Incorporated by reference in its entirety is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: 3 kilobytes ASCII (Text) file named “343969_ST25.txt,” created on Sep. 7, 2021.
Breast cancer is the second most common cancer diagnosed in women in the United States. Currently, the average risk of a woman in the United States developing breast cancer sometime in her life is about 13% (1 in 8 woman) thus about 276,480 new cases of invasive breast cancer will be diagnosed in year 2020. Studies showed that incidence rates of breast cancer were significantly higher in the military population across race and gender, likely due to differences between military woman and general population in reproductive history (age at first birth and the use of contraceptives) and exposure to hazardous chemicals. Despite a decades-long decline in the breast cancer death rate owing to treatment advances, earlier detection through screening and increased awareness, each year over 40,000 patients die from the disease in the U.S.
Regulatory T cells (Tregs) play a central role in maintaining immune system homeostasis and negatively regulate immune-mediated inflammation such as autoimmune diseases, asthma and allergy. However, Tregs also suppress effective immunity against chronic infections and tumors. A systematic review and meta-analysis of 15 published studies comprising 8666 breast cancer patients demonstrated that increased tumor-infiltrating Treg cells in breast cancer are correlated with poorer clinical outcomes including reduced overall survival, tumor malignancy, and metastasis. In contrast, depletion of Tregs with an anti-CD25 antibody (daclizumab) in combination with an experimental cancer vaccine in patients with metastatic breast cancer led to a marked and prolonged decrease in Tregs and robust priming and boosting of CD8+and CD4+T cells. Consequently, overall survival was improved in this small set of patients. In mouse breast cancer models, depletion or downregulation of Tregs resulted in enhanced anti-tumor immunity and tumor regression.
FOXP3 is a master regulator of Treg development and function. FOXP3 loss of function mutations in both humans and mice result in lethal immunodysregulation, polyendocrinopathy, enteropathy, and X-linked syndrome (IPEX) due to lack of Tregs. While the human FOXP3 gene encodes two major isoforms through mRNA alternative splicing—a long full-length isoform (FOXP3L) and a shorter isoform lacking exon 2 region (FOXP3S), mouse Foxp3 gene only encodes the Foxp3L isoform. Although the FOXP3 exon 2 region (the 2nd protein-coding exon; SEQ ID NO: 12) has been shown to be important in regulating Th17 differentiation, the two FOXP3 isoforms were both effective in directing Treg differentiation and function in vitro under forced overexpression. However, it is often noted that ectopic overexpression of FOXP3L and FOXP3S isoforms in CD4+T cells may result in supra-physiological expression levels and are not likely to represent the function of Tregs in vivo. Therefore, it remains an enigma how these two major isoforms differ in determining the functionality and biology of Tregs.
As disclosed herein the relative expression of FOXP3L and FOXP3S isoforms in CD4+T cells has been discovered to impact resistance to cancer progression and the efficacy of cancer immunotherapy.
The human FOXP3 gene encodes two major isoforms through mRNA alternative splicing—a long full-length isoform (FOXP3L) and a shorter isoform lacking exon 2 region (FOXP3S). However, the mouse Foxp3 gene only encodes the Foxp3L isoform. Thus while FOXP3L is capable of directing Treg development and function, the role of FOXP3S remains elusive. Applicant has discovered that a genetically modified mouse model that only expressed FOXP3S in Tregs conferred resistance to breast cancer progression. Additionally, applicant found that Tregs expressing FOXP3S were unstable and could transdifferentiate to helper-like T cells, thus provide enhanced anti-tumor immunity. Furthermore, patients with higher FOXP3S expression in breast cancer tissues had better overall survival than those had lower FOXP3S expression.
Therefore, in accordance with one embodiment of the present disclosure, a method is provided for enhancing antitumor immunity by promoting FOXP3S expression in Tregs, and more specifically enhancing antitumor immunity against breast cancer. In one embodiment the method comprises administering morpholino antisense oligonucleotides (MOs) to block the inclusion of the exon 2 region during pre-mRNA splicing to shift FOXP3 expression to the FOXP3S isoform. Applicant has verified that this novel MO efficiently suppressed tumor growth in preclinical mouse breast cancer model and patient derived ex vivo breast cancer model and colorectal cancer model.
In accordance with one embodiment, the present invention is directed to inducing regulatory T cells (Tregs) to transdifferentiate into helper-like T cells by modifying intracellular concentrations of FOXP3S relative to FOXP3L. More particularly, in one embodiment the intracellular concentration of FOXP3S is increased relative to FOXP3L in Tregs, optionally resulting in FOXP3S being the predominant FOXP3 isoform present in the modified Tregs. In one embodiment a method is provided for enhancing the expression of FOXP3S in Tregs, and more particularly, inducing Tregs to switch from predominantly FOXP3L expression to predominantly FOXP3S expression. In one embodiment the method comprises the step of decreasing the expression of the FOXP3L relative to FOXP3S expression, and optionally enhancing the expression of FOXP3S.
In accordance with one embodiment the expression of the FOXP3L is decreased by transfecting Tregs with an interference oligomer that inhibits or prevents the expression of FOXP3L. In one embodiment the transfection step takes place in vitro on isolated Tregs or on tumor infiltrating lymphocytes. In one embodiment the interference oligomer targets FOXP3 exon 2 and the Tregs are transfected either in vitro or in vivo. In one embodiment Tregs are transfected with both an interference oligonucleotide that targets exon 2 (present only in FOXP3L) and a nucleic acid that encodes for the FOXP3S isoform, thus simultaneously decreasing the production of FOXP3L and increasing FOXP3S production. In one embodiment the interference oligomer comprises a sequence of nucleobases targeting the following FOXP3 region TCCCTGCCCATTCACCGTCCATACCTGGTG (SEQ ID NO: 1), or a sequence of nucleobases having at least 80%, 85%, 95% or 99% sequence identity with TCCCTGCCCATTCACCGTCCATACCTGGTG (SEQ ID NO: 1), a complement thereof, or a 10, 15, 18, 20, 23 or 25 bp or larger contiguous sequence fragment of SEQ ID NO: 1 or complement thereof. In one embodiment the interference oligomer is a phosphorodiamidate morpholino. In one embodiment the interference oligomer is an interfering RNA comprising a sequence having at least 95% sequence identity with GUAUGGACGGUGAAUGG (SEQ ID NO: 10), optionally wherein the interference oligomer is a phosphorodiamidate morpholino.
In accordance with one embodiment the method of inducing Tregs to transdifferentiate into helper-like T cells can be used to treat cancer. Current treatment options for breast cancer consist of endocrine therapy, chemotherapy, radiation therapy and surgery depending on the subtypes and the stage of breast cancer. The recent advances of immunotherapy has brought in a new treatment algorithm for many types of cancer, raising the enthusiasm for using immunotherapy to treat breast cancer. Normally, Tregs infiltrate breast cancer tissues abundantly thus suppress antitumor immunity. However, applicant has discovered that Tregs modified to express enhanced intracellular concentrations of FOXP3S, relative to FOXP3L transdifferentiated to helper-like T cells, and promote the anti-tumor immune response.
Based upon this finding, FOXP3 targeting oligomers (e.g., iRNAs or morpholino oligos (MO) that target exon 2 of FOX3) can be used to shift cellular production of FOXP3L to FOXP3S expression, and the efficacy of such an approach for providing antitumor activity has been verified in a preclinical mouse model and patient-derived cancer organoid model. Accordingly, these FOXP3S-promoting MOs, or other mechanisms for altering intracellular FOXP3S/FOXP3L ratios, will serve as novel immunotherapies for breast cancer treatment, and the treatment of other solid tumors.
In accordance with one embodiment a human morpholino sequence or other interference nucleic acid is used to induce FOXP3 exon 2 skipping in vivo, and thus shift human FOXP3L isoform to FOXP3S isoform in cells targeted with the interference moieties. In one embodiment morpholinos that induce FOXP3 exon 2 skipping comprise a sequence of TCCCTGCCCATTCACCGTCCATACCTGGTG (SEQ ID NO: 1), or a sequence of nucleobases having at least 85%, 90%, 95% or 99% sequence identity with TCCCTGCCCATTCACCGTCCATACCTGGTG (SEQ ID NO: 1), or a fragment of SEQ ID NO: 1 comprising at least 10, 15, 18, 20, 23 or 25 bp or larger contiguous nucleobases of SEQ ID NO: 1. In one embodiment the interference moiety inducing FOXP3 exon 2 skipping comprises a sequence selected from the group consisting of
In one embodiment a composition comprising one or more iRNAs or morpholinos selected from the group of SEQ ID NOs: 1-8 are used to skip exon 2 of FOX3L and shift cellular production of FOXP3L to FOXP3S expression in targeted cells.
In accordance with one embodiment a human morpholino sequence or other interference nucleic acid is used to block translation of FOXP3L or induce degradation of FOXP3 mRNA containing the exon 2 region thus altering FOXP3S/FOXP3L ratios in cells targeted with the interference moieties. In one embodiment morpholinos or other interference nucleic acids comprise a fragment of CTGCCCACACTGCCCCTAGTCATGGTGGCACCCTCCGGGGCACGGCTGGG CCCCTTGCCCCACTTACAGGCACTCCTCCAGGACAGGCCACATTTCATGCA CCAG (SEQ ID NO: 12) that is at least 10 bp or larger contiguous nucleobases of SEQ ID NO: 12, or a complement thereof, and having at least 85%, 90%, 95% or 99% sequence identity with SEQ ID NO: 12.
In describing and claiming the invention, the following terminology will be used in accordance with the definitions set forth below.
The term “about” as used herein means greater or lesser than the value or range of values stated by 10 percent but is not intended to limit any value or range of values to only this broader definition. Each value or range of values preceded by the term “about” is also intended to encompass the embodiment of the stated absolute value or range of values.
As used herein, the term “purified” and like terms relate to the isolation of a molecule or compound in a form that is substantially free of contaminants normally associated with the molecule or compound in a native or natural environment. As used herein, the term “purified” does not require absolute purity; rather, it is intended as a relative definition.
The term “isolated” requires that the referenced material be removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide present in a living animal is not isolated, but the same polynucleotide, separated from some or all of the coexisting materials in the natural system, is isolated.
Tissue nanotransfection (TNT) is an electroporation-based technique capable of delivering nucleic acid sequences and proteins into the cytosol of cells at nanoscale. More particularly, TNT uses a highly intense and focused electric field through arrayed nanochannels, which benignly nanoporates the juxtaposing tissue cell members, and electrophoretically drives cargo (e.g., nucleic acids or proteins) into the cells.
As used herein a “control element” or “regulatory sequence” are non-translated regions of a functional gene, including enhancers, promoters, 5′ and 3′ untranslated regions, which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. “Eukaryotic regulatory sequences” are non-translated regions of a functional gene, including enhancers, promoters, 5′ and 3′ untranslated regions, which interact with host cellular proteins of a eukaryotic cell to carry out transcription and translation in a eukaryotic cell including mammalian cells.
As used herein a “promoter” is a sequence or sequences of DNA that function when in a relatively fixed location in regard to the transcription start site of a gene. A “promoter” contains core elements required for basic interaction of RNA polymerase and transcription factors and can contain upstream elements and response elements.
As used herein an “enhancer” is a sequence of DNA that functions independent of distance from the transcription start site and can be either 5′ or 3′ to the transcription unit. Furthermore, enhancers can be within an intron as well as within the coding sequence itself. They are usually between 10 and 300 bp in length, and they function in cis Enhancers function to increase transcription from nearby promoters. Enhancers, like promoters, also often contain response elements that mediate the regulation of transcription. Enhancers often determine the regulation of expression.
The term “identity” as used herein relates to the similarity between two or more sequences. Identity is measured by dividing the number of identical residues by the total number of residues and multiplying the product by 100 to achieve a percentage. Thus, two copies of exactly the same sequence have 100% identity, whereas two sequences that have amino acid or nucleotide deletions, additions, or substitutions relative to one another have a lower degree of identity. Those skilled in the art will recognize that several computer programs, such as those that employ algorithms such as BLAST (Basic Local Alignment Search Tool, Altschul et al. (1993) J. Mol. Biol. 215:403-410) are available for determining sequence identity.
The term “stringent conditions” is functionally defined with regard to the hybridization of a first nucleic acid to a second target nucleic acid (i.e., to a particular nucleic-acid sequence of interest) by the specific hybridization procedure discussed in Sambrook et al., 1989, at 9.52-9.55. See also, Sambrook et al., 1989 at 9.47-9.52 and 9.56-9.58. In one embodiment stringent conditions include conducting hybridization of a first and second nucleic acid using a solution comprising about 0.02 M to about 0.15 M NaCl at temperatures of about 50° C. to about 70° C., followed by washing with a high-stringency wash buffer (0.2X SSC, 0.1% SDS, 65° C.).
As used herein, the term “pharmaceutically acceptable carrier” includes any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions such as an oil/water or water/oil emulsion, and various types of wetting agents. The term also encompasses any of the agents approved by a regulatory agency of the US Federal government or listed in the US Pharmacopeia for use in animals, including humans.
As used herein, the term “treating” includes alleviation of the symptoms associated with a specific disorder or condition and/or preventing or eliminating said symptoms.
As used herein an “effective” amount or a “therapeutically effective amount” of a drug refers to a nontoxic but enough of the drug to provide the desired effect. The amount that is “effective” will vary from subject to subject or even within a subject overtime, depending on the age and general condition of the individual, mode of administration, and the like. Thus, it is not always possible to specify an exact “effective amount.” However, an appropriate “effective” amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.
As used herein the term “patient” without further designation is intended to encompass any warm blooded vertebrate domesticated animal (including for example, but not limited to livestock, horses, cats, dogs and other pets) and humans.
The term “carrier” means a compound, composition, substance, or structure that, when in combination with a compound or composition, aids or facilitates preparation, storage, administration, delivery, effectiveness, selectivity, or any other feature of the compound or composition for its intended use or purpose. For example, a carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.
The term “inhibit” defines a decrease in an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.
The term “vector” or “construct” designates a nucleic acid sequence capable of transporting into a cell another nucleic acid to which the vector sequence has been linked. The term “expression vector” includes any vector, (e.g., a plasmid, cosmid or phage chromosome) containing a gene construct in a form suitable for expression by a cell (e.g., linked to a transcriptional control element). “Plasmid” and “vector” are used interchangeably, as a plasmid is a commonly used form of vector. Moreover, the invention is intended to include other vectors which serve equivalent functions.
The term “operably linked to” refers to the functional relationship of a nucleic acid with another nucleic acid sequence. Promoters, enhancers, transcriptional and translational stop sites, and other signal sequences are examples of nucleic acid sequences that can operably linked to other sequences. For example, operable linkage of DNA to a transcriptional control element refers to the physical and functional relationship between the DNA and promoter such that the transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA.
As used herein the term “transfection” or “transfect” defines a process where a nucleic acid sequence, or a mimetic analog thereof, is internalized by eukaryotic cells, passing across the cell membrane and into the cytoplasm. Transfection can include passive mean (e.g. receptor mediated uptake, passage through cell pores) as well as external assisted means including by physical (e.g., electroporation), chemical (e.g., cationic lipid or calcium phosphate), or molecular modification (modifications to enhance cellular delivery or in vivo stability) mechanisms.
As used herein “interference oligomer” is any nucleic acid oligonucleotide or analog thereof that participates in post-transcriptional gene regulation such as silencing and splicing. Examples of interference oligomers includes, but is not limited to, phosphorodiamidate morpholino, double stranded RNA (dsRNA), small interfering RNA (siRNA), and microRNA (miRNA) that are comprised of sense and/or antisense strands.
As used herein the term “phosphorodiamidate morpholino” designates a DNA/RNA analog wherein the phosphodiester backbone of the DNA/RNA is substituted with a backbone of morpholine rings connected by phosphorodiamidate linkages.
As used herein the term “FOXP3S-promoting morpholino” designates a phosphorodiamidate morpholino that upon introduction to a cell will increase intracellular concentrations of FOXP3S relative to FOXP3L. In one embodiment the FOXP3S-promoting phosphorodiamidate morpholino is an oligonucleotide that targets and induces the exclusion of the FOXP3 exon 2 from FOXP3 mRNA thus promoting FOXP3S expression.
As used herein the term “transdifferentiate” defines an artificial process in which one mature somatic cell is transformed into another mature somatic cell without undergoing an intermediate pluripotent state or progenitor cell type.
The human FOXP3 gene encodes two major isoforms through mRNA alternative splicing—a long full-length isoform (FOXP3L) and a shorter isoform lacking exon 2 region (FOXP3S). However, the mouse Foxp3 gene only encodes the Foxp3L isoform. Thus while the FOXP3L promotes Treg development and function, the role of FOXP3S remains elusive. Applicant has discovered that a genetically modified mouse model that only expressed FOXP3S in Tregs conferred resistance to breast cancer and colon cancer progression. Additionally, applicant found that Tregs expressing FOXP3S were unstable and could transdifferentiate to helper-like T cells, thus provide enhanced anti-tumor immunity. Furthermore, breast cancer patients with higher FOXP3S transcripts in tumor tissues had better overall survival.
Therefore, in accordance with one embodiment of the present disclosure, a method is provided for enhancing antitumor immunity by promoting FOXP3S expression in Tregs, and more specifically enhancing antitumor immunity against breast cancer. In one embodiment the method comprises administering morpholino antisense oligonucleotides (MOs) to block the inclusion of exon 2 region during pre-mRNA splicing to shift FOXP3 expression to the FOXP3S isoform.
In accordance with one embodiment, the present disclosure is directed to inducing regulatory T cells (Tregs) to transdifferentiate to helper-like T cells by modifying intracellular concentrations of FOXP3S relative to FOXP3L. More particularly, as disclosed herein, a method is provided for inducing regulatory T cells (Tregs) to shift from FOXP3L production to FOXP3S production. In one embodiment, the method comprises the step of decreasing the expression of the FOXP3L isoform relative to FOXP3S expression. In one embodiment the expression of the FOXP3L isoform is decreased by transfecting said Tregs with a FOXP3S-promoting interference oligomer, in an amount effective to shift expression of FOXP3 in said Tregs to the FOXP3S isoform.
The method of inducing Tregs to shift expression of FOXP3 to the FOXP3S isoform can be conducted in vitro or in vivo. In one embodiment regulatory T cells (Tregs) or tumor infiltrating lymphocytes are recovered from a patient, induced to transdifferentiate into helper-like T cells in vitro (by increasing cellular FOXP3S production relative to FOXP3L production) and then are returned to the patient. In one embodiment regulatory T cells (Tregs) are recovered from a patient and contacted with one or more interference oligomers that target FOXP3 under conditions wherein the interference oligomers are taken up by the cell and inhibit and/or silence FOXP3 expression. Silencing or inhibiting the FOXP3L isoform expression with a morpholino or iRNA will induce tumor reactive Tregs to become helpers-like T cells. Such in vitro induced T helper cells can be reintroduced into the patient either alone or in conjunction with other standard anticancer therapies, including for example, co-administration with in vitro expanded tumor infiltrating lymphocytes.
In one embodiment Tregs are transfected in vivo with nucleic acid sequences, or nucleic acid sequence analogs, that target exon 2 of FOXP3 in a patient in need of enhanced immunotherapeutic efficacy. Interference oligomers can be placed in contact with Tregs in vivo under conditions where Tregs take up the exon 2 targeting interference oligomers and increase intracellular concentrations of FOXP3S relative to FOXP3L, optionally by shifting expression from FOXP3L to FOXP3S expression in the Tregs contacted with the interference oligomers. In one embodiment oligomers suitable for targeting exon 2 of FOXP3 comprise a sequence that specifically binds to the sequence CCATTCACCGTCCATAC (SEQ ID NO: 2) or CCAUUCACCGUCCAUAC (SEQ ID NO: 9) or a sequence having at least 90%, 95% or 99% sequence identity with SEQ ID NO: 2 or SEQ ID NO: 9. In one embodiment interference oligomers suitable for targeting exon 2 of FOXP3 comprise a sequence that binds to the sequence CCATTCACCGTCCATAC (SEQ ID NO: 2) or CCAUUCACCGUCCAUAC (SEQ ID NO: 9) under stringent hybridization conditions. In accordance with one embodiment an interference oligomers suitable for excluding exon 2 of FOXP3 comprises a sequence that binds to the sequence selected from the group of
In one embodiment a method is provided for enhancing the expression of FOXP3S in Tregs relative to the expression of FOXP3L, and more particularly, inducing Tregs to switch from expressing FOXP3L to predominantly expressing FOXP3S. In one embodiment the method comprises the step of decreasing the expression of the FOXP3L relative to FOXP3S expression, and optionally enhancing the expression of FOXP3S. In one embodiment the step of decreasing the expression of the FOXP3L relative to FOXP3S expression is conducted by introducing interference oligomers into the cytoplasm of Tregs wherein the interference oligomers specifically interfere with the expression of FOXP3L, optionally by targeting the sequence of exon 2. In one embodiment the expression of FOXP3S is enhanced, optionally in conjunction with interfering with FOXP3L expression, optionally by introducing into the Tregs nucleic acid sequences encoding FOXP3S.
In accordance with one embodiment the expression of the FOXP3L is decreased and the expression of FOXP3S is increased by transfecting Tregs with an interference oligomer that targets FOXP3 exon 2 and comprises a sequence that binds to
In accordance with one embodiment the expression of the FOXP3L is decreased by transfecting Tregs with an interference oligomer that targets FOXP3 exon 2 and comprises a sequence that binds to
In accordance with one embodiment Tregs are induced to transdifferentiate into helper-like T-cells as a therapeutic approach to treating cancer. Current treatment options for breast cancer consist of endocrine therapy, chemotherapy, radiation therapy and surgery depending on the subtypes and the stage of breast cancer. The recent advances of immunotherapy have brought in a new treatment algorithm for many types of cancer, raising the enthusiasm for using immunotherapy to treat breast cancer. Normally, Tregs infiltrate breast cancer tissues abundantly, and thus suppress antitumor immunity. However, applicant has discovered that Tregs expressing FOXP3S will transdifferentiated into helper-like T cells, and thus promote the anti-tumor immune response.
Based upon this finding, FOXP3 targeting oligomers (e.g., iRNAs or morpholino oligos (MO)) can be used to shift cellular production of FOXP3L to FOXP3S expression, and the efficacy of such an approach for providing antitumor activity has been verified in a preclinical mouse models and patient derived organoid models. Accordingly, these FOXP3S-promoting MOs, or other mechanisms for altering intracellular FOXP3S/ FOXP3L ratios, will serve as novel immunotherapies for breast cancer treatment, and the treatment of other solid tumors.
In one embodiment the method of treating cancer in a patient comprises the step of increasing the relative concentration of FOXP3S expressing regulatory T cells relative to FOXP3L expressing regulatory T cells. In accordance with one embodiment a method for treating a patient having a solid tumor is provided wherein the number of Tregs expressing FOXP3S is increased in said patient, optionally wherein the increased number of Tregs expressing FOXP3S is localized to the site of the tumor. In one embodiment the number of Tregs expressing FOXP3S is increased due to the introduction of Tregs expressing FOXP3S to said patient. In one embodiment T cells are recovered form a patient, and the T cells are treated to induce regulatory T cells to shift from FOXP3L isoform to FOXP3S expression before the T cells are reintroduced into the patient. In one embodiment the in vitro induced cells are infused intravenously into the patient or injected into the tissues harboring the solid tumor.
In one embodiment a method of treating solid tumors, including breast cancer comprises a step of inducing regulatory T cells (Tregs) to differentiate into tumor-reactive helper-like T cells by transfecting said Tregs with an interference oligomer in an amount effective to increase FOXP3S expression in said Tregs. In one embodiment Tregs are transfected with one or more interference oligomers that interfere with FOXP3L expression and/or nucleic acids sequences that encode for the FOXP3S isoform. In one embodiment the Tregs are transfected with an interference oligomer that targets exon 2 of FOXP3. In one embodiment the Tregs are transfected in vivo using standard techniques, including for example tissue nanotransfection. In one embodiment interference oligomer is introduced into the cytosol of Tregs wherein the oligomer is a FOXP3S-promoting morpholino.
In one embodiment the method of treating cancer by inducing the transdifferentiation of Tregs to helper-like T cells is conducted in conjunction with other known immunological or other cancer treatment therapies. In one embodiment the method of treating cancer disclosed herein further comprises the step of administering to said patient an immune checkpoint blockade antibody.
In accordance with embodiment 1, a method for altering regulatory T cells
(Tregs) activity is provided wherein said method comprises the step of modifying intracellular concentrations of FOXP3 isoforms FOXP3L and FOXP3S in said Tregs, wherein the amount of the FOXP3S isoform is increased relative to the FOXP3L isoform.
In accordance with embodiment 2, the method of embodiment 1 is provided wherein the expression of the FOXP3L is inhibited relative to FOXP3S by transfecting Tregs with an interference oligomer that targets FOXP3L, optionally by excluding exon 2 of FOXP3.
In accordance with embodiment 3, the method of embodiment 1 or 2 is provided wherein the expression of the FOXP3S is enhanced relative to FOXP3L by transfecting Tregs with a nucleic acid encoding for the FOXP3S isoform.
In accordance with embodiment 4, the method of any one of embodiments 1-3 is provided wherein said Tregs are transfected with an amount of said interference oligomer sufficient to induce the isolated Tregs to transdifferentiate into helper-like T cells.
In accordance with embodiment 5, the method of any one of embodiments 1-4 is provided wherein the relative expression of the FOXP3L isoform is decreased by transfecting said Tregs with a FOXP3L targeting interference oligomer comprising
In accordance with embodiment 6, the method of any one of embodiments 1-5 is provided wherein said targeting interference oligomer comprises the nucleobase sequence of TGCCCATTCACCGTCCATACCTGGT (SEQ ID NO: 8), or a complement thereof.
In accordance with embodiment 6.5, the method of any one of embodiments 1-10 6 is provided wherein the expression of FOXP3L is decreased by transfecting Tregs with an interference oligomer that targets FOXP3 exon 2 and comprises a sequence that binds to
In accordance with embodiment 7, the method of any one of embodiments 1-6.5 is provided wherein the relative expression of the FOXP3L isoform is decreased by transfecting said Tregs with a FOXP3L targeting interference oligomer that specifically binds to a sequence comprising CCATTCACCGTCCATAC (SEQ ID NO: 2) or CCAUUCACCGUCCAUAC (SEQ ID NO: 9).
In accordance with embodiment 8, the method of any one of embodiments 1-7 is provided wherein the interference oligomer specifically binds to a sequence selected from the group consisting of
In accordance with embodiment 9, the method of any one of embodiments 1-8 is provided wherein the interference oligomer is an interfering RNA comprising a sequence having at least 95% sequence identity with GUAUGGACGGUGAAUGG (SEQ ID NO: 10).
In accordance with embodiment 10, the method of any one of embodiments 1-9 is provided wherein said interference oligomer is a phosphorodiamidate morpholino.
In accordance with embodiment 11, the method of any one of embodiments 1-10 is provided further comprising the step of transfecting said Tregs with a gene that encodes the FOXP3S isoform.
In accordance with embodiment 12, a method of treating cancer in a patient is provided wherein the method comprises the steps of increasing the intracellular concentrations of FOXP3S relative to FOXP3L in regulatory T cells (Tregs).
In accordance with embodiment 13, a method of treating cancer is provided wherein the increased relative concentration of FOXP3S is achieved by transfecting isolated Tregs in vitro with an interference oligomer that targets FOXP3 in said isolated Tregs; and reintroducing said transfected Tregs into said patient.
In accordance with embodiment 14, a method of treating cancer is provided wherein regulatory T cells (Tregs) are transfected in vivo with an interference oligomer in an amount effective to increase the intracellular concentrations of FOXP3S relative to FOXP3L in said Tregs.
In accordance with embodiment 15, a method of embodiments 12 or 14 is provided wherein the interference oligomer is a FOXP3S-promoting morpholino.
In accordance with embodiment 16, the method of any one of embodiments 1-15 is provided wherein the interference oligomer comprises a sequence of nucleobases selected from the group consisting of
In accordance with embodiment 17, the method of any one of embodiments 12-16 is provided wherein said interference oligomer is a phosphorodiamidate morpholino, optionally wherein the interference oligomer is an iRNA.
In accordance with embodiment 18, the method of any one of embodiments 1-17 is provided further comprising the step of transfecting said Tregs with a gene that encodes the FOXP3S isoform.
As disclosed herein 4 patients have been identified by the applicant as carrying deletion mutations in the exon 2 of FOXP3 (
To study the functional difference of the two FOXP3 isoforms, we deleted exon 2 in the mouse Foxp3 gene (Foxp3S) that encodes the Foxp3 short isoform lacking exon 2, while leaving intact the intronic regulatory elements. Foxp3S mice were viable and morphologically normal with unaffected thymocyte and thymic Treg development. Compared with WT mice that only express the Foxp3L isoform, Foxp3S mice developed anti-dsDNA IgG (
Since Foxp3S mice failed to maintain self-tolerance, we wondered whether Treg expressing only the Foxp3S isoform are defective in their ability to suppress effector T helpers. To our surprise, in an in vitro suppressive assay, Foxp3S Tregs and Foxp3L Tregs suppressed the proliferation of responder CD4+T cells equally well (data not shown). We examined Tregs in heterozygous Foxp3 exon 2 deletion (Foxp3S/L) female mice that had no signs of autoimmune diseases. Due to random X chromosome inactivation, these mice have two populations of Tregs, expressing either the Foxp3S isoform or the Foxp3L isoform, but not both. While ˜50% of Tregs in the thymus of Foxp3S/L female mice expressed the Foxp3S isoform, their frequencies were greatly reduced to ˜20% (
The much reduced frequency in the periphery (
The fact that thymus-derived Tregs express self-reactive TCRs suggests the possibility that Foxp3S Tregs could become autoreactive effectors upon loss of Foxp3 expression, leading to autoimmunity. To test this hypothesis, we transferred purified Foxp3S and/or Foxp3L Tregs into Tcrb-deficient recipient mice as depicted in
Our data that Foxp3S Tregs could differentiate into helper T cells and mediate autoreactive immune responses (
We performed single cell RNA sequencing to phenotype the immune cells infiltrating the EO771 tumors in WT and Foxp3S mice. We found that majority of the Foxp3S Tregs infiltrating the tumors showed T helper 2-like phenotypes while majority of the Foxp3L Tregs remained Treg phenotypes (
We next analyzed transcriptomic data sets of breast cancer tissues from The Cancer Genome Atlas (TCGA) to assess clinical relevance of FOXP3S isoform in human breast cancer. In triple negative breast cancer (TNBC) cases, FOXP3S expression in the breast cancer tissues is correlated with better overall survival (
The efficacy of Foxp3 isoform shifting morpholino in triple negative breast cancer treatment is tested in mouse models. 1×106 EO771 TNBC cells were orthotopically injected into the 4th mammary fat pad on both sides of the wild type mice. On days 12, 14, 16 and 18, 30 nmol/mouse of Foxp3 isoform shifting morpholino (E2 MO) or control MO was injected intratumorally into the tumors at the left side while leave the tumors at the right side as internal controls (
The results from the intratumoral injection of E2 MO (
To further test the ability of Foxp3S Tregs to boost antitumor immunity, Foxp3S Tregs and Foxp3LTregs were isolated from donor mice and injected intratumorally 2×106 Tregs into the EO771 TNBC on day 12 post inoculation (
Peripheral blood mononuclear cells were obtained from healthy donors and cultured in vitro with 1 μM isoform-shifting morpholino (E2 MO) for 3 weeks. Flow cytometry analysis demonstrated that majority of the Tregs expressed only FOXP3S isoform (positive for pan-FOXP3 antibody staining but negative for FOXP3 exon 2-specific antibody staining) while Tregs without MO treatment expressed FOXP3L (positive for FOXP3 exon 2-specific antibody staining) (
We next tested whether tumor infiltrating lymphocytes expanded in the presence of E2 MO have enhanced killing of autologous tumor cells. Tumor resections from breast cancer and colon cancer patients were obtained. Tumor organoids were generated from tumor cells from the resections and tumor infiltrating lymphocytes (TIL) were expanded in vitro in the presence or absence of E2 MO. After 7 days expansion, TILs were co-cultured with autologous tumor organoids and the size of the organoids was measured. 3 out of 4 E2 MO expanded TILs from breast cancer patients (
This application claims priority to U.S. Provisional Patent Application No. 63/083,452 filed on Sep. 25, 2020, the disclosure of which is expressly incorporated herein.
This invention was made with government support under AI085046 and CA203737 awarded by national institutes of health. The government has certain rights in the invention.
Filing Document | Filing Date | Country | Kind |
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PCT/US2021/049606 | 9/9/2021 | WO |
Number | Date | Country | |
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63083452 | Sep 2020 | US |