration is typically intramuscular.

[0067] Tumor tissue preparation for the chamber The tumor cells used for autologous vaccination are surgically removed from the subject. In an embodiment, the tumor cells are removed from the patient using a tissue morcellator. The extraction device preferably combines a high-speed reciprocating inner cannula within a stationary outer cannula and electronically controlled variable suction. The outer cannula has a diameter of 1.1 mm, 1.9 mm, 2.5 mm, or 3.0 mm and a length of 10 cm, 13 cm, or 25 cm. The device also relies on side mouse cutting and an aspiration aperture located 0.6 mm from the blunt dissector tip. The combination of a weak forward pressure of the aperture into the tissue to be removed and suction draws the desired tissue into the side aperture, enabling precise tissue excision controlled via the reciprocating cutting action of the inner cannula. An important feature is the absence of a rotating blade, which prevents unintended tissue from being drawn into the aperture. An example of a suitable device is the Myriad® Tissue Aspirator (NICO Corporation®, Indianapolis, Indiana), which is a minimally invasive surgical system that can be used for soft tissue retrieval in combination with direct, microscopic, or endoscopic visualization. The shaved tissue is aspirated, collected in a collection chamber, and then collected in a sterile tissue trap. Blood is removed from the preparation when the tissue is collected into the sterile tissue trap. Preferably, the sterile trap contains a collection dish at the bottom of the trap and a stem that provides access to the trap. The trap structure may also contain an inner ladle-shaped structure removable from the trap to facilitate removal of the tissue from the trap.

[0068] Preferably, the morselator does not generate heat at the resection site or along its shaft and does not require ultrasonic energy for tissue removal. Thus, in certain embodiments, the tumor tissue is minced tumor tissue (i.e., tumor shaving tissue obtained by side mouse cutting in the absence of heat and optionally in the absence of sonication). Advantageously, the aspirate extract and minced tissue have a higher viability than tissue removed by other methods. The extraction process is thought to maintain higher tumor cell viability, at least in part because exposure of tumor cells to high temperatures during removal is limited. For example, the methods herein do not expose tumor cells to temperatures greater than 25° C. during removal. Thus, the cells are not exposed to temperatures above body temperature, i.e., about 37° C.

[0069] The amount of tumor tissue obtained from a subject can vary. Preferably, the amount is at least 1 gram, at least 2 grams, at least 3 grams, or at least 4 grams in terms of wet tumor tissue obtained from the patient. The tissue is removed from the sterile tissue trap and disaggregated by pipetting with a sterile pipette to break up large tissue pieces. The disaggregated cell suspension is then placed in a serum-containing medium on a sterile tissue culture plate and incubated in a tissue culture incubator. This plating step serves to enrich for desired functional cells by adhesion and helps to remove debris from the preparation. Thus, the tumor cells used in the treatments described herein preferably consist essentially of or consist of adherent cells derived from tumor tissue.

[0070] After a defined incubation time (e.g., after 6, 12, 24, or 48 hours), the cells are removed from the plate. The cells can be removed by scraping, by chemical methods (e.g., EDTA), or by enzymatic treatment (e.g., trypsin). The cells are placed in one or more diffusion chambers. In some embodiments, the cells are distributed among 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, or more diffusion chambers. Often, 20 chambers are used. In some embodiments, each diffusion chamber contains an equal number of cells. In some embodiments, the first diffusion chamber contains more cells than the second chamber.

[0071] In some embodiments, the cells are sorted before being placed in the chamber. In some embodiments, the cells are enriched by selecting one or more cell markers before being placed in the chamber. The selection can be performed, for example, using beads or by cell sorting techniques known to those skilled in the art. In some embodiments, the cells placed in the chamber are concentrated for one or more markers.

[0072] In some embodiments, implantation of the in vivo diffusion chamber over a therapeutically effective period reduces or eliminates cancer recurrence in the subject. In certain aspects, implantation of the in vivo diffusion chamber causes a reduction in tumor volume associated with cancer in the subject. In still other embodiments, implantation of the in vivo diffusion chamber over a therapeutically effective period induces elimination of the tumor in the subject. In some embodiments, implantation of the chamber inhibits tumor regrowth for at least 3 months, at least 6 months, at least 12 months, at least 36 months, or indefinitely.

[0073] The biocompatible diffusion chamber can be implanted, for example, subcutaneously, intraperitoneally, and intracranially in a subject, but is not limited thereto. In certain embodiments, the diffusion chamber is implanted in a receptive site of the body having good lymphatic drainage and / or vascular supply, such as the rectus sheath. In other embodiments, a refillable chamber may be utilized so that the diffusion chamber can be emptied after treatment and reused for treatment. In certain aspects, multiple, preferably 5 to 20, diffusion chambers can be used in a single subject.

[0074] In certain embodiments, at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, or at least about 50 chambers are implanted in the subject. In some embodiments, 10 to 20 chambers are implanted in the subject. Preferably, about 20 chambers are implanted in the subject. In certain embodiments, the tumor cells are equally distributed among each chamber.

[0075] Typically, the chamber is removed after a period of time. For example, the chamber can be implanted in the subject for about 24 hours, about 48 hours, about 72 hours, or about 96 hours. Implantation for about 48 hours is associated with beneficial treatment results. Thus, the preferred implantation time is about 48 hours. In certain embodiments, the vaccination procedure is performed once per patient. In other embodiments, the vaccination procedure is performed multiple times per patient. In embodiments, the vaccination procedure is performed 2, 3, 4, 5, 6, 7, or 8 times in a single patient. In embodiments, vaccination is repeated every 7 days, every 14 days, every 28 days, or every month, every 3 months, or every 6 months over a given period. In further embodiments, the vaccination procedure is repeated regularly until the patient's cancer disappears.

[0076] Although not bound by theory, implantation of the biocompatible diffusion chamber is thought to cause elimination or reduction of M2 cells at or near the implantation site such that an immune response to tumor antigens diffusing from the chamber is achieved. In certain embodiments, elimination or reduction of M2 cells at the implantation site results in enhanced presentation of autologous tumor antigens by antigen presenting cells (APCs) to CD4 T cells, leading to production of interferon gamma (IFNγ) and induction of type 1 tumor immunity. In certain embodiments, production of IFNγ by tumor antigen-specific CD4 T cells and the anti-M2 effect of IGF-1R AS ODN promote type 1 anti-tumor immunity and loss of anti-inflammatory M2 cells from the circulation and tumor microenvironment, indirectly impeding tumor growth. In some embodiments, production of IFNγ by tumor antigen-specific CD4 T cells and the anti-M2 effect of IGF-1R AS ODN lift suppression of effector-mediated damage to tumor cells and the tumor microenvironment (M2 cells), initiating a longer process of programming memory T cells that recognize tumor antigens. In certain embodiments, the anti-tumor adaptive immune response maintains the continuation of tumor regression.

[0077] Optionally, the cells introduced into the chamber can be enriched for a particular cell type. Nestin, an intermediate filament (IF) protein of cytoskeletal associated class VI, has long been known for its importance as a neural stem cell marker. We have discovered that nestin-positive cells (nestin+ cells) are enriched in certain brain tumor samples compared to benign tissue and that this correlate corresponds to improved treatment response. Thus, in certain embodiments, it is possible to perform a biopsy of the subject's tumor to assess the degree of nestin expression, and thus, in certain embodiments, the chamber cells are enriched for nestin-positive (“+”) cells compared to benign tissue. Without being bound by theory, nestin is thought to provide a marker associated with a suitable antigen useful for eliciting an anti-tumor immune response. Thus, the cells implanted into the chamber can be enriched for nestin+ cells as a whole compared to the tumor cell population when extracted from the subject. In some embodiments, enhanced immune response is obtained when a nestin-enriched tumor sample is used to stimulate the response.

[0078] Systemic administration Instead of or in addition to chamber implantation, IGF-1R AS ODN can be systemically administered. Thus, in embodiments, IGF-1R AS ODN is provided in a pharmaceutical composition for systemic administration. In addition to IGF-1R AS ODN, the pharmaceutical composition can include, for example, physiological saline (0.9% sodium chloride). The composition can include a phospholipid. In some embodiments, the phospholipid is uncharged or has a neutral charge at physiological pH. In some embodiments, the phospholipid is a neutral phospholipid. In certain embodiments, the neutral phospholipid is phosphatidylcholine. In certain embodiments, the neutral phospholipid is dioleoylphosphatidylcholine (DOPC). In some embodiments, the phospholipid is essentially cholesterol-free.

[0079] In some embodiments, the phospholipid and the oligonucleotide are present in a molar ratio of about 5:1 to about 100:1 or any ratio derivable therefrom. In various embodiments, the phospholipid and the oligonucleotide are present in a molar ratio of about 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, or 100:1. In some embodiments, the oligonucleotide and the phospholipid form an oligonucleotide-lipid complex such as a liposome complex. In some embodiments, at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the liposomes have a diameter of less than 5 microns. In various embodiments, the composition further comprises at least one surfactant such as polysorbate 20. In some embodiments, at least about 5% of the total liposomal antisense formulation consists of surfactant and at least about 90% of the liposomes have a diameter of less than 5 microns. In some embodiments, at least about 15% of the total liposomal antisense formulation consists of surfactant and at least about 90% of the liposomes have a diameter of less than 3 microns. In some embodiments, the population of oligonucleotides is incorporated into the population of liposomes.

[0080] In some embodiments, the pharmaceutical composition is a liquid pharmaceutical composition. In other embodiments, the pharmaceutical composition is a solid pharmaceutical composition. The dosage of systemic administration of antisense in a human subject can be about 0.025 g / kg, about 0.05 g / kg, about 0.1 g / kg, about 0.15 g / kg, or about 0.2 g / kg. In certain embodiments, the dosage of systemic administration can be from 0.025 g / kg to 0.2 g / kg. In some embodiments, the dosage is about 0.2 g / kg. In other embodiments, the dosage is from 0.004 g / kg to 0.01 g / kg. In other embodiments, the dosage is less than 0.01 g / kg. In further embodiments, the dosage is not from 0.01 g / kg to 0.2 g / kg. In a particular aspect, the antisense is supplied as a lyophilized powder and resuspended prior to administration. When resuspended, the concentration of the antisense can be in the range of about 50 mg / ml, about 100 mg / ml, about 200 mg / ml, about 500 mg / ml, about 1000 mg / ml, or in the range between those amounts.

[0081] In certain embodiments, the AS ODN can be systemically administered preoperatively, for example, to reduce tumor burden. For example, the AS ODN can be administered up to 24 hours, 36 hours, 48 hours, or 72 hours preoperatively. In a particular aspect, the pharmaceutical composition can be administered about 48 to about 72 hours preoperatively. Typically, in such situations, the administration is by intravenous bolus.

[0082] Combination therapy Historically, cancer therapy has involved treating the subject with radiation therapy, chemotherapy, or both. Many reports of such approaches exist. Advantageously, however, the chamber implantation method disclosed herein can be used to treat a subject having cancer as a monotherapy. Thus, it is preferred that the methods disclosed herein do not include chemotherapy or radiation therapy. However, despite the excellent effects achieved by the monotherapy herein, in certain situations, it may be beneficial to combine the chamber method with other therapies such as radiation therapy. In certain embodiments, radiation therapies include, but are not limited to, brachytherapy, external beam radiation therapy, and systemic radioisotope radiation therapy. In certain aspects, the radiation therapy is external beam radiation therapy. In some embodiments, external beam radiation therapies include, but are not limited to, gamma ray therapy, X-ray therapy, intensity modulated radiation therapy (IMRT), and image guided radiation therapy (IGRT). In certain embodiments, the external beam radiation therapy is gamma ray therapy. Irradiation can be performed before or after chamber implantation, for example, as salvage therapy. Typically, such salvage therapy is not performed until cancer recurrence is determined.

[0083] Thus, in certain combination methods, the chamber method, systemic method, and compositions described herein can all be used in the same subject, either alone or in combination with radiation therapy or chemotherapy. In the combination methods described herein, chamber implantation is preferably used as the first-line therapy. It is desirable to use chamber implantation first because the subject's immune system can be inhibited by other therapies, reducing the therapeutic effect of chamber implantation.

[0084] Optionally, systemic administration can be performed prior to chamber implantation. Such methods can be used to enhance the subject's immune system as a priming method. The priming method can be particularly advantageous when the subject's immune system has been compromised as a result of prior therapy.

[0085] When used in combination with systemic administration, the AS ODN can be systemically administered at least 2 weeks before, at least 1 week before, at least 3 days before, or at least 1 day before the treatment of the patient with the autologous cancer cell vaccine. In other embodiments, the AS ODN can be systemically administered at least 1 day after, at least 3 days after, at least 1 week after, or at least 2 weeks after the treatment of the patient with the autologous cancer cell vaccine or chamber.

[0086] Optionally, the subject can be re-vaccinated with a chamber using the described method after the first vaccination. For subsequent booster vaccinations, tumor cells collected and stored from the subject at the time of tissue removal can be used. Optionally, for subsequent booster vaccinations, fresh tumor tissue removed from the subject and processed as described herein can be used. Any tumors remaining in the subject can act as a depot and provide restimulation since they can express the same antigen. However, recurrent tumors can generate new antigens and thus provide additional options for stimulating an anti-tumor response. Subsequent vaccinations can occur after the first treatment is completed and the tumor has recurred or when the subject no longer responds to the first treatment.

[0087] Subjects treated with IGF-1R AS ODN Suitable subjects are animals having cancer, and typically, the subject is a human. Brain tumors such as glioblastoma are particularly effective with the methods disclosed herein, but the methods are applicable to cancer in general. Accordingly, the present disclosure provides methods of treating cancer, including those selected from the group consisting of glioma, astrocytoma, liver cancer, breast cancer, head and neck squamous cell carcinoma, lung cancer, renal cell carcinoma, hepatocellular carcinoma, gallbladder cancer, classical Hodgkin lymphoma, esophageal cancer, uterine cancer, rectal cancer, thyroid cancer, melanoma, colorectal cancer, prostate cancer, ovarian cancer, and pancreatic cancer. In a specific embodiment, the cancer is glioma. In a particular aspect, the glioma is a recurrent malignant glioma. In some embodiments, the cancer is astrocytoma. In a particular embodiment, the subject being considered for treatment has a tumor of WHO grade II, WHO grade III, or WHO grade IV. In some aspects, the tumor is astrocytoma. In a particular embodiment, the tumor is selected from grade II astrocytoma, AIII (IDH1 R132H mutant grade III astrocytoma), AIII-G (IDH1 wild-type grade III with astrocytoma characteristics of glioblastoma multiforme), or grade IV astrocytoma.

[0088] Grade IV astrocytoma is the highest grade glioma and is synonymous with glioblastoma (GBM). With an annual incidence of 3 or 4 cases per 100,000, GBM is the most common malignant primary brain tumor in adults. Standard care therapies (typically a combination of radiation therapy and chemotherapy with temozolomide) do not function well, and the average survival of GBM patients remains poor at 15 - 17 months. Advantageously, the methods herein can be used to treat newly diagnosed brain cancer and can also be used, for example, to treat recurrent glioblastoma in patients previously treated with standard care therapies. Thus, in certain embodiments, the subject can be a newly diagnosed GBM subject or a recurrent GBM subject. The subject is preferably not previously treated with any immunosuppressive therapy. In certain embodiments, eligible subjects are over 18 years of age and have a Karnofsky score of 60 or greater. Optionally, the subject does not have bilateral hemisphere disease and / or does not have an autoimmune disease.

[0089] Optionally, a subject eligible for treatment can be identified by performing a tumor biopsy on the subject. In some embodiments, the subject's tumor is assayed for the presence of monocytes. In certain embodiments, monocytes include, but are not limited to, CD11b+, CD14+, CD15+, CD23+, CD64+, CD68+, CD163+, CD204+, or CD206+ monocytes. The presence of monocytes in the tumor can be assayed using immunohistochemistry. In certain embodiments, a subject eligible for treatment exhibits greater than about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% of CD163+ M2 cells of the subject's total peripheral blood mononuclear cells (PBMC). In certain embodiments, the subject exhibits greater than about 20% of CD163+ M2 cells of the subject's total PBMC.

[0090] In still other embodiments, a subject being a candidate for treatment is identified by the presence of one or more cytokines in the subject's serum. Such cytokines include, but are not limited to, CXCL5, CXCL6, and CXCL7, IL6, IL7, IL8, IL10, IL11, IFN-γ, and HSP-70.

[0091] In still other embodiments, a subject being a candidate for treatment is identified by the presence of one or more growth factors in the subject's serum. Such growth factors include, but are not limited to, FGF-2, G-CSF, GM-CSF, and M-CSF.

[0092] In some embodiments, a subject being a candidate for treatment with a biocompatible diffusion chamber is identified by measuring the levels of a particular set of cytokines. In some embodiments, the subject has an elevation in the levels of these cytokines compared to a healthy subject. As used herein, the term "healthy subject" means a subject who does not suffer from cancer or any other disease and does not require treatment with a biocompatible diffusion chamber.

[0093] In certain embodiments, cytokines can be added to the chamber to supplement the anti-tumor immune response. For example, the cytokines added to the chamber can be selected from the group consisting of CCL19, CCL20, CCL21, and CXCL12 and combinations thereof.

[0094] In certain embodiments, the circulating CD14+ monocytes have elevated CD163 levels compared to healthy subjects. In some aspects, the CD163 levels on the circulating CD14+ monocytes are elevated by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, or at least about 100-fold compared to healthy subjects. In certain embodiments, the CD163 levels on the circulating CD14+ monocytes are elevated by about 2-fold compared to healthy subjects.

[0095] In other embodiments, the subject being a candidate for treatment has serum that polarizes undifferentiated monocytes towards M2 cells. In certain embodiments, incubation of the subject's serum with undifferentiated monocytes, although not limited to, induces the expression of one or more cell surface markers on monocytes including, but not limited to, CD11b, CD14, CD15, CD23, CD64, CD68, CD163, CD204, and / or CD206. In other embodiments, incubation of the subject's serum with undifferentiated monocytes increases the expression of one or more cell surface markers on the monocytes as compared to monocytes not incubated with the subject's serum. In certain embodiments, cell surface markers include, but are not limited to, CD11b, CD14, CD15, CD23, CD64, CD68, CD163, CD204, and / or CD206. In some embodiments, the level of one or more surface markers is increased by at least about 1.3-fold, at least about 1.5-fold, at least about 1.8-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, or at least about 100-fold as compared to undifferentiated monocytes not incubated with the subject's serum. In a particular embodiment, the level of one or more surface markers is increased by about 2-fold as compared to undifferentiated monocytes not incubated with the subject's serum. Monocytes polarized by the subject's serum can be measured using FACS.

[0096] Target cell Although not bound by theory, AS ODNs are thought to reduce the target M2 cells and / or inhibit the polarization of cells into M2 cells by downregulating IGF-1R expression. In some embodiments, IGF-1R expression in M2 cells is downregulated by at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% compared to cells not treated with antisense. IGF-1R expression in M2 cells can be measured by quantitative RT-PCR.

[0097] In some embodiments, IGF-1R expression in M2 cells is maintained in a downregulated state in a subject for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, or at least about 6 weeks after a single administration of antisense.

[0098] In some embodiments, downregulation of IGF-1R expression in M2 cells causes selective reduction of the target M2 cells compared to cells that do not express IGF-1R. In certain embodiments, the target M2 cells are reduced by at least about 2%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% compared to a subject not treated with antisense. In other embodiments, the M2 cell population is eliminated. For example, after implantation of a biocompatible diffusion chamber, the M2 cell population can be about 1%, about 2%, about 5%, or about 10% of the population prior to implantation of the biocompatible diffusion chamber. The target M2 cells can be measured using FACS. In certain embodiments, after treatment, the M2 cells are eliminated; i.e., undetectable by FACS. In other embodiments, the decrease in M2 cells can be measured using a proxy assay. For example, serum can be obtained from a subject before and after treatment and its ability to polarize to M2 cells can be evaluated. After treatment with the methods disclosed herein, the ability of the serum to polarize to M2 cells is reduced by about 80% to about 100%, about 20% to about 60%, or about 10% to about 50%.

[0099] In some embodiments, targeting IGF-1R expression in M2 cells causes cell death in the M2 cells. In certain embodiments, the cell death is necrosis. In other embodiments, the cell death is apoptosis. Apoptosis, for the purposes of the present disclosure, is defined as programmed cell death and includes, but is not limited to, regression of primary and metastatic tumors. Apoptosis is a widespread phenomenon of programmed cell death that plays a very important role in a vast number of physiological and pathological processes. Necrosis, in contrast, is accidental cell death that is a cell's reaction to various adverse conditions and toxic substances. In still other embodiments, targeting IGF-1R expression in M2 cells causes cell cycle arrest in the M2 cells.

[0100] Kit Fabrication of the complete chamber requires multiple components and multiple steps. In another aspect of the present disclosure, a kit containing components for implementing the methods disclosed herein is provided. In certain embodiments, the kit includes a chamber body that may be present in one part or two halves. Also included may be items for sealing the chamber, including one or more membranes, adhesives, and solvents (e.g., alcohol or dichloroethane). Optionally, the membrane may be ultrasonically welded to the chamber to form a seal. The kit includes antisense ODN. Optionally, the ODN may be distributed into two parts: a first part for treating cells after surgically removing them from a subject and a second part for combining with cells when introducing them into the subject. Other optional kit items include a medium for culturing cells and an antibiotic for preventing bacterial growth in the medium.

[0101] Optionally, the chambers of the kit may be pre-connected to each other (e.g., by suturing) using an inlet or other device adapted to be attached to the chamber to receive connection materials. Advantageously, pre-connecting multiple chambers allows a surgeon to easily introduce or remove a desired number of chambers.

[0102] In addition to the various aspects and embodiments disclosed herein, the following embodiments are specifically contemplated. 1. A method comprising determining MGMT methylation and / or T cell function in a subject having cancer and administering IGF-1R AS ODN to the subject.

[0103] 2. A method comprising determining MGMT methylation in a subject having cancer and administering IGF-1R AS ODN to the subject. 3. A method comprising determining MGMT methylation in a subject having cancer and administering IGF-1R AS ODN to the subject only if the subject has methylated MGMT.

[0104] 4. A method for determining T cell function in a subject having cancer and administering IGF-1R AS ODN to the subject. 5. A method for determining T cell function in a subject having cancer and administering IGF-1R AS ODN to the subject only when the subject has good T cell function.

[0105] 6. A method for identifying a subject having cancer and likely to respond to IGF-1R AS ODN and administering IGF-1R AS ODN to the subject. 7. A method for identifying a subject having cancer and likely to respond to IGF-1R AS ODN and administering IGF-1R AS ODN to the subject, wherein the likelihood of high responsiveness to IGF-1R AS ODN is evaluated by determining MGMT methylation and / or T cell function in the subject.

[0106] 8. A method for identifying a subject having cancer and likely to respond to IGF-1R AS ODN and administering IGF-1R AS ODN to the subject, wherein the likelihood of high responsiveness to IGF-1R AS ODN is evaluated by determining MGMT methylation and / or T cell function in the subject, and the likelihood of high responsiveness is established by identifying MGMT methylation in the subject and / or by determining good T cell function in the subject.

[0107] 9. A method for identifying a subject having cancer and likely to respond to IGF-1R AS ODN and administering IGF-1R AS ODN to the subject, wherein the likelihood of high responsiveness to IGF-1R AS ODN is evaluated by determining MGMT methylation, and the likelihood of high responsiveness is established by identifying MGMT methylation in the subject.

[0108] 10. A method comprising identifying a subject having cancer and likely to respond to IGF-1R AS ODN, and administering IGF-1R AS ODN to the subject, wherein the likelihood of responding to IGF-1R AS ODN is evaluated by determining T cell function in the subject, and the likelihood is established by determining good T cell function in the subject.

[0109] 11. A method for predicting the prognosis of a subject with cancer in response to IGF-1R AS ODN, the method comprising determining MGMT methylation in the subject and / or determining T cell function in the subject.

[0110] 12. A method for predicting the prognosis of a subject having cancer in response to IGF-1R AS ODN, the method comprising determining MGMT methylation in the subject and / or determining T cell function in the subject, wherein methylated MGMT and / or good T cell function in the subject indicate a good prognosis.

[0111] 13. A method for predicting the prognosis of a subject having cancer in response to IGF-1R AS ODN, the method comprising determining MGMT methylation in the subject, wherein methylated MGMT in the subject indicates a good prognosis.

[0112] 14. A method for predicting the prognosis of a subject having cancer in response to IGF-1R AS ODN, the method comprising determining T cell function in the subject, wherein good T cell function in the subject indicates a good prognosis.

[0113] 15. A method for predicting the prognosis of a subject having cancer in response to IGF-1R AS ODN, the method comprising determining MGMT methylation in the subject and / or determining T cell function in the subject, wherein unmethylated MGMT and / or insufficient T cell function in the subject indicate a poor prognosis.

[0114] 16. A method for predicting the prognosis in a subject having cancer in response to IGF-1R AS ODN, comprising determining MGMT methylation in the subject, wherein unmethylated MGMT in the subject indicates a poor prognosis.

[0115] 17. A method for predicting the prognosis in a subject having cancer in response to IGF-1R AS ODN, comprising determining T cell function in the subject, wherein insufficient T cell function in the subject indicates a poor prognosis.

[0116] 18. The method according to any of the preceding embodiments, wherein the T cell function is determined by evaluating the number of IFN-γ expressed in response to non-specific stimulation. The method according to any of the preceding embodiments, wherein the T cell function is determined by evaluating the number of IFN-γ expressed in response to non-specific stimulation, and the expression of IFN-γ by a number of T cells equal to or greater than the median in response to specific stimulation is classified as good T cell function, and the expression of IFN-γ by a number of T cells less than the median or fewer in response to non-specific stimulation is classified as insufficient T cell function.

[0117] 20. The method according to any of the preceding embodiments, wherein the IGF-1R AS ODN is administered to the subject before temozolomide is administered to the subject. 21. The method according to any of the preceding embodiments, wherein the IGF-1R AS ODN is administered to the subject at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; or at least 8 weeks before temozolomide is administered to the subject.

[0118] 22. The method according to any of the preceding embodiments, wherein the IGF-1R AS ODN is administered to the subject as an autologous cancer cell vaccine. 23. The method according to any of the preceding embodiments, wherein the IGF-1R AS ODN is administered to the subject as a fully formulated in vivo diffusion chamber.

[0119] 24. If present, the in vivo diffusion chamber is prepared by: (a) encapsulating tumor cells obtained from a subject into the in vivo diffusion chamber in the presence of IGF-1R AS ODN, where the ratio of tumor cells:IGF-1R AS ODN in the chamber is in the range of about 3.75×10 5 :1 g to about 6.25×10 5 :1 μg, and the tumor cells are obtained from the subject using a tissue morcellator, and further (b) irradiating the in vivo diffusion chamber with radiation. The method according to any of the preceding embodiments.

[0120] 25. The method according to any of the preceding embodiments using an in vivo diffusion chamber, wherein the tumor cells are enriched for nestin expression before being placed in the in vivo diffusion chamber. 26. The method according to any of the preceding embodiments using an in vivo diffusion chamber, wherein the tumor cells in the chamber are enriched for adherent cells compared to the tumor cells obtained from the subject.

[0121] 27. The method according to any of the preceding embodiments using an in vivo diffusion chamber, wherein the tumor cells consist essentially of adherent cells. 28. The method according to any of the preceding embodiments using an in vivo diffusion chamber, wherein the cells are treated with IGF-1R AS ODN before being encapsulated in the chamber.

[0122] 29. The method according to any of the preceding embodiments using an in vivo diffusion chamber, wherein IGF-1R AS ODN is present at about 2 mg to about 6 mg per million cells during the pre-encapsulation treatment.

[0123] 30. The method according to any of the preceding embodiments using an in vivo diffusion chamber, wherein IGF-1R AS ODN is present at about 4 mg per million cells during the pre-encapsulation treatment. 31. The method according to any of the preceding embodiments using an in vivo diffusion chamber, wherein the treatment with IGF-1R AS ODN before encapsulation is up to about 18 hours.

[0124] 32. Any method of any of the foregoing embodiments using a diffusion chamber for a living body, wherein the treatment with IGF-1R AS ODN before encapsulation is from about 12 hours to about 18 hours. 33. Any method of any of the foregoing embodiments, wherein the IGF-1R AS ODN has the sequence of SEQ ID NO: 1.

[0125] 34. Any method of any of the foregoing embodiments using a diffusion chamber for a living body, wherein about 2 μg of IGF-1R AS ODN is present in the chamber. 35. Any method of any of the foregoing embodiments using a diffusion chamber for a living body, wherein the irradiated tumor cells are present in the range of about 750,000 to about 1,250,000 per chamber.

[0126] 36. Any method of any of the foregoing embodiments using a diffusion chamber for a living body, wherein about 1,000,000 irradiated tumor cells are present per chamber. 37. Any method of any of the foregoing embodiments using a diffusion chamber for a living body, comprising implanting into two or more diffusion chambers for a living body.

[0127] 38. Any method of any of the foregoing embodiments using a diffusion chamber for a living body, wherein about 10 to about 30 diffusion chambers for a living body are implanted into the subject. 39. Any method of any of the foregoing embodiments using a diffusion chamber for a living body, wherein about 10 to about 20 diffusion chambers for a living body are implanted into the subject.

[0128] 40. Any method of any of the foregoing embodiments using a diffusion chamber for a living body, wherein the diffusion chamber is implanted into the subject for 48 hours. 41. Any method of any of the foregoing embodiments, wherein the cancer is a brain cancer.

[0129] 42. Any method of any of the foregoing embodiments, wherein the brain cancer is selected from grade II astrocytoma, grade AIII astrocytoma, grade AIII-G astrocytoma, and grade IV astrocytoma (glioblastoma multiforme).

[0130] The method of any of the foregoing embodiments, wherein the brain cancer is grade IV glioblastoma (glioblastoma multiforme). 43. The method of any of the foregoing embodiments, wherein the subject is human.

[0131] Examples Example 1 Introduction IGV-001, a novel combination vaccine, was evaluated in newly diagnosed adults with glioblastoma. IGV-001 consists of autologous glioblastoma tumor cells and antisense oligodeoxynucleotides against insulin-like growth factor type 1 receptor (IGF-1R) DNA / mRNA (IMV-001; formerly called NOBEL) and is co-administered via a biocompatible diffusion chamber implanted in the abdomen. IGV-001 is thought to promote tumor immunity through the release of tumor antigens with stimulation of antigen presentation. This phase 1b study was based on a phase 1a study in patients with recurrent World Health Organization grade III or IV astrocytomas, and 8 out of 12 patients showed improvement on radiographs.

[0132] Methods Study design Adults with newly diagnosed malignant gliomas confirmed radiologically were enrolled, with the condition that the Karnofsky performance status score be at least 60. Patients were randomly assigned to one of four types of IGV-001 exposure: minimum (10 chambers implanted for 24 hours); low (10 chambers implanted for 48 hours); high (20 chambers implanted for 24 hours); and maximum (20 chambers implanted for 48 hours).

[0133] During the craniotomy for tumor resection, the Myriad tissue aspirator was used to aspirate and pulverize the tumor tissue, maintaining the viability of the tumor cells according to the standard of care (SOC). The surgeon created an abdominal acceptor site between the rectus sheath and the rectus muscle for subsequent implantation of the diffusion chamber. The harvested tumor cells were treated ex vivo with IMV-001 for 4 - 8 hours and then encapsulated in 10 or 20 (depending on randomization) 1.4 cm diffusion chambers for living organisms together with additional IMV-001 and irradiated with 5 Gy. The radiation of the cells treated with IMV-001 causes the release of immunostimulatory antigens. The chamber was implanted into the abdominal acceptor site within 24 hours of the craniotomy. The chamber was removed 24 or 48 hours later (depending on randomization), and the abdomen was closed. Due to a higher than expected incidence of deep vein thrombosis (DVT) in previous experience with IMV-001, prophylactic enoxaparin was administered daily for 3 months, and the patients were monitored for DVT by compression ultrasound twice a week during the first hospitalization and then monthly for 3 months. SOC (i.e., radiation and temozolomide [TMZ]) was started 4 - 6 weeks after surgery and continued for 6 weeks. The patients received an additional 12 cycles of TMZ as maintenance therapy.

[0134] The highest exposure cohort showed the highest levels in circulating, potentially therapeutic, pro-inflammatory cytokines and in clinical and radiological improvement after treatment, so randomization was stopped after patient 23. The protocol was amended, and subsequent patients received that highest exposure. This amendment reflects the transition from a protocol that mainly aimed to record safety together with related exploratory biomarker evaluations to a protocol with clinical exploratory endpoints including overall survival (OS) and progression-free survival (PFS).

[0135] Evaluation Adverse events (AEs) and serious AEs were recorded from the time of chamber implantation until 30 days after the end of the trial, and for at least 6 weeks after treatment. AEs were evaluated and classified according to the Common Terminology Criteria for Adverse Events v4.03 of the US National Cancer Institute. Asymptomatic grade 1 and grade 2 laboratory values were not considered AEs unless judged clinically significant by the treating physician. Magnetic resonance imaging (MRI) was performed within 14 days before surgery and at least up to 24 months after surgery. MRI scans were read by a neuroradiologist blinded to the patient's corticosteroid dosage and clinical status. Radiographic responses were based on the Response Assessment in Neuro-Oncology (RANO) 2 and Immunotherapy RANO (iRANO) criteria. Time to progression was evaluated from the day of surgery until the first observation of objective disease progression measured by MRI. Evidence of disease progression had to be confirmed by an independent radiographic review committee. Progression-free survival (PFS) was measured from the day of surgery until progression or discontinuation (discontinuation refers to excluding the patient from the clinical trial for any of various criteria). Overall survival (OS) was the elapsed time from the day of surgery until the most recent follow-up or death. Patients considered to have withdrawn from the study were followed for OS.

[0136] Since the mechanism of action of IGV-001 is thought to depend largely on the immune response, subsets of circulating lymphocytes and monocytes, serum cytokines and chemokines, and T cell function (based on the number of T cells expressing the pro-inflammatory cytokine interferon-γ [IFN-γ] in response to non-specific stimulation) were quantified before and after treatment.

[0137] Statistical analysis The intention-to-treat (ITT) population included all enrolled patients who did not fail screening and were used for the evaluation of safety and clinical outcomes. The number and percentage of subjects with AEs were summarized overall, by severity grade, and by association with the investigational drug or SOC and the desired period. For multiple AEs per subject during the desired period, only the most serious one was reported. Time-to-event data (PFS and OS) were analyzed using the product-limit method and graphed at the combined points using a step function. SAS version 9.4 (SAS Institute; Cary, North Carolina) was used for all analyses. Patient-level data from the SOC arms of published studies with similar enrollment criteria were imputed using the method described by Guyot et al. (2012). The OS and PFS of these SOC arms were compared with the IGV-001 treatment cohort using the log-rank test.

[0138] T cell assay protocol Samples of whole blood or leukapheresis from patients in glioblastoma clinical trials were separated, and PBMCs were cryopreserved in DMSO. These PBMC samples were utilized for T cell isolation and stimulation. Downstream analysis of triggered cells was performed using ELISPOT.

[0139] Isolation of naive T cells PBMC samples were thawed, washed with warmed RPMI medium supplemented with 10% FBS, 1% L-glutamine, and 1% penicillin / streptomycin to remove DMSO. The pellet was resuspended in a known volume of medium, and the initial cell number was obtained using a CountessII FL automated cell counter (ThermoFisher Scientific). Isolation of naive T cells using magnetic beads was performed by negative selection using the Easysep Human T Cell Isolation Kit (StemCell Technologies #17951). T cell isolation was performed simultaneously in 96-well plates for multiple samples from clinical trials.

[0140] In vitro stimulation of T cells - ELISPOT assay A polyvinylidene fluoride membrane Elispot plate (96-well plate, Merck Millipore #S2EM004M99) was coated with anti-IFN-γ monoclonal antibody (Mabtech #3420-3-1000) and incubated overnight at 4°C for 14 - 16 hours. The antibody solution was discarded, and the plate was blocked using serum-free medium. After incubation at room temperature for 2 hours, the medium was decanted, and 50,000 T cells were added to each well together with anti-CD3 / CD28 / CD2 (Immunocult, StemCell Technologies #10910) T cell activator and incubated at 37°C, 5% CO2 for 20 hours. The medium containing these T cell activators was used as a negative control (NC), and PBMC from healthy donors was used as a positive control (PC). After incubation, the supernatant was aspirated, and the wells were washed twice with deionized water and then with wash buffer A (1×PBS containing 0.05% Tween-20). The wells were incubated with the detection antibody (diluted 1:250 in 1×PBS containing 10% FBS, Mabtech #3420-6-250) at room temperature for 2 hours. Next, the wells were washed with wash buffer A and incubated with streptavidin-horseradish peroxidase (diluted 1:100 in 1×PBS containing 10% FBS, BD Bioscience #557630) at room temperature for 1 hour. The streptavidin-horseradish peroxidase solution was discarded, and the wells were washed with wash buffer A and wash buffer B (1×PBS). AEC substrate solution (1 drop of AEC chromogen per 1 ml of AEC substrate, BD Bioscience #551951) was added to each well, and spot generation was monitored for 5 minutes to a maximum of 20 minutes. The reaction was stopped using deionized water, and the plate was air-dried before counting the spots using an Elispot reader. The obtained data were analyzed using Graphpad Prism version 7.

[0141] Results Patients A total of 33 patients were treated within a period of 31 months. The demographic and baseline clinical characteristics are shown in Table 2. Six, five, five, and 17 patients received the lowest, low, high, and highest exposures, respectively.

[0142] [Table 2]

[0143] Safety IGV-001 was generally well tolerated. As of the date more than 41 months after the first patient was treated, there were five AEs related to laparotomy, one grade 3 hematoma, three grade 2 hematomas, and one grade 1 wound complication. There were no recorded abdominal wound infections. There were eight AEs that may be related to the treatment, two grade 3 seizures, one grade 3 DVT, one grade 3 hydrocephalus, one grade 3 alanine aminotransferase (ALT) elevation, one grade 3 aspartate aminotransferase (AST) elevation, one grade 3 encephalopathy, and one grade 2 DVT.

[0144] Eleven out of 22 deaths were not due to the progression of glioblastoma. Seven out of these 11 deaths occurred within 12 months of treatment with IGV-001, and the details are shown in Table 3.

[0145] [Table 3]

[0146] The median (range) follow-up period of patients administered IGV-001 was 13 months (4 - 40). The radiographic response included the persistent absence of anatomical enhancement after total resection, persistent shrinkage from the initial postoperative tumor volume, and persistent shrinkage starting within approximately 6 months after surgery following an increase in the anatomical tumor volume after partial resection. Two patients also showed spontaneous regression of the recurrent tumor (not published). Tumor recurrence was usually on the same side and was considered local (within 2 cm from the center of origin). Of the 11 surviving patients, 6 were progression-free according to the RANO criteria at the last observation.

[0147] Clinical Outcome The current standard of care (SOC) in cases suspected of glioblastoma starts with maximal safe resection. Pathologically confirmed cases then receive radiotherapy and temozolomide (TMZ) concurrently, followed by maintenance with TMZ.6. This SOC was established by a 2005 study conducted by the European Organization for Research and Treatment of Cancer under the leadership of Roger Stupp. This trial showed that adding temozolomide (TMZ) to radiotherapy extended overall survival (OS) compared to radiotherapy alone (14.6 months vs. 12.1 months). There is also significant consistency in OS among the SOC arms of large randomized clinical trials published in top peer-reviewed journals over more than 8 years.

[0148] As of the date when more than 41 months had passed since the first patient was treated, the 11 patients treated with IGV-001 were alive and had normal physical function. The median OS was 17.3 months (Figure 1A), and the OS rate at 24 months was 31%. The OS benefit of IGV-001 was enhanced when the analysis was limited to patients who received the highest exposure to IGV-001 (median, 21.9 months, Figure 1B).

[0149] OS is summarized in Table 4 by exposure group. Favorable survival rates were associated with the highest exposure to IGV-001 (i.e., 48 hours in 20 chambers).

[0150]

Table 4

[0151] Stupp et al. showed that tumor progression occurred before death in 95% of all patients who received SOC for newly diagnosed glioblastoma. In our study, 14 patients died within the first year, and among them, 7 (50%) had no disease progression (Table 2). The OS of patients treated with IGV-001 was evaluated independent of the mortality rate that might be caused by SOC by excluding deaths due to disease progression. The resulting median OS was 22.1 months, which is favorably contrasted with the published estimated value of SOC.

[0152] In addition, other trial subgroups with clinical characteristics that might favorably affect better OS were evaluated. Methylation of the O6-methylguanine-DNA methyltransferase (MGMT) promoter halts the ability of cells to demethylate the methyl group of O6 guanine and enhances the therapeutic effect of TMZ compared with patients with an unmethylated MGMT promoter. In the companion paper of the Stupp trial, the effect of the MGMT promoter on the survival outcome of patients receiving SOC was examined. The median OS of patients with a methylated MGMT promoter was 21.7 months, whereas it was 12.7 months in patients without methylation. Similarly, the favorable survival rate (median OS, 30.9 months vs. 11.3 months) in patients with a methylated MGMT promoter treated with SOC following IGV-001 was noteworthy (Figure 3).

[0153] As of the date when more than 41 months had passed since the first patient was treated, 15 patients had no progression at the primary site, and among them, 6 survived. The median PFS of patients treated with IGV-001 was 10.4 months, and the PFS rate at 6 months was 87% (Table 5).

[0154]

Table 5

[0155] Since PFS data can be obtained earlier than OS data, we compared our PFS results with the SOC arms of two large randomized clinical trials published in top peer-reviewed journals. Compared with SOC, there was a significant improvement in PFS with IGV-001 (Figure 4).

[0156] Hegi, Gilbert, and Stupp reported that the median PFS was 10.3, 10.5, and 10.7 months, respectively, in patients with methylated MGMT promoter who were administered SOC only. The median PFS of patients with methylated MGMT promoter who were administered IGV-001 was 30.9 months (Table 6), which was comparable to that of SOC (p = 0.004; Figure 5).

[0157]

Table 6

[0158] Initial investigation of predictive biomarkers In a preliminary analysis, good pre-treatment T cell function (i.e., the number of T cells expressing IFN-γ in response to non-specific stimulation being at or above the median) was associated with longer OS than when T cell function was insufficient (i.e., below the median; Table 7). This suggests the involvement of the immune system in the mechanism of action of IGV-001.

[0159]

Table 7

[0160] Conclusion The results of this Phase 1b clinical trial of IGV-001 in newly diagnosed glioblastoma patients are compelling. The combination vaccine was implanted in 33 patients, and the tolerability was generally good. Among the 14 patients who died in the first year, 7 had no disease progression, and all deaths were unrelated to the treatment. The median OS and PFS were comparable to those reported for SOC in large clinical trials. The highest exposure to IGV-001, methylation of the MGMT promoter, and good pre-treatment T cell function were associated with longer survival.

[0161] References

[0162]

Table 8

[0163] Incorporation by reference All patents and publications referenced herein are hereby incorporated by reference in their entirety.

Claims

1. 1. A method comprising: identifying a subject having cancer and likely to respond to treatment with an IGF-1R AS ODN; and administering the IGF-1R AS ODN to the subject, wherein likely to respond to the IGF-1R AS ODN is assessed by determining MGMT methylation and / or T cell function in the subject.

2. 1. A method comprising identifying a subject having cancer and having a high likelihood of responding to treatment with an IGF-1R AS ODN and administering the IGF-1R AS ODN to said subject, wherein the high likelihood of responding to the IGF-1R AS ODN is assessed by determining MGMT methylation and / or T-cell function in said subject, wherein the high likelihood is established by identifying MGMT methylation in said subject and / or determining good T-cell function in said subject.

3. 1. A method comprising: identifying a subject having cancer and likely to be responsive to an IGF-1R AS ODN; and administering the IGF-1R AS ODN to the subject, wherein the likely to be responsive to the IGF-1R AS ODN is assessed by determining MGMT methylation in the subject, and wherein the likely is established by identifying MGMT methylation in the subject.

4. 1. A method comprising: identifying a subject having cancer and likely to respond to treatment with an IGF-1R AS ODN; and administering the IGF-1R AS ODN to the subject, wherein the likely to respond to the IGF-1R AS ODN is assessed by determining T cell function in the subject, and wherein the likely is established by determining good T cell function in the subject.

5. A method for predicting the prognosis of a subject suffering from cancer in response to an IGF-1R AS ODN, the method comprising determining MGMT methylation and / or determining T cell function in said subject.

6. 1. A method of predicting prognosis in response to an IGF-1R AS ODN in a subject having cancer, the method comprising determining MGMT methylation and / or determining T cell function in the subject, wherein methylated MGMT and / or good T cell function in the subject indicates a good prognosis.

7. 1. A method for predicting a prognosis in response to an IGF-1R AS ODN in a subject having cancer, the method comprising determining MGMT methylation in the subject, wherein methylated MGMT in the subject indicates a good prognosis.

8. 1. A method of predicting a prognosis in response to an IGF-1R AS ODN in a subject having cancer, the method comprising determining T cell function in the subject, wherein better T cell function in the subject indicates a better prognosis.

9. 1. A method of predicting prognosis in response to an IGF-1R AS ODN in a subject having cancer, the method comprising determining MGMT methylation and / or determining T cell function in the subject, wherein unmethylated MGMT and / or insufficient T cell function in the subject indicates a poor prognosis.

10. A method for predicting the prognosis of a subject having cancer in response to an IGF-1R AS ODN, comprising determining MGMT methylation in said subject, wherein unmethylated MGMT in said subject indicates a poor prognosis.

11. 1. A method for predicting the prognosis of a subject having cancer in response to an IGF-1R AS ODN, the method comprising determining T cell function in the subject, wherein insufficient T cell function in the subject indicates a poor prognosis.

12. The method of any one of claims 1 to 11, wherein the T cell function is determined by assessing the amount of IFN-γ expressed in response to a non-specific stimulus.

13. 13. The method of any one of claims 1 to 12, wherein the T cell function is determined by assessing the number of IFN-γ expressed in response to a non-specific stimulus, wherein a median or greater number of T cells expressing IFN-γ in response to a specific stimulus is classified as good T cell function, and a below-median or lower number of T cells expressing IFN-γ in response to a non-specific stimulus is classified as poor T cell function.

14. The method of any one of claims 1 to 13, wherein an IGF-1R AS ODN is administered to the subject before temozolamide is administered to the subject.

15. 15. The method of any one of claims 1 to 14, wherein the IGF-1R AS ODN is administered to the subject at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; or at least 8 weeks prior to temozolamide being administered to the subject.

16. The method according to any one of claims 1 to 15, wherein the IGF-1R AS ODN is administered to the subject as an autologous cancer cell vaccine.

17. The method of any one of claims 1 to 16, wherein the IGF-1R AS ODN is administered to the subject as a fully formulated biological diffusion chamber.

18. When present, the biocompatible diffusion chamber comprises: (a) encapsulating tumor cells obtained from a subject in a biocompatible diffusion chamber in the presence of IGF-1R AS ODN, wherein the ratio of tumor cells:IGF-1R AS ODN in the chamber is about 3.75×10 ５ : 1 μg ~ approx. 6.25×10 ５ 18. The method of any one of claims 1 to 17, wherein the tumor cells are obtained from a subject using a tissue morcellator and further prepared by (b) irradiating the biological diffusion chamber.

19. 19. The method of any one of claims 1 to 18, using an in vivo diffusion chamber, wherein the tumor cells are enriched for nestin expression prior to being placed in the in vivo diffusion chamber.

20. 20. The method of any one of claims 1 to 19, using a biological diffusion chamber, wherein the tumor cells in the chamber are enriched for adherent cells compared to tumor cells obtained from the subject.

21. The method according to any one of claims 1 to 20, using a biological diffusion chamber, in which the tumor cells consist essentially of adherent cells.

22. The method of any one of claims 1 to 21, using a biological diffusion chamber, in which the cells are treated with the IGF-1R AS ODN before being enclosed in the chamber.

23. 23. The method of any one of claims 1 to 22, using a biological diffusion chamber, wherein the IGF-1R AS ODN is present at about 2 mg to about 6 mg per million cells during processing prior to encapsulation.

24. The method of any one of claims 1 to 23, using a biological diffusion chamber, wherein the IGF-1R AS ODN is present at about 4 mg per million cells during processing prior to encapsulation.

25. The method of any one of claims 1 to 24, using a biological diffusion chamber, wherein the treatment with IGF-1R AS ODN prior to encapsulation is for up to about 18 hours.

26. The method of any one of claims 1 to 25, using a biological diffusion chamber, wherein the treatment with the IGF-1R AS ODN prior to encapsulation is for about 12 hours to about 18 hours.

27. The method of any one of claims 1 to 26, wherein the IGF-1R AS ODN has the sequence of SEQ ID NO:

1.

28. The method of any one of claims 1 to 27, which uses a biological diffusion chamber, wherein the IGF-1R AS ODN in the chamber is present at about 2 µg.

29. 29. The method of any one of claims 1 to 28, using a biological diffusion chamber, wherein the irradiated tumor cells are present in the range of about 750,000 to about 1,250,000 cells per chamber.

30. 30. The method of any one of claims 1 to 29, using a biological diffusion chamber, in which the irradiated tumor cells are present at about 1,000,000 cells per chamber.

31. 31. The method of any one of claims 1 to 30, using a biological diffusion chamber, comprising implanting two or more biological diffusion chambers in a subject.

32. 32. The method according to any one of claims 1 to 31, using biocompatible diffusion chambers, wherein about 10 to about 30 biocompatible diffusion chambers are implanted in the subject.

33. 33. The method of any one of claims 1 to 32 using biocompatible diffusion chambers, wherein about 10 to about 20 biocompatible diffusion chambers are implanted in the subject.

34. 34. The method of any one of claims 1 to 33, using an in vivo diffusion chamber, wherein the diffusion chamber is implanted in the subject for 48 hours.

35. The method of any one of claims 1 to 34, wherein the cancer is brain cancer.

36. 10. The method of any of the preceding claims, wherein the brain cancer is selected from grade II astrocytoma, grade AIII astrocytoma, grade AIII-G astrocytoma, and grade IV astrocytoma (glioblastoma multiforme).

10. The method of any preceding claim, wherein the brain cancer is grade IV astrocytoma (glioblastoma multiforme).

37. The method of any one of claims 1 to 36, wherein the subject is a human.

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