Patent Application
20160060314
The present invention pertains to (i) improved cell-permeable SOCS3 (iCP-SOCS3) proteins as protein-based biotherapeutics, which are well-enhanced in their ability to transport biologically active SOCS3 proteins across the plasma membrane, to increase in its solubility and manufacturing yield, and to induce anti-tumor effect in solid tumors; (ii) polynucleotides that encode the same, and (iii) anti-solid tumor compositions that comprise the same.
The Janus kinase signal transducers and activators of transcription signaling (JAK/STAT) plays important roles in immune responses, including oncogenesis. So many investigations demonstrated that STAT-3, an important member of STAT proteins, was considered as a protooncogene in various types of disorder. STAT-3 is phosphorylated and dimerizes by the Janus kinase (JAK), and its overexpression and constitutive activation can significantly induce cell proliferation, tumor angiogenesis, invasion. Meanwhile, inhibition of JAK-STAT signaling led to suppress the cancer cell growth and induce apoptosis. Suppressor of cytokine signaling-3 (SOCS3), a kind of endogenous protein inhibitor of JAK/STAT pathway, was identified to be inversely associated with the STAT3 expression and phosphorylation in vivo and in vitro and aberrant expression of SOCS3 protein was observed in human solid tumors including gastric, colorectal and breast cancer, and glioblastoma.
Gastric cancer remains the second leading cause of cancer-related death in the world. Advances in early detection and decreased chronic Helicobacter pylori infection rates have led to a substantial reduction in gastric cancer rates worldwide. However, effective treatment regimens for gastric cancers, especially advanced gastric cancer, are still lacking; therefore, the prognosis of patients with this disease remains poor. SOCS3 mRNA levels are higher in adjacent normal mucosal tissues, however, gastric cancer patients with high simultaneous expression of SOCS3 have a better overall survival than those with low simultaneous expression. Based on this, SOCS3 may represent new therapeutic target to treat gastric cancer.
Colorectal cancer is one of the most fatal neoplastic diseases worldwide and a serious global health problem, with over one million new cases and half million mortalities worldwide each year. It has been reported as being relevant to some inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis. The pathogenesis of colorectal carcinoma is complex, with the involvement of multiple cellular transduction pathways including IL-6/STAT3 signaling. Reduced or silenced SOCS3 has been found in many human types of cancer including colorectal cancer, and restoring SOCS3 expression in the cancer cells inhibits IL-6-mediated STAT3 activation, induces tumor cell apoptosis and decreases cell proliferation. Therefore, suppression of the IL-6/STAT3 pathway via modulation of SOCS3 has been a promising strategy for anti-colon/colorectal cancer therapy.
Glioblastoma, the most common neoplasm among diffuse infiltrating astrocytomas, is notorious for its ability to evade immune-surveillance as well as for its invasive and angiogenic properties. Gliomas are the most common type of primary brain tumors are highly malignant and are associated with a very poor prognosis. Glioblastoma is a very aggressive subtype of glioma with very short life expectancy and limited treatment options. A hallmark of this lethal disorder is the presence of activated STAT3. Because SOCS3 is a negative regulator of STAT-3 activation, it hypothesized that SOCS3 may function as a tumor suppressor in glioblastoma tissues.
Breast cancer is a disease that arises from the accumulation of alterations in the genome of cells that make up the mammary gland. Breast cancer is the most common type of cancer among women, with an estimated 1.38 million new cases of cancer diagnosed in 2008 (23% of all cancers), and the second most common type of cancer overall (10.9% of all cancers). Expression of SOCS3 protein is significantly down-regulated in breast cancer specimens and replacing of SOCS3 protein may directly influence the treatment of breast cancer.
Cytokine signaling is strictly regulated by the SOCS family proteins induced by different classes of agonists, including cytokines, hormones and infectious agents. Among them, SOCS1 and SOCS3 are relatively specific to STAT1 and STAT3, respectively. SOCS1 inhibits JAK activation through its N-terminal kinase inhibitory region (KIR) by the direct binding to the activation loop of JAKs, while SOCS3 binds to janus kinases (JAKs)-proximal sites on the receptor through its SH2 domain and inhibits JAK activity that blocks recruitment of STAT3. Both promote anti-inflammaory effects due to the suppression of inflammation-inducing cytokine signaling. Furthermore, the SOCS box, another domain in SOCS proteins, interacts with E3 ubiquitin ligases and/or couples the SH2 domain-binding proteins to the ubiquitin—proteasome pathway. Therefore, SOCSs inhibit cytokine signaling by suppressing JAK kinase activity and degrading the activated cytokine receptor complex.
In connection with SOCSs and various solid tumors including gastric, colorectal and breast cancer, and glioblastoma, the SOCS1 gene has been implicated as an anti-oncogene in the tumor development. Previous studies have reported that aberrant methylation in the CpG island of SOCS1 induces its transcriptional silencing in cancer cell lines, and SOCS1 heterozygous mice are hypersensitive to various cancers. In addition, abnormalities of SOCS3 are also associated with the solid tumors. Hypermethylation of CpG islands in the SOCS3 promoter is correlated with its transcriptional silencing in tumors cell lines. SOCS3 overexpression down-regulates active STAT3, induces apoptosis, and suppresses growth in cancer cells. The importance of STAT3 to inflammation-associated carcinogenesis is underlined by the previous study that cancer-specific deletion of SOCS3 in a mouse carcinoma model results in larger and more numerous tumors. This means that SOCS3 plays a major role in the negative regulation of the JAK/STAT pathway in carcinogenesis and contributes to the suppression of tumor development by protecting the tissue cells.
To negatively control JAK/STAT signaling, recombinant SOCS3 proteins that contain a cell-penetrating peptide (CPP)—membrane-translocating motif (MTM) from fibroblast growth factor (FGF)-4 has been reported. These recombinant SOCS3 proteins inhibited STAT phosphorylation, inflammatory cytokines production and MHC-II expression in cultured and primary macrophages. In addition, SOCS3 fused to MTM protected mice challenged with a lethal dose of the SEB super-antigen, by suppressing apoptosis and hemorrhagic necrosis in multiple organs. However, the SOCS3 proteins fused to FGF4-derived MTM displayed extremely low solubility, poor yields and relatively low cell- and tissue-permeability. Therefore, the MTM-fused SOCS3 proteins were not suitable for further clinical development as therapeutic agents. To overcome these limitations, improved SOCS3 recombinant proteins (iCP-SOCS3) fused to the combination of novel hydrophobic CPPs, namely advanced macromolecule transduction domains (aMTDs) to greatly improve the efficiency of membrane penetrating ability in vitro and in vivo with solubilization domains to increase in their solubility and manufacturing yield when expressed and purified from bacteria cells.
In this new art of invention, aMTD/SD-fused iCP-SOCS3 proteins (iCP-SOCS3), much improved physicochemical characteristics (solubility & yield) and functional activity (cell-/tissue-permeability) compared with the protein fused only to FGF-4-derived MTM. In addition, the newly developed iCP-SOCS3 proteins have now been demonstrated to have therapeutic application in treating the tumors, exploiting the ability of SOCS3 to suppress JAK/STAT signaling. The present invention represents that macromolecule intracellular transduction technology (MITT) enabled by the new hydrophobic CPPs that are aMTD may provide novel protein therapy through SOCS3-intracellular protein replacement against the various cancer cells. These findings suggest that restoration of SOCS3 by replenishing the intracellular SOCS3 with iCP-SOCS3 protein creates a new paradigm for anti-cancer therapy, and the intracellular protein replacement therapy with the SOCS3 recombinant protein fused to the combination of aMTD and SD pair may be useful to treat the various tumors.
An aspect of the present invention relates to improved cell-permeable SOCS3 (iCP-SOCS3) capable of mediating the transduction of biologically active macromolecules into live cells.
iCP-SOCS3 fused to novel hydrophobic CPPs—namely advanced macromolecule transduction domains (aMTDs)—greatly improve the efficiency of membrane penetrating ability in vitro and in vivo of the recombinant proteins.
iCP-SOCS3 fused to solubilization domains (SDs) greatly increase in their solubility and manufacturing yield when they are expressed and purified in the bacteria system.
An aspect of the present invention also, relates to its therapeutic application for delivery of a biologically active molecule to a cell, involving a cell-permeable SOCS3 recombinant protein, where the aMTD is attached to a biologically active cargo molecule.
Other aspects of the present invention relate to an efficient use of aMTD sequences for drug delivery, protein therapy, intracellular protein therapy, protein replacement therapy and peptide therapy.
The present invention provides improved cell-permeable SOCS3 as a biotherapeutics having improved solubility/yield, cell-/tissue-permeability and anti-tumor effect in solid tumors. Therefore, this would allow their practically effective applications in drug delivery and protein therapy including intracellular protein therapy and protein replacement therapy.
The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings.
In this invention, it has been hypothesized that exogenously administered SOCS3 proteins could compensate for the apparent inability of endogenously expressed members of this physiologic regulator to interrupt constitutively active cancer-initiating JAK/STAT signaling and excessive cell cycle, resulting in the inhibition of the tumorigenesis. To prove our hypothesis, the SOCS3 recombinant proteins were fused to novel hydrophobic CPPs called aMTDs to improve their cell-/tissue-permeability, additionally adopted solubilization domains to increase their solubility/yield in physiological condition, and then tested whether exogenous administration of SOCS3 proteins can reconstitute their endogenous stores and restore their basic function as the negative feedback regulator that attenuates JAK/STAT signaling. This art of invention has demonstrated “intracellular protein therapy” by designing and introducing cell-permeable form of SOCS3 that has a great potential of anti-cancer therapeutic applicability in solid tumors.
To address the limitation of previously developed hydrophobic CPPs, novel sequences have been developed. To design new hydrophobic CPPs for intracellular delivery of cargo proteins such as SOCS3, identification of optimal common sequence and/or homologous structural determinants, namely critical factors (CFs), had been crucial. To do it, the physicochemical characteristics of previously published hydrophobic CPPs were analyzed. To keep the similar mechanism on cellular uptake, all CPPs analyzed were hydrophobic region of signal peptide (HRSP)-derived CPPs (e.g. MTS and MTD).
These 17 hydrophobic CPPs published from 1995 to 2014 have been analyzed for their 11 different characteristics—sequence, amino acid length, molecular weight, pI value, bending potential, rigidity/flexibility, structural feature, hydropathy, residue structure, amino acid composition, and secondary structure of the sequences. Two peptide/protein analysis programs were used (ExPasy: http://web.expasy.org/protparam/, SoSui: http://harrier.nagahama-i-bio.ac.jp/sosui/sosui_submit.html) to determine various indexes, structural features of the peptide sequences and to design new sequence. Followings are important factors analyzed.
Average length, molecular weight and pI value of the peptides analyzed were 10.8±2.4, 1,011±189.6 and 5.6±0.1, respectively.
Bending potential (Bending or No-Bending) was determined based on the fact whether proline (P) exists and/or where the amino acid(s) providing bending potential to the peptide in recombinant protein is/are located. Proline differs from the other common amino acids in that its side chain is bonded to the backbone nitrogen atom as well as the alpha-carbon atom. The resulting cyclic structure markedly influences protein architecture which is often found in the bends of folded peptide/protein chain. Eleven out of 17 were determined as ‘Bending’ peptide which means that proline should be present in the middle of sequence for peptide bending and/or located at the end of the peptide for protein bending. As indicated above, peptide sequences could penetrate the plasma membrane in a “bent” configuration. Therefore, bending or no-bending potential is considered as one of the critical factors for the improvement of current hydrophobic CPPs.
Since one of the crucial structural features of any peptide is based on the fact whether the motif is rigid or flexible, which is an intact physicochemical characteristic of the peptide sequence, instability index (II) of the sequence was determined. The index value representing rigidity/flexibility of the peptide was extremely varied (8.9-79.1), but average value was 40.1±21.9 which suggested that the peptide should be somehow flexible, but not too rigid or flexible.
Alanine (V), valine (V), leucine (L) and isoleucine (I) contain aliphatic side chain and are hydrophobic—that is, they have an aversion to water and like to cluster. These amino acids having hydrophobicity and aliphatic residue enable them to pack together to form compact structure with few holes. Analyzed peptide sequence showed that all composing amino acids were hydrophobic (A, V, L and I) except glycine (G) in only one out of 17 and aliphatic (A, V, L, I, and P). Their hydropathic index (Grand Average of Hydropathy: GRAVY) and aliphatic index (AI) were 2.5±0.4 and 217.9±43.6, respectively.
As explained above, the CPP sequences may be supposed to penetrate the plasma membrane directly after inserting into the membranes in a “bent” configuration with hydrophobic sequences adopting an α-helical conformation. In addition, our analysis strongly indicated that bending potential was crucial. Therefore, structural analysis of the peptides conducted to determine whether the sequence was to form helix or not. Nine peptides were helix and 8 were not. It seems to suggest that helix structure may not be required.
In the 11 characteristics analyzed, the following 6 are selected namely “Critical Factors (CFs)” for the development of new hydrophobic CPPs—advanced MTDs: i) amino acid length, ii) bending potential (proline presence and location), iii) rigidity/flexibility (instability index: II), iv) structural feature (aliphatic index: AI), v) hydropathy (GRAVY) and vi) amino acid composition/residue structure (hydrophobic and aliphatic A/a).
Since the analyzed data of the 17 different hydrophobic CPPs (analysis A) previously developed during the past 2 decades showed high variation and were hard to make common- or consensus—features, additional analysis B and C was also conducted to optimize the critical factors for better design of improved CPPs—aMTDs.
In analysis B, 8 CPPs used with each cargo in vivo were selected. Length was 11±3.2, but 3 out of 8 CPPs possessed little bending potential. Rigidity/Flexibility was 41±15, but removing one [MTD85: rigid, with minimal (II: 9.1)] of the peptides increased the overall instability index to 45.6±9.3. This suggested that higher flexibility (40 or higher II) is potentially be better. All other characteristics of the 8 CPPs were similar to the analysis A, including structural feature and hydropathy.
To optimize the ‘Common Range and/or Consensus Feature of Critical Factor’ for the practical design of aMTDs and the random peptides, which were to prove that the ‘Critical Factors’ determined in the analysis A, B and C were correct to improve the current problems of hydrophobic CPPs—protein aggregation, low solubility/yield, and poor cell/tissue-permeability of the recombinant proteins fused to the MTS/MTM or MTD, and non-common sequence and non-homologous structure of the peptides, empirically selected peptides were analyzed for their structural features and physicochemical factor indexes.
The peptides which did not have a bending potential, rigid or too flexible sequences (too low or too high Instability Index), or too low or too high hydrophobic CPP were unselected, but secondary structure was not considered because helix structure of sequence was not required. 8 selected CPP sequences that could provide a bending potential and higher flexibility were finally analyzed. Common amino acid length is 12 (11.6±3.0). Proline should be presence in the middle of and/or the end of sequence. Rigidity/Flexibility (II) is 45.5-57.3 (Avg: 50.1±3.6). AI and GRAVY representing structural feature and hydrophobicity of the peptide are 204.7±37.5 and 2.4±0.3, respectively. All peptides are consisted with hydrophobic and aliphatic amino acids (A, V, L, I, and P). Therefore, analysis C was chosen as a standard for the new design of new hydrophobic CPPs (TABLE 1).
1-3. Determination of Critical Factors for Development of aMTDs
For confirming the validity of 6 critical factors providing the optimized cell-/tissue-permeability. All 240 aMTD sequences have been designed and developed based on six critical factors (TABLES 2-1 to 2-6). (The aMTD amino sequences are SEQ ID NOS: 1 to 240, and the aMTD nucleotide sequences are SEQ ID NOS: 241 to 480.) All 240 aMTDs (hydrophobic, flexible, bending, aliphatic and helical 12 a/a-length peptides) were practically confirmed by their quantitative and visual cell-permeability. To determine the cell-permeability of aMTDs and random peptides which do not satisfy one or more critical factors have also been designed and tested. Relative cell-permeability of 240 aMTDs to the negative control (random peptide, hydrophilic & non-alipatic 12A/a length peptide) was significantly increased by up to 164 fold, with average increase of 19.6±1.6. Moreover, compared with reference CPPs (MTM and MTD), novel 240 aMTDs averaged of 13±1.1 (maximum 109.9) and 6.6±0.5 (maximum 55.5) fold higher cell-permeability, respectively. As a result, there were vivid association of cell-permeability of the peptides and critical factors. According to the result from the newly designed and tested novel 240 aMTDs, the empirically optimized critical factors are provided below.
These examined critical factors are within the range that we have set for our critical factors; therefore, we were able to confirm that the aMTDs that satisfy these critical factors have much higher cell-permeability (TABLE 3) and intracellular delivery potential compared to reference hydrophobic CPPs reported during the past two decades.
2. Development of SOCS3 Recombinant Proteins Fused to aMTD and Solubilization Domain
2-1. Design of Novel Hydrophobic CPPs—aMTDs for Development of Recombinant SOCS3 Proteins
Based on these six critical factors proven by experimental data, newly designed advanced macromolecule transduction domains (aMTDs) have been developed, and optimized for their practical therapeutic usage to facilitate protein translocation across the membrane. For this present invention, cell-permeable SOCS3 recombinant proteins have been developed by adopting aMTD165 (TABLE 4) that satisfied all 6 critical factors (TABLE 5).
In the previous study, recombinant cargo (SOCS3) proteins fused to hydrophobic CPP could be expressed in bacteria system and purified with single-step affinity chromatography; however, protein dissolved in physiological buffers (e.q. PBS, DMEM or RPMI1640 etc.) was highly insoluble and had extremely low. Therefore, an additional non-functional protein domain (solubilization domain: SD; TABLE 6) has been fused to the recombinant proteins at their C terminus to improve low solubility/yield and to enhance relative cell-/tissue-permeability.
According to the specific aim, solubilization domain A (SDA) and B (SDB) were first selected. We hypothesize that fusion of SOCS3 with SDs and novel hydrophobic CPP, aMTD, would greatly increase solubility/yield and cell-/tissue-permeability of recombinant cargo proteins—SOCS3—for the clinical application. SDA is a soluble tag, a tandem repeat of 2 N-terminal domain (NTD) sequences of CP—000113.1, which is a very stable soluble protein present in a spore surface coat of Myxococcus xanthus. SDB, a heme-binding part of cytochrome, provides a visual aid for estimating expression level and solubility. Bacteria expressing SDB containing fusion proteins appears red when the fused proteins are soluble.
Histidine-tagged human SOCS3 proteins were designed (
PCR primers for SOCS3 and SDA and/or SDB fused to SOCS3 are summarized in TABLES 7, 8 and 9, respectively. The cDNA and amino acid sequences of histidine tag are provided in SEQ ID NO: 481 and 482, and cDNA and amino acid sequences of aMTDs are indicated in SEQ ID NOs: 483 and 484, respectively. The cDNA and amino acid sequences are displayed in SEQ ID NOs: 485 and 486 (SOCS3); SEQ ID NOs: 487 and 488 (SDA); and SEQ ID NOs: 489 and 490 (SDB), respectively.
The SOCS3 recombinant proteins were expressed in E. coli BL21-CodonPlus (DE3) cells, grown to an OD600 of 0.6 and induced for 3 hrs with 0.6 mM isopropyl-β-D-thiogalactopyranoside (IPTG). The proteins were purified by Ni2+ affinity chromatography and dissolved in a physiological buffer such as DMEM medium.
The histidine-tagged SOCS3 proteins were expressed, purified, and prepared in soluble form (
SOCS3 recombinant proteins containing aMTD165 and solubilization domain (HM165S3A and HM165S3B) had little tendency to precipitate whereas recombinant SOCS3 proteins lacking a solubilization domain (HS3 and HM165S3) were largely insoluble. Solubility of aMTD/SD-fused SOCS3 proteins was scored on a 5 point scale compared with that of SOCS3 proteins lacking the solubilization domain (
Yields per L of E. coli for each recombinant protein (mg/L) ranged from 1 to 47 mg/L (
3. aMTD/SD-Fused SOCS3 Recombinant Proteins Significantly Increase Cell- and Tissue-Permeability
3-1. aMTD/SD-Fused SOCS3 Recombinant Proteins are Cell-Permeable
To examine protein uptake, SOCS3 recombinant proteins were conjugated to 5/6-fluorescein isothiocyanate (FITC). RAW 264.7 (
3-2. aMTD/SD-Fused SOCS3 Recombinant Proteins Enhance the Systemic Delivery to a Variety of Tissues
To further investigate in vivo delivery of SOCS3 recombinant proteins, FITC-labeled SOCS3 proteins were monitored following intraperitoneal (IP) injections in mice. Tissue distributions of fluorescence-labeled-SOCS3 proteins in different organs was analyzed by fluorescence microscopy (
3-3. aMTD-Mediated Intracellular Delivery is Bidirectional Mode
SOCS3 recombinant proteins lacking SD (HS3 and HM165S3) were less soluble, produced lower yields, and showed tendency to precipitate when they were expressed and purified in E. coli. Therefore, we additionally designed (
We next investigated how of aMTD165-mediated intracellular delivery was occurred. The aMTD-mediated intracellular delivery of SOCS3 protein did not require protease-sensitive protein domains displayed on the cell surface (
Moreover, we also tested whether cells treated with aMTD165-fused SOCS3 protein could transfer the protein to neighboring cells. For this, cells transduced with FITC-HM165S3B (green) were mixed with CD14-labeled cells (red), and cell-to-cell protein transfer was assessed by flow cytometry, scoring for CD14/FITC double-positive cells. Efficient cell-to-cell transfer of HM165S3B, but not HS3 or HS3B (
4. aMTD/SD-Fused SOCS3 Protein Efficiently Inhibits Cellular Processes
4-1. aMTD/SD-Fused SOCS3 Protein Inhibits the Activation of STATs Induced by INF-γ
The ultimate test of cell-penetrating efficiency is a determination of intracellular activity of SOCS3 proteins transported by aMTD. Since endogenous SOCS3 are known to block phosphorylation of STAT1 and STAT3 by IFN-γ-mediated Janus kinases (JAK) 1 and 2 activation, we demonstrated whether cell-permeable SOCS3 inhibits the phosphorylation of STATs. All SOCS3 recombinant proteins containing aMTD (HM165S3, HM165S3A and HM165S3B), suppressed IFN-γ-induced phosphorylation of STAT1 and STAT3 (
4-2. aMTD/SD-Fused SOCS3 Recombinant Protein Inhibits the Secretion of Inflammatory Cytokines TNF-α and IL-6
We next investigated the effect of cell-permeable SOCS3 proteins on cytokines secretion. Treatment of C3H/HeJ primary peritoneal macrophages with SOCS3 proteins containing aMTD165 suppressed TNF-α and IL-6 secretion induced by the combination of IFN-γ and LPS by 50-90% during subsequent 9 hrs of incubation (
5. iCP-SOCS3 Suppresses Pro-Tumorigenic Functions in Solid Cancer Cells
5-1. iCP-SOCS3 Enhances the Cellular Uptake into Various Cancer Cells and Systemic Delivery to Various Tissues
Although solid tumor is one of the most cancers with a high mortality rate, there are few drugs for treating this lethal disorder. Since constitutive activation of STAT3 is found in various types of tumors and SOCS3 is closely related to the development of various solid tumors including gastric, colorectal and breast cancer, and glioblastoma, we first chose the various tumors as a primary indication of the iCP-SOCS3 as an anti-cancer agent.
To determine the cell-permeability of iCP-SOCS3 in the solid cancer cells, cellular uptake of FITC-labeled SOCS3 recombinant proteins was quantitatively evaluated by flow cytometry. FITC-HM165S3B recombinant protein (iCP-SOCS3) promoted the transduction into cultured cancer cells (
5-2. iCP-SOCS3 Inhibits Viability of Cancer Cells
Since the endogenous level of SOCS3 protein is reduced in solid tumor—gastric, colorectal and breast cancer, and glioblastoma—patients, and SOCS3 negatively regulates cell growth and motility in cultured tumor cells, we investigated whether iCP-SOCS3 inhibits cell viability through SOCS3 intracellular delivery in solid tumor cells. As shown in
5-3. iCP-SOCS3 Protein Induces Apoptosis in Colorectal Cancer Cells
To further determine the effect of iCP-SOCS3 on the tumorigenicity of various cancers, we subsequently investigated whether iCP-SOCS3 regulates apoptosis in HCT116 colorectal cancer cells. HM165S3B protein (iCP-SOCS3) was a considerably efficient inducer of apoptosis in HCT116 cells, as assessed either by a fluorescent terminal dUTP nick-end labeling (TUNEL) assay (
5-4. iCP-SOCS3 Inhibits Migration/Invasion of Gastric, Colorectal and Breast Cancer, and Glioblastoma
We next examined the ability of iCP-SOCS3 to influence cell migration to various cancer cells, such as gastric (AGS), colorectal (HCT116) and breast cancer (MDA-MB-231), and glioblastoma (U-87 MG) cells. These cells were treated with recombinant proteins for 2 hrs, the monolayers were wounded, and cell migration in the wound was monitored after 24 or 48 hrs (
6. iCP-SOCS3 Suppresses Pro-Tumorigenic Functions in Various Cancer Cells
6-1. iCP-SOCS3 Suppresses the Gastric and Colorectal Cancer, and Glioblastoma Xenograft
We assessed the anti-tumor activity of iCP-SOCS3 against human cancer xenografts. Balb/c nu/nu mice were subcutaneously implanted with tumor block (1 mm3) of tumor cells into the left side of the back. Tumor-bearing mice were intravenously administered HM165S3B or control proteins (HS3B; 600 μg/head, respectively) for 21 days and observed for 2 weeks following the termination of the treatment (
6-2. iCP-SOCS3 Regulates the Expression of Tumor-Associated Markers in Human Tumor Xenograft
The anti-tumor activity of HM165S3B at day 35 was accompanied by changes in the expression of biomarkers linked to SOCS3 signaling, including p21, Bax, cleaved caspase-3, CD31, and VEGF (
The following examples are presented to aid practitioners of the invention, to provide experimental support for the invention, and to provide model protocols. In no way are these examples to be understood to limit the invention.
H-regions of signal sequences (HRSP)-derived CPPs (MTM, MTS and MTD) do not have a common sequence, a sequence motif, and/or a common structural homologous feature. In this invention, the aim is to develop improved hydrophobic CPPs formatted in the common sequence and structural motif that satisfy newly determined ‘critical factors’ to have a ‘common function’, to facilitate protein translocation across the membrane with similar mechanism to the analyzed CPPs. 6 critical factors have been selected to artificially develop novel hydrophobic CPP, namely advanced macromolecule transduction domain (aMTD). These 6 critical factors include the followings: amino acid length of the peptides (ranging from 9 to 13 amino acids), bending potentials (dependent with the presence and location of proline in the middle of sequence (at 5′, 6′, 7′ or 8′ amino acid) and at the end of peptide (at 12′)), instability index (II) for rigidity/flexibility (II: 40-60), grand average of hydropathy (GRAVY) for hydropathy (GRAVY: 2.1-2.4), and aliphatic index (AI) for structural features (AI: 180-220). Based on these standardized critical factors, new hydrophobic peptide sequences, namely advanced macromolecule transduction domain peptides (aMTDs), in this invention have been developed and selected to be fused with the cargo protein, SOCS3, to develop improved cell-permeable SOCS3 recombinant protein (iCP-SOCS3).
Histidine-tagged human SOCS3 proteins were constructed by amplifying the SOCS3 cDNA (225 amino acids) for aMTD fused to SOCS3 cargo. The PCR reactions (100 ng genomic DNA, 10 pmol each primer, each 0.2 mM dNTP mixture, 1× reaction buffer and 2.5 U Pfu(+) DNA polymerase (Doctor protein, Korea)) were digested on the restriction enzyme site between Nde I (5′) and Sal I (3′) involving 35 cycles of denaturing (95° C.), annealing (62° C.), and extending (72° C.) for 45 sec each. For the last extension cycle, the PCR reactions remained for 10 min at 72° C. The PCR products were subcloned into 6× His expression vector, pET-28a(+) (Novagen). Coding sequence for SDA or SDB fused to C terminus of his-tagged aMTD-SOCS3 was cloned at BamHI (5′) and SalI (3′) in pET-28a(+) from PCR-amplified DNA segments and confirmed by DNA sequence analysis of the resulting plasmids.
The recombinant proteins were purified from E. coli BL21-CodonPlus (DE3) cells grown to an A600 of 0.6 and induced for 3 hrs with 0.6 mM IPTG. Denatured recombinant proteins were purified by Ni2+ affinity chromatography as directed by the supplier (Qiagen, Hilden, Germany). After purification, they were dialyzed against a refolding buffer (0.55 M guanidine HCl, 0.44 M L-arginine, 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 100 mM NDSB, 2 mM reduced glutathione, and 0.2 mM oxidized glutathione) and changed to a physiological buffer such as DMEM medium.
For quantitative cell-permeability, recombinant SOCS3 proteins were conjugated to 5/6-fluorescein isothiocyanate (FITC) according to the manufacturer's instructions (Sigma-Aldrich, St. Louis, Mo.). RAW 264.7 cells were treated with 10 μM FITC-labeled recombinant proteins for 1 hr at 37° C., washed three times with cold PBS, and treated with proteinase K (10 μg/mL) for 20 min at 37° C. to remove cell-surface bound proteins. Cell-permeability of these recombinant proteins was analyzed by flow cytometry (Guava, Millipore, Darmstadt, Germany) using the Flowio cytometric analysis software.
For visual cell permeability, NIH3T3 cells were cultured on coverslips in 24-well plates and with 10 μM FITC-conjugated recombinant proteins for 1 hr at 37° C. These cells on coverslips were washed with PBS, fixed with 4% formaldehyde for 10 min, and washed three times with PBS at room temperature. Coverslips were mounted with VECTASHIELD Mounting Medium (Vector laboratories, Burlingame, Calif.) with DAPI (4′,6-diamidino-2-phenylindole) for nuclear staining. Intracellular localization of fluorescent signal was determined by confocal laser scanning microscopy (LM700, Zeiss, Germany).
ICR mice (6-week-old, female) were injected intraperitoneally (600 μg/head) with either FITC only or FITC-conjugated SOCS3 recombinant proteins. After 2 hrs, the liver, kidney, spleen, lung, heart, and brain were isolated, washed with an O.C.T. compound (Sakura), and frozen on dry ice. Cryosections (20 μm) were analyzed by fluorescence microscopy (Carl Zeiss, Gottingen, Germany).
RAW264.7 cells were pretreated with different agents to assess the effect of various conditions on protein uptake: (i) 5 μg/ml proteinase K for 10 min, (ii) 20 μM Taxol for 30 min, (iii) 10 μM antimycin in the presence or absence of 1 mM ATP for 2 hrs, (iv) incubation on ice (or maintained at 37° C.) for 60 min, and (v) 100 mM EDTA for 3 hrs. These agents were used at concentrations known to be active in other applications. The cells were then incubated with 10 μM FITC-labeled proteins for 1 hr at 37° C., washed three times with ice-cold phosphate-buffered saline, treated with proteinase K (10 μg/ml for 5 min at 37° C.) to remove cell-surface bound proteins, and analyzed by flow cytometry. To assess cell-to-cell protein transfer, RAW264.7 cells containing FITC-conjugated protein were prepared in the same way and mixed with untreated cells labeled with PreCP-Cy5.5-CD14 antibody for 2 hrs. Cell-to-cell protein transfer, resulting in FITC-Cy5.5 double-positive cells, was monitored by flow cytometry.
PANC-1 cells (Korean Cell Line Bank, Seoul, Korea) were cultured in modified Eagle's medium (DMEM; Welgene, Daege, Korea) supplemented with 10% (v/v) FBS, penicillin (100 units/ml), and streptomycin (10 μg/ml, Gibco BRL) and pretreated with 10 μM of SOCS3 recombinant proteins for 2 hrs followed by exposing the cells to agonists (100 ng/ml IFN-γ) for 15 min. Cells were lysed with RIPA lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate, 10 mM NaF, and 2 mM Na3VO4) containing a protease inhibitor cocktail and then centrifuged at 13,000×g for 15 min at 4° C. Equal amounts of lysates were resolved by SDS-PAGE, transferred onto PVDF membranes, and probed with phospho (pY701)-specific STAT1 (Cell Signaling, Danvers, Mass.).
Peritoneal macrophages were obtained from C3H/HeJ mice. Peritoneal macrophages were incubated with 10 μM recombinant proteins (1:HS3, 2:HM165S3, 3:HM165S3A and 4:HM165S3B, respectively) for 1 hr at 37° C. and then stimulated them with LPS (500 ng/ml) and/or IFN-γ (100 U/ml) without removing iCP-SOCS3 proteins for 3, 6, or 9 hrs. The culture media were collected, and the extracellular levels of cytokine were measured by a cytometric bead array (BD Biosciences, Pharmingen) according to the manufacturer's instructions.
Cells originated from human cancer cells and mouse fibroblast (NIH3T3) were purchased (ATCC, Manassas, Va.) and maintained as recommended by the supplier. These cells (3×103/well) were seeded in 96 well plates. The next day, cells were treated with DMEM (vehicle) or recombinant SOCS3 proteins for 96 hrs in the presence of serum (2%). Proteins were replaced daily. Cell growth and survival were evaluated with the CellTiter-Glo Cell Viability Assay (Promega, Madison, Wis.). Measurements using a Luminometer (Turner Designs, Sunnyvale, Calif.) were conducted following the manufacturer's protocol.
Apoptotic cells were analyzed using terminal dUTP nick-end labeling (TUNEL) assay with In Situ Cell Death Detection kit TMR red (Roche, 4056 Basel, Switzerland). Cells were treated with either 10 μM SOCS3 recombinant protein or buffer alone for 16 hrs with 2% fetal bovine serum. Treated cells were washed with cold PBS two times, fixed in 4% paraformaldehyde (PFA, Junsei, Tokyo, Japan) for 1 hr at room temperature, and incubated in 0.1% Triton X-100 for 2 min on the ice. Cells were washed with cold PBS twice, and treated TUNEL reaction mixture for 1 hr at 37° C. in dark, washed cold PBS three times and observed by fluorescence microscopy (Nikon, Tokyo, Japan).
Annexin V/7-Aminoactinomycin D (7-AAD) staining was performed using flow cytometry according to the manufacturer's guidelines. Briefly, 1×106 cells were washed three times with ice-cold PBS. The cells were then resuspended in 100 μl of binding buffer and incubated with 1 μl of 7-AAD and 1 μl of annexin V-PE for 30 min in the dark at 37° C. Flow cytometric analysis was immediately performed using a guava easyCyte™ 8 Instrument (Merck Millipore).
Cells were treated with either DMEM (vehicle) or 10 μM SOCS3 recombinant proteins, lysed in RIPA lysis buffer containing proteinase inhibitor cocktail, incubated for 15 min at 4° C., and centrifuged at 13,000 rpm for 10 min at 4° C. Equal amounts of lysates were separated on 15% SDS-PAGE gels and transferred to a nitrocellulose membrane. The membranes were blocked using 5% skim milk or 5% Albumin in TBST and incubated with the following antibodies: anti-Bcl-2 (Santa Cruz biotechnology) and anti-Cleaved Caspase 3 (Cell Signaling Technology), then HRP conjugated anti-mouse or anti-rabbit secondary antibody.
Cells were seeded into 12-well plates, grown to 90% confluence, and incubated with 10 μM HS3, HM165S3A, HM165S3A or HM165S3B in serum-free medium for 2 hrs prior to changing the growth medium. The cells were washed twice with PBS, and the monolayer at the center of the well was “wounded” by scraping with a pipette tip. Cells were cultured for an additional 72 hrs and cell migration was observed by phase contrast microscopy. The migration is quantified by counting the number of cells that migrated from the wound edge into the clear area.
The lower surface of Transwell inserts (Costar) was coated with gelatin (10 μg/ml), and the membranes were allowed to dry for 1 hr at room temperature. The Transwell inserts were assembled into a 24-well plate, and the lower chamber was filled with growth media containing 10% FBS and FGF2 (10 μg/ml). Cells (5×105) were added to each upper chamber, and the plate was incubated at 37° C. in a 5% CO2 incubator for 24 hrs. Migrated cells were stained with 0.6% hematoxylin and 0.5% eosin and counted.
The lower surface of Transwell inserts (Costar) was coated with gelatin (10 μg/ml), the upper surface of Transwell inserts was coated with matrigel (40 μg per well; BD Biosciences), and the membranes were allowed to dry for 1 hr at room temperature. The Transwell inserts were assembled into a 24-well plate, and the lower chamber was filled with growth media containing 10% FBS and FGF2 (10 μg/ml). Cells (5×105) were added to each upper chamber, and the plate was incubated at 37° C. in a 5% CO2 incubator for 24 hrs. Migrated cells were stained with 0.6% hematoxylin and 0.5% eosin and counted.
Female Balb/c nu/nu mice were subcutaneously implanted with NCI-N87, HCT116 or U-87 MG tumor block (1 mm3) into the left back side of the mouse. Tumor-bearing mice were intravenously administered with iCP-SOCS3 or the control proteins (600 μg/head) for 21 days and observed for 2 weeks following the termination of the treatment. Tumor size was monitored by measuring the longest (length) and shortest dimensions (width) once a day with a dial caliper, and tumor volume was calculated as width2×length×0.5.
After protein treatment, mice were killed, and six organs (brain, heart, lung, liver, kidney, and spleen) from each were collected and kept in a suitable fixation solution until the next step.
Tissue samples were fixed in 4% Paraformaldehyde (Duksan) for 3 days, dehydrated, cleared with xylene and embedded in Paraplast. Sections (6 μm thick) of tumor were placed onto poly-L-lysine coated slides. To block endogenous peroxidase activity, sections were incubated for 15 min with 3% H2O2 in methanol. After washing three times with PBS, slides were incubated for 30 min with blocking solution (5% fetal bovine serum in PBS). Rabbit anti-p21 antibody (sc-397, SantaCruz), mouse anti-Bax antibody (sc-7480, SantaCruz) and rabbit anti-VEGF (ab46154, abcam) were diluted 1:1000 (to protein concentration 0.1 μg/ml) in blocking solution, applied to sections, and incubated at 4° C. for 24 hrs. After washing three times with PBS, sections were incubated with biotinylated mouse and rabbit IgG (Vector Laboratories) at a 1:1000 dilution for 1 hr at room temperature, then incubated with avidin-biotinylated peroxidase complex using a Vectorstain ABC Kit (Vector Laboratories) for 30 min at room temperature. After the slides are reacted with oxidized 3,3-diaminobenzidine as a chromogen, they were counterstained with Harris hematoxylin (Sigma-Aldrich). Permanently mounted slides were observed and photographed using a microscope equipped with a digital imaging system (ECLIPSE Ti, Nikon, Japan).
All data are presented as mean±s.d. Differences between groups were tested for statistical significance using Student's t-test and were considered significant at p<0.05 or p<0.01.
It will be apparent to those skilled in the art that various modifications can be made to the above-described exemplary embodiments of the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention covers all such modifications provided that they come within the scope of the appended claims and their equivalents.
This application claims the benefit of the filing date of U.S. Provisional Application No. 62/042,493, filed on Aug. 27, 2014, in the United States Patent and Trademark Office, the disclosure of which is incorporated herein in its entirety by reference.
| Number | Date | Country | |
|---|---|---|---|
| 62042493 | Aug 2014 | US |
