Patent Application
20240309063
The present invention refers to an isoform of the TGF-B receptor II, codifying polynucleotides, vectors, cells, transformed peptides, and fusion peptides, method and uses. More specifically, it refers to an isoform of the TGF-beta receptor II comprising a sequence of about 80 amino acids and lacking a transmembrane domain. The isoform comprises the amino acid sequence of SEQ ID No. 12. The isoform may have the amino acid sequence set forth in SEQ ID No. 2 or sequences having at least 85% sequence identity to the sequence set forth in SEQ ID No. 2.
Transforming growth factor-beta (TGF-β) is abundant in bone matrix and has been shown to regulate the activity of osteoblasts and osteoclasts in vitro and in vivo. Human Adipose derived Mesenchymal Stromal Cells (ASC) are precursors of osteoblasts, adipoblasts and chondroblasts. Thus, studies initially focused on the secretion of cytokines by ASC which have a profound effect in bone remodeling, such as Tgf-β1, Osteoprotegerin (OPG) and Hepatocyte Growth Factor (HGF).
TGF-β1 concentrations are high in subchondral bone from humans with osteoarthritis. High concentrations of TGF-β1 induced formation of nestin-positive mesenchymal stem cell (MSC) clusters, leading to formation of marrow osteoid islets accompanied by high levels of angiogenesis (Zhen G, et al. (Nat Med. 19: 704-12, 2013). It has been found that transgenic expression of active TGF-β1 in osteoblastic cells induced osteoarthritis, whereas inhibition of TGF-β activity, by means of a TβRII dominant negative receptor, in subchondral bone, attenuated the degeneration of articular cartilage leading to less development of osteoarthritis. It has also been reported that mice expressing a dominant negative type II TGF-β receptor (TβRII-DN) in osteoblasts, show decreased TGF-β responsiveness in osteoblasts and increased bone volume, demonstrating that endogenous TGF-beta acts directly on osteoblasts to regulate bone remodeling, structure and biomechanical properties (Filvaroff, E. et al. Development, 126: 4267-4279, 1999). In addition, TGF-β also regulates osteoclastogenesis and osteoclast survival, in part through the induction of osteoprotegerin (OPG), a protein known to inhibit osteoclast formation and function (Thirunavukkarasu K, et al. J. Biol. Chem. 276:36241-36250, 2001).
Transgenic mice that overexpress the dominant-negative type II TGF-β receptor (dnTgfbr2) in skeletal tissue exhibit progressive skeletal degeneration (Buckwalter J A, et al. Clin Orthop Relat Res 423: 7-16, 2004). The articular chondrocytes in the superficial zone of cartilage tissue become hypertrophic with increased type X collagen expression. Loss of proteoglycan and progressive degradation of cartilage tissue have been observed in 6-month-old mice which strongly resemble human osteoarthritis (OA) (OA-like) (Serra R, et al. J Cell Biol 139: 541-552, 1997). TGF-β signaling plays a critical role not only in the regulation of chondrocyte homeostasis during cartilage destruction, but also in the manipulation of subchondral bone cell behavior during osteophyte formation, another feature of OA (van der Kraan P M, et al. Osteoarthr Cartilage 15: 237-244, 2007).
The role of the TGF-β signaling pathway in osteophyte formation was further explored by blocking studies using specific TGF-β inhibitors. Several groups demonstrated that ablation of endogenous TGF-β activity, by intra-articular overexpression of soluble TGF-β type II receptor extracellular domain or Smad7, suppresses osteophyte formation in experimental murine OA models (Scharstuhl A, et al. J Immunol 169: 507-514, 2002). These observations clearly demonstrate that TGF-β plays a dominant role in the induction of osteophytes, at least in murine OA models.
In vivo, TGF-β1 also induces angiogenesis (Madri J A, et al. J Cell Biol. 106: 1375-1384, 1988; Roberts A B, Proc Natl Acad Sci USA. 83: 4167-4171, 1986; Yang E Y, et al. J Cell Biol. 111: 731-741, 1990.). In OA, high TGF-β1 levels are also accompanied by high levels of angiogenesis. Hepatocyte growth factor (HGF) is a potent mitogen, morphogen, and motogen for a variety of cells, mainly epithelial cells. Increased expression of the HGF/HGF-receptor system in osteoarthritic cartilage, suggest a regulatory role in the homeostasis and pathogenesis of human joint cartilage (Pfander D, et al. Osteoarthritis Cartilage. 7: 548-59, 1999).
Previous studies have shown that TGF-β can promote angiogenesis and tumor invasion via stimulation of HGF expression (Chu S H, et al. J Neurooncol., 85: 33-38, 2007; Lewis M P, et al. Br J Cancer 90: 822-832, 2004)). Conversely, TGF-β has also been shown to inhibit HGF transcription, potentially through binding of a TGF-β inhibitory element located approximately 400 bp upstream of the HGF transcription start site (Liu Y, et. al. J Biol Chem., 269: 4152-4160, 1994; Plaschke-Schlütter A, et al. J Biol Chem., 270: 830-836, 1995), and abrogation of this effect leads to cancer development (Cheng N, et al. Cancer Res. 67: 4869-4877, 2007).
Quinolones (QNs) antibiotics such as Ciprofloxacin (CPFX) were widely used in clinical practice owing to their wide spectrum antibacterial activity and high degree of bioavailability. They were not approved for use in children and adolescents due their toxic effects on joint cartilage of immature animals (Cuzzolin L, et al. Expert Opin Drug Saf 1: 319-24, 2002). Quinolones, administered systemically, caused arthropathy and tendinopathy when given during the growth phase (Sendzik J, et al. Int J Antimicrob Agents 33: 194-200, 2009.). It was reported that Ciprofloxacin decreased thickness of articular cartilage of the femoral condyle, inhibit proliferation of cultivated chondrocytes and secretion of soluble proteoglycans in a concentration- and time-dependant manner in juvenile rats (Li, P. et al. Arch. Pharmacol. Sin. 25: 1262-1266, 2004).
Chondrocyte cluster formation is a feature of all mechanical and chemical OA models (Moriizumi T, et al. Virchows Arch B Cell Pathol Incl Mol Pathol., 51: 461-474, 1986; van der Kraan P M, et al. Am J Pathol., 135:1001-1014, 1989). Animals with quinolone arthropathy showed cavities in the middle zone of the articular cartilage containing necrotic chondrocytes. After 14 days, many of the lacunae in defective areas contained chondrocyte clusters. When treated for 14 days, and after a 14-day recovery period, territorial matrix had been deposited around individual chondrocytes within the clusters, indicating that in immature joints there is a certain degree of spontaneous repair by cluster cells (Sharpnack D D, et al. Lab Anim Sci., 44: 436-442, 1994). It has been shown that TGF-β1 is activated in the subchondral bone in response to altered mechanical loading in an anterior cruciate ligament transection (ACLT) osteoarthritis mouse model (Zhen G, et al. Nat Med. 19: 704-12, 2013). Additionally, CPFX was found to up-regulate TGF-β1 production by HT-29 cells and its anti-proliferative effect was abolished when TGF-β1 was blocked (Bourikas L A, et al. Br J Pharmacol. 157: 362-70, 2009).
Adipose derived stem cells (hASCs) express cytokines such as IL-6, GM-CSF and Flt3-ligand (Tholpady S S, et al. Clin Plast Surg 33: 55-62, 2006; Katz A J, et al. Stem Cells. 23: 412-23, 2005; Schäfer A, et al. Stem Cells 25: 818-827, 2007). These cytokines are regulated by TGF-β1 either negatively (GM-CSF, SCF and Flt3-ligand) (Jacobsen S E, et al. J Immunol., 151: 4534-4544, 1993; Jacobsen S E, et al. Blood 87: 5016-5026, 1996) or positively (IL-6, TPO) (Ramsfjell V, et al. J Immunol. 158: 5169-5177, 1997.). Recently, overexpression of a dominant negative mutant of the human TβRII receptor (TβRII-DN) in mammalian cells has been shown to be very effective in blocking TGF-β1 action. This mutant, based on the isoform A of the receptor, is capable to bind TGF-β1 but signaling is disrupted due to the absence of a serine/threonine kinase domain. TβRIIA-DN has been shown to disrupt TGF-β1 mediated signaling allowing the study of the behavior of different cell types in the absence of either a paracrine or an autocrine effect of the cytokine (Fan X, et al. The Journal of Immunology 168: 755-762, 2002.).
Various documents disclosing different TGF-β1 receptors, chimerics, fusion proteins, domains, are known, for example, EP0975771, WO 2008/157367, US 2006/0247198, U.S. Pat. No. 6,001,969, and WO 94/09815.
The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 1850233_sequence_listing.xml, created Feb. 20, 2024, which is 13, 660 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.
A soluble isolated isoform of the TGF beta II receptor is provided comprising a sequence of about 80 amino acids and lacking the transmembrane domain; wherein the isoform would be acting as a TGFβ-1 agonist. In a preferred embodiment, the amino acid sequence of the isoform has at least 85%, 90%, 95%, or 99% identity with the amino acid sequence set forth in SEQ ID No. 2. The isoform comprises within its sequence the peptide disclosed in SEQ ID No. 12.
A polynucleotide encoding a soluble isoform of the TGF beta II receptor is provided, which in a preferred embodiment has at least 90%, 95%, or 99% identity with the nucleotide sequence of SEQ ID No. 1. In another preferred embodiment, the polynucleotide further comprises a Kozak sequence.
A fusion peptide is provided comprising an isoform of the TGF beta II receptor fused to a ligand. In a preferred embodiment the isoform is an amino acid sequence having at least 85% sequence identity to SEQ ID No. 2 and the ligand is the Fc of an immunoglobulin.
An antibody binding the soluble isoform of the TGF beta II receptor is provided. In a preferred embodiment, the antibody binds the amino acid sequence shown in SEQ ID No. 12.
A method of treating diseases associated to TGF-β dysregulation is provided, comprising administering to a mammal in need thereof the soluble isoform of the TGF beta receptor.
A method of treating diseases associated to TGF-β dysregulation is provided, comprising administering to a mammal in need thereof an antibody binding the soluble isoform of the TGF beta II receptor. In a preferred embodiment the antibody recognizes and binds the amino acid sequence shown in SEQ ID No. 12. The associated diseases may be selected from any disorder related to dysregulation of TGF-β signals, such as cancer, fibrosis, and cardiovascular diseases; metabolic and musculoskeletal defects, mutations in TβRII (TGFBR2 gene), for example, Loeys-Dietz syndrome (LDS), Marfan syndrome type 2 (MFS2), or different aneurisms (FTAAD).
FIG. 7 shows TβRII splicing variant mRNA profiles in human leukocyte subsets, such as granulocytes, T-lymphocytes (CD3+), B lymphocytes (CD19+), and monocytes (CD14+);
FIG. 9A shows overexpression of TβRII-SE in A549 cellsas results of a flow cytometry analysis showing the percentage of eGFP expressing A549 cells transduced with a lentiviral vector encoding TβRII-SE (Lt-TβRII-SE) and control vectors;
FIG. 10A shows the results of a proliferative MTT assay. A): A549 cells untransduced (UT) and transduced with Lt-TβRII-SE, Lt-TβRIIA-DN, and Lt-eGFP, treated with 0.4 nM TGFB-1 and untreated;
A variant or isoform of the TGF beta receptor II is disclosed, which is expressed in human cells referred to herein as endogenous soluble TβRII (TβRII-SE) and that contrarily to other isoforms acts like a TGF-β1 agonist.
By using specific primers, a region of the human TβRII mRNA from T-lymphocytes only encoding the extracellular (ECD) and the transmembrane (TMD) domains and excluding the intracellular domain (ICD) was initially amplified by RT-PCR, (
After the PCR reaction, DNA products were cloned into the pGEM-T Easy plasmid. Plasmids were digested with Agel and Sall and revealed in an agarose gel the presence of clones with inserts of three different sizes (
DNA sequencing and BLAST alignment (NCBI) of all clones indicated that clones 3, 7, 8, 11, and 12 (582 bp) were identical to human TGF β receptor II variant A (TβRII-A). Additionally, clone 2 (657 bp) showed 100% identity with the isoform TβRII-B. Clone 10 (433 bp) was similar to the TβRII-A sequence but with an additional 149 bp deletion. In this clone, the last 62 bp encoded by exon II and the first 88 bp encoded by exon III were absent, TβRII-SE (SEQ ID No. 1) (
The alignment of the predicted amino acid sequence of all three isoforms (
This isoform differs in 12 amino acids at the carboxyl end compared to the membrane bound variants of TβRII (isoforms A and B). Due to this, and according to the predicted amino acid sequence, the TβRII-SE isoform of clone 10 lacks pivotal sites for the productive action of TGF-β such as amino acid 176 of SEQ ID No. 3 that contributes to the ligand-receptor binding through hydrophobic contact; amino acid E142 of SEQ ID No. 3 which forms hydrogen bonds with R25 of TGF-β increased affinity and determined binding specificity and amino acid C71 of SEQ ID No. 3 which forms a disulfide bridge with C54 of the same receptor (see
As previously mentioned, the new isoform is referred to as TβRII Soluble Endogenous (TβRII-SE). The TβRII-SE isoform is different from the secretable genetically engineered TβRII isoform. The latter is an artificial TβRII receptor with a truncated TβRII-A fused to the Fc region of human IgM and blocks the effects of TGF-β, thus acting as an antagonist (reference, R. J Akhurst. J. Clin. Invest. 109: 1533-3610, 2002).
To determine the theoretical molecular weight of the TβRII-SE isoform, post-translational modifications (PTM) predicted from the amino acid sequence (SEQ ID No. 2) were established by using different computer programs (Table 1). In this analysis, three glycation sites at K46, K52 and K78 (NetGlycate program) (Johansen, M. B.; Glycobiology 16: 844-853, 2006); three phosphorylation sites at S31, S59 and Y73 (NetPhos program) (Blom, N.; Journal of Molecular Biology 294: 1351-1362, 1999) and one site for sumoylation in K46 (SUMOplot™ program, ABGENT, CA, USA) were identified. On the other hand, sites for sulfonation, C-mannosylation, O-GaINAC glycosilation, O-glycosilation, N-glycosilation, myristoylation, and palmitoylation were not found in TβRII-SE. In this study it was estimated that the molecular weight of the mature TβRII-SE isoform was of about 18.4 kDa.
To confirm whether TβRII-SE mRNA was also present in human cells other than lymphocytes, we amplified by RT-PCR using the same set of primers various human cell lines and primary cultures (
To check whether TβRII-SE is also present in leukocytes different from T-lymphocytes, granulocytes, monocytes, B-cells and T-cells were purified from human peripheral blood by density gradient and subsequent magnetic immune-purification with specific monoclonal antibodies, to high purity (
To determine whether TβRII-SE may be secreted to the extra cellular medium, TβRII-SE cDNA was cloned downstream from the ubiquitous promoter CMV in a self-inactivating (SIN) bicistronic lentiviral vector also expressing eGFP, as described in the examples, to generate the Lt-TβRII-SE vector. As a control, two lentiviral vectors were used: one bicistronic encoding a dominant negative TβRII mutant together with eGFP (Lt-TβRIIA-DN) and another encoding eGFP alone (Lt-eGFP), also under the action of the CMV promoter (
With these lentiviral vectors, shown in
The molecular weight of TβRII-SE detected by Western blot is in agreement with the predicted molecular weight, after the addition of post-translational modifications (18 kDa) (Table 1). This is the first evidence ever that there exists a new secretable TβRII receptor variant or isoform in human cells.
To show the function of the TβRII-SE isoform, functional assays were carried out wherein untransduced, expressing nearly undetectable levels of TβRII-SE, transduced with lentiviral vectors encoding eGFP alone, or bicistronics together with either TβRII-SE or the dominant negative (DN) mutant of the TβRIIA variant known to work as a TGF-β1 antagonist, A549 cells were used.
Initially, MTT ((3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; thiazolyl blue) assays were performed to evaluate if overexpression of TβRII-SE inhibits or not cell proliferation in the presence of 0.4 nM TGFβ-1 (
Additionally, to check whether TβRII-SE acts as a TGFβ-1 agonist, A459 cells either overexpressing TβRII-SE or not (untransduced cells or UT) were incubated in the presence of increasing concentrations of TGFβ-1 (
To further assess the agonistic role of the TβRII-SE isoform, hASCs were transduced with Lt-TβRII-SE, Lt-TβRIIA-DN, and Lt.eGFP, at an MOI of 150 as described in the examples. Seventy two hours after transduction the percentage of eGFP expressing cells was measured by flow cytometry (
RT-PCR performed on poly A+ mRNA from either transduced or untransduced hASC cells showed the pattern of TβRII isoforms expression depicted in
mRNA levels of all three isoforms of Type II TGF-β receptor were also quantified by qRT-PCR (
This compensation effect was also verified by addition of exogenous TGF-β1 and analysis of mRNA levels of the TβRII variants in hASCs cells (
According to this, it was also found that mRNA of both TβRII-A and TβRII-B are highly upregulated (40- and 50-fold increase, respectively) in cells overexpressing Lt-TβRII-SE in the presence of physiological concentrations of TGF-β1 compared to levels of mRNA produced in the absence of exogenous TGF-β1, further confirming the role of TβRII-SE acting as a TGF-β1 agonist by increasing the expression of membrane-bound receptors TβRII and TβRII-B (
Furthermore, the effect of TβRII-SE recombinant isoform was measured on a panel of 80 cytokines secreted by hASCs cells (
The results obtained with cytokine arrays are shown in Table 2. Increase or decrease of cytokines levels are referred to the levels secreted by cells transduced with the control vector Lt.eGFP either in the presence (paracrine) or absence (autocrine) of exogenous TGF-β1. UC: unchanged levels with respect to cells transduced with the control vector Lt.eGFP. Abs: absent in mock transducer cells control. Dark grey boxes: decreased to undetected levels or absent in the supernatant of cells transduced with control vector Lt.eGFP.
Light gray boxes: cytokines present.
It is shown that in ASC cells overexpressing TβRII-DN with a high TGF-β1 concentration, OPG secretion remains unchanged with respect to the values obtained in Lt.eGFP-transduced control cells, making cells insensitive to TGF-β1.
On the other hand, high TGF-β1 concentrations caused a dramatic drop of OPG secretion in TβRII-SE overexpressing cells compared to control cells (Lt.eGFP-transduced). The TβRII-SE isoform acts oppositely to the TGF-β1 inhibitor (TβRII-DN) and seems to favor osteoclastogenesis.
Table 3 summarizes the results obtained by other authors, and those compared to the results disclosed in the present application regarding the cytokine array and the relationship with osteoarthritis (OA).
It is shown that in cells overexpressing TβRII-SE HGF secretion is highly upregulated both in the presence (4.16 times) or absence (7.65 times) of exogenous TGF-β1, whereas in cells overexpressing the dominant negative mutant TβRII-DN, HGF secretion decreases 1.81 times or is absent, in the absence and presence of exogenous TGF-β1, respectively. These results show that the TβRII-SE isoform is involved in the positive regulation of HGF.
Increased TGF-β1 acts differently in animals depending on whether injections were applied in normal or osteoarthritic models. In normal animals, either TGF-β1 protein or adenovirus TGF-β1 injection generates increased synthesis and content of proteoglycan and osteophyte formation. On the other hand, in osteoarthritis (OA)-induced models, increases in the TGF pathway help to decrease cartilage damage, proteoglycan and osteophyte formation. Thus, the effect of the TβII-SE isoform was analyzed either in CPFX-treated juvenile rats (24 days old) or untreated rats, by intra-articular injections of lentiviral vectors encoding a recombinant protein of the codon-optimized (co) TβRII-SE fused to the constant fragment (Fc) of the human immunoglobulin 1 (IgG1) (Lt.coTβRII-SE/Fc) or the enhanced green fluorescent protein (Lt.eGFP).
Seven days after injecting the vector into rats treated with ciprofloxacin (CPFX), only articulations overexpressing the fusion peptide or a fused coTβRII-SE/Fc isoform showed radiolucent images with irregular borders in the femoral condyle, consistent with intraosteal geodes (
When compared to serum levels of urea, creatinine, total proteins, albumin, alkaline phosphatase, alanine transaminase (ALT), and aspartate transaminase (AST), a statistically significant difference was only found for the latter. An increase in aspartate transaminase (AST) was only observed in serum of rats treated with CPFX and intra-articularly injected with Lt.coTβRII-SE (
In the present application, the generation of a new recombinant TβRII-SE protein expressed in human cells is shown. It is known that in nature, the concentration of soluble receptors is very low, thus, to increase the levels of the recombinant TβRII-SE protein, the original coding sequence was codon optimized, and a Kozak sequence was included (Epoch Biolabs Inc., Texas, USA) referred to herein as coTβRII-SE (SEQ ID No. 4) and encoded by SEQ ID No. 5 (
As can be observed,
As can be observed,
Subsequently, the recombinant coTβRII-SE/Fc cDNA was inserted between the Agel and EcoRV sites of a SIN lentiviral vector (
To check recombinant protein production, A549 cells were transduced at an MOI=300 either with the control vector Lt.eGFP (93% of eGFP expressing cells) or Lt.coTβRII.SE/Fc (47.53% of eGFP expressing cells) and Mock transduced (
To verify the presence of human IgG1 mRNA in Lt.coTβRII-SE/Fc transduced cells, total mRNA of Mock transduced (vehicle), Lt.eGFP transduced and Lt.coTβRII-SE/Fc transduced cells was extracted and RT-PCR assays were performed using specific primers for human IgG1-Fc (
Additionally, to verify the presence of the TβRII-SE/Fc protein both in cell lysates and supernatants, total proteins from Mock, Lt.eGFP and Lt.coTβRII-SE/Fc transduced cells lysates and supernatants were western blotted (
A method to treat liver fibrosis was developed employing the lentiviral vector encoding the fusion protein TβRII-SE/Fc of the invention.
To study the effect of TβRII-SE/Fc expression on liver fibrogenesis, a rat model of carbon tetrachloride (CCL4) induced liver fibrosis was used. After animal euthanasia, liver gross appearance was evaluated macroscopically.
Effect of TβRII-SE/Fc expression on body weight and liver to body weight ratio: body weight was controlled in all rats throughout the experiment. It was observed that CCl4 treatment during eight weeks caused a growth retardation of rats, evidenced by the decrease of final body weight gain compared to rats of the vehicle group. Injection of Lv.TβRII-SE/Fc partially reversed the BW loss induced by this hepatotoxic agent. This beneficial effect was more evident after 4 weeks of CCl4 administration (
Effect of TβRII-SE/Fc expression on serum liver enzymes: to evaluate liver injury, AST and ALT serum levels were determined. As it is shown in
Effect of TβRII-SE/Fc expression on liver architecture: histological sections were stained with H&E to evaluate the general architecture of the liver. This analysis revealed that animals that received vehicle instead of CCl4, presented livers with a conserved architecture with cords of hepatocytes radiating from central veins (
Effect of TβRII-SE expression on liver fibrosis: collagen deposition was evaluated by Sirius Red staining in liver sections from different experimental group rats. CCl4 administration induced extensive deposition of collagen fibers evidenced by the observation of bridging fibrosis.
Use of the Lv.TβRII-SE/Fc vector to treat cancer: it was observed that intratumoral TβRII-SE/Fc overexpression inhibits tumor growth (
Assays were conducted to determine rheumatoid arthritis (RA) disease activity by means of measuring TβRII-SE by flow cytometry, with the TβRII-SE monoclonal antibody of the invention, conjugated with ATTO647N. The percentage of neutrophils expressing TβRII-SE (
When the percentage of neutrophils expressing TβRII-SE of each patient was correlated with its matching disease activity score (DAS28-ESR) value, it could be observed a negative correlation (Spearman's rank correlation coefficient rs=−0.69), statistically significant (p=0.0009), (
Also, experiments were carry out to detect intracellular TβRII-SE concentration by In-cell ELISA in neutrophils from patients (N=5) with diferent RA activity levels. (Table 4).
Relative intracellular TβRII-SE protein levels in neutrophils from RA patients were correlated with their matching DAS28-ESR score (Table 5).
When both sets of data were analyzed by the Spearman's Rank correlation test, a negative correlation was observed between TβRII-SE levels and DAS28-ESR (
This invention is better illustrated in the following examples, which should not be construed as limiting the scope thereof. On the contrary, it should be clearly understood that other embodiments, modifications and equivalents thereof may be possible after reading the present description, which may be suggested to a person of skill without departing from the spirit of the present invention and/or the scope of the appended claims.
Human adipose derived mesenchymal stromal cells (hASC) were obtained from 20 g subcutaneous fat following the protocol described by Zuk et al. (Zuk PA, et al. Mol Biol Cell 13: 4279-95, 2002) and cultured in the presence of DMEM supplemented with 10% human serum and 1% L-glutamine. Epstein Barr Virus immortalized lymphoblastoid cells were generated from peripheral blood mononuclear cells as described (Protocols in Immunology) and cultured with RPMI medium. Human A459 (lung adenocarcinoma), HT1080 (fibrosarcoma), Caco-2 (colorectal carcinoma), Hep 3B (hepatocellular carcinoma), Jurkat (acute lymphoblastoid leukemia), HEK293 (human embryonic kydney), and 293T cell lines were cultured in DMEM supplemented with 10% FCS and 1% penicillin/streptomycin. The cells were cultured in a humidified 5% CO2 incubator at 37° C.
Granulocytes, lymphocytes and monocytes were isolated from heparinized peripheral blood by Ficoll-Paque™ PLUS (GE Healthcare Bio-Sciences AB) gradient centrifugation. After centrifugation two fractions were obtained, one containing granulocytes/erythrocytes and another with peripheral blood mononuclear cells (PBMC). To obtain granulocytes, erythrocytes were lysed with KCI 0,6 M. PBMCs were labelled with anti CD3+, CD14+, and CD19+monoclonal antibodies conjugated with magnetic microbeads (Miltenyi Biotech) and separated using MS columns (Miltenyi Biotech) in a MiniMACS magnet (Miltenyi Biotech). Viable cells were determined by Trypan blue dye exclusion and counted in an hemocytometer. The purity of B- and T-lymphocyte and monocyte sub-populations was determined by flow cytometric analysis using a FACSCalibur flow cytometer (BD Biosciences). Cell sub-populations homogenized in RNA Lysis Buffer (SV Total RNA Isolation System, Promega) were stored at −80° C. until RNA extraction.
TβRII PCR fragments were cloned by insertion into the pGEM-T Easy plasmid (Promega Corporation WI, USA) under the conditions established by the manufacturers and E. coli transformation.TβRII PCR fragments were sequenced by using M13 forward and direct primers in a DNA sequencer ABI 3130 (Applied Biosystems Inc, CA, USA).
The TβRII-SE coding sequence containing an Agel site was codon optimized, the stop codon was deleted and a Kozak sequence included (Epoch Biolabs Inc. Texas, USA). The human IgG1 Fc coding sequence was obtained by RT-PCR from total blood mRNA using specific oligonucleotides as primers (forward: 5′AGA TCT GAC AAA ACT CAC ACA TGC 3′ (SEQ ID No. 8) and reverse: 5′ GAT ATC TTT ACC CGG AGA CAG G 3′ (SEQ ID No. 9)), containing a Bg/II site (forward primer) and EcoRV (reverse primer), to allow in frame fusion to TβRII-SE and to the lentiviral vector, respectively. The fusion construct (coTβRII-SE/Fc) of 951 bp Agel/EcoRV comprises 258 bp of the coTβRII-SE fused in frame with 693 bp of the human IgG1-Fc.
The cDNA encoding the three human TβRII isoforms were cloned into the pRRLsin18.cPPT.WPRE lentiviral vector, , generating the transfer vectors pRRLsin18.cPPT.CMV-TβRII-SE.ireseGFP.WPRE, pRRLsin18.cPPT.CMV-TβRII-DN.ireseGFP.WPRE, and pRRLsin18.cPPT.CMV-coTβRII-SE/Fc.ireseGFP.WPRE. Vesicular Stomatitis Virus G protein-pseudotyped lentiviruses (VSV-G) were generated by transient transfection of the transfer vectors together with the envelope plasmid (pCMV-VSVG), the packaging plasmid (pMDLg/pRRE) and Rev plasmid (pRSV-REV), into the 293T cell line, as previously described (R. A. Dewey, et al. Experimental Hematology 34: 1163-1171, 2006). The supernatant was harvested once every 12 hours for 48 hours and frozen in aliquots. Viral titers were determined by transducing A549 cells (yielding 107 infectious particles per milliliter). The pRRLsin18.cPPT.CMV-eGFP.WPRE lentiviral vector was used as control.
Total RNA from different primary cultures and cell lines was isolated using the Absolutely RNA kit (Stratagene, La Jolla, CA, USA). First-strand cDNA was synthesized by mixing 1 μg of DNA free total RNA, 50 pmol primer p(DT)15 (Roche Diagnostics GmbH, Mannheim, Germany), 0.5 mM deoxyribonucleotide triphosphate, 5 mM dithiothreitol, and 1 U Expand Reverse Transcriptase (Roche Diagnostics GmbH). cDNA corresponding to different isoforms of TβRII receptor was detected by PCR amplification in the presence of Expand High Fidelity polymerase (Roche Diagnostics GmbH), 0.2 mM dNTPS, and 0,5 μM of each primer (forward: 5′ACCGGTATGGGTCGGGGGCTGCTC3′ (SEQ ID No. 10) and reverse: 5′GTCGACTCAGTAG CAGTAGAAGATG3′ (SEQ ID No. 11) for 35 cycles using the following PCR conditions: 1 min. at 95° C., 1 min. at 55° C., and 1 min. at 95° C.
Quantitative RT-PCR was performed on diluted cDNA samples with FastStart Universal SYBR Green Master (Rox) (Roche Applied Science) using the Mx3005P™ Real-Time PCR Systems (Stratagene) under universal cycling conditions (95° C. for 10 min; 40 cycles of 95° C. for 15 s; then 60° C. for 1 min). All results were normalized to GAPDH mRNA levels and further the results were analyzed using the MxPro™ QPCR computer program and Infostat statistical computer program (Di Rienzo J. A., et al. InfoStat versión 2010. Grupo InfoStat, FCA, National University of Cordoba, Argentina. URL, http://www.infostat.com.ar)
A549 cells were transduced with lentiviral vectors at a multiplicity of infection (MOI) of 50 in the presence of 8 μg/ml polybrene. Percentage of eGFP positive cells was measured in a FACscalibur (Becton Dikinson) cytometer.
Cells were harvested, counted, and inoculated at the appropriate concentrations into 96-well plates using a multichannel pipette. After 24 hr, TGF-β1 (10 ng/ml and 20 ng/ml; Sigma) was added to the culture wells, and cultures were incubated for 24 hr and 48 hr at 37° C., under an atmosphere of 5% CO2. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma) solution at a concentration of 5 mg/ml was added to the media and the cells were further incubated for 4 hr. After replacing 100 μl of supernatant with 100 μl of DMSO, the absorbance of each well was determined at 540 nm with a SEAC (Sirio S) photometer (Italy). The percentage of cell survival was defined as the relative absorbance of treated versus untreated cells.
A549 and hASC cells were transduced at an MOI of 50 and 200 respectively, with the different lentiviral constructs, in the presence of 8 μg/ml polybrene (Sigma). Forty-eight hours after transduction, cells were harvested, washed in phosphate-buffered saline (PBS) supplemented with 10% fetal calf serum and the percentage of eGFP positive cells was analyzed by flow cytometry (FACscalibur, BD)
For Western blot analysis, both 20 μl and 100 μl of cell supernatant were loaded on 10% SDS-polyacrylamide gels, separated by electrophoresis and blotted onto Immovilon PVDF membranes (Millipore Corporation, Bedford, MA, USA). The membrane was exposed to anti-TβRII monoclonal primary antibody (clone C-4) (Santa Cruz, Biotechnology) diluted 1/200, or the monoclonal antibody IM 0577 (unprotected)], capable of specifically detecting TβRII-SE, diluted 1/500. Horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (Becton Dickinson GmbH) diluted 1/10000 was used as secondary antibody. Protein detection was performed with the Amersham ECL Plus Western blotting detection reagents (Amersham Buchler GmbH, Germany) in a Typhoon 9410, Variable Mode Imager (GE Healthcare Bio-Sciences AB, Uppsala, Sweden).
cDNA sequences belonging to the different TβRII isoforms were used and the predicted protein sequences and statistics were obtained using the EditSeq software (DNAstar, Inc. Madison, WI, USA). Both the DNA and the predicted protein sequences belonging to the TβRII-SE cDNA were aligned to known isoforms of the human TβRII receptor (A and B) using the MegAlign software (DNASTAR, Inc. Madison, WI, USA).
A cytokine/chemokine array kit G5 (Ray Biotech Inc., Norcross, GA) was used to detect a panel of 80 secreted cytokines as recommended by the manufacturer. hASCs P7 untransduced or transduced with lentiviral vectors were grown for 72 h in a medium supplemented with 0.1% BSA. Supernatants were collected, filtered and frozen after collection. For densitometry analysis of the arrays, Typhoon 9410 Variable mode Imager (GE Healthcare Life Sciences) was used, and signal intensity values were measured using the Image analysis software ImageQuant TL 7.0 (GE Healthcare Life Sciences). Microarray data were analyzed with RayBio® Antibody Array Analysis Tool. Good data quality and adequate normalization were ensured using internal control normalization without background. Any ≥1.5-fold increase or ≤0.65-fold decrease in signal intensity for a single analyte between samples or groups may be considered a measurable and significant difference in expression, provided that both sets of signals are well above background (Mean background+3 standard deviations, accuracy≈99%).
Antibodies were generated by Rheabiotech, Campinas, Brazil. Immunization of both rabbit (polyclonal antibody) or mice (monoclonal antibody), was performed using a Multiple Antigene Peptide System (MAPS) with 8 identical copies of a peptide containing the 13 amino acids (FSKVHYEGKKKAW) (SEQ ID No. 12), which are only found in TβRII-SE and not in the other splicing variants of the receptor. The monoclonal antibody IM-0577 was developed in mice and purified by protein G affinity chromatography.Antibodies specificity was assayed by indirect ELISA by sensitization with antigen at a concentration of 5 μg/ml in Carbonate Buffer 0.2 M, blocked by PBS/BSA and detected with serial dilutions (1:1000-1:64000) of the specific antibody. The ELISA test was developed with a Horseradish peroxidase (HRP)-conjugated secondary antibody together with H2O2/OPD as chromogenic substrate, and detected by absorbance at 492 nM.
Male 24-day-old Wistar rats were housed under controlled conditions at 21±1° C. with 50%±5% relative humidity and a constant light-dark schedule (light, 8 a.m. to 8 p.m.). Food and tap water was provided ad libitum. The rats received ciprofloxacin hydrochloride on day 24 by oral administration of 200 mg/kg of body weight during 10 days. The animals were examined for clinical abnormalities including motility alterations and weighted during the treatment period.
On day 14 after ciprofloxacin treatment, 50 μl viral vectors were injected intra-articularly with either Lt.coTβRII-SE/Fc (2.35×106 transducing Units, TU) or Lt.eGFP (6×106 TU). Control animals without ciprofloxacin were treated in the same manner.
Male Wistar rats weighting 150-200 g were housed at Mar del Plata National University Laboratory Animal Unit at a mean constant temperature of 22° C. with a 12 h light-dark cycle, and free access to standard pellet chow and water. All experiments were performed according to the ‘Guide for the Care and Use of Laboratory Animals’ and approved by the Institutional Animal Care and Use Committee (CICUAL) of Mar del Plata National University. The experimental groups were designed as follows (n=7 per group): (I) Control group received intraperitoneal (ip) injection of vehicle of CCl4; (II) CCl4 group received ip injection of CCl4; (III) Lv.TβRII-SE/Fc+CCl4 group received intrahepatic (ih) injection of Lv.TβRII-SE/Fc (week 0) before treatment with CCl4.
In vivo Liver Transduction
Animals were ih injected with Lv.TβRII-SE/Fc (5−10×107 transduction units/ml) a week before the induction of liver fibrosis (
Liver fibrosis was induced by ip injection of carbon tetrachloride (CCl4) 1 ml in oil (1:1), per kg of body weight (BW), twice a week, for 8 weeks (
Body weight (BW) measurements were taken before each CCl4 ip injection, and after completion of the experiment. These data were used to calculate BW gain, which was expressed as the percentage (%) of increase respect to the initial BW. After euthanasia, livers were harvested and weighted to calculate the liver to body weight ratio (LW/BW), also expressed as percentage.
Liver enzyme levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) were determine in serum with an automatic analyzer BT300 plus (Biotecnica), according to the manufacturer's recommendations.
Livers fixed in 10% neutral buffered formalin were embedded in paraffin. Liver sections (5 μm) were stained with Hematoxylin and Eosin (H&E), for liver architecture visualization. For liver fibrosis assessment, sections were stained with 0.1% Sirius Red. Quantification of Sirius Red-positive areas was performed in at least ten microscopy fields per histological section using the software ImageJ. Results were expressed as mean percentage of Sirius Red-positive area per field.
For immunohistochemical analysis, 5 μm sections were dewaxed and rehydrated. Endogenous peroxidase activity was blocked with 3% H2O2 3% in methanol (10 min, at room temperature). Antigen retrieval was performed using the heat induced epitope retrieval (HIER) method with 0.1 M citrate buffer, pH 6. Tissue sections were then incubated for 12-16 h at 4° C. with rabbit anti-α-smooth muscle actin (anti-α-SMA, 1:500, Cell Signaling Technology, Danvers, MA). After two washes with PBS, slides were incubated with HiDef Detection amplifier Mouse and Rabbit (Cell Marque, Rocklin, CA) for 10 min, at room temperature. Sections were further washed with PBS and incubated with HiDef Detection HRP Polymer Detector (Cell Marque, Rocklin, CA) for 10 min, at room temperature. Finally, sections were washed twice with PBS, and immunohistochemical staining was obtained using the DAB Chromogen kit (Cell Marque, Rocklin, CA) by 5 min. incubation at room temperature, and counterstained with Hematoxylin. Dehydrated sections were mounted and imaged on a Nikon Eclipse E200 microscope.
Data were analyzed using two-way ANOVA followed by the Fisher's Least Significant Difference (LSD) test. Statistical significance was set at <0.05. Results are expressed as mean±SD.
TN60 murine mammary carcinoma cells were injected subcutaneously into syngenic C3H/S mice (N=6-7 per group), as it is described by García M. et al., 2015 Biological Rhythm Research 46: 573-578. Ten days after, 1.5×106 transduction units of a lentiviral vectors encoding TβRII-SE/Fc (Lv.TβRII-SE/Fc) (N=7), or the control vector Lv. TβRII-DN (dominant negative) (N=6) were intratumorally injected. As an additional control, mice were intratumorally injected with the same volume of culture medium (vehicle).
Tumor diameter was determine every 2-3 days by measuring the tumor perimeter with a digital caliper, Tumor mean volume was determine by the formula V=4/3 (p×r3). Two weeks after tumor implantation, mice were euthanized by cervical dislocation.
Peripheral blood was collected by venipuncture from 19 RA patients diagnosed according to the ACR/EULAR 2010 criteria. All procedures were approved by CER Medical Institute Research Ethics Committee, and the Comisión Conjunta de Investigación en Salud, Department of Health, Buenos Aires Province, Argentina, registered under the number 2919/653/13. All procedures were performed after signing off a voluntary informed consent, by the donors. Exclusion criteria included severe anaemia, autoimmune diseases different from RA, any other disease/condition able to increase ESR, treatment with biological drugs, treatment with disease-modifying anti-rheumatic drugs (DMARDs) except methotrexate, and with drugs with known effect on the TGF-β signalling cascade (losartan).
Detection of TβRII-SE in neutrophils by Flow Cytometry: both neutrophils and peripheral blool mononuclear cells (PBMC) were isolated by Ficoll-Paque™ PLUS density gradient. Red blood cells were eliminated from the neutrophil fraction by incubation with a hypertonic buffer (0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM EDTA). To determine the percentage of cells expressing TβRII-SE, 1×106 of both, neutrophils and PBMC were fixed and permeabilized with the Cytofix/Cytoperm Kit (BD Biosciences, USA) Subsequently, cells were incubated with 0.5 μg of the anti-TβRII-SE monoclonal antibody of the invention conjugated with the fluorochrome ATTO 647N. Cells were resuspended in 100 μl of PBS and were analyzed by Flow Cytometry in a FACSCalibur device (BD Biosciences, USA), using Flowjo software (BD Biosciences, USA). The percentage of neutrophils expressing TβRII-SE was determined by taking as cut off the fluorescence value obtained with lymphocytes of each patient, as reference. TβRII-SE fluorescence values in neutrophils were correlated with DAS28-ESR disease activity scores by the Spearman's rank correlation test of the OriginPro 8.5.1 software (Origin Lab Corporation, Northampton, MA, USA).
To develop a method to quantify intracellular TβRII-SE in leukocytes by In-cell ELISA in RA pacientes, 2.6×106 células/cm2, in saline solution+2 (0.9% NaCl, 1 mM MgCl2, 1 mM CaCl2), were incubated in 96 well plates for 20 minutes at room temperature, to allow cell adherence to plastic. Subsequently, cells were washed twice with 1×PBS, and fixed and permeabilized with 100 μL of Fix/Perm solution (BD Cytofix/Cytoperm™, USA) for 20 min. at 4° C. After two washes with 250 μL of 1×BD Perm/Wash buffer (BD Perm/Wash™, USA), adhered cells were incubated with the anti-TβRII-SE antibody (10 μg/mL in 50 μL of BD Perm/Wash buffer) for 30 minutes to 16 hours at 4° C. As control, cells were also incubated without the above mentioned antibody. After two aditional washes with 250 μL of 1×BD Perm/Wash Buffer, cells were incuabated with 1 μg/mL secondary antibody (Anti Mouse HRP conjugated-Promega, USA), in 50 μL de 1×BD Perm/Wash Buffer, for 90 minutes. Subsequently, cells were incubated with 100 μL of quenching solution (10% V/V H2O2 in 1×BD Perm/Wash Buffer. After 3 washes with 250 μL of 1×BD Perm/Wash Buffer, cells were incubated with 100 μL of TMB substrate (Life Technologies, EEUU), in the dark, and 655 nm absorbance was determined every 5 minutes for 30 minutes, in a microplate reader (Biotek, SYNERGY™ H1, USA). In addition, the number of adhered cells was determined by cristal violet staining, to be used as In-cell ELISA normalizer. To this end, each well was washed four times with 200 μL 1×PBS and cells were incubated with 50 μl crystal violet solution containing 2 g de crystal violet (Sigma, USA), 20 ml 95% ethanol, 0.8 g amonium oxalate, and 80 ml distiled water, for 30 minutes at room temperature. After wahing the wells with abundant tap water, cells were incubated with 100 μL of 1% SDS for 60 minutes at room temperatura. Finally, absorbance at 595 nm was determined in a microplate reader (Biotek, SYNERGY™ H1, USA).
Intracellular TβRII-SE relative concentration values were determined as follows:
AbsNn=Absn655/Absn595
AbsNT=AbsT655/AbsT595
TβRII-SE relative concentration=(AbsNT−AbsNn)* 100
AbsNT=normalized absorbance of the well containing Anti TβRII-SE primary antibody.
AbsT655=Absorbance at 655 nm of the well containing Anti TβRII-SE primary antibody.
AbsT595=Absorbance at 595 nm of the well containing Anti TβRII-SE primary antibody.
AbsNn=normalized absorbance of the well without primary antibody (negative).
Absn655=Absorbance at 655 nm of the well without primary antibody (negative).
Absn595: Absorbance at 595 nm of the well without primary antibody (negative).
TβRII-SE relative concentration in plastic adhered leukocytes from RA patients was correlated with their matching DAS28-ESR value using the Spearman rank correlation test of the OriginPro 8.5.1 software (Origin Lab Corporation, Northampton, MA, USA).
This application is a Divisional of U.S. application Ser. No. 17/228,348 filed Apr. 12, 2021, pending, entitled TGF-β receptor II isoform, fusion peptide, methods of treatment and methods in vitro, which is a Divisional of U.S. application Ser. No. 16/173,426 filed Oct. 29, 2018, now U.S. Pat. No. 11,072,647 issued Jul. 27, 2021, entitled, TGF-β receptor II isoform, fusion peptide, methods of treatment and methods in vitro, which is a continuation-in-part of U.S. application Ser. No. 15/105,162 filed Jun. 16, 2016, entitled, ISOFORM OF THE TGF-BETA RECEPTOR II, now U.S. Pat. No. 10,233,227 issued Mar. 19, 2019, which is a national stage entry of PCT/US2014/071338 filed Dec. 19, 2014, under the International Convention claiming priority over U.S. Provisional Patent Application No. 61/917,974 filed Dec. 19, 2013, the content of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 61917974 | Dec 2013 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17228348 | Apr 2021 | US |
| Child | 18502333 | US | |
| Parent | 16173426 | Oct 2018 | US |
| Child | 17228348 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15105162 | Jun 2016 | US |
| Child | 16173426 | US |
