Methods and compositions for treating breast cancer using antisense

Abstract

To provide compositions and methods for treating breast cancer using antisense (AS) nucleic acids directed against Insulin-like Growth Factor 1 Receptor (IGF-1R).SOLUTION: An AS may be administered to 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 metastatic breast cancer.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure relates to compositions and methods for treating breast cancer. [Background technology]

[0002] Breast cancer is the most common cancer in women in the United States. Although the number of deaths from breast cancer has declined over time, it is still the second leading cause of cancer deaths among women overall. Approximately 237,000 cases of breast cancer are diagnosed in women and 2,100 in men in the United States each year. Approximately 41,000 women and 450 men die from breast cancer each year in the United States. Although most breast cancers are found in women over the age of 50, breast cancer also affects younger women. Approximately 11% of all new cases of breast cancer in the United States are found in women under the age of 45.

[0003] Breast cancer treatments may include surgery (lumpectomy, mastectomy), hormonal therapy, biological therapy, radiation therapy, and / or chemotherapy. If breast cancer is not detected / treated in the early stages, it can gradually develop into widespread metastatic disease.

[0004] Breast cancer varies widely between patients in terms of aggressiveness and genetic status. For this reason, treatment options depend on the type of breast tumor identified and must be tailored for each patient. In most cases, patients will receive a combination of two to five therapies. While effective in some cases, such multiple therapies are costly and take a toll on the patient's overall health. Breast cancer treatments can cause side effects that may continue or appear months or years after the end of treatment. These side effects include lung inflammation, lymphedema, heart failure, blood clots, premature menopause, and secondary cancers such as leukemia. Summary of the Invention [Problem to be solved by the invention]

[0005] Thus, there is a need in the art for new and improved treatments for breast cancer, including metastatic breast cancer. [Means for solving the problem]

[0006] The present disclosure relates to a method for treating insulin-like growth factor 1 (insulin-like growth factor 1) receptor (IGFR). We demonstrate that antisense oligodeoxynucleotides (AS-ODNs) targeting IGF-1R (insulin-like growth factor receptor-1, IGF-1R) effectively stimulate a subject-specific response to treat breast cancer when used in the therapeutic approach described herein. In certain embodiments, the method is effective for treating breast cancer in patients alone as part of an autologous cancer cell vaccine, or optionally with systemic administration. In a preferred approach, the method disclosed herein provides effective breast cancer therapy as a monotherapy, i.e., without chemotherapy and without radiation therapy.

[0007] In some embodiments, the present disclosure provides a biodiffusion chamber for implantation into a subject suffering from breast cancer, the biodiffusion chamber comprising irradiated tumor cells and irradiated insulin-like growth factor 1 receptor antisense oligodeoxynucleotide (IGF-1R AS ODN). In some embodiments, the tumor cells are removed from a resection site in the subject.

[0008] In embodiments, the present disclosure provides a diffusion chamber containing irradiated IGF-1R AS ODN and irradiated, adherent-enriched, minced tumor cells, the biodiffusion chamber comprising a membrane that is impermeable to the cells and permeable to the IGF-1R AS ODN.

[0009] In embodiments, the tumor cells are removed from the resection site using an endoscopic device. In further embodiments, the tumor cells are 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 opening, and the tumor cells are drawn into the side opening by electronically controlled variable suction. In embodiments, the tissue morcellator does not generate heat at the resection site. In further embodiments, the tumor cells are enriched for expression of nestin prior to being placed in the biodiffusion chamber. In some embodiments, implantation of the chamber inhibits tumor regrowth 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, or at least 36 months.

[0010] In additional embodiments, the present disclosure provides a method for preparing a biodiffusion chamber for implantation into a subject suffering from breast cancer, the method comprising placing tumor cells in the biodiffusion chamber in the presence of an IGF-1R AS ODN and irradiating the biodiffusion chamber, the tumor cells being removed from a resection site of the subject using a tissue morcellator that does not generate heat at the resection site. Typically, multiple chambers are used. For example, about 10 chambers, or about 20 chambers may be used. Advantageously, optimal anti-tumor responses are obtained when the number of cells in the chamber is about 750,000 to about 1,250,000, e.g., about 1,000,000 per chamber when 20 chambers are implanted.

[0011] 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 additional embodiments, the outer cannula includes a side opening, and the tumor cells are drawn into the side opening by electronically controlled variable suction.

[0012] In embodiments, the present disclosure provides a method of treating a subject suffering from breast cancer, the method comprising implanting one or more biodiffusion chambers into the subject, the one or more biodiffusion chambers containing irradiated tumor cells, irradiated insulin-like growth factor 1 receptor antisense oligodeoxynucleotide (IGF-1R AS ODN), and the tumor cells are removed from a resection site in the subject using a non-heat generating tissue morcellator at the resection site. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 shows the experimental procedure used to obtain the data presented in Example 1. C57BL / 6 mice were vaccinated with various chambers as described in Table 2. The chambers were then explanted 48 hours later. The animals were then inoculated with a breast cancer cell line (E0771) in the right fourth fat pad. The animals were monitored for tumor development, growth and survival. [Figure 2A] Figure 2A shows a graph demonstrating the effect of vaccination on animal survival. Results presented represent the average of two independent experiments. Survival is defined as the end-point sacrifice, i.e., when animals developed tumors to ±1200 mm3 or if animals did not develop tumors, at 100 days after chamber implantation. Figure 2A shows the Kaplan-Meier curve showing overall survival. [Figure 2B] Figure 2B shows a graph demonstrating the effect of vaccination on animal survival. Results presented represent the average of two independent experiments. Survival is defined as the end-point sacrifice, i.e., when animals develop tumors of ±1200 mm3 or if animals do not develop tumors, at 100 days after chamber implantation. Figure 2B shows the median survival time. [Figure 3A] FIG. 3A is a graphical representation showing the effect of vaccination on tumor development. Results presented represent the average of two independent experiments. FIG. 3A shows the median survival rate. [Figure 3B]Graph showing the effect of vaccination on tumor development. Results presented represent the average of two independent experiments. Figure 3B shows the percentage and number of animals that remained tumor-free at the end of the experiment. [Figure 4] Graphical representation of the effect of vaccination on tumor progression. Animals in the "fast growing" category reached endpoint sacrifice before 70 days post vaccination (dpv). Animals in the "slow growing" category were sacrificed after 70 dpv. [Diagram 5] Figure 1: Graph showing IgG1 optical density (405 nm) measured in an E0771-based cell ELISA in sera from C57Bl / 6 mice implanted for 48 hours in diffusion chambers containing PBS (n=20, "control"), mice vaccinated by implantation for 48 hours in diffusion chambers containing 1x106 E0771 breast tumor cells mixed with 4 mg IGF-1R AS-ODN (n=2-, "mixed"), and mice vaccinated by implantation for 48 hours in diffusion chambers containing 1x106 E0771 cells treated overnight with 4 mg IGF-1R AS-ODN, washed, and added to the chamber with an additional 10 μg IGF-1R AS-ODN (n=20, "o / n+mixed"). sac=sera of mice collected on different days of euthanasia between animals. Asterisks indicate statistical significance. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Disclosed herein are compositions and methods for personalized therapy of breast cancer.More specifically, the disclosure provides a therapeutic approach for breast cancer that aims to reduce the length and number of post-surgery treatments, especially hormonal therapy, and thereby reduce the possibility of side effects.The data presented herein show that prior vaccination with IGF-1R antisense-treated breast cancer cells can prevent tumor growth.Thus, the disclosure provides a promising new treatment for breast cancer patients, including those who are in advanced stage or triple negative cancer.Specifically, the disclosure provides a single post-surgery autologous vaccine for the treatment of breast cancer patients.

[0015] definition All terms not defined herein have their common art recognized meaning.

[0016] As used herein, terms such as "a," "an," and "the" include singular and plural referents unless the context clearly dictates otherwise.

[0017] As used herein, the term "about" when used before a numerical value indicates a range of that value plus or minus 10%. For example, "about 100" includes 90 and 110. As used herein, the term "autologous" refers to cells or tissues obtained from the same individual.

[0018] As used herein, the term "autologous cancer cell vaccine" refers to a therapeutic agent that is produced in part by isolating tumor cells from an individual and processing these tumor cells ex vivo. The cells, or cells derived therefrom, are then readministered to the individual from whom the tumor cells were isolated. In embodiments, the autologous cancer cell vaccine may include additional components, such as buffers and / or antisense nucleic acids, in addition to tumor cells. In embodiments, the "autologous cancer cell vaccine" may refer to a biodiffusion chamber that includes tumor cells and one or more additional components. In some embodiments, the "autologous cancer cell vaccine" may be a "fully formulated chamber" and is referred to herein as a "fully formulated biodiffusion chamber."

[0019] As used herein, the term "fully formulated chamber" or "fully formulated biodiffusion chamber" refers to a biodiffusion chamber containing autologous tumor cells and other cells contained within the tumor microenvironment (TME), which may or may not be treated with a first amount of IGF-1R AS ODN prior to encapsulation within the chamber. The cells are encapsulated with an exogenously added second amount of IGF-1R AS ODN, for example at least 2 μg, at least 4 μg, at least 6 μg, at least 8 μg, or at least 10 μg, and then the chamber is irradiated with 5 Gy of gamma radiation.

[0020] As used herein, the term "small molecule" includes nucleic acids, peptides, proteins, and other chemicals (such as cytokines and growth hormones produced by cells), but does not include cells, exosomes, or microvesicles.

[0021] As used herein, the terms "targeting IGF-1R expression" or "targeting IGF-1R expression" refer to administering an antisense nucleic acid having a sequence designed to bind to IGF-1R.

[0022] As used herein, the term "systemic administration" refers to achieving delivery of a substance throughout the body of a subject. Typical systemic administration routes include parenteral, transdermal, intraperitoneal, intravenous, subcutaneous, and intramuscular administration.

[0023] Other routes of administration include oral, nasal, topical, ocular, buccal, sublingual, intravaginal, intrahepatic, intracardiac, intrapancreatic, by inhalation, and via an implantable pump.

[0024] breast cancer There are several types of breast cancer, including angiosarcoma, ductal carcinoma in situ (DCIS), inflammatory breast cancer, invasive lobular carcinoma, and Paget's disease of the breast. Breast cancer most often begins in cells within the milk-producing ducts (invasive ductal carcinoma). Breast cancer can also begin in glandular tissue called lobules (invasive lobular carcinoma) or other cells or tissues within the breast.

[0025] The most common symptom of breast cancer is a new lump or mass. Other possible symptoms include swelling of all or part of the breast (even if no obvious lump can be felt), inflamed or dimpling skin, breast or nipple pain, nipple retraction (turning inward), redness / scaling / or thickening of the nipple or breast skin, and nipple discharge.

[0026] It is estimated that approximately 5-10% of breast cancers are associated with inherited genetic mutations. Several inherited genetic mutations have been identified that may increase the likelihood of breast cancer, including BRCA1, BRCA2, ATM, CDH1, CHEK2, NBN, NF1, PALB2, PTEN, STK11, and TP53 (p53). BRCA1 gene mutation carriers have a 55-65 percent chance of developing breast cancer by age 70, and BRCA2 gene mutation carriers have approximately a 45 percent chance of developing breast cancer by age 70. Women with BRCA1 mutations have an increased risk of triple-negative cancers (i.e., estrogen receptor negative, progesterone receptor negative, HER2 negative). BRCA2 carriers tend to have breast cancers that are estrogen receptor positive.

[0027] Treatment options for breast cancer depend on the stage of the cancer, but also on the hormone receptor status of the tumor tissue. Hormone receptor positive cancers respond fairly well to hormone or targeted therapy, such as tamoxifen. However, these treatments are costly and time-consuming (hormonal therapy with tamoxifen for up to 5 years). However, triple-negative breast cancers (i.e., estrogen receptor negative, progesterone receptor negative, HER2 negative), hormone receptor negative cancers (i.e., estrogen receptor negative, progesterone receptor negative), and advanced cancers with metastasis represent a population that does not respond well to current hormone or targeted therapy and therefore requires new treatments.

[0028] The risk of breast cancer and associated risk factors include female gender, increasing age, personal history of breast cancer, family history of breast cancer, inherited genes that increase the risk of cancer, radiation exposure, obesity, and alcohol consumption.

[0029] Antisense molecules Antisense molecules are nucleic acids that function by binding to target complementary sequences of mRNA by Watson and Crick base pairing rules. Translation of target mRNA is inhibited by active and / or passive mechanisms when hybridization occurs between complementary strands. In passive mechanisms, hybridization between mRNA and exogenous nucleotide sequences leads to double strand formation that prevents ribosome complexes from reading the message. In active mechanisms, hybridization promotes the binding of RNaseH, which destroys the RNA but leaves the antisense intact to hybridize to another complementary mRNA target. Either or both mechanisms inhibit the translation of proteins that contribute to or perpetuate the malignant phenotype. As therapeutic agents, antisense molecules are much more selective and, as a result, more effective and less toxic than conventional drugs.

[0030] The methods and compositions disclosed herein include the use of antisense molecules for cancer therapy. Typically, the antisense molecule is an antisense oligodeoxynucleotide (AS-ODN). In some embodiments, the antisense molecule comprises a modified phosphate backbone. In some embodiments, the phosphate backbone modification makes the antisense more resistant to nuclease degradation. In some embodiments, the modification is a locked antisense. In other embodiments, the modification is a phosphorothioate bond. In some embodiments, the antisense comprises one or more phosphorothioate bonds. In some embodiments, the phosphorothioate bond stabilizes the antisense molecule by conferring nuclease resistance, thereby increasing its half-life. In some embodiments, the antisense may be partially phosphorothioate bonded. 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 phosphorothioate-linked.In some embodiments, the antisense is fully phosphorothioate-linked.In other embodiments, phosphorothioate linkage can be substituted for phosphodiester linkage.In some embodiments, the antisense has at least one terminal phosphorothioate monophosphate.

[0031] In some embodiments, the antisense molecule comprises one or more CpG motifs. In other embodiments, the antisense molecule does not comprise a CpG motif. In some embodiments, the one or more CpG motifs are methylated. In other embodiments, the one or more CpG motifs are unmethylated. In some embodiments, when the antisense molecule is administered to a subject, the one or more unmethylated CpG motifs induce an innate immune response. In some embodiments, the innate immune response is mediated by the binding of the unmethylated CpG-containing antisense molecule to Toll-like receptors (TLRs).

[0032] In certain embodiments, the antisense molecule comprises at least one terminal modification or "cap". The cap can be a 5' and / or 3'-cap structure. The term "cap" or "terminal-cap" includes chemical modifications at either end of the oligonucleotide (with respect to the terminal ribonucleotide), including modifications at the bond 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 compromising the molecular interaction with the target sequence or intracellular machinery. Such modifications can be selected based on their increased potency in vitro or in vivo. The cap can be at the 5'-end (5'-cap) or 3'-end (3'-cap), or can be at both ends. In certain embodiments, the 5'- and / or 3'-caps are independently selected from phosphorothioate monophosphate, abasic residue (moiety), phosphorothioate linkage, 4'-thionucleotide, carbocyclic nucleotide, phosphorodithioate linkage, inverted nucleotide or inverted abasic moiety (2'-3' or 3'-3'), phosphorodithioate monophosphate, and methylphosphonate moiety. The phosphorothioate or phosphorodithioate linkage, when part of the cap structure, is generally located between the two terminal nucleotides at the 5' end and between the two terminal nucleotides at the 3' end.

[0033] In preferred embodiments, the antisense molecule targets the expression of the 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. When activated by its ligands (IGF-I, IGF-II and insulin), IGF-1R regulates a wide range of cellular functions, including proliferation, transformation and cell survival. IGF-1R is not an absolute requirement for normal growth, but is essential for growth in anchorage-independent conditions that can occur in malignant tissues. A review of the role of IGF-1R in tumors is provided in Baserga et al., Vitamins and Hormones, 53:65-98 (1997), which is incorporated herein by reference in its entirety.

[0034] In certain embodiments, the antisense molecule is an oligonucleotide directed against the DNA or RNA of a growth factor or growth factor receptor, such as, for example, IGF-1R.

[0035] In one embodiment, the antisense is a deoxynucleotide to IGF-1R (IGF-1R AS ODN). The full length coding sequence of IGF-1R is provided as SEQ ID NO: 15 (see, e.g., PCT / US2016 / 26970, which is incorporated by reference in its entirety).

[0036] In some embodiments, the antisense molecule comprises a nucleotide sequence that is complementary to the IGF-1R signal sequence and comprises either RNA or DNA. The IGF-1R signal sequence is a 30 amino acid sequence. In other embodiments, the antisense molecule comprises a nucleotide sequence that is complementary to a portion of the IGF-1R signal sequence and comprises either RNA or DNA. In some embodiments, the antisense molecule comprises a nucleotide sequence that is complementary to codons 1-309 of IGF-1R and comprises either RNA or DNA. In other embodiments, the antisense molecule comprises a nucleotide sequence that is complementary to a portion of codons 1-309 of IGF-1R and comprises either RNA or DNA.

[0037] In some embodiments, 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, IGF-1R AS ODN is about 15 nucleotides to about 22 nucleotides in length.In some embodiments, IGF-1R AS ODN is about 18 nucleotides in length.

[0038] In one embodiment, the IGF-1R AS ODN forms a secondary structure at 18° C., but does not form a secondary structure at about 37° C. In another embodiment, the IGF-1R AS ODN does not form a secondary structure at about 18° C., or at about 37° C. In yet another embodiment, the IGF-1R AS ODN does not form a secondary structure at any temperature. In another embodiment, the IGF-1R AS ODN does not form a secondary structure at 37° C. In a particular embodiment, the secondary structure is a hairpin loop structure.

[0039] In some embodiments, the IGF-1R AS ODN comprises the nucleotide sequence of SEQ ID NO: 1, or a fragment thereof. In some 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 with SEQ ID NO: 1, or a fragment thereof. In some embodiments, the IGF-1R AS ODN comprises one or more phosphorothioate bonds (see, for example, SEQ ID NO: 16).

[0040] In one embodiment, the IGF-1R AS ODN consists of SEQ ID NO: 1. NOBEL is an 18-mer oligodeoxynucleotide with a phosphorothioate backbone and a sequence complementary to codons 2-7 of the IGF-1R gene. Thus, NOBEL is an antisense oligonucleotide for IGF-1R (IGF-1R AS ODN). At the 5' end, the NOBEL sequence derived as a complementary sequence to the IGF-1R gene is 5'-TCCTCCGGAGCCAGACTT-3' (SEQ ID NO: 1) It is.

[0041] NOBEL has a stable shelf life and is resistant to nuclease degradation due to its phosphorothioate backbone. Administration of NOBEL can be provided in any of the standard methods for introducing oligodeoxynucleotides known to those skilled in the art. Advantageously, the AS ODNs disclosed herein, including NOBEL, can be administered with little / no toxicity. Even at a level of about 2g / kg (scaled) based on mouse tests (40 μg in tail vein), no toxicity issues were noted. NOBEL can be manufactured according to the general procedures known to those skilled in the art.

[0042] Antisense molecules, such as the NOBEL sequence of SEQ ID NO:1, may also comprise one or more p-ethoxy backbone modifications disclosed in U.S. Patent No. 9,744,187, the entirety of which is incorporated herein by reference.In some embodiments, the nucleic acid backbone of antisense molecules comprises at least one p-ethoxy backbone bond.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 antisense molecules may be p-ethoxy bonded.The remaining bond may be phosphodiester bond or phosphorothioate bond or a combination thereof. In a preferred embodiment, between 50% and 80% of the phosphate backbone linkages in each oligonucleotide are p-ethoxy backbone linkages and between 20% and 50% of the phosphate backbone linkages in each oligonucleotide are phosphodiester backbone linkages.

[0043] Various IGF-1R antisense sequences are biologically active in some or all of the multimodal effects of NOBEL sequence.18-mer NOBEL sequence has both IGF-1R receptor downregulation activity and TLR agonist activity, and further experiments in mice suggest that both activities are necessary for antitumor immune activity in vivo.AS ODN molecule has antitumor activity, but does not have antitumor activity, although its complementary sense sequence also has CpG motif.

[0044] In certain embodiments, the sequence of the antisense is selected from the group consisting of SEQ ID NOs: 1-14, as shown in Table 1. In some embodiments, the antisense has 90% sequence identity to one or more of SEQ ID NOs: 1-14. In some embodiments, the antisense has 80% sequence identity to one or more of SEQ ID NOs: 1-14. In some embodiments, the antisense has 70% sequence identity to one or more of SEQ ID NOs: 1-14.

[0045] [Table 1] In some embodiments, the IGF-1R AS ODN comprises the nucleotide sequence of any one of SEQ ID NOs: 1-14, or a fragment thereof. In some 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 SEQ ID NOs: 1-14, or a fragment thereof.

[0046] In some embodiments, the antisense molecule downregulates expression of a downstream gene of the IGF-1R pathway in the cell. In some embodiments, the downstream gene is hexokinase (Hex II). In some embodiments, the antisense molecule downregulates expression of a housekeeping gene in the cell. In some embodiments, the housekeeping gene is L13.

[0047] In some embodiments, the IGF-1R AS ODN is chemically synthesized. In some embodiments, the IGF-1R AS ODN is produced by solid-phase organic synthesis. In some embodiments, the synthesis of the IGF-1R AS ODN is carried out in a synthesizer equipped with a closed chemical column reactor using flow-through technology. In some embodiments, each synthesis cycle sequence on a solid support consists of multiple steps that are carried out sequentially until a full-length IGF-1R AS ODN is obtained. In some embodiments, the IGF-1R AS ODN is stored in liquid form. In other embodiments, the IGF-1R AS ODN is lyophilized before storage. In some embodiments, the lyophilized IGF-1R AS ODN is dissolved in water before use. In other embodiments, the lyophilized IGF-1R AS ODN is dissolved in an organic solvent before use. In still other embodiments, the lyophilized IGF-1R AS ODN is formulated into a pharmaceutical composition. In some embodiments, the pharmaceutical composition is a liquid pharmaceutical composition. In other embodiments, the pharmaceutical composition is a solid pharmaceutical composition. Additional antisense nucleic acids are also described in US 2017 / 0056430, which is incorporated by reference in its entirety.

[0048] Autologous cancer cell vaccine introduction Immunotherapy is currently used to target hematological malignancies that have one common cellular antigen. Unfortunately, solid tumors are much more complex and represent an epigenetic progression of genetic changes to a malignant state with an unidentifiable number of tumor-specific targets. To make matters even more challenging, within the cancer group of WHO diagnosis, there is a significant variation in tumor phenotype. An autologous cell vaccine could encompass all such variations and all such targets, and would be an ideal subject-specific immunotherapy for solid tumor cancer. However, autologous cancer cell vaccines cannot be obtained from primary cell cultures, since successive passaging alters the tumor phenotype and thus reduces the set of tumor-specific antigens. This would also require lot release qualification, which is difficult to achieve at each passaging. The present disclosure removes these concerns by plating freshly excised and morselized tumor cells and reimplanting them within 24 hours as depot antigens. In some embodiments, the superior results achieved herein are obtained by ensuring that an adequate number of cells are present in the chamber, among other details described herein.

[0049] Previous studies have designed autologous cell vaccines through the use of antigen-presenting cells instead of autologous tumor cells. In this paradigm, the subject's monocytes are harvested from pre-treatment plasma leukopheresis and differentiated ex vivo into autologous dendritic cells (DCs). The dendritic cells are then presented to crude lysates of the subject's tumor to induce DC activation / maturation, and at a later time point, the mature dendritic cells cross-primed with tumor antigens are injected into the subject as a DC vaccine. However, ex vivo differentiation is missing some important stimulatory components that only occur in vivo. In addition, differentiation of DCs from hematopoietic precursors requires extensive in vitro manipulation with labor-intensive cell processing in expensive facilities. The present disclosure circumvents these concerns by providing immunomodulatory and immunostimulatory antisense oligodeoxynucleotides (AS-ODNs) that promote the endogenous DC maturation process and the generation of appropriate immune responses. More specifically, the present disclosure provides a biodiffusion chamber containing dispersed tumor cells from a patient and irradiated antisense molecules, which is implanted into the patient for a therapeutic period. Without being bound by theory, it is believed that the combination of irradiated tumor cells, antisense, and the biodiffusion chamber act in concert to simulate a local immune response, preventing immune system depression by reducing or eliminating M2 cells, and enhancing the immune response.

[0050] Thus, the present disclosure shows that irradiated implantable biodiffusion chambers containing freshly excised tumor cells and IGF-1R AS ODN can safely function as an effective, subject-specific autologous cell vaccine for breast cancer immunotherapy. Thus, the use of the claimed implantable biodiffusion chambers to mount an immune response that selectively targets tumor cells in a subject provides a new and important approach for the treatment of breast cancer, including metastatic breast cancer.

[0051] Biodiffusion Chamber A representative diffusion chamber includes a chamber barrel having two ends, a first end and a second end. In some embodiments, the biodiffusion chamber is a small ring capped on both sides by a porous, cell-impermeable membrane, such as the Duropore membrane manufactured by Millipore Corporation. Optionally, one of the ends may be closed as part of the chamber body, leaving only one end open and sealed using a porous membrane. The membrane may be made of plastic, Teflon, polyester, or any inert material that is strong, flexible, and can withstand chemical treatments. The chamber may be made of any material, such as, but not limited to, plastic, Teflon, Lucite, titanium, plexiglass, or an inert material that is non-toxic and well tolerated by humans. In addition, the chamber should be able to withstand sterilization. In some embodiments, the diffusion chamber is sterilized with ethylene oxide before use. Other suitable chambers are described in U.S. Provisional Application No. 62 / 621,295, filed January 24, 2018, U.S. Pat. No. 6,541,036, PCT / US16 / 26970, and U.S. Pat. No. 5,714,170, which are incorporated by reference in their entireties.

[0052] In certain embodiments, the membrane allows the passage of small molecules but not cells (i.e., cells cannot enter or leave the chamber). In some embodiments, the diameter of the pores in the membrane allows diffusion of nucleic acids and other chemicals (e.g., cytokines produced by cells, etc.) from the chamber but does not allow cells to pass between the chamber and the subject in which the chamber is implanted. However, biodiffusion chambers useful in the present disclosure, including any chamber that does not allow cells to pass between the chamber and the subject in which the chamber is implanted, are provided that allow exchange and passage of factors between the chamber and the subject. Thus, in certain embodiments, the pore size is greater than or equal to 100 μm in volume. 3The membrane has a cut-off that prevents passage of more than 1 μm of material into or out of the chamber. In some embodiments, the pores in the membrane have a diameter of about 0.25 μm or less. For example, the pores may have a diameter of about 0.1 μm. In certain embodiments, the pores range from 0.1 μm to 0.25 μm in diameter. See also Lange et al., J. Immunol., 1994, 153, 205-211 and Lanza et al., Transplantation, 1994, 57, 1371-1375, which are incorporated by reference in their entireties. This pore diameter prevents passage of cells into or out of the chamber. In certain embodiments, the diffusion chamber is constructed from a 14 mm lucite ring with a hydrophilic Durapore membrane (Millipore Corporation, Bedford, Massachusetts) with a 0.1 μm pore size.

[0053] In certain embodiments, the biodiffusion chamber includes a membrane that allows the IGF-1R AS ODN to diffuse out of the chamber. In some embodiments, about 50% of the IGF-1R AS ODN diffuses out of the chamber within about 12 hours, and about 60% of the IGF-1R Within approximately 24 hours, the AS ODNs diffused out of the chamber, and approximately 80% of the IGF-1R AS The ODNs diffuse out of the chamber within about 48 hours and / or about 100% of the IGF-1R AS ODNs diffuse out of the chamber within about 50 hours.

[0054] In an exemplary approach, to assemble the biodiffusion chamber, a first porous membrane is attached to one side of the first diffusion chamber using adhesive and pressure to create a seal. A second porous membrane is similarly attached to a second diffusion chamber ring. The membrane can also be secured in place with a rubber gasket, which may provide a better seal. The diffusion chamber ring is allowed to dry overnight (minimum 8 hours). The first and second diffusion chamber rings are then attached to each other using adhesive and allowed to dry overnight (minimum 8 hours). In a preferred embodiment, the bonding process of the first and second chamber rings includes using dichloroethane as a solvent to facilitate adhesion between the two rings. In an alternative approach, the chamber may have only one side that includes a porous membrane.

[0055] On the barrel portion of the chamber, one or more openings (e.g., ports) are provided that can be covered by a cap, which, when the chamber is implanted, can be accessed from outside the subject's body, thus allowing the diffusion chamber to be refilled. The openings allow multiple and consecutive sampling of the contents without contamination and without harming the subject, thus greatly reducing the number of implantation procedures performed on the subject. Prior to implantation in the patient, the one or more openings can be sealed with bone wax, a port plug, or a cap made, for example, from PMMA. The cap can be a self-sealing rubber screw-on type, adapted to the opening. In some configurations, the diffusion chamber can include two or more injection openings or ports. Sampling of the chamber contents can be performed by accessing the openings by removing the cap outside the subject's body and inserting a common needle and syringe. In some embodiments, the chamber can further include a removal device. Such a device facilitates removal of the chamber from the patient.

[0056] In some embodiments, the chamber serves as an antigen depot designed to allow tumor antigens to diffuse out of the chamber for the purpose of promoting a therapeutic host immune response. Exogenous IGF-1R AS ODN and ex vivo irradiation promote a proinflammatory response. This combination represents a novel autologous cell vaccine with exogenous active pharmaceutical ingredient (API) and radiation that is associated with clinical and radiographic improvements, and increased survival in the protocol, which applicants interpret as inducing or enhancing tumor immune effects. Furthermore, low concentrations of IGF-1R The addition of AS ODN is important for the proinflammatory response.

[0057] In one embodiment, the disclosure provides a biodiffusion chamber containing (a) tumor cells and (b) an effective amount of an antisense molecule for implantation into a subject suffering from cancer. In another embodiment, a method of treating cancer in a subject is provided, comprising (a) obtaining a biodiffusion chamber containing tumor cells and an effective amount of an antisense nucleic acid, (b) irradiating the biodiffusion chamber and contents, and (c) implanting the irradiated biodiffusion chamber into the subject for an effective therapeutic period.

[0058] In some embodiments, the IGF-1R AS ODN is present in the biodiffusion chamber in an amount ranging from about 0.5 μg to about 10 μg. In some embodiments, the IGF-1R AS ODN is present in an amount ranging from about 1 μg to about 5 μg per chamber, or from about 2 μg to about 4 μg per chamber. For example, the IGF-1R may be present in an amount of about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, or about 5 μg per chamber. In certain embodiments, the IGF-1R AS ODN is present in an amount of about 2 μg per chamber. In certain embodiments, the IGF-1R AS ODN is present in an amount of about 4 μg per chamber. In some embodiments, the IGF-1R AS ODN is present in the biodiffusion chamber in an amount ranging from about 10 μg to about 500 μg. In some embodiments, the IGF-1R AS ODN is present in an amount ranging from about 40 μg to about 400 μg per chamber, from about 40 to about 70 μg per chamber, or from about 100 μg to about 200 μg per chamber. In certain embodiments, the IGF-1R AS ODN is present in an amount of about 100 μg per chamber. In certain embodiments, the IGF-1R AS ODN is present in an amount of about 200 μg per chamber. In some embodiments, the IGF-1R AS ODN is present in an amount of about 200 μg per chamber. The ODN is present in an amount ranging from about 1 mg to about 100 mg per chamber, or from about 2 mg to about 10 mg per chamber. In certain embodiments, the IGF-1R AS ODN is present in an amount of about 2 mg per chamber. In certain embodiments, the IGF-1R AS ODN is present in an amount of about 4 mg per chamber. Without being bound by theory, it is believed that these levels avoid M2 immunostimulatory responses in the subject while promoting an enhanced Th1 response in the subject.

[0059] In some embodiments, tumor cells are not treated with IGF-1R AS ODN before encapsulation in the chamber. However, tumor cells are usually treated with IGF-1R AS ODN before encapsulation in the chamber. The time of treating cells before encapsulation can vary. For example, tumor cells can be treated with IGF-1R AS ODN ex vivo 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 immediately before encapsulation. Usually, tumor tissue can be treated ex vivo for about 12 hours to about 18 hours before encapsulation. Advantageously, cells can be encapsulated after pretreatment lasting up to overnight. Without being bound by theory, it is believed that preencapsulation treatment plays a desirable role in stimulating the production of tumor antigens.

[0060] The amount of IGF-1R AS ODN used for the pre-encapsulation treatment can be in the range of about 1 mg to 8 mg per million cells, for example, 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 pre-encapsulation treatment is about 4 mg per million cells.

[0061] In some embodiments, the IGF-1R AS ODN for ex vivo treatment of tumor cells is used at a concentration ranging from at least about 2 mg / ml to at least about 5 mg / ml. In some embodiments, the IGF-1R AS ODN is used at a concentration of at least 4 mg / ml. In certain embodiments, the IGF-1R AS ODN is used at a concentration of 4 mg / ml.

[0062] In some embodiments, the IGF-1R AS ODN used for ex vivo tumor cell treatment is the same as the IGF-1R AS ODN present in the chamber. In other embodiments, the IGF-1R AS ODN used for ex vivo tumor cell treatment is different from the IGF-1R AS ODN present in the chamber. In some embodiments, the IGF-1R AS ODN used for ex vivo tumor cell treatment 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 for ex vivo tumor cell treatment is about 15 nucleotides to about 22 nucleotides in length. In some embodiments, the IGF-1R AS ODN used for ex vivo tumor cell treatment is about 18 nucleotides in length.

[0063] In one embodiment, 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 another embodiment, the IGF-1R AS ODN used to treat tumor cells does not form a secondary structure at about 18° C., or at about 37° C. In yet another embodiment, the IGF-1R AS ODN used to treat tumor cells ex vivo does not form a secondary structure at any temperature. In another embodiment, the IGF-1R AS ODN used to treat tumor cells does not form a secondary structure at 37° C. In a particular embodiment, the secondary structure is a hairpin loop structure.

[0064] In some embodiments, the IGF-1R AS ODN used for tumor cell treatment comprises the nucleotide sequence of SEQ ID NO: 1, or a fragment thereof. In some embodiments, the IGF-1R AS ODN used for tumor cell treatment 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 with SEQ ID NO: 1, or a fragment thereof. In some embodiments, the IGF-1R AS ODN used for tumor cell treatment is SEQ ID NO: 1.

[0065] After treating the tumor cells with AS-ODN for a period of time, the AS-ODN is removed and new AS-ODN is added to the chamber, which is then irradiated before implantation into a subject. In some embodiments, the biodiffusion chamber is treated with 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 some embodiments, the radiation dose is not more than about 5 Gy. In other embodiments, the radiation dose is at least about 5 Gy. In some embodiments, the radiation dose is 5 Gy. In some embodiments, the biodiffusion 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 less than about 24 hours before implantation into a subject. In other embodiments, the chamber is irradiated about 24 hours before implantation into a subject. In still other embodiments, the chamber is irradiated at least about 24 hours before implantation into a subject. In still other embodiments, the chamber is irradiated about 48 hours or less before implantation into a subject. In yet other embodiments, the chamber is irradiated for at least about 48 hours prior to implantation into the subject.

[0066] Usually, tumor cells are killed before transplantation, for example by radiation, but the cells do not have to be killed, and in fact it may be advantageous to keep the cells viable to facilitate release of antigens. Thus, in some embodiments, the cells may not be irradiated before transplantation. However, for safety reasons, it is desirable to prevent the release of live tumor cells into the subject.

[0067] Tumor cells can be placed in the chamber in various numbers. In one embodiment, about 1×10 4 ~Approx. 5×10 6 In another embodiment, about 1×10 tumor cells are placed in each diffusion chamber. 5 ~Approx. 1.5×10 6 In yet another embodiment, about 5×10 tumor cells are placed in the diffusion chamber. 5 ~1×10 6 A number of tumor cells are placed in the chamber. The method may be used with a subject. Applicants have discovered that the number of tumor cells may affect the subject's anti-tumor response and that an appropriate range should be selected to increase the chances of obtaining the desired outcome. In some embodiments, the anti-tumor immune response is optimal in the range of about 750,000 to about 1,250,000 cells in the chamber, with a peak at about 1 million cells / chamber. Multiple chambers containing irradiated tumor cells are administered, with the number of cells per chamber preferably being maintained in that range to maintain an optimal immune response. Preferably, the tumor cells are intact and not autolyzed or otherwise damaged as described herein.

[0068] In some embodiments, it may be preferable to maintain the ratio of cells to AS ODN in the chamber. Thus, in one embodiment, the chamber may contain about 2 μg of AS ODN and 750,000 to 1,250,000 cells, e.g., 1,000,000 cells. The ratio of cells to AS ODN is therefore about 3.75×10 5 ~Approx. 6.25×10 5 / μg AS ODN range, e.g., about 5.0×10 5 cells / μg. Thus, in a typical patient receiving 20 chambers, the total dose of AS ODN is approximately 40 μg.

[0069] Typically, administration is in a chamber as described herein.However, in some embodiments, irradiated cells and IGF-1R AS ODN can be co-administered to a subject without being physically contained together in a chamber or another container.In some methods using this approach, irradiated cells IGF-1R AS ODN are thus dispersed, diffused, or metabolized in the body limited by the subject's physiology.Thus, in some embodiments, for example, tumor cells for use can be prepared as described herein for a chamber and administered with IGF-1R AS ODN, but administration may not be contained in a physical container.Such administration is usually intramuscular administration.

[0070] Tumor tissue preparation for chambers Tumor cells used for autologous vaccination are surgically removed from the subject. In some embodiments, the tumor cells are removed from the patient using a tissue morcellator. The excision device preferably combines a high-speed reciprocating inner cannula and electronically controlled variable suction within a stationary outer cannula. 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 instrument also relies on a side-opening resection and suction opening located 0.6 mm from the blunt end of the desiccator. The combination of gentle forward pressure of the opening on the tissue to be removed and suction draws the desired tissue into the side opening, allowing for controlled and precise tissue resection by the reciprocal 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 opening. One example of a suitable device is the Myriad® Tissue Aspirator (NICO Corporation®, Indianapolis, IN), a minimally invasive surgical system that can be used for the removal of soft tissue under direct, microscopic, or endoscopic visualization. The scraped tissue is aspirated, collected in a collection chamber, and collected in a sterile tissue trap. Blood is removed from the preparation during collection of the tissue into the sterile tissue trap. Preferably, the sterile trap includes a collection dish at the base of the trap and a stem that provides access to the trap. The trap structure may also include a ladle-shaped internal structure that is removable from the trap to facilitate tissue removal from the trap.

[0071] Preferably, the morcellator 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 morcellated tumor tissue (i.e., tumor tissue scraped off by a side-opening resection without heat, and optionally without sonication). Advantageously, the aspirator resection and morcellated tissue have a higher viability than tissue removed by other methods. The resection process is believed to maintain a higher tumor cell viability, in part, by limiting exposure of tumor cells to high temperatures during the resection period. For example, the methods herein do not expose tumor cells to more than 25°C during resection. Thus, the cells are not exposed to temperatures above body temperature, i.e., about 37°C.

[0072] In some embodiments, the tumor cells are viable when removed from the patient, for example, using a tissue morcellator. In some embodiments, removal of cells from the patient using a tissue morcellator does not kill the cells. In some embodiments, at least 60%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98, or at least 99% of the tumor cells removed from the patient using a tissue morcellator are viable.

[0073] The amount of tumor tissue obtained from a subject may vary. Preferably, the amount is at least 1, at least 2, at least 3 grams or at least 4 grams of wet tumor tissue obtained from a patient. The tissue is removed from a sterile tissue trap and disaggregated by pipetting with a sterile pipette to break up large tissue fragments. The suspension of disaggregated cells is then placed in a sterile tissue culture plate in serum-containing medium and incubated in a tissue culture incubator. This plating step serves to enrich for the desired functional cells by adhesion and also aids in removing debris from the preparation. Thus, the tumor cells used in the processes described herein preferably consist essentially of or consist of adherent cells from tumor tissue.

[0074] After a predetermined incubation time (e.g., 6, 12, 24, or 48 hours), the cells are removed from the plate. The cells may 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 divided between 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 the same number of cells. In some embodiments, the first diffusion chamber contains more cells than the second chamber.

[0075] In some embodiments, the cells are sorted before being placed in the chamber. In some embodiments, the cells are enriched by selecting with one or more cell markers before being placed in the chamber. Selection can be performed, for example, using beads or using cell sorting techniques known to those skilled in the art. In some embodiments, the cells placed in the chamber are enriched with one or more markers.

[0076] In some embodiments, implantation of the biodiffusion chamber during the therapeutic benefit period reduces or eliminates the recurrence of cancer in the subject. In some embodiments, implantation of the biodiffusion chamber causes a reduction in tumor volume associated with cancer in the subject. In yet other embodiments, implantation of the biodiffusion chamber during the therapeutic benefit period induces the elimination of tumors 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. Biodiffusion chambers may be implanted into a subject in the following non-limiting ways: subcutaneous, intraperitoneal, and intracranial. In certain embodiments, the diffusion chamber is implanted into a recipient site with good lymphatic drainage and / or vascular supply, such as the rectus sheath. In other embodiments, a refillable chamber may be used, allowing the diffusion chamber to be reused for treatment and emptied after treatment. In some embodiments, multiple diffusion chambers, preferably 5-20 diffusion chambers, may be used in a single subject.

[0077] In some 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 into the subject. In some embodiments, 10-20 chambers are implanted into the subject. Preferably, about 20 chambers are implanted into the subject. In some embodiments, the tumor cells are divided evenly between each chamber.

[0078] Typically, the chamber is removed after a period of time. For example, the chamber may 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 therapeutic outcomes. Thus, the preferred time of implantation is about 48 hours. In some embodiments, the vaccination procedure is performed once per patient. In other embodiments, the vaccination procedure is performed multiple times per patient. In some embodiments, the vaccination procedure is performed 2, 3, 4, 5, 6, 7, or 8 times in a single patient. In some embodiments, the vaccination is repeated for a period of time, every 7, 14, or 28 days, or every 1, 3, or 6 months. In further embodiments, the vaccination procedure is repeated periodically until the patient is cancer-free.

[0079] Without being bound by theory, it is believed that implantation of the biodiffusion chamber causes the elimination or reduction of M2 cells at or near the implantation site, and an immune response to tumor antigens diffusing from the chamber is achieved. In some embodiments, the elimination or reduction of M2 cells at the implantation site leads to enhanced presentation of self-tumor antigens by antigen-presenting cells (APCs) to CD4 T cells, leading to the production of interferon gamma (IFNγ) and induction of type 1 tumor immunity. In some embodiments, the production of IFNγ by tumor antigen-specific CD4 T cells and the anti-M2 effect of IGF-1R AS ODN promotes type 1 anti-tumor immunity and the loss of anti-inflammatory M2 cells from the circulation and tumor microenvironment, which indirectly interfere with tumor growth. In some embodiments, the production of IFNγ by tumor antigen-specific CD4 T cells and the anti-M2 effect of IGF-1R AS ODN cause effector-mediated damage of tumor cells and 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 sustains ongoing tumor regression.

[0080] Optionally, the cells introduced into the chamber can be enriched for certain cell types. Nestin, a cytoskeleton-associated class VI intermediate filament (IF) protein, has traditionally been noted for its importance as a neural stem cell marker. Applicants have discovered that nestin-positive cells (nestin+ cells) are enriched in certain tumor samples compared to benign tissue, and that this corresponds to improved therapeutic response. Thus, in some embodiments, the subject's tumor can be harvested to assess the degree of nestin expression, and thus, in some embodiments, the chamber cells are enriched for nestin-positive ("+") cells compared to benign tissue. Without being bound by theory, it is believed that nestin provides a marker associated with a suitable useful antigen that generates an anti-tumor immune response. Thus, the cells implanted into the chamber can be enriched for nestin+ cells overall compared to the tumor cell population when removed from the subject.

[0081] composition In some embodiments, the disclosure provides a composition comprising cancer cells (e.g., breast cancer cells) and an antisense (e.g., IGF-1R AS ODN). In some embodiments, the composition comprises minced cancer cells (e.g., breast cancer cells) and an antisense (e.g., IGF-1R AS ODN). In some embodiments, the composition comprises minced cancer cells (e.g., breast cancer cells) and an antisense (e.g., IGF-1R AS ODN), where one or both of the cancer cells and the antisense are irradiated. In some embodiments, the composition comprises minced and irradiated cancer cells (e.g., breast cancer cells) and an irradiated antisense (e.g., IGF-1R AS ODN).

[0082] In some embodiments, the composition comprises about 1×10 4 , about 5×10 4 , about 1×10 5 , about 5×10 5 , about 1×10 6 , about 5×10 6 , about 1×10 7 , about 5×10 7 , about 1×10 8 , about 5×10 8 , about 1×10 9 , about 5×10 9 , about 1×10 10 , about 5×10 10In some embodiments, the composition comprises about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg, about 10 μg, about 20 μg, about 30 μg, about 40 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg, about 100 μg, about 250 μg, about 500 μg, about 750 μg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, or about 10 mg antisense (e.g., IGF-1R AS-ODN). In some embodiments, the composition comprises about 1 μg / ml, about 2 μg / ml, about 3 μg / ml, about 4 μg / ml, about 5 μg / ml, about 6 μg / ml, about 7 μg / ml, about 8 μg / ml, about 9 μg / ml, about 10 μg / ml, about 20 μg / ml, about 30 μg / ml, about 40 μg / ml, about 50 μg / ml, about 60 μg / ml, about 70 μg / ml, about 80 μg / ml, about 90 μg / ml, about 100 μg / ml, about 250 μg / ml, about 500 μg / ml, about 750 μg / ml, or about 1 mg / mL antisense (e.g., IFG-1R AS-ODN).

[0083] In some embodiments, the composition comprises a cancer cell (eg, a breast cancer cell) and an antisense (eg, an IGF-1R AS ODN), and further comprises a pharma- ceutically acceptable carrier, buffer, stabilizer, or excipient.

[0084] A biodiffusion chamber containing the composition of the present disclosure is also provided. Systemic administration As an alternative to or as a supplement to implantation of the chamber, the IGF-1R AS ODN may be administered systemically. Thus, in some embodiments, the IGF-1R AS ODN is provided in a pharmaceutical composition for systemic administration. In addition to the IGF-1R AS ODN, the pharmaceutical composition may include, for example, saline (0.9% sodium chloride). The composition may include a phospholipid. In some embodiments, the phospholipid is uncharged or neutrally charged at physiological pH. In some embodiments, the phospholipid is a neutral phospholipid. In some embodiments, the neutral phospholipid is a phosphatidylcholine. In some embodiments, the neutral phospholipid is dioleoylphosphatidylcholine (DOPC). In some embodiments, the phospholipid is essentially cholesterol-free.

[0085] 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 therein. In various embodiments, the phospholipid and the oligonucleotide are present in a molar ratio of about 5:1, about 10:1, about 15:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1, about 45:1, about 50:1, about 55:1, about 60:1, about 65:1, about 70:1, about 75:1, about 80:1, about 85:1, about 90:1, about 95:1, or about 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 are less than 5 microns (5 μm) in diameter. In various embodiments, the composition further comprises at least one surfactant, such as, for example, polysorbate 20. In some embodiments, at least about 5% of the total liposomal antisense drug product is comprised of surfactant and at least about 90% of the liposomes are less than 5 microns (5 μm) in diameter. In some embodiments, at least about 15% of the total liposomal antisense drug product, comprised of surfactant and at least about 90% of the liposomes, is less than 3 microns (3 μm) in diameter. In some embodiments, the population of oligonucleotides is incorporated into the population of liposomes.

[0086] In some embodiments, the pharmaceutical composition is a liquid pharmaceutical composition. In other embodiments, the pharmaceutical composition is a solid pharmaceutical composition. The dosage for systemic administration of the antisense in a human subject may 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 for systemic administration may be 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 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 0.01 g / kg to 0.2 g / kg. In some embodiments, the antisense is provided as a lyophilized powder and resuspended prior to administration. When resuspended, the concentration of the antisense may be about 50 mg / ml, about 100 mg / ml, about 200 mg / ml, about 500 mg / ml, about 1000 mg / ml, or a range between these amounts.

[0087] In certain embodiments, the AS ODN may be administered systemically prior to surgery, such as prior to surgery to reduce tumor burden. For example, the AS ODN may be administered up to 24 hours, up to 36 hours, up to 48 hours, or up to 72 hours prior to surgery. In certain embodiments, the pharmaceutical composition may be administered about 48 to about 72 hours prior to surgery. Typically, in such situations, administration is by intravenous bolus.

[0088] Combination therapy Breast cancer therapy has traditionally required treating subjects with radiation, chemotherapy, biological therapy, hormone therapy, or a combination thereof. Such approaches have well-documented challenges. Advantageously, however, the chamber implantation method disclosed herein can be used to treat subjects with cancer as a monotherapy. Thus, it is preferred that the method disclosed herein does not include chemotherapy, radiation therapy, biological therapy, and / or hormone therapy. However, despite the superior efficacy achieved by the monotherapy approach herein, it may be beneficial under certain circumstances to combine the chamber method with other therapies, such as radiation therapy. In certain embodiments, radiation therapy includes, but is not limited to, internal source radiation therapy, external beam radiation therapy, and total body radioisotope radiation therapy. In certain aspects, radiation therapy is external beam radiation therapy. In some embodiments, external beam radiation therapy includes, but is not limited to, gamma radiation therapy, x-ray therapy, intensity modulated radiation therapy (IMRT), and image-guided radiation therapy (IGRT). In certain embodiments, external beam radiation therapy is gamma radiation therapy. Radiation may be administered prior to or after chamber implantation, for example, as salvage therapy. Typically, such salvage therapy approaches are not undertaken until it is determined that the cancer has recurred.

[0089] Therefore, in a combination approach, both the chamber method and the systemic method and composition described herein can be used alone or in combination with one or more of radiation therapy, chemotherapy, hormone therapy, and biological therapy in the same subject.In the combination approach described herein, chamber transplantation is preferably used as the first-line therapy.It is desirable to use chamber transplantation first because other therapies may suppress the subject's immune system, reducing the therapeutic benefit of chamber transplantation.

[0090] Optionally, systemic administration of AS ODN can be performed before chamber implantation.This approach can be used as a priming approach to enhance the immune system of the subject.The priming approach can be particularly advantageous when the immune system of the subject is impaired by previous therapy.

[0091] When systemic administration is used in combination, the AS ODN may be administered systemically at least 2 weeks, at least 1 week, at least 3 days, or at least 1 day prior to treatment of the patient with the autologous cancer cell vaccine. In other embodiments, following treatment of the patient with the autologous cancer cell vaccine, i.e., the chamber, the AS ODN may be administered systemically at least 1 day, at least 3 days, at least 1 week, or at least 2 weeks.

[0092] Optionally, the subject may be revaccinated in the chamber using the method described herein following the first vaccination. The second or further booster vaccination may use tumor cells that have been collected from the subject during tissue removal and stored. Optionally, the second or further booster vaccination may use fresh tumor tissue that has been removed from the subject and processed as described herein. Any tumors remaining in the subject may express the same antigens, thus acting as a depot and providing restimulation. However, recurrent tumors may develop new antigens, thus providing an additional option to stimulate anti-tumor responses. Subsequent vaccinations may be after the first treatment is completed and the tumor recurs, or if the subject fails to respond to the first treatment.

[0093] Targets for treatment with IGF-1R AS ODN A suitable subject is an animal having breast cancer, and typically the subject is a human. The present disclosure provides a method of treating breast cancer, including metastatic breast cancer. The breast cancer may be, for example, angiosarcoma, ductal carcinoma in situ (DCIS), inflammatory breast cancer, invasive lobular carcinoma, Paget's disease of the breast, male breast cancer, invasive ductal carcinoma. In some embodiments, the patient may suffer from triple-negative breast cancer (i.e., estrogen receptor negative, progesterone receptor negative, HER2 negative). In some embodiments, the patient may suffer from hormone receptor negative breast cancer (i.e., estrogen receptor negative, progesterone receptor negative). In some embodiments, the subject who is a candidate for treatment suffers from stage I, stage II, stage III, or stage IV breast cancer.

[0094] In some embodiments, the subject may have a genetic mutation known to be associated with an increased risk of breast cancer, for example, a mutation in one or more of the following genes: BRCA1, BRCA2, ATM, CDH1, CHEK2, NBN, NF1, PALB2, PTEN, STK11, or TP53 (p53).

[0095] The subject is preferably one who has not been previously treated with any immunosuppressive therapeutic approach. In certain embodiments, eligible subjects are over 18 years old and have a Karnofsky score of 60 or greater.

[0096] Optionally, subjects who are candidates for treatment may be identified by performing a tumor biopsy on the subject. In some embodiments, tumors from the subject are analyzed for the presence of monocytes. In some 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 may be analyzed using immunohistochemistry. In some embodiments, subjects who are candidates for treatment exhibit more than about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% CD163+ M2 cells of the subject's total peripheral blood mononuclear cells (PBMCs). In some embodiments, subjects exhibit more than about 20% CD163+ M2 cells of the subject's total PBMCs.

[0097] In yet other embodiments, subjects who are candidates for treatment are identified by the presence of one or more cytokines in the subject's serum, including, but not limited to, CXCL5, CXCL6, and CXCL7, IL6, IL7, IL8, IL10, IL11, IFN-γ, and HSP-70.

[0098] In yet other embodiments, subjects who are candidates for treatment are identified by the presence of one or more growth factors in the subject's serum, including, but not limited to, FGF-2, G-CSF, GM-CSF, and M-CSF.

[0099] In some embodiments, subjects who are candidates for treatment in a biodiffusion chamber are identified by measuring the levels of a particular set of cytokines. In some embodiments, the subjects have elevated levels of these cytokines compared to healthy subjects. As used herein, the term "healthy subject" refers to a subject who is not suffering from cancer or any other disease and is not in need of treatment in a biodiffusion chamber.

[0100] In certain embodiments, cytokines can be added to the chamber to enhance anti-tumor immune responses. For example, the cytokines added to the chamber can be selected from the group consisting of CCL19, CCL20, CCL21, and CXCL12, and combinations thereof.

[0101] In some embodiments, compared with healthy subjects, circulating CD14+ monocytes have elevated levels of CD163.In some embodiments, compared with healthy subjects, the level of CD163 of circulating CD14+ monocytes is elevated 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.In certain embodiments, compared with healthy subjects, the level of CD163 of circulating CD14+ monocytes is elevated about 2-fold.

[0102] In other embodiments, the subject who is a candidate for treatment has serum that polarizes undifferentiated monocytes into M2 cells.In some embodiments, incubation of the subject's serum with undifferentiated monocytes induces the expression of one or more cell surface markers of 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 of monocytes compared to monocytes that are not incubated with the subject's serum.In some embodiments, the 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 times, at least about 1.5 times, at least about 1.8 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, or at least about 100 times, compared to undifferentiated monocytes not incubated with subject serum.In certain embodiments, the level of one or more surface markers is increased by about 2 times, compared to undifferentiated monocytes not incubated with subject serum.Monocytes polarized by subject serum can be measured using FACS.

[0103] target cell Without being bound by theory, it is believed that AS ODN reduces M2 cells of a subject and / or inhibits the polarization of cells into M2 cells by downregulating IGF-1R expression.In some embodiments, the 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.The IGF-1R expression in M2 cells can be measured by quantitative RT-PCR.

[0104] In some embodiments, after receiving a single dose of antisense, IGF-1R expression in M2 cells remains downregulated in the 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.

[0105] In some embodiments, downregulation of expression of IGF-1R in M2 cells causes a selective reduction of M2 cells in the subject compared to cells not expressing IGF-1R. In certain embodiments, the M2 cells in the subject 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 the antisense. In other embodiments, the M2 cell population is eliminated. For example, after implantation of the biodiffusion chamber, the M2 cell population can be about 1%, about 2%, about 5%, or about 10% of the population before implantation of the biodiffusion chamber. The M2 cells in the subject can be measured using FACS. In certain embodiments, the M2 cells are eliminated after treatment, i.e., are not detectable by FACS. In other embodiments, the reduction of M2 cells is measured using a proxy assay. The M2 cell polarization capacity of the serum may be measured using a ELISA (immunoassay). For example, serum from a subject may be obtained before and after treatment for assessment of its M2 cell polarization capacity. After treatment with the methods disclosed herein, the M2 cell polarization capacity of the serum is reduced by about 80% to about 100%, about 20% to about 60%, or about 10% to about 50%.

[0106] In some embodiments, targeting the expression of IGF-1R in M2 cells causes cell death of M2 cells. In some embodiments, the cell death is necrosis. In other embodiments, the cell death is apoptosis. For the purposes of this disclosure, apoptosis is defined as programmed cell death, including but not limited to the regression of primary and metastatic tumors. Apoptosis is a widespread phenomenon of programmed cell death that plays an important role in a myriad of physiological and pathological processes. In contrast, necrosis is an accidental cell death that is a cellular response to a variety of harmful conditions and toxic substances. In yet other embodiments, targeting the expression of IGF-1R in M2 cells causes cell cycle arrest of M2 cells.

[0107] kit The preparation of a complete chamber requires many components and many steps. In another embodiment of the present disclosure, a kit is provided that includes components for carrying out the methods disclosed herein. In an embodiment, the kit includes a chamber body, which may be one part or two halves. Items for sealing the chamber, including one or more membranes, adhesives, and solvents (e.g., alcohol, or dichloroethane), may also be included. Optionally, the membrane may be ultrasonically welded to the chamber to create a seal. The kit includes an antisense ODN. Optionally, the ODN may be divided into two parts. A first part for treating the cells after surgical removal from the subject, and a second part for combining with the cells when introduced into the subject. Other optional kit items include media for cell culture, and antibiotics to prevent bacterial growth in the media.

[0108] Optionally, the chambers in the kit may be pre-connected to one another (e.g., by sutures) with grommets or other devices attached to the chambers and adapted to receive a connecting material. Advantageously, by pre-connecting multiple chambers, a desired number of chambers may be easily introduced and removed by the surgeon.

[0109] Working Example Example 1 - NOBEL inhibits breast cancer growth in mice Experimental procedure The experimental protocol is shown in Figure 1. C57BL / 6 female mice were vaccinated with chambers filled with various contents as shown in Table 2. The chambers were then explanted 48 hours later. The animals were then inoculated with a breast cancer cell line (E0771) into the right fourth fat pad. The animals were monitored for tumor development, growth and survival.

[0110] Each chamber contains 1 x 10 6E0771 cells that were either untreated, treated with 4 mg of NOBEL Antisense at the time of loading of the chamber, or treated with 4 mg of NOBEL Antisense overnight followed by 10 μg of NOBEL Antisense at the time of loading of the chamber. Irradiation at the time of loading consisted of a dose of 5 Gray.

[0111] [Table 2] Effect of vaccination on animal survival As shown in Figure 2A and Figure 2B, the effect of vaccination on animal survival was measured. The results presented represent the average of two independent experiments. Mice vaccinated with chambers containing tumor cells and AS-ODN (NOBEL), with or without radiation, had the highest survival rate.

[0112] The effect of vaccination on tumorigenesis (appearance of tumors) The effect of vaccination on tumor development was also measured, as shown in Figure 3A and Figure 3B. The results presented represent the average of two independent experiments. Mice vaccinated with chambers containing tumor cells and AS-ODN (NOBEL), with or without radiation, also had fewer tumors.

[0113] The effect of vaccination on tumor progression (speed of growth) The impact of vaccination on tumor progression was also studied, as shown in Figure 4. Vaccination with chambers containing tumor cells and AS-ODN (with or without radiation) significantly ameliorated tumor progression.

[0114] conclusion Vaccination with AS-ODN and inactivated breast cancer cells reduces tumor appearance, improves survival, and limits tumor progression in mice. Therefore, chamber vaccination is a promising treatment for patients with breast cancer, especially metastatic and triple-negative breast cancer.

[0115] Example 2 - Vaccination increases breast cancer-specific IgG1 levels in vivo (1) PBS as a control, (2) 1 × 10 mixed with 4 mg of NOBEL IGF-1R AS ODN 6 E0771 breast tumor cells, or (3) 1 × 10 cells that were treated with 4 mg of NOBEL overnight, washed, and added to the chamber with an additional 10 μg of NOBEL. 6 Biodiffusion chambers containing either E0771 cells or IgG1 cells were implanted into C57Bl / 6 mice. Blood samples were collected at the time of implantation (day 0) and at days 8, 21, and 43 post-implantation. Blood was also collected on the day of euthanasia, which varied among animals. Levels of E0771-specific IgG1 antibodies were measured using an E0771-based cell ELISA.

[0116] As shown in Figure 5, vaccination with a chamber containing E0771 breast tumor cells mixed with 4 mg of NOBEL led to a statistically significant increase in E0771-specific IgG1 levels compared to controls. Vaccination with a chamber containing E0771 cells that had been treated overnight with NOBEL, washed, and added to the chamber with additional NOBEL led to an even greater increase in E0771-specific IgG1 levels. Thus, this data demonstrates that vaccination with the described biodiffusion chamber increases breast cancer-specific IgG1 levels in vivo.

[0117] Example 3 - Vaccination of human patients with cancer 1. Preparation of Biodiffusion Chambers Tumor tissue is surgically removed from breast cancer patients using a tissue aspirator (NICO Myriad®) and placed into a sterile tissue trap, which is then transferred to a designated BSL-2 facility where the tumor tissue is processed and placed into a biodiffusion chamber. The biodiffusion chamber contains autologous tumor cells removed during surgery. Prior to being added to the biodiffusion chamber, the cells are pretreated overnight (approximately 12-18 hours) with a first amount (4 mg / ml) of an 18-mer IGF-1R AS ODN having the sequence 5'-TCCTCCGGAGCCAGACTT-3' (NOBEL) (SEQ ID NO: 1). Based on data indicating that AS ODNs have immunomodulatory properties, a second amount (2 μg) of exogenous NOBEL antisense is added to the chamber (Cv), and the chamber is subsequently irradiated. The ratio of tumor cells to IGF-1R AS ODN in the chamber is approximately 3.75 x 10 5 :1μg~approx.6.25×10 5 :1μg range.

[0118] Vaccination with autologous tumor cells and IGF-1R AS ODN in patients with breast cancer Breast cancer patients will be identified before, after, or concurrently with treatment using standard of care. Each patient will meet the following criteria: age >18, Karnofsky Performance Score ≥60, and no comorbidities that would prevent treatment.

[0119] Biodiffusion chambers are prepared as described above. The day after surgery to remove tumor tissue, 2-20 irradiated biodiffusion chambers are implanted into the patient's rectus sheath. After 24-48 hours, the chambers are removed. Optionally, antisense nucleotides against IGFR-1 are administered simultaneously.

[0120] Radiologic evaluations were used to monitor clinical efficacy over time. Serial imaging evaluations may be performed on Philips 1.5T and 3T MRI scanners or GE 1.5T MRI scanner. Routine anatomical MRI features may be assessed. Physiological MRI techniques of dynamic susceptibility weighted (DSC) MR perfusion and 15-directional diffusion tensor imaging (DTI) may also be utilized. MR perfusion and DTI post-processing were performed on a Nordic Ice workstation (v.2.3.14). rCBV may be calculated relative to contralateral normal white matter. Mean diffusion coefficient (mean diffusivity) may be calculated from the DTI data.

[0121] Treatment may be repeated as necessary to inhibit and / or eliminate tumor growth. Incorporation by Reference All patents and publications referenced herein are hereby incorporated by reference in their entirety for all purposes.

[0122] Numbered Embodiments of the Invention Without limiting the scope of the appended claims, the present disclosure includes the following numbered embodiments.

[0123] 1. A method of preparing a biodiffusion chamber for implantation into a subject with breast cancer, comprising: (a) encapsulating tumor cells obtained from a subject in a biodiffusion chamber in the presence of an IGF-1R AS ODN; the tumor cells are obtained from the subject using a tissue morcellator; (b) irradiating the biodiffusion chamber. and

[0124] 2. The method of embodiment 1, wherein said breast cancer is selected from hormone receptor negative and triple negative breast cancer. 3. The method of embodiment 2, wherein said breast cancer is triple-negative breast cancer.

[0125] 4. The method of embodiment 1, wherein said breast cancer is metastatic breast cancer. 5. The method of embodiment 1, wherein the subject has a genetic mutation in one or more of the following genes: BRCA1, BRCA2, ATM, CDH1, CHEK2, NBN, NF1, PALB2, PTEN, STK11, TP53 (p53).

[0126] 6. The method according to any one of the preceding embodiments, wherein the tumor cells are dispersed prior to being added to the chamber. 7. The method of any one of embodiments 1-6, wherein the cells are not exposed to temperatures above body temperature during removal from the subject.

[0127] 8. The method of any one of embodiments 1-7, wherein the cells are not exposed to temperatures above 37° C. during removal from the subject. 9. The method of any one of embodiments 1-8, wherein the tissue morcellator comprises a sterile trap.

[0128] 10. The method of any one of embodiments 1-9, wherein the tissue morcellator comprises a high speed reciprocating inner cannula within a stationary outer cannula. 11. The method of any one of embodiments 1-10, wherein the outer cannula comprises a side opening, and further wherein the tumor cells are drawn into the side opening by electronically controlled variable suction.

[0129] 12. The method of any one of embodiments 1-11, wherein said tumor cells are enriched for nestin expression prior to being placed in said biodiffusion chamber.

[0130] 13. The method of any one of embodiments 1-12, wherein the tumor cells in the chamber are enriched for adherent cells compared to the tumor cells obtained from the subject. 14. The method of embodiment 13, wherein said tumor cells consist essentially of adherent cells.

[0131] 15. The method according to any one of embodiments 1-14, wherein the cells are treated with an IGF-1R AS ODN prior to encapsulation in the chamber. 16. The method of embodiment 15, wherein said IGF-1R AS ODN is present at about 2 mg to about 6 mg per million cells during said process prior to encapsulation.

[0132] 17. The method of embodiment 16, wherein said IGF-1R AS ODN is present at about 4 mg per million cells during said process prior to encapsulation. 18. The method of embodiment 15, wherein said treatment with IGF-1R AS ODN prior to encapsulation is for up to about 18 hours.

[0133] 19. The method of embodiment 15, wherein said treatment with IGF-1R AS ODN prior to encapsulation is for about 12 hours to about 18 hours. 20. The method of embodiment 1, wherein the IGF-1R AS ODN has the sequence of SEQ ID NO:1.

[0134] 21. A method for treating a subject having breast cancer, comprising implanting two or more biodiffusion chambers in said subject according to any one of embodiments 1-20. 22. The method of embodiment 21, wherein about 10 to about 30 biodiffusion chambers are implanted into the subject.

[0135] 23. The method of embodiment 22, wherein about 10 to about 20 biodiffusion chambers are implanted into the subject. 24. The method of any one of embodiments 30-32, wherein the diffusion chamber is implanted in the subject for 48 hours.

[0136] 25. The method of any one of embodiments 21-24, wherein the breast cancer is selected from hormone receptor negative and triple negative breast cancer. 26. The method of embodiment 25, wherein the breast cancer is triple-negative breast cancer.

[0137] 27. The method of any one of embodiments 21-24, wherein said breast cancer is metastatic breast cancer. 28. The method according to any one of embodiments 21 to 27, wherein the subject has a genetic mutation in one or more of the following genes: BRCA1, BRCA2, ATM, CDH1, CHEK2, NBN, NF1, PALB2, PTEN, STK11, TP53 (p53).

[0138] 29. The method according to any one of embodiments 21-28, wherein the method is carried out without chemotherapy, without radiation therapy, or without both. 30. The method of embodiment 21, comprising a second implantation of the chamber following the first implantation.

[0139] 31. The method of embodiment 30, wherein said second transplant uses tumor cells obtained from the first administration and those cells obtained from the subject at the same time. 32. The method of embodiment 30, wherein said second transplant uses tumor cells obtained from said subject whose tumor recurred after completion of a first treatment or who did not respond to a first treatment. 33. A method of vaccinating a subject with breast cancer, comprising: (i) obtaining minced tumor tissue from the subject; (ii) collecting the comminuted tissue in a sterile trap; (iii) harvesting adherent cells from the minced tissue; (iv) encapsulating the harvested cells with an insulin-like growth factor 1 receptor antisense oligodeoxynucleotide (IGF-1R AS ODN) having the sequence of SEQ ID NO:1 in a biodiffusion chamber; (v) irradiating the chamber; and (vi) implanting the chamber in the subject; and and obtaining an immune response against said breast cancer.

[0140] 34. The method of embodiment 33, wherein the breast cancer is selected from hormone receptor negative and triple negative breast cancer. 35. The method of embodiment 34, wherein the breast cancer is triple-negative breast cancer.

[0141] 36. The method of embodiment 33, wherein the breast cancer is metastatic breast cancer. 37. The method of embodiment 33, wherein the subject has a genetic mutation in one or more of the following genes: BRCA1, BRCA2, ATM, CDH1, CHEK2, NBN, NF1, PALB2, PTEN, STK11, TP53 (p53).

[0142] 38. The method according to any one of embodiments 33-37, comprising treating said adherent cells with an IGF-1R AS ODN for up to 18 hours prior to encapsulation. 39. The method according to any one of embodiments 33-38, wherein the subject is vaccinated with 20 chambers for about 48 hours.

[0143] 40. The method according to any one of embodiments 33-39, wherein the tumor cells are not exposed to temperatures above body temperature. 41. A biodiffusion chamber for implantation into a subject having breast cancer, comprising: (a) irradiated tumor cells, the tumor cells include adherent cells obtained from tumor tissue of the subject; the tumor cells are pre-incubated with an insulin-like growth factor 1 receptor antisense oligodeoxynucleotide (IGF-1R AS ODN) prior to encapsulation in the chamber; (b) an irradiated IGF-1R AS ODN, The IGF-1R AS ODN has the sequence of SEQ ID NO: 1 and is an irradiated IGF-1R AS ODN. a biodiffusion chamber comprising:

[0144] 42. A biodiffusion chamber as described in embodiment 41, wherein the breast cancer is selected from hormone receptor negative breast cancer and triple negative breast cancer. 43. A biodiffusion chamber as described in embodiment 42, wherein the breast cancer is triple-negative breast cancer.

[0145] 44. A biodiffusion chamber as described in embodiment 41, wherein the breast cancer is metastatic breast cancer. 45. The biodiffusion chamber of embodiment 41, wherein the subject has a genetic mutation in one or more of the following genes: BRCA1, BRCA2, ATM, CDH1, CHEK2, NBN, NF1, PALB2, PTEN, STK11, TP53 (p53).

[0146] 46. ​​A biodiffusion chamber according to any one of embodiments 41 to 45, wherein the tumor cells in the chamber are enriched for nestin-positive cells relative to the tumor tissue obtained from the subject.

[0147] 47.About 10 6 A biodiffusion chamber according to any one of embodiments 41-46, wherein a number of tumor cells are present in said chamber. 48. A biodiffusion chamber according to any one of embodiments 41 to 47, wherein the tumor cells are obtained from the subject using a tissue morcellator.

[0148] 49. A biodiffusion chamber as described in embodiment 48, wherein the tissue morcellator includes a high speed reciprocating inner cannula within a stationary outer cannula. 50. The biodiffusion chamber of embodiment 48, wherein the tissue morcellator does not generate heat when the tumor tissue is obtained from the subject.

Claims

1. 1. A method of preparing a biodiffusion chamber for implantation into a subject with breast cancer, comprising: (a) encapsulating tumor cells obtained from the subject, the tumor cells being obtained from the subject using a tissue morcellator, in the biodiffusion chamber in the presence of an IGF-1R AS ODN; (b) irradiating the biodiffusion chamber. and

2. 2. The method of claim 1, wherein the breast cancer is selected from hormone receptor negative and triple negative breast cancer.

3. The method of claim 2, wherein the breast cancer is triple-negative breast cancer.

4. The method of claim 1, wherein the breast cancer is metastatic breast cancer.

5. 2. The method of claim 1, wherein the subject has a genetic mutation in one or more of the following genes: BRCA1, BRCA2, ATM, CDH1, CHEK2, NBN, NF1, PALB2, PTEN, STK11, TP53 (p53).

6. The method of any one of claims 1 to 5, wherein the tumor cells are dispersed before being added to the chamber.

7. The method of any one of claims 1 to 6, wherein the cells are not exposed to temperatures above body temperature during removal from the subject.

8. The method of any one of claims 1 to 7, wherein the cells are not exposed to temperatures above 37°C during removal from the subject.

9. The method of any one of claims 1 to 8, wherein the tissue morcellator comprises a sterile trap.

10. The method of any one of claims 1 to 9, wherein the tissue morcellator comprises a high speed reciprocating inner cannula within a stationary outer cannula.

11. The method of any one of claims 1 to 10, wherein the outer cannula comprises a side opening, and further wherein the tumor cells are drawn into the side opening by electronically controlled variable suction.

12. The method of any one of claims 1 to 11, wherein the tumor cells are enriched for nestin expression prior to being placed in the biodiffusion chamber.

13. The method of any one of claims 1 to 12, wherein the tumor cells in the chamber are enriched for adherent cells compared to the tumor cells obtained from the subject.

14. The method of claim 13, wherein the tumor cells consist essentially of adherent cells.

15. The method of any one of claims 1 to 14, wherein the cells are treated with an IGF-1R AS ODN prior to encapsulation in the chamber.

16. 16. The method of claim 15, wherein said IGF-1R AS ODN is present at about 2 mg to about 6 mg per million cells during said treatment prior to encapsulation.

17. 17. The method of claim 16, wherein said IGF-1R AS ODN is present at about 4 mg per million cells during said treatment prior to encapsulation.

18. 16. The method of claim 15, wherein said treatment with IGF-1R AS ODN prior to encapsulation is for up to about 18 hours.

19. The method of claim 15, wherein said treatment with IGF-1R AS ODN prior to encapsulation is for about 12 hours to about 18 hours.

20. The method of claim 1, wherein the IGF-1R AS ODN has the sequence of SEQ ID NO:

1.

21. A method for treating a subject having breast cancer, comprising implanting into said subject two or more biodiffusion chambers according to the method of any one of claims 1 to 20.

22. 22. The method of claim 21, wherein about 10 to about 30 biodiffusion chambers are implanted in the subject.

23. 23. The method of claim 22, wherein about 10 to about 20 biodiffusion chambers are implanted in the subject.

24. The method of any one of claims 30 to 32, wherein the diffusion chamber is implanted in the subject for 48 hours.

25. The method according to any one of claims 21 to 24, wherein the breast cancer is selected from hormone receptor negative and triple negative breast cancer.

26. 26. The method of claim 25, wherein the breast cancer is triple-negative breast cancer.

27. The method according to any one of claims 21 to 24, wherein the breast cancer is metastatic breast cancer.

28. The method according to any one of claims 21 to 27, wherein the subject has a genetic mutation in one or more of the following genes: BRCA1, BRCA2, ATM, CDH1, CHEK2, NBN, NF1, PALB2, PTEN, STK11, TP53 (p53).

29. The method of any one of claims 21 to 28, wherein the method is performed without chemotherapy, without radiation therapy, or without both.

30. 22. The method of claim 21, comprising a second implantation of the chamber subsequent to the first implantation.

31. 31. The method of claim 30, wherein the second transplant uses tumor cells obtained from the subject simultaneously with those cells obtained from the first administration.

32. 31. The method of claim 30, wherein the second transplant uses tumor cells obtained from a subject whose tumor recurred after completion of a first treatment or who did not respond to a first treatment.

33. 1. A method of vaccinating a subject with breast cancer, comprising: (i) obtaining minced tumor tissue from said subject; (ii) collecting the morselized tissue in a sterile trap; (iii) harvesting adherent cells from the minced tissue; (iv) encapsulating the harvested cells with an insulin-like growth factor 1 receptor antisense oligodeoxynucleotide (IGF-1R AS ODN) having the sequence of SEQ ID NO:1 in a biodiffusion chamber; (v) irradiating the chamber; and (vi) implanting the chamber in the subject; and and obtaining an immune response against said breast cancer.

34. 34. The method of claim 33, wherein the breast cancer is selected from hormone receptor negative and triple negative breast cancer.

35. 35. The method of claim 34, wherein the breast cancer is triple-negative breast cancer.

36. 34. The method of claim 33, wherein the breast cancer is metastatic breast cancer.

37. 34. The method of claim 33, wherein the subject has a genetic mutation in one or more of the following genes: BRCA1, BRCA2, ATM, CDH1, CHEK2, NBN, NF1, PALB2, PTEN, STK11, TP53 (p53).

38. 38. The method of any one of claims 33 to 37, comprising treating the adherent cells with IGF-1R AS ODN for up to 18 hours prior to encapsulation.

39. 39. The method of any one of claims 33 to 38, wherein the subject is vaccinated with 20 chambers for about 48 hours.

40. 40. The method of any one of claims 33 to 39, wherein the tumor cells are not exposed to temperatures above body temperature.

41. 1. A biodiffusion chamber for implantation into a subject having breast cancer, comprising: (a) irradiated tumor cells, the tumor cells include adherent cells obtained from tumor tissue of the subject; the irradiated tumor cells, which have been pre-incubated with an insulin-like growth factor 1 receptor antisense oligodeoxynucleotide (IGF-1R AS ODN) prior to encapsulation in the chamber; (b) an irradiated IGF-1R AS ODN, said IGF-1R AS ODN having the sequence of SEQ ID NO:1; a biodiffusion chamber comprising:

42. 42. The biodiffusion chamber of claim 41, wherein the breast cancer is selected from hormone receptor negative breast cancer and triple negative breast cancer.

43. 43. The biodiffusion chamber of claim 42, wherein the breast cancer is triple negative breast cancer.

44. 42. The biodiffusion chamber of claim 41, wherein the breast cancer is metastatic breast cancer.

45. 42. The biodiffusion chamber of claim 41, wherein the subject has a genetic mutation in one or more of the following genes: BRCA1, BRCA2, ATM, CDH1, CHEK2, NBN, NF1, PALB2, PTEN, STK11, TP53 (p53).

46. A biodiffusion chamber according to any one of claims 41 to 45, wherein the tumor cells in the chamber are enriched for nestin-positive cells compared to the tumor tissue obtained from the subject.

47. about 10 ６ 47. The biodiffusion chamber of any one of claims 41 to 46, wherein a single tumor cell is present within the chamber.

48. The biodiffusion chamber of any one of claims 41 to 47, wherein the tumor cells are obtained from the subject using a tissue morcellator.

49. 49. The biodiffusion chamber of claim 48, wherein the tissue morcellator includes a high speed reciprocating inner cannula within a stationary outer cannula.

50. 49. The biodiffusion chamber of claim 48, wherein the tissue morcellator does not generate heat when obtaining the tumor tissue from the subject.

Images