Patent Application
20250188457
The text of the computer readable sequence listing filed herewith, titled “NWEST-42252_202_SequenceListing.xml”, created Sep. 13, 2024, having a file size of 29,261 bytes, is hereby incorporated by reference in its entirety.
Provided herein are inhibitors of the TFPI2-CD51-STAT6 signaling axis and methods for the treatment of cancer (e.g., glioblastoma) therewith. In particular, inhibitors of expression or activity of components of the TFPI2-CD51-STAT6 signaling axis are provided, with or without an immune checkpoint inhibitor, for the treatment of glioblastoma.
Glioblastoma (GBM) is a devastating primary brain tumor. The median survival of GBM patients is approximately 14-16 months following initial diagnosis despite aggressive standard-of-care (SOC) treatment, which includes maximal surgical resection followed by chemo/radiotherapy (Ref. 1; incorporated by reference in its entirety). Poor prognosis and treatment recurrence of GBM are in part due to the presence of glioblastoma stem cells (GSCs), a subpopulation of cancer cells that are featured by stem cell-like capabilities (Ref. 2; incorporated by reference in its entirety). Mice implanted with GSCs produce tumors that are less responsive to chemotherapy than tumors established from more differentiated GBM cells: a finding that supports a crucial role of GSCs in treatment resistance (Ref. 3; incorporated by reference in its entirety). GSCs only account for ˜10% of total cancer cells in tumor tissues. However, the GSC subpopulation is known for self-renewal that is important for tumor growth and recurrence. Given the significant inverse correlation between GSC stemness and survival (Ref. 4; incorporated by reference in its entirety), targeting GSCs holds a great potential for GBM therapy (Refs. 2, 5-6; incorporated by reference in its entirety).
Despite advances in the understanding of GSC biologic properties, significant knowledge gaps remain, including those involving relationships between GSCs and additional cell populations of the tumor microenvironment (TME) (Refs. 2, 7; incorporated by reference in its entirety). Within the GBM TME, tumor-associated macrophages and microglia (TAMs) are abundant and account for up to 50% of total cells in the tumor mass (Ref. 8; incorporated by reference in its entirety). TAMs are usually polarized towards an immunosuppressive phenotype that can inhibit the infiltration and anti-tumor activation of cytotoxic T cells and induce immunotherapy resistance. Increasing evidence supports a symbiotic interaction between GSCs and TAMs (Refs. 2, 7, 9-10; incorporated by reference in their entireties). For instance, recent studies have demonstrated that GSC expression of CLOCK and its heterodimeric partner BMAL1 transcriptionally upregulates olfactomedin-like 3 (OLFML3) and legumain (LGMN) that, in turn, promote microglia infiltration and immunosuppressive polarization (Refs. 11-12; incorporated by reference in their entireties).
Provided herein are inhibitors of the TFPI2-CD51-STAT6 signaling axis and methods for the treatment of cancer (e.g., glioblastoma) therewith. In particular, inhibitors of expression or activity of components of the TFPI2-CD51-STAT6 signaling axis are provided, with or without an immune checkpoint inhibitor, for the treatment of glioblastoma.
In some embodiments, provided herein are methods of treating cancer in a subject, wherein the subject has an inhibited TFPI2-CD51-STAT6 signaling axis, the method comprising administering to the subject an immune checkpoint inhibitor (ICI). In some embodiments, the subject has been previously administered a TFPI2-CD51-STAT6 signaling axis inhibitor (e.g., TFPI2 inhibitor).
In some embodiments, provided herein are methods of treating cancer in a subject comprising administering a TFPI2 inhibitor to the subject.
In some embodiments, provided herein are methods of treating cancer in a subject comprising co-administering to the subject: (a) a TFPI2-CD51-STAT6 signaling axis inhibitor, and (b) an immune checkpoint inhibitor (ICI).
In some embodiments, the subject suffers from glioblastoma.
In some embodiments, an ICI targets an immune checkpoint molecule/protein selected from the group consisting of CTLA4, PD-1, PD-L1, PD-L2, A2AR, B7-H3, B7-H4, BTLA, KIR, LAG3, TIM-3, VISTA, or a ligand/receptor thereof. In some embodiments, an ICI is selected from nivolumab, pembrolizumab, atezolizumab, pidilizumab, AMP-224, AMP-514, STI-A1110, TSR-042, RG-7446, BMS-936559, BMS-936558, MK-3475, MPDL3280A, MEDI-4736, MSB-0020718C, AUR-012 and STI-A1010.
In some embodiments, a TFPI2-CD51-STAT6 signaling axis inhibitor is a STAT6 inhibitor. In some embodiments, the STAT6 inhibitor is an inhibitor of STAT6 expression. In some embodiments, the STAT6 inhibitor is an inhibitor of STAT6 activity.
In some embodiments, a TFPI2-CD51-STAT6 signaling axis inhibitor is a CD51 inhibitor. In some embodiments, the CD51 inhibitor is an inhibitor of CD51 expression. In some embodiments, the CD51 inhibitor is an inhibitor of CD51 activity.
In some embodiments, a TFPI2-CD51-STAT6 signaling axis inhibitor is a TFPI2 inhibitor. In some embodiments, the TFPI2 inhibitor is an inhibitor of TFPI2 expression. In some embodiments, the TFPI2 inhibitor is an inhibitor of TFPI2 activity.
In some embodiments, an inhibitor of TFPI2, CD51, or STAT6 expression is an shRNA, a miRNA, a morpholino, a ribozyme, an antisense nucleic acid molecule, or a CRISPR-based construct.
In some embodiments, an inhibitor of TFPI2, CD51, or STAT6 activity is a small molecule, peptide, antibody, or antibody fragment.
In some embodiments, administration or co-administration is by one or more of oral, intravenous, or other acceptable routes.
Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments described herein, some preferred methods, compositions, devices, and materials are described herein. However, before the present materials and methods are described, it is to be understood that this invention is not limited to the particular molecules, compositions, methodologies or protocols herein described, as these may vary in accordance with routine experimentation and optimization. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the embodiments described herein.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. However, in case of conflict, the present specification, including definitions, will control. Accordingly, in the context of the embodiments described herein, the following definitions apply.
As used herein and in the appended claims, the singular forms “a” “an” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “a TFPI2 inhibitor” is a reference to one or more TFPI2 inhibitors and equivalents thereof known to those skilled in the art, and so forth.
As used herein, the term “comprise” and linguistic variations thereof denote the presence of recited feature(s), element(s), method step(s), etc. without the exclusion of the presence of additional feature(s), element(s), method step(s), etc. Conversely, the term “consisting of” and linguistic variations thereof, denotes the presence of recited feature(s), element(s), method step(s), etc. and excludes any unrecited feature(s), element(s), method step(s), etc., except for ordinarily-associated impurities. The phrase “consisting essentially of” denotes the recited feature(s), element(s), method step(s), etc. and any additional feature(s), element(s), method step(s), etc. that do not materially affect the basic nature of the composition, system, or method. Many embodiments herein are described using open “comprising” language. Such embodiments encompass multiple closed “consisting of” and/or “consisting essentially of” embodiments, which may alternatively be claimed or described using such language.
As used herein, the term “pharmaceutically acceptable carrier” refers to non-toxic solid, semisolid, or liquid filler, diluent, encapsulating material, formulation auxiliary, excipient, or carrier conventional in the art for use with a therapeutic agent for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. The pharmaceutically acceptable carrier is appropriate for the formulation employed. For example, if the therapeutic agent is to be administered orally, the carrier may be a gel capsule. A “pharmaceutical composition” typically comprises at least one active agent (e.g., the copolymers described herein) and a pharmaceutically acceptable carrier.
As used herein, the term “effective amount” refers to the amount of a composition (e.g., pharmaceutical composition) sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.
As used herein, the term “administration” refers to the act of giving a drug, prodrug, or other agent, or therapeutic treatment (e.g., pharmaceutical compositions of the present invention) to a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs. Exemplary routes of administration to the human body can be through the eyes (e.g., intraocularly, intravitreally, periocularly, ophthalmic, etc.), mouth (oral), skin (transdermal), nose (nasal), lungs (inhalant), oral mucosa (buccal), ear, rectal, by injection (e.g., intravenously, subcutaneously, intratumorally, intraperitoneally, etc.) and the like.
As used herein, the terms “co-administration” and “co-administer” refer to the administration of at least two agent(s) or therapies to a subject. In some embodiments, the co-administration of two or more agents or therapies is concurrent (e.g., in the same or separate formulations). In other embodiments, a first agent/therapy is administered prior to a second agent/therapy. Those of skill in the art understand that the formulations and/or routes of administration of the various agents or therapies used may vary. The appropriate dosage for co-administration can be readily determined by one skilled in the art. In some embodiments, when agents or therapies are co-administered, the respective agents or therapies are administered at lower dosages than appropriate for their administration alone. Thus, co-administration is especially desirable in embodiments where the co-administration of the agents or therapies lowers the requisite dosage of a potentially harmful (e.g., toxic) agent(s).
As used herein, the term “subject” broadly refers to any animal, including but not limited to, human and non-human animals (e.g., dogs, cats, cows, horses, sheep, poultry, fish, crustaceans, etc.). As used herein, the term “patient” typically refers to a subject that is being treated for a disease or condition.
As used herein, the term “antibody” refers to a whole antibody molecule or a fragment thereof (e.g., fragments such as Fab, Fab′, and F(ab′)2), unless specified otherwise; an antibody may be polyclonal or monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, etc.
As used herein, the term “antibody fragment” refers to a portion of a full-length antibody, including at least a portion antigen binding region or a variable region. Antibody fragments include, but are not limited to, Fab, Fab′, F(ab′)2, Fv, scFv, Fd, diabodies, and other antibody fragments that retain at least a portion of the variable region of an intact antibody. See, e.g., Hudson et al. (2003) Nat. Med. 9:129-134; herein incorporated by reference in its entirety. In certain embodiments, antibody fragments are produced by enzymatic or chemical cleavage of intact antibodies (e.g., papain digestion and pepsin digestion of antibody) produced by recombinant DNA techniques, or chemical polypeptide synthesis.
Provided herein are inhibitors of the TFPI2-CD51-STAT6 signaling axis and methods for the treatment of cancer (e.g., glioblastoma) therewith. In particular, inhibitors of expression or activity of components of the TFPI2-CD51-STAT6 signaling axis are provided, with or without an immune checkpoint inhibitor, for the treatment of glioblastoma.
Glioblastoma (GBM) tumors contain multiple cell populations, including self-renewing glioblastoma stem cells (GSCs) and immunosuppressive microglia. Experiments conducted during development of embodiments herein identified Kunitz-type protease inhibitor TFPI2 as a critical factor connecting these cell populations and their associated GBM hallmarks of stemness and immunosuppression. TFPI2 promotes GSC self-renewal and tumor growth via activation of the c-Jun N-terminal kinase (JNK)-signal transducer and activator of transcription 3 (STAT3) pathway. Secreted TFPI2 interacts with its functional receptor CD51 on microglia to trigger the infiltration and immunosuppressive polarization of microglia through activation of STAT6 signaling. Inhibition of the TFPI2-CD51-STAT6 signaling axis activates T cells and synergizes with anti-PD1 therapy in GBM mouse models. In human GBM, TFPI2 correlates positively with stemness, microglia abundance, immunosuppression, and poor prognosis. Experiments conducted during development of embodiments herein reveal unexpected roles of TFPI2 in GBM biology and supports therapeutic targeting of TFPI2 and/or the TFPI2-CD51-STAT6 signaling axis as an effective strategy for this deadly disease.
Experiments conducted during development of embodiments herein identified tissue factor pathway inhibitor 2 (TFPI2) has been identified as a key effector of the symbiotic interaction between GSCs and TAMs. TFPI2 is a member of the Kunitz-type serine proteinase inhibitor family. Despite its name and structural similarity to TFPI1, a potent inhibitor for factors Xa and VIIa/TF (tissue factor) complex in the extrinsic pathway of blood coagulation, TFPI2 has little inhibitory effect on TFs (Refs. 13-14; incorporated by reference in their entireties). Instead, TFPI2 is recognized as a critical factor that can inhibit extracellular matrix (ECM) degradation via suppressing plasmin-dependent activation of pro-matrix metalloproteinase 1 (MMP1) and pro-MMP13. Since ECM degradation is important for tumor growth and metastasis (ref. 15; incorporated by reference in its entirety), TFPI2 has been considered as a tumor suppressor (Refs. 16-17; incorporated by reference in its entirety). However, contrasting results indicate a pro-tumor activity of TFPI2 in multiple cancers, including hepatocellular carcinoma (Ref. 18; incorporated by reference in its entirety), melanoma (Refs. 19-20; incorporated by reference in their entireties), and ovarian clear cell carcinoma (Ref. 21; incorporated by reference in its entirety). These findings suggest that the role of TFPI2 in tumor biology may be context and cancer type dependent. In GBM, TFPI2 has been shown to inhibit the growth, survival, migration, and invasion of differentiated GBM cells (Refs. 22-24; incorporated by reference in their entireties).
Experiments conducted during development of embodiments herein identified an unexpected function for TFPI2 in supporting maintenance of stemness and tumor growth via activation of the c-Jun N-terminal kinase (JNK)-signal transducer and activator of transcription 3 (STAT3) pathway in GSCs. Moreover, secreted TFPI2 contributes to an immunosuppressive TME by promoting microglia infiltration and polarization via activation of CD51 (also known as integrin alpha V, ITGAV)-STAT6 signaling pathway in microglia. Inhibition of the TFPI2-CD51-STAT6 pathway in GBM mouse models activates antitumor immunity that extends the survival of tumor-bearing animal subjects. This anti-tumor immune activation can be coupled with immune checkpoint inhibitor (ICI) to further extend survival. In combination with results from analysis of patient tumor and plasma samples, experiments conducted during development of embodiments herein demonstrate that TFPI2 as a therapeutic target for treating GBM.
In some embodiments, compositions and methods described herein employ a TFPI2 inhibitor or a composition/method for the inhibition of TFPI2 activity or expression.
In some embodiments, compositions and methods described herein employ a CD51 inhibitor or a composition/method for the inhibition of CD51 activity or expression.
In some embodiments, compositions and methods described herein employ a STAT6 inhibitor or a composition/method for the inhibition of STAT6 activity or expression.
In some embodiments, provided herein are inhibitors of TFPI2, CD51, and/or STAT6 activity. In some embodiments, an inhibitor is a small molecule, peptide, antibody, antibody fragment, a genome editing agent, etc. that upon administration to a cell, reduces the activity of TFPI2, CD51, and/or STAT6.
In some embodiments, a TFPI2, CD51, and/or STAT6 inhibitor is an antibody or antibody fragment. In some embodiments, a TFPI2, CD51, and/or STAT6 inhibitor is an antibody or antibody fragment that binds to TFPI2, CD51, or STAT6 and reduces TFPI2, CD51, and/or STAT6 activity. In some embodiments, an anti-TFPI2, anti-CD51, or anti-STAT6 antibody or antibody fragment is provided that reduced the activity of TFPI2, CD51, or STAT6 and/or reduces binding of TFPI2, CD51, or STAT6 to a ligand thereof. In some embodiments, an anti-CD51 antibody is selected from Abituzumab, Anti-ITGAV-DM4 ADC (ADC-W-109), and Anti-ITGAV (clone MF-T)-SPDB-DM4 ADC (ADC-W-422). An anti-STAT6 is selected from Phospho-STAT6-Y641 Antibody Blocking Peptide (BP3270a) and Eblasakimab.
In some embodiments, a TFPI2 inhibitor is a small molecule, protein (e.g., antibody) or peptide that binds to TFPI2 and inhibits its activity.
In some embodiments, a CD51 inhibitor is a small molecule, protein (e.g., antibody) or peptide that binds to TFPI2 and inhibits its activity. In some embodiments, a CD51 inhibitor is selected from cilengitide, MK-0429, Cyclo(-RGDfK), Cyclo(RGDyK) trifluoroacetate, Cyclo(-RGDfK) TFA, Cyclo RGDfC, HSDVHK-NH2, Cyclo(Arg-Gly-Asp-D-Phe-Cys) TFA, Echistatin, Nesvategrast, Cyclo(RGDyK), JNJ-26076713, Bexotegrast, EMD527040, and abituzumab.
In some embodiments, a STAT6 inhibitor is a small molecule, protein (e.g., antibody) or peptide that binds to STAT6 and inhibits its activity. STAT6 activity has been inhibited by small molecules (Nagashima, et al. Bioorg. Med. Chem. 2007, 15, 1044-1055; Nagashima, et al. Bioorg. Med. Chem. 2008, 16, 6509-6521.; Nagashima, et al. Bioorg. Med. Chem. 2009, 17, 6926-6936.; Ohga, et al. Eur. J. Pharmacol. 2008, 590, 409-416.; Chiba, et al. Am. J. Respir. Cell Mol. Biol. 2009, 41, 516-524.; incorporated by reference in their entireties) and antibodies (Walsh, G. M. Expert Opin. Emerg. Drugs 2012, 17, 37-42.; and Blease. Curr. Opin. Investig. Drugs 2008, 9, 1180-1184.; incorporated by reference in their entireties). STAT6 expression has been inhibited by, for example, siRNA (Darcan-Nicolaisen, Y. et al. J. Immunol. 2009, 182, 7501-7508; incorporated by reference in its entirety). Examples of STAT6 inhibitors include AS 1571499, AS 1617612, AS 1810722, (R)-76, (R)-84, and those described in, for example, U.S. Pub. No. 2016/0145279; incorporated by reference in its entirety. In some embodiments, a STAT6 inhibitor is selected from AS 1571499, AS 1617612, AS 1810722, (R)-76, (R)-84, STAT6-IN-1, STAT6-IN-2, STAT6-IN-3, PM-43I, PM-81I, and Picroside I.
In some embodiments, provided herein are inhibitors of TFPI2, CD51, and/or STAT6 expression. In particular embodiments, an inhibitor TFPI2, CD51, and/or STAT6 expression is a nucleic acid-based inhibitor. In some embodiments, the inhibitor is a small molecule, an aptamer, a siRNA, a shRNA, a miRNA, a morpholino, a ribozyme, an antisense nucleic acid molecule, a CRISPR-Cas9-based construct, a CRISPR-Cpf1-based construct, a meganuclease, a zinc finger nuclease, a transcription activator-like (TAL) effector (TALE) nuclease, etc.
In some embodiments, the inhibitor TFPI2, CD51, and/or STAT6 expression is a small interfering RNA (siRNA), also known as short interfering RNA or silencing RNA. In some embodiments, an siRNA is an 18 to 30 nucleotide, preferably 19 to 25 nucleotide, most preferred 21 to 23 nucleotide or even more preferably 21 nucleotide-long double-stranded RNA molecule. siRNA is involved in the RNA interference (RNAi) pathway where the siRNA interferes with the expression of a specific gene (e.g., TFPI2, CD51, or STAT6). siRNAs naturally found in nature have a well-defined structure: a short double-strand of RNA (dsRNA) with 2-nt 3′ overhangs on either end. Each strand has a 5′ phosphate group and a 3′ hydroxyl (—OH) group. This structure is the result of processing by dicer, an enzyme that converts either long dsRNAs or small hairpin RNAs into siRNAs. siRNAs can also be exogenously (artificially) introduced into cells to bring about the specific knockdown of a gene of interest (e.g., TFPI2, CD51, or STAT6). Essentially any gene for which the sequence is known can thus be targeted based on sequence complementarity with an appropriately tailored siRNA. The double-stranded RNA molecule or a metabolic processing product thereof is capable of mediating target-specific nucleic acid modifications, particularly RNA interference and/or DNA methylation. Exogenously introduced siRNAs may be devoid of overhangs at their 3′ and 5′ ends, however, in some embodiments at least one RNA strand has a 5′- and/or 3′-overhang. Preferably, one end of the double-strand has a 3′-overhang from 1 to 5 nucleotides, more preferably from 1 to 3 nucleotides and most preferably 2 nucleotides. The other end may be blunt-ended or has up to 6 nucleotides 3′-overhang. In general, any RNA molecule suitable to act as siRNA and inhibit TFPI2, CD51, or STAT6 is envisioned in the present invention. In some embodiments, siRNA duplexes are provided composed of 21-nt sense and 21-nt antisense strands, paired in a manner to have a 2-nt 3′-overhang. The sequence of the 2-nt 3′ overhang makes a small contribution to the specificity of target recognition restricted to the unpaired nucleotide adjacent to the first base pair. 2′-deoxynucleotides in the 3′ overhangs are as efficient as ribonucleotides, but are often cheaper to synthesize and probably more nuclease resistant. Delivery of siRNA may be accomplished using any of the methods known in the art, for example by combining the siRNA with saline and administering the combination intravenously or intranasally or by formulating siRNA in glucose (such as for example 5% glucose) or cationic lipids and polymers can be used for siRNA delivery in vivo through systemic routes either intravenously (IV) or intraperitoneally (IP). In some embodiments, provided herein are siRNA molecules that target and inhibit the expression (e.g., knock down) of TFPI2, CD51, or STAT6.
A short hairpin RNA (shRNA) is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression (e.g., TFPI2, CD51, or STAT6) via RNA interference. In some embodiments, shRNA uses a vector introduced into cells and utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). The RISC binds to and cleaves RNAs which match the siRNA that is bound to (e.g., comprising the sequence of TFPI2, CD51, or STAT6). In some embodiments, si/shRNAs to be used in the present invention are chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer. In some embodiments, provided herein are shRNA molecules that target and inhibit the expression (e.g., knock down) of TFPI2, CD51, or STAT6.
Further molecules effecting RNAi (and useful herein for the inhibition of expression of TFPI2, CD51, or STAT6) include, for example, microRNAs (miRNA). Said RNA species are single-stranded RNA molecules. Endogenously present miRNA molecules regulate gene expression by binding to a complementary mRNA transcript and triggering of the degradation of said mRNA transcript through a process similar to RNA interference. Accordingly, exogenous miRNA may be employed as an inhibitor of TFPI2, CD51, or STAT6 after introduction into target cells. In some embodiments, provided herein are miRNA molecules that target and inhibit the expression (e.g., knock down) of the TFPI2, CD51, or STAT6.
Morpholinos (or morpholino oligonucleotides) are synthetic nucleic acid molecules having a length of about 20 to 30 nucleotides and, typically about 25 nucleotides. Morpholinos bind to complementary sequences of target transcripts (e.g., TFPI2, CD51, or STAT6) by standard nucleic acid base-pairing. They have standard nucleic acid bases which are bound to morpholine rings instead of deoxyribose rings and linked through phosphorodiamidate groups instead of phosphates. Due to replacement of anionic phosphates into the uncharged phosphorodiamidate groups, ionization in the usual physiological pH range is prevented, so that morpholinos in organisms or cells are uncharged molecules. The entire backbone of a morpholino is made from these modified subunits. Unlike inhibitory small RNA molecules, morpholinos do not degrade their target RNA molecules. Rather, they sterically block binding to a target sequence within a RNA and prevent access by molecules that might otherwise interact with the RNA. In some embodiments, provided herein are morpholino oligonucleotides that target and inhibit the expression (e.g., knock down) of TFPI2, CD51, or STAT6.
A ribozyme (ribonucleic acid enzyme, also called RNA enzyme or catalytic RNA) is an RNA molecule that catalyzes a chemical reaction. Many natural ribozymes catalyze either their own cleavage or the cleavage of other RNAs, but they have also been found to catalyze the aminotransferase activity of the ribosome. Non-limiting examples of well-characterized small self-cleaving RNAs are the hammerhead, hairpin, hepatitis delta virus, and in vitro-selected lead-dependent ribozymes, whereas the group I intron is an example for larger ribozymes. The principle of catalytic self-cleavage is well established. Since it was shown that hammerhead structures can be integrated into heterologous RNA sequences and that ribozyme activity can thereby be transferred to these molecules, catalytic antisense sequences can be engineered for almost any target sequence can be created, provided the target sequence contains a potential matching cleavage site. The basic principle of constructing hammerhead ribozymes is as follows: A region of interest of the RNA (e.g., a portion of TFPI2, CD51, or STAT6), which contains the GUC (or CUC) triplet, is selected. Two oligonucleotide strands, each usually with 6 to 8 nucleotides, are taken and the catalytic hammerhead sequence is inserted between them. In some embodiments, provided herein are ribozyme inhibitors of the TFPI2, CD51, or STAT6.
In some embodiments, TFPI2, CD51, or STAT6 is inhibited by modifying the TFPI2, CD51, or STAT6 sequence in target cells. In some embodiments, the alteration of TFPI2, CD51, or STAT6 is carried out using one or more DNA-binding nucleic acids, such as alteration via an RNA-guided endonuclease (RGEN). For example, the alteration can be carried out using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins. In general, “CRISPR system” refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), and/or other sequences and transcripts from a CRISPR locus. The CRISPR/Cas nuclease or CRISPR/Cas nuclease system can include a non-coding RNA molecule (guide) RNA, which sequence-specifically binds to DNA, and a Cas protein (e.g., Cas9), with nuclease functionality (e.g., two nuclease domains). One or more elements of a CRISPR system can derive from a type I, type II, or type III CRISPR system, e.g., derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes. In some aspects, a Cas nuclease and gRNA (including a fusion of crRNA specific for the target sequence (e.g., a sequence within TFPI2, CD51, or STAT6) and fixed tracrRNA) are introduced into the cell. In general, target sites at the 5′ end of the gRNA target the Cas nuclease to the target site using complementary base pairing. The target site may be selected based on its location immediately 5′ of a protospacer adjacent motif (PAM) sequence, such as typically NGG, or NAG. In this respect, the gRNA is targeted to the desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of the guide RNA to correspond to the target DNA sequence (e.g., sequence within the TFPI2, CD51, or STAT6 gene). In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence. Typically, “target sequence” generally refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between the target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. The CRISPR system can induce double stranded breaks (DSBs) at the SRC-3 target site, followed by disruptions or alterations as discussed herein. In other embodiments, Cas9 variants, deemed “nickases,” are used to nick a single strand at the target site (e.g., within the TFPI2, CD51, or STAT6 gene). Paired nickases can be used, e.g., to improve specificity, each directed by a pair of different gRNAs targeting sequences such that upon introduction of the nicks simultaneously, a 5′ overhang is introduced. In other embodiments, catalytically inactive Cas9 is fused to a heterologous effector domain such as a transcriptional repressor or activator, to affect gene expression (e.g., to inhibit expression of TFPI2, CD51, or STAT6). In some embodiments, the CRISPR system is used to alter TFPI2, CD51, or STAT6, inhibit expression of TFPI2, CD51, or STAT6, and/or to inactivate the expression product of the TFPI2, CD51, or STAT6.
The term “antisense nucleic acid molecule” or “antisense oligonucleotide” as used herein, refers to a nucleic acid which is complementary to a target nucleic acid. An antisense molecule in accordance with the invention is capable of interacting with the target nucleic acid, more specifically it is capable of hybridizing with the target nucleic acid. Due to the formation of the hybrid, transcription of the target gene(s) and/or translation of the target mRNA is reduced or blocked. Standard methods relating to antisense technology have been described (see, e.g., Melani et al., Cancer Res. (1991) 51:2897-2901). In some embodiments, provided herein are antisense oligonucleotides capable of inhibiting expression of TFPI2, CD51, or STAT6 when administered to cell or subject. In some embodiments, the antisense oligonucleotides are antisense DNA- and/or RNA-oligonucleotides. In some embodiments, provided herein are modified antisense oligonucleotides, such as, antisense 2′-O-methyl oligo-ribonucleotides, antisense oligonucleotides containing phosphorothiaote linkages, antisense oligonucleotides containing Locked Nucleic Acid LNA(R) bases, morpholino antisense oligonucleotides, PPAR-gamma agonists, antagomirs. In some embodiments, ASOs comprise Locked Nucleic Acid (LNA) or 2′-methoxyethyl (MOE) modifications (internucleotide linkages are phosphorothioates interspersed with phosphodiesters, and all cytosine residues are 5′-methylcytosines).
In some embodiments, an inhibitor of the TFPI2-CD51-STAT6 signaling axis is administered for the treatment of a cancer. Certain embodiments herein are directed to administration of an inhibitor of the TFPI2-CD51-STAT6 signaling axis to a subject with cancer, in remission from cancer, or at elevated risk of cancer. In some embodiments, an inhibitor of the TFPI2-CD51-STAT6 signaling axis is administered as part of therapeutic or prophylactic regimen for the treatment or prevention of acute myeloid leukemia, cancer in adolescents, adrenocortical carcinoma childhood, AIDS-related cancers (e.g., Lymphoma and Kaposi's Sarcoma), anal cancer, appendix cancer, astrocytomas, atypical teratoid, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain stem glioma, brain tumor, bronchial tumors, burkitt lymphoma, carcinoid tumor, atypical teratoid, embryonal tumors, germ cell tumor, primary lymphoma, cervical cancer, childhood cancers, chordoma, cardiac tumors, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myleoproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, extrahepatic ductal carcinoma in situ (DCIS), embryonal tumors, CNS cancer, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, fibrous histiocytoma of bone, gall bladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), germ cell tumor, gestational trophoblastic tumor, hairy cell leukemia, head and neck cancer, heart cancer, liver cancer, hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors, pancreatic neuroendocrine tumors, kidney cancer, laryngeal cancer, lip and oral cavity cancer, liver cancer, lobular carcinoma in situ (LCIS), lung cancer, lymphoma, metastatic squamous neck cancer with occult primary, midline tract carcinoma, mouth cancer multiple endocrine neoplasia syndromes, multiple myeloma/plasma cell neoplasm, mycosis fungoides, myelodysplastic syndromes, myelodysplastic/myeloproliferative neoplasms, multiple myeloma, merkel cell carcinoma, malignant mesothelioma, malignant fibrous histiocytoma of bone and osteosarcoma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-hodgkin lymphoma, non-small cell lung cancer (NSCLC), oral cancer, lip and oral cavity cancer, oropharyngeal cancer, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pleuropulmonary blastoma, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, transitional cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, stomach (gastric) cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, T-Cell lymphoma, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, trophoblastic tumor, unusual cancers of childhood, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, or viral-induced cancer. In particular embodiments, an inhibitor of the TFPI2-CD51-STAT6 signaling axis is administered for the treatment of glioblastoma (e.g., primary glioblastoma, secondary glioblastoma, etc.).
In some embodiments, an inhibitor of the TFPI2-CD51-STAT6 signaling axis is co-administered with one or more additional agents (e.g., for the treatment of cancer).
In some embodiments, an inhibitor of the TFPI2-CD51-STAT6 signaling axis is co-administered with an immune checkpoint inhibitor. Immune checkpoint inhibitors are a class of drugs designed to enhance the body's natural immune response against cancer and other diseases by blocking certain molecules (checkpoints) that regulate immune responses. Immune checkpoint molecules are ac component of systems that helps maintain immune balance and prevent excessive immune activation. Cancers often exploit immune checkpoint pathways to evade detection and attack by the immune system. By inhibiting specific immune checkpoints, immune checkpoint inhibitors can reactivate the immune response, enabling immune cells to recognize and attack cancer cells more effectively. Immune checkpoint inhibitors work by targeting specific cell surface molecules on immune cells or cancer cells. These molecules are often ligands or receptors that, when engaged, suppress immune responses. By blocking the interactions between these molecules, checkpoint inhibitors can enhance immune responses against cancers. In some embodiments, an inhibitor of the TFPI2-CD51-STAT6 signaling axis is co-administered with an immune checkpoint inhibitor (e.g., antibody or fragment thereof) that targets an immune checkpoint molecule/protein selected from the group consisting of CTLA4, PD-1, PD-L1, PD-L2, A2AR, B7-H3, B7-H4, BTLA, KIR, LAG3, TIM-3, VISTA, or a ligand/receptor thereof. In some embodiments, an inhibitor of the TFPI2-CD51-STAT6 signaling axis is co-administered with an immune checkpoint inhibitor selected from nivolumab, pembrolizumab, atezolizumab, pidilizumab, AMP-224, AMP-514, STI-A1110, TSR-042, RG-7446, BMS-936559, BMS-936558, MK-3475, MPDL3280A, MEDI-4736, MSB-0020718C, AUR-012 and STI-A1010.
In some embodiments, an inhibitor of the TFPI2-CD51-STAT6 signaling axis (alone or co-administered with an immune checkpoint inhibitor) is co-administered along with administration of a chemotherapy agent. In some embodiments, the chemotherapeutic is selected from the group consisting of mitotic inhibitors, alkylating agents, anti-metabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzyme inhibitors, topoisomerase inhibitors, protein-protein interaction inhibitors, biological response modifiers, anti-hormones, angiogenesis inhibitors, and anti-androgens.
Non-limiting examples are chemotherapeutic agents, cytotoxic agents, and non-peptide small molecules such as Gleevec® (Imatinib Mesylate), Velcade® (bortezomib), Casodex (bicalutamide), Iressa® (gefitinib), and Adriamycin as well as a host of chemotherapeutic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, Casodex™, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxanes, e.g., paclitaxel (TAXOL™, Bristol-Myers Squibb Oncology, Princeton, N.J.) and docetaxel (TAXOTERE™, Rhone-Poulenc Rorer, Antony, France); retinoic acid; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included as suitable chemotherapeutic cell conditioners are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, (Nolvadex™), raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; camptothecin-11 (CPT-11); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO). Where desired, the compounds or pharmaceutical composition of the present invention can be used in combination with commonly prescribed anti-cancer drugs such as Herceptin®, Avastin®, Erbitux®, Rituxan®, Taxol®, Arimidex®, Taxotere®, ABVD, AVICINE, Abagovomab, Acridine carboxamide, Adecatumumab, 17-N-Allylamino-17-demethoxygeldanamycin, Alpharadin, Alvocidib, 3-Aminopyridine-2-carboxaldehyde thiosemicarbazone, Amonafide, Anthracenedione, Anti-CD22 immunotoxins, Antineoplastic, Antitumorigenic herbs, Apaziquone, Atiprimod, Azathioprine, Belotecan, Bendamustine, BIBW 2992, Biricodar, Brostallicin, Bryostatin, Buthionine sulfoximine, CBV (chemotherapy), Calyculin, cell-cycle nonspecific antineoplastic agents, Dichloroacetic acid, Discodermolide, Elsamitrucin, Enocitabine, Epothilone, Eribulin, Everolimus, Exatecan, Exisulind, Ferruginol, Forodesine, Fosfestrol, ICE chemotherapy regimen, IT-101, Imexon, Imiquimod, Indolocarbazole, Irofulven, Laniquidar, Larotaxel, Lenalidomide, Lucanthone, Lurtotecan, Mafosfamide, Mitozolomide, Nafoxidine, Nedaplatin, Olaparib, Ortataxel, PAC-1, Pawpaw, Pixantrone, Proteasome inhibitor, Rebeccamycin, Resiquimod, Rubitecan, SN-38, Salinosporamide A, Sapacitabine, Stanford V, Swainsonine, Talaporfin, Tariquidar, Tegafur-uracil, Temodar, Tesetaxel, Triplatin tetranitrate, Tris(2-chloroethyl)amine, Troxacitabine, Uramustine, Vadimezan, Vinflunine, ZD6126 or Zosuquidar.
In some embodiments, an inhibitor of the TFPI2-CD51-STAT6 signaling axis (alone or co-administered with an immune checkpoint inhibitor) is co-administered with an amount of one or more substances selected from anti-angiogenesis agents, signal transduction inhibitors, antiproliferative agents, glycolysis inhibitors, or autophagy inhibitors.
Anti-angiogenesis agents, such as MMP-2 (matrix-metalloproteinase 2) inhibitors, MMP-9 (matrix-metalloprotienase 9) inhibitors, and COX-11 (cyclooxygenase 11) inhibitors, can be used in conjunction with an inhibitor of the TFPI2-CD51-STAT6 signaling axis (alone or co-administered with an immune checkpoint inhibitor). Anti-angiogenesis agents include, for example, rapamycin, temsirolimus (CCI-779), everolimus (RAD001), sorafenib, sunitinib, and bevacizumab. Examples of useful COX-II inhibitors include CELEBREX™ (alecoxib), valdecoxib, and rofecoxib. Examples of useful matrix metalloproteinase inhibitors are described in WO 96/33172 (published Oct. 24, 1996), WO 96/27583 (published Mar. 7, 1996), European Patent Application No. 97304971.1 (filed Jul. 8, 1997), European Patent Application No. 99308617.2 (filed Oct. 29, 1999), WO 98/07697 (published Feb. 26, 1998), WO 98/03516 (published Jan. 29, 1998), WO 98/34918 (published Aug. 13, 1998), WO 98/34915 (published Aug. 13, 1998), WO 98/33768 (published Aug. 6, 1998), WO 98/30566 (published Jul. 16, 1998), European Patent Publication 606,046 (published Jul. 13, 1994), European Patent Publication 931, 788 (published Jul. 28, 1999), WO 90/05719 (published May 31, 1990), WO 99/52910 (published Oct. 21, 1999), WO 99/52889 (published Oct. 21, 1999), WO 99/29667 (published Jun. 17, 1999), PCT International Application No. PCT/IB98/01113 (filed Jul. 21, 1998), European Patent Application No. 99302232.1 (filed Mar. 25, 1999), Great Britain Patent Application No. 9912961.1 (filed Jun. 3, 1999), U.S. Provisional Application No. 60/148,464 (filed Aug. 12, 1999), U.S. Pat. No. 5,863,949 (issued Jan. 26, 1999), U.S. Pat. No. 5,861,510 (issued Jan. 19, 1999), and European Patent Publication 780,386 (published Jun. 25, 1997), all of which are incorporated herein in their entireties by reference. Preferred MMP-2 and MMP-9 inhibitors are those that have little or no activity inhibiting MMP-1. More preferred, are those that selectively inhibit MMP-2 and/or AMP-9 relative to the other matrix-metalloproteinases (e.g., MAP-1, MMP-3, MMP-4, MMP-5, MMP-6, MMP-7, MMP-8, MMP-10, MMP-11, MMP-12, and MMP-13). Some specific examples of MMP inhibitors useful in the invention are AG-3340, RO 32-3555, and RS 13-0830.
Autophagy inhibitors include, but are not limited to chloroquine, 3-methyladenine, hydroxychloroquine (Plaquenil™), bafilomycin A1, 5-amino-4-imidazole carboxamide riboside (AICAR), okadaic acid, autophagy-suppressive algal toxins which inhibit protein phosphatases of type 2A or type 1, analogues of cAMP, and drugs which elevate cAMP levels such as adenosine, LY204002, N6-mercaptopurine riboside, and vinblastine. In addition, antisense or siRNA that inhibits expression of proteins including but not limited to ATG5 (which are implicated in autophagy), may also be used.
In some embodiments, medicaments which are administered in conjunction with an inhibitor of the TFPI2-CD51-STAT6 signaling axis (alone or co-administered with an immune checkpoint inhibitor) include any suitable drugs usefully delivered by inhalation for example, analgesics, e.g., codeine, dihydromorphine, ergotamine, fentanyl or morphine; anginal preparations, e.g., diltiazem; antiallergics, e.g., cromoglycate, ketotifen or nedocromil; anti-infectives, e.g., cephalosporins, penicillins, streptomycin, sulphonamides, tetracyclines or pentamidine; antihistamines, e.g., methapyrilene; anti-inflammatories, e.g., beclomethasone, flunisolide, budesonide, tipredane, triamcinolone acetonide or fluticasone; antitussives, e.g., noscapine; bronchodilators, e.g., ephedrine, adrenaline, fenoterol, formoterol, isoprenaline, metaproterenol, phenylephrine, phenylpropanolamine, pirbuterol, reproterol, rimiterol, salbutamol, salmeterol, terbutalin, isoetharine, tulobuterol, orciprenaline or (-)-4-amino-3,5-dichloro-α-[[[6-[2-(2-pyridinyl)ethoxy]hexyl]-amino]methyl]benzenemethanol; diuretics, e.g., amiloride; anticholinergics e.g., ipratropium, atropine or oxitropium; hormones, e.g., cortisone, hydrocortisone or prednisolone; xanthines e.g., aminophylline, choline theophyllinate, lysine theophyllinate or theophylline; and therapeutic proteins and peptides, e.g., insulin or glucagon. Exemplary therapeutic agents useful for a combination therapy with an inhibitor of the TFPI2-CD51-STAT6 signaling axis (alone or co-administered with an immune checkpoint inhibitor) include but are not limited to agents as described above, radiation therapy, hormone antagonists, hormones and their releasing factors, thyroid and antithyroid drugs, estrogens and progestins, androgens, adrenocorticotropic hormone; adrenocortical steroids and their synthetic analogs; inhibitors of the synthesis and actions of adrenocortical hormones, insulin, oral hypoglycemic agents, and the pharmacology of the endocrine pancreas, agents affecting calcification and bone turnover: calcium, phosphate, parathyroid hormone, vitamin D, calcitonin, vitamins such as water-soluble vitamins, vitamin B complex, ascorbic acid, fat-soluble vitamins, vitamins A, K, and E, growth factors, cytokines, chemokines, muscarinic receptor agonists and antagonists; anticholinesterase agents; agents acting at the neuromuscular junction and/or autonomic ganglia; catecholamines, sympathomimetic drugs, and adrenergic receptor agonists or antagonists; and 5-hydroxytryptamine (5-HT, serotonin) receptor agonists and antagonists.
Other suitable therapeutic agents for coadministration with an inhibitor of the TFPI2-CD51-STAT6 signaling axis (alone or co-administered with an immune checkpoint inhibitor) also include agents for pain and inflammation such as histamine and histamine antagonists, bradykinin and bradykinin antagonists, 5-hydroxytryptamine (serotonin), lipid substances that are generated by biotransformation of the products of the selective hydrolysis of membrane phospholipids, eicosanoids, prostaglandins, thromboxanes, leukotrienes, aspirin, nonsteroidal anti-inflammatory agents, analgesic-antipyretic agents, agents that inhibit the synthesis of prostaglandins and thromboxanes, selective inhibitors of the inducible cyclooxygenase, selective inhibitors of the inducible cyclooxygenase-2, autacoids, paracrine hormones, somatostatin, gastrin, cytokines that mediate interactions involved in humoral and cellular immune responses, lipid-derived autacoids, eicosanoids, β-adrenergic agonists, ipratropium, glucocorticoids, methylxanthines, sodium channel blockers, opioid receptor agonists, calcium channel blockers, membrane stabilizers and leukotriene inhibitors.
Additional therapeutic agents contemplated for co-administration with an inhibitor of the TFPI2-CD51-STAT6 signaling axis (alone or co-administered with an immune checkpoint inhibitor) include diuretics, vasopressin, agents affecting the renal conservation of water, rennin, angiotensin, agents useful in the treatment of myocardial ischemia, anti-hypertensive agents, angiotensin converting enzyme inhibitors, 0-adrenergic receptor antagonists, agents for the treatment of hypercholesterolemia, and agents for the treatment of dyslipidemia.
Other therapeutic agents contemplated for co-administration with an inhibitor of the TFPI2-CD51-STAT6 signaling axis (alone or co-administered with an immune checkpoint inhibitor) include drugs used for control of gastric acidity, agents for the treatment of peptic ulcers, agents for the treatment of gastroesophageal reflux disease, prokinetic agents, antiemetics, agents used in irritable bowel syndrome, agents used for diarrhea, agents used for constipation, agents used for inflammatory bowel disease, agents used for biliary disease, agents used for pancreatic disease. Therapeutic agents used to treat protozoan infections, drugs used to treat Malaria, Amebiasis, Giardiasis, Trichomoniasis, Trypanosomiasis, and/or Leishmaniasis, and/or drugs used in the chemotherapy of helminthiasis. Other therapeutic agents include antimicrobial agents, sulfonamides, trimethoprim-sulfamethoxazole quinolones, and agents for urinary tract infections, penicillins, cephalosporins, and other, β-lactam antibiotics, an agent comprising an aminoglycoside, protein synthesis inhibitors, drugs used in the chemotherapy of tuberculosis, Mycobacterium avium complex disease, and leprosy, antifungal agents, antiviral agents including nonretroviral agents and antiretroviral agents.
Examples of therapeutic antibodies that can be combined with an inhibitor of the TFPI2-CD51-STAT6 signaling axis (alone or co-administered with an immune checkpoint inhibitor) include but are not limited to anti-receptor tyrosine kinase antibodies (cetuximab, panitumumab, trastuzumab), anti CD20 antibodies (rituximab, tositumomab), and other antibodies such as alemtuzumab, bevacizumab, and gemtuzumab.
Moreover, therapeutic agents used for immunomodulation, such as immunomodulators, immunosuppressive agents, tolerogens, and immunostimulants are contemplated by the methods herein. In addition, therapeutic agents acting on the blood and the blood-forming organs, hematopoietic agents, growth factors, minerals, and vitamins, anticoagulant, thrombolytic, and antiplatelet drugs.
HMC3 microglia, 293T and CT2A cells, SIM-A9 microglia, as well as SB28 and GL261 cells were cultured in Eagle's Minimum Essential Medium (ATCC, #30-2003), Dulbecco's Modified Eagle's Medium (DMEM; Gibco, #11995-065), DMEM-Ham's F12 medium (Gibco, #10565-018), and RPMI 1640 medium (RPMI; Gibco, #11875093), respectively, containing 10% fetal bovine serum (FBS, Fisher Scientific, #16140071) and 1:100 antibiotic-antimycotic (Gibco, #15140-122). For stemness maintenance, CT2A cells were cultured in neural stem cell (NSC) proliferation media (Millipore, #SCM005) containing 20 ng/ml epidermal growth factor (EGF; PeproTech, #AF-100-15) and basic fibroblast growth factor (bFGF; PeproTech, #100-18B). Similarly, human (GSC2, GSC17, GSC20, GSC23, GSC262, GSC272) and mouse (QPP7 and 005 GSC) GSCs were cultured in NSC proliferation media containing 20 ng/ml EGF and bFGF. Human GSCs were gifted by Dr. Frederick F. Lang from the Brain Tumor Center (The University of Texas MD Anderson Cancer Center). QPP7 and 005 GSCs were provided by Dr. Jian Hu (The University of Texas MD Anderson Cancer Center) and Dr. Samuel D. Rabkin (Massachusetts General Hospital), respectively. JURKAT cells were cultured in human plasma-like medium (Thermo Fisher Scientific, #A4899101) containing 10% FBS and 1:100 antibiotic-antimycotic. All cells were confirmed to be mycoplasma-free and were maintained at 37° C. and 5% CO2. Conditioned media (CM) were collected from number-matched control, TFPI2 shRNA knockdown, or TFPI2-overexpressing cells after culturing for another 24 hrs in FBS- and growth factor-free culture medium.
Primary mouse CD8+ T cells were isolated from the spleens of C57BL/6 mice using a CD8+ T cell isolation kit (Miltenyi Biotec, #130-104-075) according to manufacturer's instructions. Isolated CD8+ T cells were cultured in RPMI 1640 media (Gibco, #22400-089) containing 10% FBS, 50 μM 2-Mercaptoethanol (Sigma, #M7522), and 1:100 antibiotic-antimycotic. Then, primary mouse CD8+ T cells were expanded by 30 U/ml mouse IL-2 (Miltenyi Biotec, #130-120-662) and activated by Dynabeads® mouse T-activator CD3/CD28 (Gibco, #11456D) at bead-to-cell ratio of 1:1 before analysis and co-culture studies.
Adult human primary microglia (PrhMG) were isolated (Ref. 38; incorporated by reference in its entirety). Fresh cortical tissues from frontal poles of postmortem human brains were harvested at autopsy. Brain tissues were rinsed with cold RPMI 1640 medium on ice. After removing meninges and visible blood vessels, blocks of cortex were minced and incubated in a 0.25% trypsin EDTA solution for 1 hr. The suspension was passed through 100 mm and 40 mm Nitex mesh filters and resuspended in microglia media (ScienCell Research Laboratories, #1901) with 5% FBS, 100 mg/ml antibiotic-antimycotic, 1.5 ml/500 ml Primocin (InvivoGen, #ant-pm-05), 1% microglia growth supplement (ScienCell, #1952), and 10 ng/ml granulocyte-macrophage colony-stimulating factor (PeproTech, #315-03). Cells were then labeled with CD11b magnetic beads and MACS method was used to select CD11b+ cells. After selection, cells were cultured in poly-D-lysine coated plates.
Brains of neonate mice were homogenized in 10 ml HBSS on ice. Brain-derived microglia were then purified using 30/70 Percoll (GE Healthcare, #17-0891-01) with gradient separation for 30 min at 4° C. The isolated microglia were cultured in poly-D-lysine coated plates with microglia media containing 5% FBS, 100 mg/ml antibiotic-antimycotic, 1.5 ml/500 ml Primocin, 1% microglia growth supplement, and 10 ng/ml granulocyte-macrophage colony-stimulating factor. The purity of microglia was determined by flow cytometry analysis, in which more than 90% of cells are CX3CR1 positive.
shRNAs targeting human TFPI2, mouse Tfpi2, and mouse Itgav in the pLKO.1 vector (Sigma, #SHC001) were used. Lentiviral particles were generated (Ref. 61; incorporated by reference in its entirety). The following mouse and human shRNA sequences (TFPI2: #1 TRCN0000373908, #2 TRCN0000373822, #3 TRCN0000072725; Tfpi2: #2 TRCN0000271824, #3 TRCN0000271717; and Itgav: #2 TRCN0000066588, #5 TRCN0000066590, #6 TRCN0000066591) were selected following validation. Lentiviral particles were generated (Refs. 11-12, 61; incorporated by reference in their entireties). 8 mg of the shRNA and packaging plasmids (4 mg psPAX2 and 2 mg pMD2.G) were transfected using Lipofectamine 2000 (Invitrogen, #13778150) into 293T cells. Supernatant with lentiviral particles was collected at 48 and 72 hrs after transfection. GSCs or microglia were infected with lentiviral supernatant containing 10 mg/ml polybrene (Millipore, #TR1003-G) and then selected using 2 mg/ml puromycin (Millipore, #540411). Proteins were extracted to assess the expression of TFPI2 or CD51 by immunoblots. For rescue experiments, TFPI2 shRNA knockdown QPP7 cells were transfected with a human TFPI2 construct that is resistant to TFPI2 shRNAs.
CRISPR KO of TFPI2 in human GSC272 cells was produced from the transfection of sgRNA (GenScript, #SC1969) targeting the TFPI2 protein sequence (TTCTCCGTTACTACTACGAC) and Cas9 Nuclease (GenScript, #Z03621-0.5). 50 pmol of Cas9 Nuclease and 100 pmol of sgRNA were incubated in 125 ml of Opti-MEM media (Gibco, #31985088) for 10 mins at room temperature before combining with an additional 125 ml of Opti-MEM media containing Lipofectamine™ CRISPRMAX™ (Invitrogen, #CMAX00001) transfection reagent. GSC272 cells were incubated with the cocktail of sgRNA, Cas9 nuclease, and transfection reagent for 48 hrs. Following the incubation period, transfected GSC272 cells were expanded in culture, then taken for GFP tag cell-sorting using FACS ARIA 4-Laser Sorter. GFP+ cells were returned to normal cell culture conditions for expansion. CRISPR KO efficiency was verified using immunoblot to assess TFPI2 expression.
For overexpression, GSCs were transfected with a TFPI2 overexpression plasmid generated via a cloning methodology. A pCMV6-Entry vector containing the TFPI2 (Myc-DDK-tagged) sequence (Origene, #RC202760) and a pLenti-C-mGFP Lentiviral Gene Expression Vector (Origene, #PS100071) were digested using restriction enzymes corresponding to the open reading frames. Restriction enzymes Mlul (Promega, #R6381) and Sgfl (Promega, #R7103) were mixed with Restriction Digest Buffer C to enhance digestion efficiency. Following digestion, each mixture was incubated with antarctic phosphatase for 3 hrs to prevent self-ligation. T4 DNA ligase (Thermo Scientific, #B69) was included in a ligation mixture to induce the ligation of the TFPI2 insert into the pLenti-C-mGFP Lentiviral Gene Expression Vector. Ligation mixture was transformed into high-efficiency chemically competent E. coli cells (Thermo Scientific, #C737303), and recovered in LB broth (Fisher BioReagents, #BP9723). Post-recovery, LB broth containing E. coli transformants were plated on LB selection plates containing 34 μg/ml chloramphenicol (Fisher BioReagents, #BP904) for selection of clones containing the TFPI2 ORF in the pLent-C-mGFP Lentiviral Gene Expression Vector. After 16 hrs of incubation at 37° C., three colonies were picked from the selection plates for inoculation in LB Broth supplemented with 34 μg/ml chloramphenicol to maintain selection, and then further purified for plasmid DNA using a QIAprep Spin Miniprep Kit (Qiagen, #27106). Purified plasmids were then transfected into GSCs using lentiviral transfection methodology (Ref. 12; incorporated by reference in its entirety).
For transwell migration assay, HMC3 microglia, SIM-A9 microglia, and PrhMG were suspended in serum-free culture medium and seeded into transwell 24-well plates with permeable polycarbonate membrane inserts (5.0 μm, Corning, #07-200-149). GSC conditioned media (CM), or basal cell culture medium with TFPI2 recombinant protein (BioVision, #7481-10) were added to the remaining receiver wells. The STAT6 inhibitor AS151749 (Selleck Chemicals, #S8685) was used to study the role of STAT6 in microglia migration. The CD51 inhibitor MK-0429 (MedChemExpress, #HY-15102) was used to study the role of CD51 in TFPI2-induced microglia migration. After 10 hrs, the migrated microglia were fixed and stained with crystal violet (Sigma, #C-3886). The number of transferred cells was counted using ImageJ (NIH, Bethesda, ML).
The scratch-wound assay was performed (Ref. 74; incorporated by reference in its entirety). For tracking single cell movement, microglia were cultured in 96-well plates (Corning, #3599) with different treatments. Incucyte Live Cell Analysis System was used to record cell movement over time. The time-lapse images were reconstructed and analyzed using TrackMate (Refs. 75-76; incorporated by reference in its entirety). The speed of each individual cell was calculated by processing images with LoG detector and Linear Assignment Problem (LAP) tracker.
The protein expression in cells was determined by western blotting analysis (Ref. 61; incorporated by reference in its entirety). Cells were homogenized in RIPA lysis buffer containing phosphatase/protease cocktail inhibitors (Cell Signaling Technology, #5870). The protein concentration was determined by BCA Protein Assay Kit (Thermo Fisher Scientific, #P123225). Protein solution was mixed with the Leammli sample buffer and heated at 95° C. before loading to SurePAGE gels (GenScript, #M00653). The gels were run at a constant voltage of 80 V and then transferred to 0.2 μm nitrocellulose membrane (Bio-Rad, #1620112) using the Trans-Blot Turbo system (Bio-Rad) with a preprogrammed standard protocol for 30 min. Nitrocellulose membrane was incubated with 5% dry milk to block any unspecific staining. Primary antibodies, including TFPI2 (Abcam, #ab186747), CD133 (Abeam, #ab19898), SOX2 (Abcam, #ab97959), cleaved caspase-3 (Cell Signaling Technology, #9661), P-STAT6 (Abcam, #ab28829), STAT6 (Cell Signaling Technology, #9362), P-STAT3 (Cell Signaling Technology, #9145), STAT3 (Cell Signaling Technology, #9139), P-AKT (Cell Signaling Technology, #4058S), AKT (Bio-Rad, #VMA00253), P-JNK (Cell Signaling Technology, #4668S), JNK (Cell Signaling Technology, #9252S), PLCγ1 (Cell Signaling Technology, #5690S), P-PLCγ1 (Cell Signaling Technology, #8713S), PKCz (Cell Signaling Technology, #9368S), P-PKCz (Cell Signaling Technology, #9378S), Integrin aV/CD51 (Cell Signaling Technology, #4711S), and Actin (Cell Signaling Technology, #3700), were incubated with the membrane overnight. HRP-linked, anti-mouse (Cell Signaling Technology, #7076) or anti-rabbit (Cell Signaling Technology, #7074S) secondary antibodies were applied to the nitrocellulose membrane accordingly. After washing, nitrocellulose membrane was incubated with ECL substrate and imaged under ChemiDoc Touch Imaging System (Bio-Rad).
Co-IP was performed using Invitrogen™ Dynabeads™ Protein G Immunoprecipitation Kit (Thermo Fisher Scientific, #10007D). Briefly, human or mouse microglia were treated with 20 ng/ml TFPI2 recombinant protein for 1 hr. RIPA lysis buffer was then used to extract protein after washing with PBS. Magnetic beads (1.5 mg) were separated from the solution using a magnetic stand. 10 mg TFPI2 antibody (Abcam, #ab186747), CD51 antibody (Cell Signaling Technology, #4711S), or rabbit IgG (isotype control, Cell Signaling Technology, #2729) was prepared in 200 ml of antibody binding and washing Buffer. Antibody or IgG was incubated with magnetic beads for 10 min at room temperature with rotation. After removing the supernatant, antibody/IgG-conjugated magnetic beads were incubated with cell lysis buffer for 2 hrs at room temperature with rotation to immunoprecipitate TFPI2- or CD51-interacting antigens. The magnetic bead-antibody-antigen complex was washed three times using 200 ml washing buffer and then eluted with the elution buffer. The complex was loaded into 4-12% SurePAGE gels to evaluate target protein expression after Co-IP.
PrhMG were incubated with 20 ng/ml TFPI2 recombinant protein for 1 hr to allow the binding between TFPI2 and its potential receptors. After incubation, cells were pelleted, and membrane-associated proteins were extracted using the Mem-PER™ Plus Membrane Protein Extraction Kit (Thermo Fisher Scientific, #89842). 5×106 PrhMG were resuspended by scraping. After centrifugation at 300 g for 5 min, cells were suspended in cell wash solution and recentrifuged, and then were incubated with 0.75 ml permeabilization buffer for 10 min at 4° C. with constant mixing. Permeabilized cells were centrifuged for 15 min at 16,000 g to remove the supernatant containing cytosolic proteins. The cell pellets were then incubated with 0.5 ml solubilization buffer at 4° C. for 30 min with constant mixing. Solubilized membrane and membrane-associated proteins were isolated by centrifuging at 16,000 g for 15 min at 4° C. Extracted membrane-associated proteins were proceeded for IP-MS pull-down assay. A total of 50 mg cell membrane lysate was incubated with 10 ml TFPI2 antibody (Abcam, #ab186747) or IgG control (Cell Signaling Technology, #2729) for 12 hrs with gentle rotation at 4° C. 100 ml Protein A/G PLUS-Agarose (#sc-2003, Santa Cruz Biotechnology) were mixed with each sample on ice. The lysate bead mixture was incubated at 4° C. under rotary agitation for 4 hrs. The tubes were centrifuged to remove supernatant. The beads were then washed in lysis buffer three times to remove non-specific binding. After removing wash buffer, the complex was washed with pre-urea wash buffer (50 mM Tris pH 8.5, 1 mM EGTA, 75 mM KCl) to remove all residual supernatant. The protein was eluted with the elution buffer (20 mM Tris pH 7.5, and 100 mM NaCl) and sent to the Northwestern Proteomics Core for protein identification. Samples were in-gel digested (trypsin) and subject to mass spectrometry analysis. IP pulled-down samples were analyzed by LC-MS/MS using a Dionex UltiMate 3000 Rapid Separation nanoLC coupled to the Orbitrap Elite Mass Spectrometer (Thermo Fisher Scientific Inc, San Jose, CA). Raw files were searched against a human database and submitted to Mascot and Scaffold searches.
Calcium mobilization in microglia was determined using the Fluo-4 Direct™ Calcium Assay Kit (Thermo Fisher Scientific, #F10472). Briefly, microglia with or without CD51 knockdown were seeded into 96-well plates overnight at a concentration of 5000 cells/well with 50 ml complete cell culture media. CD51 inhibitor MK-0429 at distinct concentrations was added into corresponding wells for 1 hr before the testing. 2× Fluo-4 Direct™ calcium reagent loading solution (50 ml) was directly added to wells containing cells in culture media. The plate was incubated at 37° C. for 30 min. 20 ng/ml TFPI2 recombinant protein was added to the plate. Fluorescence kinetic was immediately measured in a microplate reader using instrument settings for excitation at 494 nm and emission at 516 nm.
Cells were pelleted, and RNA was isolated with the RNeasy Mini Kit (Qiagen, #74106). RNA was quantified by NanoDrop spectrophotometers and then reverse-transcribed into cDNA by using the All-In-One 5×RT MasterMix (Applied Biological Materials, #G592) in T00 Thermal Cycler (Bio-Rad). RT-qPCR was performed using SYBR Green PCR Master Mix (Bio-Rad, #1725275) in CFX Connect Real-Time PCR Detection System (Bio-Rad). The expression of each gene was normalized to housekeeping genes. RT-PCR primers are listed in Table 2.
Immunohistochemistry and immunofluorescence were performed using a standard protocol (Ref. 61; incorporated by reference in its entirety). Paraffin sections were baked at 65° C. for 2 hrs prior to staining. Sections were then subjected to xylene and ethanol in gradient concentrations for deparaffinization and rehydration. Permeabilization buffer containing 0.3% (v/v) Triton X-100 and 1% (v/v) H2O2 was added for 30 min to block endogenous peroxidase. Antigen retrieval was conducted by boiling sections in the sodium citrate buffer (0.01 M, pH=6). After washing with PBS, tissues were blocked by 5% goat serum for 30 min. Sections were incubated with primary antibody for 1 hr at room temperature and then overnight at 4° C. Sections were subjected to corresponding secondary antibodies or rabbit-on-rodent HRP-polymer (Biocare, #RMR 622) for 1 hr at room temperature. For immunofluorescence, cell nucleus was counterstained with DAPI/anti-fade mounting medium (Vector Laboratories, #H-1200-10). Protein signal was captured using Nikon AX/AX R Confocal Microscope System in the Center for Advanced Microscopy (CAM) at Northwestern University. The relative intensity of protein was determined by Image J. For immunohistochemistry, tissues were incubated with DAB Quanto (Epredia, #TA125QHDX). The nucleus was then stained by hematoxylin. After dehydration, the images were captured by using EVOS Cell Imaging System. ImageJ with IHC profiler plug-in was used for scoring positive signals (Ref. 77; incorporated by reference in its entirety).
H&E staining was performed using the staining kit (Abcam, #ab245880) Paraffin sections were baked at 65° C. for 2 hrs prior to staining. Sections were then subjected to xylene and ethanol in gradient concentrations for deparaffinization and rehydration. After washing two times in distilled water, sections were incubated with hematoxylin, Mayer's (Lillie's Modification) for 5 min. Slides were rinsed in distilled water to remove excess stains. Sections were incubated with the Bluing Reagent for 15 sec, and then the Eosin Y Solution (Modified Alcoholic) for 3 min. After washing, slides were dehydrated in three changes of absolute alcohol. The images of tissue sections were captured by TissueFAXS in the Center for Advanced Microscopy (CAM) at Northwestern University.
After starvation for 24 hrs followed by TFPI2 recombinant protein treatment for 1 hr, HMC3 cell lysate was prepared using a lysis buffer with 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 10 mg/ml pepstatin. Phosphorylation profiles of kinases were determined using Proteome Profiler Human Phospho-Kinase Array Kit (Bio-Techne, #ARY003C) as the instruction manual. Signal was detected under ChemiDoc Touch Imaging System (Bio-Rad).
At the endpoint of experiments, mice were sacrificed to harvest their brains and spleens. Immune cells in the brain and spleen were isolated (Ref. 11; incorporated by reference in its entirety). After perfusion with PBS, brains and spleens were homogenized in 10 ml HBSS or 2% FBS RPMI 1640 media on ice, respectively. Brain/spleen-derived immune cells were then purified using 30/70 Percoll (GE Healthcare, #17-0891-01) with gradient separation for 30 min at 4° C. Spleen cells were treated with 1 ml ACK buffer (Thermo Fisher, #A1049201) for 5 min to remove red blood cells. After deactivation in complete RPMI 1640 media, cells were filtered through 70 μm strainers (Thermo Fisher, #08-771-2) and ready for further analysis.
T cell-mediated cytotoxicity was determined by Cytotox96 non-radioactive cytotoxicity assay kit (Promega, #G1780) based on the release of lactate dehydrogenase (LDH). After expansion and activation, mouse primary CD8+ T cells or human JURKAT T cells were collected. About 105 T cells were co-cultured with SIM-A9 microglia or PrhMG (1:1 ratio) treated with TFPI2 recombinant protein in the presence or absence of STAT6 inhibitor AS1517499 for 24 hrs. T cells (effector cells) were then collected for further co-culturing with CT2A cells or GSC272 cells (target cells) at different ratios (10000:1000, 5000:1000, 2500:1000, 1250:1000, 620:1000, 310:1000, 160:1000, 80:1000, 40:1000, 20:1000) for 5 hrs. Experimental groups and corresponding control groups, including effector cell spontaneous LDH release, target cell spontaneous LDH release, target cell maximum LDH release, volume correction control, and culture medium background control were set up following the manufacturer's instructions to evaluate T cell-mediated cytotoxicity on GSCs. Optical density (OD) was measured by a Biotek Synergy 2 SL Microplate Reader. The cytotoxicity was calculated by the formula: (experimental OD value-effector spontaneous OD value-target spontaneous OD value)/(target maximum OD value-target spontaneous OD value).
Cell apoptosis was evaluated using Apotracker Green (BioLegend, #427402) (Ref. 78; incorporated by reference in its entirety). 105 cells were collected and stained with Apotracker (1:10 dilution) in 100 ml cell staining buffer. 5 ml propidium iodide (PI) solution (BioLegend, #421301) was added for labeling late apoptotic and necrotic cells. After washing with PBS twice, FITC and PI signals were analyzed in BD FACSymphony flow cytometer. The gating strategy for apoptosis analysis is shown in
For membrane protein staining, the single-cell suspensions were washed with PBS and then incubated with fixable viability dye (Invitrogen, #5211229035) on ice for 10 min. Following washing with PBS, cells were incubated with the anti-CD16/CD32 cocktail (BioLegend, #103132) in 2% BSA PBS to block Fc receptors for 30 min on ice. Different antibody combinations, including Percp/Cy5.5 anti-mouse CD45 (BioLegend, #103132), AF488 anti-mouse CD3 (BioLegend, #100210), BV421 anti-mouse CX3CR1 (BD Bioscience, #567531), BUV395 anti-mouse CD4 (BD Bioscience, #740208), BV711 anti-mouse CD8 (BioLegend, #100747), PE/Cy7 anti-mouse/human CD11b (BioLegend, #101216), PE anti-mouse CD68 (BD Bioscience, #566386), PE anti-mouse PD1 (BioLegend, #135205), AF647 anti-mouse CD206 (BD Bioscience, #565250), AF647 anti-human CD206 (BioLegend, #321116), PE anti-human CD69 (BioLegend, #310906), PE/Cy7 anti-mouse CD69 (BioLegend, #104512), and BV650 anti-human HLA-DR (BioLegend, #307650) were added to cell suspensions for 30 min on ice. CD51 (Cell Signaling Technology, #4711S) was added to cell suspension with corresponding antibody combinations for 30 min on ice, followed by incubation of goat anti-rabbit IgG cross-adsorbed secondary antibody (AF594) for 30 min. After washing in PBS, cells were fixed in fixation buffer (BioLegend, #420801) overnight. Samples were read in BD FACSymphony flow cytometer and analyzed in FlowJo v10.8.1.
Intracellular protein staining was performed separately or followed by cell surface marker staining. Cells were fixed in fixation buffer at room temperature for 20 min. After washing in permeabilization buffer (0.1% Triton X-100 in PBS) for two times, cells were incubated with AF700 anti-human IFN-7 (BioLegend, #502520) or FITC anti-human/mouse ARG1 (BioLegend, #IC5868F) for 20 min at room temperature. Then cell suspensions were rewashed in permeabilization buffer and read in BD FACSymphony flow cytometer.
Phospho-flow samples were fixed in a fixation buffer at room temperature for 15 min. Cells were then stained with a fixable viability dye and permeabilized in 90% cold methanol for 15 min on ice. After blocking Fc receptors, cells were resuspended with P-STAT6 primary antibody for 1 hr on ice. Cells were washed in PBS three times and incubated with goat anti-rabbit IgG cross-adsorbed secondary antibody (AF594). Cells were centrifuged at 300 g for 5 min to remove supernatant and washed in PBS twice before the flow cytometry analysis. The gating strategies for flow cytometry analyses are shown in
Cell proliferation was assessed using the CellTrace CFSE Cell Proliferation Kit (Invitrogen, #C34554). 106 cells were harvested and incubated with CFSE working solution (1:1000) for 20 min at 37° C. The staining was stopped by adding complete cell culture media. After washing, cells were continuously cultured for 5 days in the dark. At the end of the incubation, cells were collected by spinning down at 1500 rpm for 5 min. The cell pellet was resuspended in cell staining buffer for flow cytometry analysis. The percentage of CFSE positive peaks over the undivided peak (generation 0) was analyzed using the ImageJ software.
The tumorsphere formation assay was performed (Ref. 79; incorporated by reference in its entirety). GSCs were treated with different concentrations of recombinant TFPI2 protein for 24 hrs with or without inhibition of JNK or STAT3, or modified with TFPI21 KO, knockdown or overexpression. After treatment or genetic modifications, cells were centrifuged at 1500 rpm for 5 min and resuspended in the plain cell culture media without additional supplements. Cells were then seeded into a 96-wells plate at 100 cells/well. The number of tumorspheres in each well was imaged and quantified after 10 days.
The levels of TFPI2 in human plasma were measured by enzyme-linked immunoassay (ELISA) using the commercial TFPI2 kit (MyBioSource, #MBS2507217) following the manufacturer's instructions. OD value was measured by a Biotek Synergy 2 SL Microplate Reader.
Bulk RNA-Seq and scRNA-Seq Data Analysis
Extracted RNA from shC and shTFPI2 GSC272 cells were processed to Oligo-dT based library and analyzed using a NovaSeq 6000 instrument to generate RNA-seq dataset at The University of Chicago Functional Genomics (RRID:SCR_019196). Raw FASTQ data from two flowcells per sample was uploaded to Galaxy server (Ref. 80; incorporated by reference in its entirety). Trimmomatic was performed to read and trim paired-end reads. Read quality reports were generated with FastQC tool. HISAT2 program was used to align the data where hg38 was selected as the reference genome. Samtools merge was performed on BAM files to merge data from different flowcells. Gene expression was then determined by FeatureCounts. AnnotateMyIDs program was executed to generate the annotation file. Limma was used to calculate differential expression genes and generate DE table. The up-regulated and downregulated pathways were determined by GSEA analysis. scRNA-seq data of GSE131928 (Ref. 30; incorporated by reference in its entirety) was used for analyzing the expression of TFPI2 and stemness signature in malignant cells, and the scRNA-seq data of EGAS00001004422 (Ref. 81; incorporated by reference in its entirety) and GSE131928 (Ref. 30; incorporated by reference in its entirety) was used for analyzing microglia signature in GBM patient tumors with TFPI2 high versus low in GBM cells.
For bioinformatics analyses of human GBM patient tumor data, the microarray gene expression and survival data of TCGA dataset or other available datasets was downloaded from GlioVis: gliovis.bioinfo.cnio.es/. The analyses of gene expression, signature expression, correlation, survival, and the GSEA of interesting gene signatures in IDH-WT GBM patients were performed (Refs. 11-12, 61; incorporated by reference in their entireties).
Female C57BL/6 (#0000664), nude mice (#007850), and B6.12952(C)-Stat6tm1Gru/J mice (STAT6-KO; #005977) at 6 weeks of age were purchased from the Jackson Laboratory. Mice were grouped by 5 animals and maintained in a pathogen-free IVC System (San Diego, CA) for a week before the experiment. All animal experiments were performed with the approval of the Institutional Animal Care and Use Committee (IACUC) at Northwestern University. The intracranial xenograft tumor models were established (Refs. 11, 61; incorporated by reference in their entireties). Ketamine (100 mg/kg) and xylazine (20 mg/kg) were used for anesthetizing mice through intraperitoneal injection. Then a dental drill was used to open a small hole in the skull 1.2 mm anterior and 3.0 mm lateral to the bregma. A total volume of 5 mL cells in FBS-free DMEM was injected into the right caudate nucleus 3 mm below the surface of the brain using a 10 mL Hamilton syringe with an unbeveled 30-gauge needle. Total three doses of 20 mg/kg meloxicam were administrated before and after surgery through subcutaneous injection. After cancer cell implantation, mice were monitored for recording survival. Mice with neurological deficits or moribund appearance were sacrificed. Following the transcardiac perfusion with PBS and 4% paraformaldehyde (PFA), brains were removed and fixed in 4% PFA, and were processed for paraffin-embedded blocks or cryosectioning.
GBM patient peripheral blood plasma samples (n=55) were collected from the Northwestern Central Nervous System Tissue Bank (NSTB). Patient tumor samples (n=60) from surgically-resected IDH-WT GBMs were collected at the NSTB. All patients were diagnosed according to the WHO diagnostic criteria by neuropathologist Dr. Craig Horbinski. Detailed patient information is provided in Table 3. Control plasma samples (n=6) from healthy human blood were purchased from Solomonpark (#4345), which are commercially available anonymized and de-identified. According to The George Washington University Institutional Review Board and based on the guidelines from the Office of Human Research Protection, the conducted research meets the criteria for exemption #4 (45 CFR 46.101(b) Categories of Exempt Human Subjects Research) and does not constitute human research.
Statistical analysis was conducted using GraphPad Prism 9 (GraphPad Software, USA). In vitro and in vivo measurement data were presented as the means±SEM or SD. Comparison between two groups was evaluated using Student's t-test, and multiple comparisons among groups were evaluated using a one-way ANOVA test in Tukey's method. The survival analysis for animal models was performed using Log-rank (Mantel-Cox) test. Correlation analysis was performed using the Pearson test to determine the R and P values. Differences with a minimum of P<0.05 were indicated as statistically significant.
Pan-cancer analyses have shown that high stemness of cancer cells is correlated positively with increased immunosuppressive pathways (Ref. 25; incorporated by reference in its entirety). To identify potential associations between GSCs and immunity, correlation analyses were performed between tumor stromal and immune cell scores (Ref. 26; incorporated by reference in its entirety) and GSC signature (Ref. 27; incorporated by reference in its entirety) using The Cancer Genome Atlas (TCGA) GBM dataset. It was found that GSC signature correlated positively with both stromal and immune scores (
To investigate a possible relationship between TFPI2 expression and GSC self-renewal, TCGA GBM data was analyzed by comparing TFPI2-amplified patient tumors, that have elevated TFPI2 expression, versus non-amplified tumors for stemness signature. The result of this analysis showed that the stemness signature was enriched in TFPI2-amplified tumors (
To address the effects of reducing cellular TFPI2 in GSC sternness maintenance, TFPI2 was targeted by shRNA-mediated knockdown (
To further investigate the role of TFPI2 in GBM tumor in vivo, the shRNA knockdown system was utilized to deplete TFPI2 in QPP7 and CT2A tumors implanted into C57BL/6 mice and in GSC272 tumors implanted into nude mice. TFPI2 depletion significantly inhibited tumor growth (
To determine the molecular basis of TFPI2's effect on GSC self-renewal, RNA-seq profiling was performed on GSC272 cells with or without TFPI2 knockdown. Gene Set Enrichment Analysis (GSEA) on oncogenic signatures resulted in identification of 8 signatures that were influenced by TFPI2 (
TCGA GBM datasets (HG-U133A and Agilent-4502A) with TFPI2-high patient tumors versus TFPI2-low patient tumors and GSC272 RNA-Seq data with shTFPI2 versus shRNA control (shC) were analyzed. GSEA on oncogenic signatures resulted in identification of two key signatures that were influenced by TFPI2 expression (
Emerging evidence demonstrates that cancer cell stemness is directly correlated with immunosuppression across cancer types, including GBM (Refs. 7, 25; incorporated by reference in its entirety). CLOCK is a key transcription factor that is amplified in about 5% of GBM cases and acts as a positive regulator of GSC stemness and microglia infiltration (Ref. 11-12; incorporated by reference in their entireties). Analysis of TCGA GBM and LGG datasets demonstrated that TFPI2 amplification is mutually exclusive with CLOCK amplification (
Since TFPI2 is a secreted protein, experiments were conducted during development of embodiments herein to determine whether GSC-derived TFPI2 promote microglia infiltration. Using transwell migration assays, we found that recombinant TFPI2-supplemented media dramatically increased the migration of distinct types of microglia (
To investigate the molecular basis of TFPI2-induced microglia migration, a human phospho-kinase antibody array was used, the results from which showed STAT6 and AKT as being activated in HMC3 microglia in association with TFPI2 recombinant protein treatment (
To further investigate the role of STAT6 in mediating TFPI2-induced microglia migration, microglia were isolated from wild-type (WT) and STAT6-KO mice and used in a transwell migration assay, the results of which showed that microglia isolated from STAT6-KO mice had impaired migration ability compared to microglia isolated from WT mice in response to TFPI2 treatment (
Microglia are a heterogeneous population of immune cells in the GBM TME and can be classified as immunostimulatory or immunosuppressive phenotype (Ref. 8; incorporated by reference in its entirety). Flow cytometry results showed that CM from GSC262 and GSC23 polarized PrhMG towards an immunosuppressive phenotype by increasing the percentage of CD206+ cells, and this effect was increased when using CM from GSC262 and GSC23 modified for increasing TFPI2 expression (
Experiments were conducted during development of embodiments herein to investigate whether TFPI2-induced microglia immunosuppressive polarization is regulated by STAT6 signaling using flow cytometry analysis for CD206 and ARG1 expression, with results showing that immunosuppressive microglia were increased by TFPI2 recombinant protein treatment. This increase was negated by genetic depletion of STAT6 in primary mouse microglia (
CD51 is a Functional TFPI2 Receptor that Mediates TFPI2-Induced Microglia Migration and Immunosuppressive Polarization.
To investigate whether TFPI2 exerts migratory and polarization effects through interaction with microglia membrane proteins, PrhMG were incubated with TFPI2 recombinant protein (
Experiments were conducted during development of embodiments herein to examine whether CD51 inhibition influences TFPI2 effects on microglia. TFPI2-induced phosphorylation of STAT6 was impaired by genetic and pharmacologic inhibition of CD51 in mouse and human microglia (
Inhibition of the TFPI2-CD51-STAT6 Signaling Axis Activates Antitumor Immune Response and Synergizes with Anti-PD1 Therapy.
Experiments were conducted during development of embodiments herein to determine whether inhibiting microglia immunosuppressive effects via blockade of the TFPI2-CD51-STAT6 signaling pathway would influence T cell-mediated anti-tumor immune responses in GBM. shC and shTfpi2 QPP7 GSCs and CT2A cells were implanted into the brains of immunocompromised nude mice. The anti-tumor effects associated with TFPI2 depletion were reduced in nude mice when compared against results from immunocompetent C57BL/6 mice (
Since the CD51-STAT6 signaling axis plays a prominent role in promoting TFPI2-induced microglia migration, immunosuppressive polarization, and anti-tumor immune response, experiments were conducted during development of embodiments herein to explore the impact of this signaling on tumor growth and ICI therapy efficacy in immunocompetent GBM mouse models. A second immunocompetent GBM mouse model, 005 GSC, that expresses relatively high levels of TFPI2 (
TFPI2 is a Prognostic Biomarker that Correlates with GSC Stemness and Microglia Abundance in GBM.
Immunohistochemistry staining for TFPI2, SOX2, CD133, CX3CR1, Ki67, and cleaved caspase 3 was performed in GBM patient tumors (
Since TFPI2 encodes a secreted protein, its protein level was analyzed in the plasma of 6 healthy controls and 55 GBM patients, with results showing that plasma levels of TFPI2 were significantly higher in GBM patients than healthy controls (
The following references, some of which are cited above, are herein incorporated by reference in their entireties.
The present application claims priority to U.S. Provisional Patent Application Ser. No. 63/582,715 filed Sep. 14, 2023, which is hereby incorporated by reference in its entirety.
This invention was made with Government support under grant numbers NS124594 and CA240896 awarded by the National Institutes of Health, and grant number W81XWH-21-1-0380 awarded by the Department of Defense. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63582715 | Sep 2023 | US |
