Patent Application
20130029907
The present invention relates to novel polypeptides that bind to IL-23 receptor and inhibit the binding of IL-23 to its corresponding receptor and cell signaling thereof. The present invention also relates to compositions containing the novel polypeptides, and use of same to treat IL-23 associated human diseases including, for example, inflammatory bowel diseases, psoriasis and Crohn's disease.
Overt production of inflammatory cytokines such as IL-23 has been implicated in a number of diseases including multiple sclerosis, asthma, rheumatoid arthritis, inflammatory bowel diseases, Crohn's disease and psoriasis. Over 10 million people in the United States are affected by these IL-23 mediated diseases. Systemic levels of IL-23 are found to be associated with rheumatoid arthritis. (Melis, et al., Ann. Rheum. Dis., 69: 618-623, 2010). IL-23 gene knock-out mice are found to be resistant to development of many inflammatory diseases, indicating a crucial role of IL-23 in the pathogenesis of the disorders. (Langowski, et al., Nature, 442:7101, 2006). Single nucleotide polymorphisms in the IL-23 receptor genes have been associated with psoriasis. (Capon et al., Hum. Genet. 122:201-6, 2007). A single nucleotide polymorphism in the IL-23 receptor gene has been found to be associated with Crohn's disease and inflammatory bowel diseases. (Duerr, et al., Science, 314:1461-1463, 2006).
At a structural level, cytokine IL-23 is a heterodimeric cytokine that consists of two subunits (i.e., p19 and p40). The mature p19 subunit contains 170 amino acid residues in a 4 α-helix bundle, and the mature p40 subunit contains 306 amino acid residues consisting of three sequentially arranged β-strands. (Oppmann, et al., Immunity 13 (5): 715-725, 2000). Binding of IL-23 to its corresponding receptor (i.e., IL-23R) mediates cell signaling. For example, binding of IL-23 to IL-23R stimulates the production of IL-17, which is a key inflammatory mediator. IL-23R is a heterodimeric receptor consisting of an IL-23Rα subunit and an IL-12Rβ1 subunit. (Parham, et al., J. Immunol., 168:5699-5708, 2002). Interestingly, the IL-12Rβ1 subunit is shared with the IL-12 receptor.
Several researchers have reported using anti-IL-23 antibodies or IL-23 agonists in an attempt to disrupt IL-23 signaling. These studies shed light on the potential clinical applications in treating IL-23 related diseases. For example, U.S. Pat. No. 7,790,862 discloses the use of anti-p19 antibodies. Anti-p19 polyclonal antibodies reduced IL-17A and IL-17F production in a murine splenocyte assay. These authors purported that such anti-p19 antibodies may have utility in treating inflammatory diseases and cancer. U.S. Pat. No. 7,282,204 discloses the use of anti-p19 and anti-p40 antibodies. The authors claimed that anti-p19 antibodies reduced cancer volume in skin tumors, while anti-p40 antibodies provoked weight gain as to reduce cachexia. U.S. Patent Publication No. 2010/0166767 discloses the use of humanized anti-IL-23R antibodies. The authors determined the equilibrium dissociation constant for the created humanized anti-human IL-23R antibodies (i.e., Kd of Hum20D7 is 131 pM). U.S. Pat. No. 7,575,741 discloses the generation of an IL-23 fusion protein comprising the p19 subunit linked to the p40 subunit. The fusion protein is found to have property of enhancing wound healing in various mouse models.
The use of anti-p40 antibodies in inhibiting IL-23 activity, however, has many drawbacks. In particular, because the p40 subunit is shared by the IL-23 and IL-12 cytokines, anti-p40 antibodies may have the unintended consequence of inhibiting IL-12, demonstrating their non-specific effects (See, e.g., Oppmann, et al., Immunity 13:715-725, 2000; Cua, et al., Nature 421:744-748, 2003). Similarly, knocking out the p40 subunit has the dual affect of knocking out both IL-23 and IL-12.
Antibody-based approaches to inhibit IL-23R mediated signaling have additional shortcomings. First, it is recognized that antibody therapy often elicits an adverse immune response to the foreign administered antibodies during treatment. Second, repeated administration of antibodies (e.g., rodent-derived antibodies) potentially leads to fatal anaphylactic responses and suffer from loss in therapeutic efficacy and potency. Third, use of chimeric antibodies (i.e., antibodies in which mouse variable regions are fused with human constant regions) still possesses unwanted immunogenicity, albeit they may attenuate some anaphylactic responses. Fourth, while use of humanized antibodies (i.e., antibodies generated by grafting rodent CDR loops to human frameworks) may remedy the immunogenicity issues, preparation of humanized antibodies often leads to less effective antibodies (i.e., reduced antibody binding activity).
Although the amino acid sequence of IL-23R is known, there has been no report relating to the crystallization study of the molecular structure of IL-23R. Relying on the linear amino acid sequence with the aid of computer modeling, Chemtob et al. generated peptides that correspond to the hinge regions within the D1, D2 and D3 of the IL-23R. These authors disclosed in the U.S. Patent Publication 2010/0190710 ('710 application) that their IL-23R based polypeptides (e.g., LPDEVTCV, TEEEQQYL, TEEEQQYL, and MEESKQLQL) may represent potential antagonists of IL-23R. In particular, the '710 application discloses certain polypeptides can inhibit IL-23R mediated cell signaling (i.e., STAT3 phosphorylation). They reported using polypeptides that mimic IL-23R and function as IL-23R antagonists.
There is a continuing need for the discovery of novel polypeptides that inhibit IL-23 mediated cell signaling and diseases. The present inventors report herein, using a novel phage display approach, the identification of novel polypeptides that can bind to IL-23R. There is also disclosed the structure of these polypeptides, its compositions and methods of treatment using same in inhibiting binding of IL-23 to its receptor thereof and the subsequent cell signaling events. The novel polypeptides have utility in clinical application in the treatment of IL-23 mediated immuno-diseases.
The present invention provides the discovery of therapeutic peptides (screened with a phage display library method) that are capable of inhibiting the binding of IL-23 to its corresponding receptor thereof. The therapeutic peptides have clinical application in the treatment of IL-23 associated immunological diseases including, in particular, Crohn's disease and inflammatory bowel diseases.
The peptides according to this invention are useful in methods and compositions for the treatment of IL-23 associated diseases. Targeting of IL-23 or the IL-23 receptor (i.e., the IL-23 axis) with the present peptides thus represents a novel therapeutic approach for autoimmune diseases including psoriasis, inflammatory bowel disease, rheumatoid arthritis and multiple sclerosis.
In another aspect, the present invention provides an isolated polypeptide having a core structure of WX1X2X3W. The X1, X2, and X3 are amino acid residues, with the proviso that when one of X1, X2 or X3 is W, the remaining X1, X2 or X3 cannot be W. The polypeptides of the present invention are characterized by its ability to inhibit the binding of IL-23 to IL-23 receptor and the IL-23-mediated signaling. For example, the polypeptides are effective in binding to IL-23R as evidenced by flow cytometry and immunoprecipitation with a soluble IL-23R (i.e., Δ9). The polypeptides are also effective in blocking IL-23 mediated signaling in cells as evidenced by the phosphor-STAT3 levels.
In one aspect, the X3 in the WX1X2X3W core structure of the polypeptides is an aromatic amino acid, such as Y, W or F. Preferably, the X3 is Y.
In one embodiment, the WX1X2YW core structure provides an isolated polypeptide that has an amino acid sequence of SEQ NO: 2, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ NO: 54, SEQ ID NO: 55, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 76, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 91, and SEQ ID NO: 126.
In another embodiment, the present invention provides an isolated polypeptide that contains a core structure which has an amino acid sequence of WX1X2YW, wherein WX1X2YW is WVQYW (SEQ ID NO: 94), WYNYW (SEQ ID NO: 95), WMTYW (SEQ ID NO: 96), WATYW (SEQ ID NO: 97), WEMYW (SEQ ID NO: 98), WKDYW (SEQ ID NO: 99), WEHYW (SEQ ID NO: 100), WQNYW (SEQ ID NO: 101), WMQYW (SEQ ID NO: 103), WHAYW (SEQ ID NO: 104), WQTYW (SEQ ID NO: 105), WYAYW (SEQ ID NO: 107), WEDYW (SEQ ID NO: 108), WLQYW (SEQ ID NO: 111), and WLNYW (SEQ ID NO: 112).
In another aspect, the present invention provides an isolated polypeptide having a core structure of WX1X2WW.
In one embodiment, the present invention provides an isolated polypeptide that has an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 20, SEQ NO: 57, SEQ ID NO: 58, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90.
In another embodiment, the present invention provides an isolated polypeptide that contains a core structure which has an amino acid sequence of WX1X2WW, wherein WX1X2WW is WEDWW (SEQ ID NO: 116), WIDWW (SEQ ID NO: 117), WDDWW (SEQ ID NO: 118), WEEWW (SEQ ID NO: 119), WQDWW (SEQ ID NO: 120), WENWW (SEQ ID NO: 121), WQNWW (SEQ ID NO: 122), WEKWW (SEQ ID NO: 123), WKDWW (SEQ ID NO: 124), and WKKWW (SEQ ID NO: 125).
In another aspect, the present invention provides an isolated polypeptide having a core structure of WX1X2FW.
In one embodiment, the present invention provides an isolated polypeptide that has an amino acid sequence of SEQ NO: 18, SEQ ID NO: 29, SEQ ID NO: 71, SEQ ID NO: 74, SEQ ID NO: 92, SEQ ID NO: 106, SEQ ID NO: 109, SEQ ID NO: 114, and SEQ ID NO: 115.
In another aspect, the present invention provides an isolated polypeptide having a core structure of WWX2X3W.
In one embodiment, the present invention provides an isolated polypeptide that has an amino acid sequence of SEQ ID NO: 102, SEQ ID NO: 110, and SEQ ID NO: 113.
In another aspect, the present invention provides an isolated polypeptide that is a cyclic peptide through a disulfide bridge between two cysteine residues.
In one embodiment, the present invention provides an isolated polypeptide that has an amino acid sequence of SEQ ID NO: 130 and SEQ ID NO: 131.
In one embodiment, the present invention provides an isolated polypeptide that has an amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 19, SEQ ID NO: 25, SEQ ID NO: 70, and SEQ ID NO: 93. The isolated polypeptide is capable of inhibiting the binding of IL-23 to IL-23 receptor and IL-23-mediated signaling.
In another aspect, the present invention provides an isolated polypeptide having a core structure of WX1X2X3W.
Preferably, the isolated polypeptide is 12 amino acids in length.
In another aspect, the present invention provides an isolated polypeptide of 9-18 amino acids in length. The isolated polypeptide comprises a core structure having an amino acid sequence of WX1X2X3W, wherein W is tryptophan, X1, X2 and X3 are amino acids, with the proviso that when one of X1, X2 or X3 is W, the remaining X1, X2 or X3 cannot be W. The isolated polypeptide binds to soluble recombinant human IL-23R and inhibits binding between IL-23 and its corresponding receptor thereof.
In another aspect, the present invention provides an isolated polypeptide having a length of 9-18 amino acids. The polypeptide comprises a core structure having an amino acid sequence of WX1X2WW or WWX2X3W where W is tryptophan and X1, X2 and X3 are amino acids.
In one embodiment, the present invention provides an isolated polypeptide having a core structure of WEDWW (SEQ ID NO: 116), WIDWW (SEQ ID NO: 117), WDDWW (SEQ ID NO: 118), WEEWW (SEQ ID NO: 119), WQDWW (SEQ ID NO: 120), WENWW (SEQ ID NO: 121), WQNWW (SEQ ID NO: 122), WEKWW (SEQ ID NO: 123), WKDWW (SEQ ID NO: 124) or WKKWW (SEQ ID NO: 125).
In another aspect, the present invention provides an isolated polypeptide having a length of 9-18 amino acids. The polypeptide comprises a core structure having an amino acid sequence of WX1X2X3W, wherein W is tryptophan and X1, X2 and X3 are amino acids, with the proviso that when one of X1, X2 or X3 is W, the remaining X1, X2 or X3 cannot be W. The isolated polypeptide binds to soluble recombinant human IL-23R and inhibits binding between IL-23 and its receptor.
In another aspect, the present invention provides isolated polypeptides that inhibit IL-23 mediated cell signaling.
In another aspect, the present invention provides a pharmaceutical composition comprising the isolated polypeptides disclosed in this application, together with a pharmaceutically acceptable carrier.
In another aspect, the present invention provides a method of inhibiting the binding of IL-23 to IL-23 receptor on a cell and IL-23 mediated signaling thereof, comprising the steps of: a) providing a cell in need of treatment; and b) exposing the cell to an isolated polypeptide.
In another aspect, the present invention provides a method of treating IL-23 associated diseases, comprising the steps of: a) identifying a human suspected of suffering an IL-23 associated disease; and b) administering a composition containing polypeptides of the present invention in an effective amount to block IL-23 binding to its receptor and subsequent cell signaling thereof.
The present invention can be better understood from the following description of preferred embodiments, taken in conjunction with the accompanying drawings. It should be apparent to those skilled in the art that the described embodiments of the present invention provided herein are merely exemplary and illustrative and not limiting.
As used herein, the term “IL-23” refers to Interleukin-23. IL-23 is a heterodimeric cytokine consisting of two subunits; namely, p19 (IL-23 alpha subunit) (nucleotide sequence—NCBI NM—016584; protein sequence—NCBI NP—057668), and p40 (IL-12 beta subunit) (nucleotide sequence—NCBI NM—002187; protein sequence—NCBI NP—002187).
As used herein, the term “IL-23R” refers to the interleukin-23 receptor. IL-23R is composed of two (2) subunits: namely, IL-23Rα (mRNA sequence NCBI-NM—144701, protein sequence NCBI NP 653302) and IL-12Rβ1 (mRNA and protein sequences NCBI—NM—005535). The IL-23Rα gene is located on chromosome 1p31.3. The full-length translated IL-23R protein is a type I cytokine receptor. Human IL-12β1, when partnered with human IL-12β2, forms a different interleukin receptor (i.e., IL-12 receptor).
As used herein, the term “Δ9” refers to the naturally-occurring truncated IL-23Rα resulting from IL-23Rα gene splicing. For purposes of this application, “Δ9 variant”, “Δ9 isoform”, and “Δ9 protein” are used interchangeably to refer to this particular naturally-occurring truncated IL-23Rα protein. The Δ9 protein has 348 amino acids plus eight (8) novel amino acid sequences unique to Δ9 protein (i.e., a total of 356 amino acids). The signal sequence (i.e., 1-23 amino acids) on the immature Δ9 protein (located inside the cells) is cleaved before the mature Δ9 protein is released outside of the cells. The mature Δ9 therefore has a total of 333 amino acids (i.e., 24-356). For purposes of this application, therefore, the term “Δ9” is intended to include both of these two (2) forms. The immature Δ9 (i.e., containing 1-23 signal peptide and amino acid residues 24-348 and the eight (8) novel amino acid sequence) has an amino acid sequence set forth in SEQ ID NO: 126.
As used herein, the term “IL-23 mediated cell signaling” refers to a detectable biological activity after IL-23 binding to the IL-23 receptor and includes, for example, STAT3 phosphorylation and the stimulation of chemokines, cytokines or pro-inflammatory molecules.
As used herein, the term “IL-23 associated disease” refers to a disease condition that results from abnormal activity of IL-23 or its receptor and includes, for example, inflammatory bowel diseases, Crohn's disease, ulcerative colitis, psoriasis, and the like.
As used herein, the term “amino acid” refers to a molecule containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. Amino acids are the structural units that make up proteins. Twenty-two amino acids are naturally incorporated into polypeptides and are called natural amino acids. Non-natural amino acids refers to those amino acids that are not found in proteins (for example carnitine, GABA, and L-DOPA), or are not produced directly and in isolation by standard cellular machinery (for example, hydroxyproline and selenomethionine). Got purposes of this application, amino acids may include non-naturally occurring amino acids including, for example, lanthionine, 2-aminoisobutyric acid, dehydroalanine, gamma-aminobutyric acid, and the like.
As used herein, “amino acid” may be represented by either the full name of the amino acid, standard three-letter code, or standard single-letter code as below: A=Ala; C=Cys; D=Asp; E=Glu; F=Phe; G=Gly; H=His; I=Ile; K=Lys; L=Leu; M=Met; N=Asn; P=Pro; Q=Gln; R=Arg; S=Ser; T=Thr; V=Val; W=Trp; and Y=Tyr.
As used herein, the term “polypeptide” refers to an amino acid chain comprising two or more amino acids. The terms “protein”, “polypeptide”, and “peptide” are used synonymously in this application.
As used herein, the term “core structure” refers to an amino acid sequence that is present in many of the isolated polypeptides screened using a Phage Display Library and human IL-23R in the present invention. The core structure comprises two tryptophan (W) residues separated by amino acid residues. An example of a core structure is WX1X2X3W (W=tryptophan and X1, X2 and X3 are amino acid residues). Another example of a core structure is WX1X2X3 X4X5X6W (W=tryptophan and X1, X2, X3, X4, X5, and X6 are amino acid residues). All of the screened isolated polypeptides have the ability to (i) bind human IL-23R, (ii) inhibit IL-23 binding to IL-23R and (iii) inhibit the IL-23 mediated subsequent cellular signaling thereof.
As used herein, the term “isolated” refers to polypeptides that are essentially free of other substances with which they may be found in vivo or as a result of being produced synthetically.
As used herein, the term “FLAG” refers to a protein tag that can be added to a protein using recombinant DNA technology. The peptide sequence of the FLAG-tag is: N-DYKDDDDK-C (1,012 Da). The insertion of a FLAG permits the protein to be isolated by affinity chromatography; for example, separating a recombinant over-expressed protein from a wild-type protein expressed by a host organism. FLAG can also be used in the isolation of protein complexes. Inserting a FLAG-tag to a protein also allows one to purify the protein with an antibody against the FLAG sequence. Examples are cellular localization studies by immunofluorescence or detection by SDS PAGE protein electrophoresis.
As used herein, the term “cyclic peptide” refers to a polypeptide chain whose amino and carboxyl termini are themselves linked together with a peptide bond that forms a circular chain (i.e., between the alpha carboxyl of one residue to the alpha amine of another). For purposes of this application, cyclic peptides may also include linkage other than a peptide bond such as non-alpha amide linkage, thioether linkage between Trp and Cys residues.
As used herein, the term “Phage Display Screening” refers to a method for studying protein-peptide interactions that uses bacteriophages that have been genetically engineered to express a set of peptides on their outer surface. Phage Display was originally invented by George P. Smith in 1985. Smith demonstrated the display of peptides on filamentous phage by fusing the peptide of interest on to gene3 of filamentous phage. The connection between genotype and phenotype enables large libraries of proteins (i.e., a phage display library) to be screened and amplified in a process called in vitro selection or bio-panning.
The term “inhibit” refers to a decrease of a biological activity. For purposes of this application, biological activity includes IL-23 cell signaling.
As used herein, the term “treat” or “treatment” as used within the context of the present invention are meant to include therapeutic as well as prophylactic treatments for the diseases. Thus, for example, it includes the administration of the peptides prior to or following the onset of the IL-23 associated diseases thereby preventing clinical signs of the IL-23 associated diseases. As another example, administration of the peptides after clinical manifestation of the diseases to combat the symptoms of the IL-23 associated disease comprises “treatment” of the diseases. Those “in need of treatment” include humans who already having the IL-23 associated diseases.
As used herein, the term “ELISA” refers to “Enzyme-Linked ImmunoSorbent Assay” and is a biochemical technique used in detecting the presence of antibody or antigen in a sample.
As used herein, the term “Competitive ELISA” refers to an ELISA performed using two ligands for a target molecule to determine relative signal and binding affinities of the two ligands.
As used herein, the term “STAT3” (also known as signal transducer and activator of transcription 3) (NCBI Accession No. NP—003141) refers to the transcription factor which in humans is encoded by the STAT3 gene.
As used herein, the term “IC50” refers to half maximal inhibitory concentration (IC50). IC50 is a measure of the effectiveness of a compound in inhibiting biological or biochemical function. This quantitative measure indicates how much of a particular drug or other substance is needed to inhibit a given biological process by half.
As noted above, the present invention is generally directed to compositions and methods for treatment of IL-23 mediated diseases in human. In one aspect, the composition includes polypeptides that inhibit binding of IL-23 to its corresponding receptor and inhibit its cellular activation thereof.
In accordance with the present invention, the present inventors used a Phage Display Screening Assay. A phage library containing a large collection of peptides (i.e., containing approximately 1×1011 pfu random peptide sequences) was screened based on binding ability towards IL-23R. In this study, we used a phage library that displayed 12-mer polypeptides. The phage library was screened using soluble IL-23R to identify polypeptides of interest (i.e., those that would bind to IL-23R). One skilled in the art would recognize that the polypeptides displayed on the phage surface are not limited to 12-mer in length. Indeed, the polypeptides of the present invention would encompass various amino acid lengths insofar as they possess the binding ability towards IL-23R. Exemplary amino acid lengths include, but not limited to, 14-mer, 16-mer, 18-mer and the like.
In order to screen polypeptides with respect to its ability to bind IL-23R, we used a soluble form of IL-23R. In one embodiment, Phage Display Screening was conducted using a Δ9 IL-23R protein (i.e., IL-23R lacking a transmembrane domain, and thus render the receptor soluble). A particular Δ9 IL-23R protein used in the assay is the soluble IL-23R protein that has an amino acid sequence set forth in SEQ ID NO: 126. This particular Δ9 protein is a soluble form of IL-23R that lacks the trans-membrane domain. To screen the bound polypeptides that bind to soluble IL-23R, the IL-23R is coupled to a Flag protein (SEQ ID NO: 128). Anti-Flag affinity gel (e.g., agarose) is then used to capture the random polypeptides that interact with Δ9 IL-23R-Flag (which is precipitated by an anti-Flag affinity agarose) (See,
The polypeptides of the present invention are screened and selected based on a unique function that is equally shared by all the screened polypeptides; that is, they bind to IL-23R. In one embodiment, the polypeptides of the present invention bind specifically to IL-23R that is integrally expressed on a cell membrane (i.e., IL-23R having a transmembrane domain). In another embodiment, the polypeptides of the present invention bind specifically to a soluble IL-23R (e.g., IL-23R lacking a transmembrane domain). Binding of the polypeptides to IL-23R (either membrane bound IL-23R or soluble IL-23R) may be determined using a binding assay specifically designed to detect the binding between a polypeptide and the IL-23R.
In one embodiment, binding of polypeptides to IL-23R can be determined using an IL-23R expressing cell. An exemplary IL-23R expressing cell may include a cell line that is stably transfected with IL-23R cDNA.
In another embodiment, soluble IL-23R may be employed to develop a binding assay with a polypeptide. An exemplary soluble IL-23R includes, but not limited to, Δ9 protein, which amino acid sequence is set forth in SEQ ID NO: 126. Another exemplary soluble IL-23R is IL-23R coupled with Fc (i.e., fusion protein) (SEQ ID NO: 127).
Other binding assays known to the art may be used to assess the binding of a polypeptide with IL-23R. One exemplary assay is immunoprecipitation. One skilled in the art would recognize that an immunoprecipitation assay often involves the use of a bead (e.g., agarose beads) to immunoprecipitate a polypeptide of interest. To better detect the binding in an immunoprecipitation assay, polypeptides are often labeled with a radioactive agent or a fluorescent compound.
Using the Flag protein approach, we have identified a total of 13 polypeptides (i.e., peptide nos. 52, 60, 61, 65, 68, 70, 71, 72, 74, 75, 77 and 79) (See,
In another embodiment, Phage Display Screening was conducted using a different soluble IL-23R. A particular soluble IL-23R used in our study is IL-23R-Fc chimera. This particular IL-23R has the full-length IL-23R amino acid sequence, but coupled with an Fc region of human IgG1 (SEQ ID NO: 127). To screen the bound polypeptides that bind to soluble IL-23R, the IL-23R is coupled with the Fc (i.e., Fc fusion protein). A protein A sepharose is then used to capture the random polypeptides that interact with the IL-23R-Fc fusion protein (which is precipitated by a protein-A sepharose).
Using the IL-23R-Fc chimera protein approach, we have identified a total of 29 polypeptides (i.e., peptide nos. 1, 2, 3, 4, 5, 6, 7, 9, 10, 12, 13, 14, 15, 16, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 and 32) (See,
It is noted that one (1) polypeptide sequence was identified both in the Flag protein approach and in the IL-23R-Fc chimera protein approach.
Accordingly, using soluble IL-23R (i.e., both Flag protein approach and IL-23R-Fc chimera protein approach) in a Phage Display Screening assay, we have identified a total of 28 novel polypeptides that bound IL-23. It is noted that the Flag protein approach generates fewer polypeptides in comparison to the IL-23R-Fc chimera approach. This may be attributed to the finding that the Flag protein approach has yielded a higher background (i.e., anti-Flag and Flag may provide non-specific screening—that is, a false positive result). Many of the screened polypeptides in fact were due to Flag sequences. On the other hand, the use of IL-23R-Fc chimera in our Phage Display Screening provides optimal results.
The polypeptides of the present invention may be made by synthetic methods. Preferably, solid phase synthesis techniques may be used to prepare the present polypeptides. Suitable techniques are well known in the art. For example, Merrifield, Chem. Polypeptides, pages 335-361 (Katsoyannis and Panayotis editors) (1973); Merrifield, J. Am. Chem. Soc., Volume 85, page 2149 (1963); Davis et al., Biochem. Intl., Volume 10, pages 394-414 (1985); Stewart and Young, Solid Phase Peptide Synthesis (1969); U.S. Pat. No. 3,941,763; Finn et al., The Proteins (3d edition), Volume 2, pages 105-253 (1976). Solid phase synthesis is the preferred technique for making individual polypeptides because of its cost-effectiveness.
In one embodiment, the present invention provides modifications of inhibitory polypeptides such as cyclic polypeptide or Fc coupled polypeptides. The modification can protect therapeutic peptides from proteolytic enzymes, increase stability as well as enhance circulation time of the peptides. Thus, the modified polypeptides possess an enhanced biological activity of the therapeutic molecule. The techniques for protein modification and Fc fusion proteins are known to one skilled in the art. (See, for example, WO 00/24782 which describes fusion proteins comprising Fc antibody domains linked to biologically active peptides and their use as pharmaceutical agents).
In one embodiment, the modified polypeptide is cyclic. For example, the polypeptide may be modified to contain two Cys residues at the C-terminus and N-terminus, which could cyclize by disulfide bond formation. The cyclization of linear peptides to obtain cyclized polypeptides having the core structure according to the present invention can be carried out by various methods known to the person skilled in the art.
In one embodiment, the inhibitory polypeptides are coupled to the “Fc” domain of an antibody. Antibodies contain two functionally independent parts, a variable domain known as “Fab”, which binds antigen, and a constant domain known as “Fc”, which functions as complement activation and binds to the Fc receptor present on the phagocytic cells (e.g., macrophages). Because of its size, an Fc domain generally has a long serum half-life as compared to that of a Fab which is short-lived. (See, Capon et al., Nature, Volume 337, pages 525-31 (1989)). The Fc domain may be fused to the N-terminus or C-terminus of the polypeptide or at both the N- and C-termini. The coupling of a polypeptide (linear or cyclic) to an Fc can provide longer half-life and offers additional advantages of Fc receptor binding and protein A binding. Preferably, the immunoglobulin source of the native Fc is of human origin (to avoid antibody-antigen reaction during human therapeutic application of the polypeptides). Preferably, the Fc may be IgG1 and IgG2. One example of a native Fc is a disulfide-bonded dimer resulting from papain digestion of an IgG. (See, Ellison et al., Nucleic Acids Res., Volume 10, pages 4071-4079 (1982).
In one embodiment, the modified polypeptides may be synthesized by well-known organic chemistry techniques. Alternatively, the modified peptides of the invention may be made in transformed host cells using recombinant DNA techniques. To do so, a recombinant DNA molecule coding for the peptide is prepared. Methods of preparing such DNA molecules are well known in the art. See, generally, Watson et al., Molecular Biology of the Gene, Volumes I and II, The Benjamin/Cummings Publishing Company, Inc., Menlo Park, Calif. (1987); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). These references are herein entirely incorporated by reference. In one embodiment, a vector capable of expressing the peptides or modified peptides in an appropriate host. The vector comprises the DNA molecule that codes for the peptides or modified peptides operatively linked to appropriate expression control sequences. Expression control sequences include promoters, activators, enhancers, operators, ribosomal binding sites, start signals, stop signals, cap signals, polyadenylation signals, and other signals involved with the control of transcription or translation. The resulting vector having the DNA molecule thereon is used to transform an appropriate host. This transformation may be performed using methods well known in the art. Preferably, microbial hosts include bacteria such as Escherichia (E. coli) and the like. Preferred strains of Escherichia coli includes, for example, HB101, (ATCC NO. 33694) DH5α, DH10, and MC1061 (ATCC NO. 53338)).
The present invention provides a total of twenty-eight (28) novel amino acid sequences (screened and isolated from our Phage Display library) that exhibit the ability to bind to IL-23R. The screened polypeptides are shown to have the ability to bind IL-23R.
For purposes of this application, the polypeptides can be linear or cyclic. Preferably, the polypeptides are cyclic. The present invention also provides methods of using the polypeptides or pharmaceutical compositions of the inventive peptides in the treatment of IL-23 associated diseases. The present polypeptides are capable of binding to IL-23R and blocking the IL-23 mediated cell signaling. The therapeutic application of the polypeptides (linear or cyclic) includes treatment of immunological diseases where overt production of IL-23 is a key feature. For example, the polypeptides can be used to suppress immunological responses in a cell or human diseases such as Crohn's disease and inflammatory bowel diseases as well as in general inflammation such as arthritis, rheumatic diseases, lupus, osteoarthritis, inflammatory bowel disorders, psoriasis, and the like. The peptides of the invention also have therapeutic value for the prevention of immunological diseases often characterized by IL-23.
The present invention also relates to the use of one or more of the peptide of the present invention in the manufacture of a medicament for the treatment of a disorder such as any one of those mentioned above.
Amino acid sequences of the screened polypeptides were determined by sequencing. When the amino acid sequences of the screened polypeptides are aligned, many polypeptides exhibit a unique feature of an amino acid sequence (which we called a “core structure”). Based on the presence of the unique feature, the screened polypeptides can be categorized into two (2) general classes.
The first class of the screened polypeptides encompasses novel polypeptides that share a core structure of amino acids. This class occupies about seventy-five percent (82%) (23/28) of the total screened polypeptides. All of the polypeptides possess a core structure that contains an amino acid motif comprising two (2) tryptophan (W) residues separated by a few amino acids. For example, the two (2) tryptophan (W) residues are separated by one (1), three (3), six (6) or eight (8) amino acids.
In one embodiment, the core structure is composed of two (2) tryptophan (W) residues separated by one (1) amino acid residue (i.e., WX1W) where X1 is an amino acid other than tryptophan. An example of this core structure includes SEQ ID NO: 16 (peptide no. 13).
In another embodiment, the core structure is composed of two (2) tryptophan (W) residues separated by three (3) amino acid residues (i.e., WX1X2X3W) where X1, X2 and X3 are three (3) separate amino acids. Examples of this core structure include SEQ ID NO: 1 (peptide no. 23, 52, 65, 71, 74, 77); SEQ ID NO: 2 (peptide no. 60, 68); SEQ ID NO: 4 (peptide no. 66, 70, 72); SEQ ID NO: 7 (peptide no. 1); SEQ ID NO: (peptide no. 4, 12, 26, 28); SEQ ID NO: 13 (peptide no. 7); SEQ ID NO: 14 (peptide no. 9); SEQ ID NO: 15 (peptide no. 10); SEQ ID NO: 17 (peptide no. 14); SEQ ID NO: 18 (peptide no. 15); SEQ ID NO: 20 (peptide no. 19); SEQ ID NO: 21 (peptide no. 20, 21); SEQ ID NO: 22 (peptide no. 22); SEQ ID NO: 23 (peptide no. 23, 52, 65, 71, 74, 77); SEQ ID NO: 24 (peptide no. 24); SEQ ID NO: 25 (peptide no. 25); SEQ ID NO: 26 (peptide no. 27); SEQ ID NO: 27 (peptide no. 29); SEQ ID NO: 28 (peptide no. 30); or SEQ ID NO: 29 (peptide no. 32).
In another embodiment, the core structure is composed of two (2) tryptophan (W) residues separated by six (6) amino acid residues (i.e., WX1X2X3X4X5X6W, where X1, X2, X3, X4, X5, and X6 are six (6) separate amino acids. Examples of this core structure include SEQ ID NO: 8 (peptide no. 2) and SEQ ID NO: 19 (peptide no. 18).
In another embodiment, the core structure is composed of two (2) tryptophan (W) residues separated by eight (8) amino acid residues (i.e., WX1X2X3X4X5X6X7, X8W, where X1, X2, X3, X4, X5, X6, X7, and X8 are eight (8) separate amino acids. Examples of this core structure include SEQ ID NO: 6 (peptide no. 79).
Polypeptides that include the core structure (e.g., WX1W, WX1X2X3W, WX1X2X3X4X5X6W, and the like) have the ability to bind IL-23R and inhibit IL-23 notwithstanding the remainder of the polypeptide sequence.
The second class of the screened polypeptides encompasses novel polypeptides that do not contain a recognizable core structure, despite having a similar ability to bind to IL-23R (as compared to the first class of polypeptides). This class occupies eighteen (18) percent (18%) (5/28) of the total screened polypeptides. Examples of these polypeptide include SEQ ID NO: 3 (peptide no. 61); SEQ ID NO: 5 (peptide no. 75); SEQ ID NO: 9 (peptide no. 3); SEQ ID NO: 11 (peptide no. 5, 16, 31); SEQ ID NO: 12 (peptide no. 6).
The novel polypeptides were tested for their ability to inhibit IL-23 using a Competitive ELISA. In such an assay, IL-23R/Fc fusion protein (SEQ ID NO: 127) was immobilized on a solid support. In this instance, the solid support was a 96-well plate, but the support may be any support that is typically used for an ELISA. The novel polypeptide and IL-23 were allowed to compete for binding to the immobilized IL-23R/Fc fusion protein. IL-23 that bound to IL-23R-Fc was detected using biotinylated anti-p40 antibody. The bound anti-p40 antibody was detected using streptavidin-horseradish peroxidase (HRP) and a tetramethylbenzidine (TMB) substrate was added to measure peroxidase activity. The color was measured at optical density (OD) 450nm. The color intensity was directly proportional to the amount of the bound IL-23 protein. The Competitive ELISA demonstrated that the novel polypeptides inhibit IL-23 binding to IL-23R. (See,
It was found that the novel peptides could effectively inhibit the biological effects of IL-23 binding to IL-23R. It is recognized that the protein STAT3 is phosphorylated in response to IL-23 binding to IL-23R. Phosphorylation of STAT3 (i.e., formation of pSTAT3) results in the production of IL-17 and IL-17F, which, in turn, produce the inflammatory response associated with diseases including, but not limited to, psoriasis, irritable bowel disease, Crohn's disease and ulcerative colitis. Thus, it is generally recognized that reduced levels of STAT3 phosphorylation indicate decreased levels of IL-23 mediated cell signaling and correspondingly, will result in reduced inflammation levels.
pSTAT3 levels in the cell lysate of DB cells that were treated with the novel synthetic polypeptide (i.e., polypeptide no. 1 (SEQ ID NO: 7), 7 (SEQ ID NO: 13), 16 (SEQ ID NO: 11)) and IL-23 was measured using polyclonal rabbit anti-p-STAT3 antibodies (Cell Signaling Technology, Beverly, Mass.). Polypeptide nos. 1 and 7, which had previously been shown to bind strongly to IL-23R and inhibit IL-23, reduced pSTAT3 levels in cell lysates. Polypeptide no. 16, which demonstrated limited ability to bind to IL-23R and inhibit IL-23, did not show perceptible pSTAT3 reduction when pSTAT3 levels were measured by Western blot. Polypeptide nos. 1 (SEQ ID NO: 7) and 7 (SEQ ID NO: 13) both belong to the first class of polypeptides that include a core structure composed of two (2) tryptophan (W) residues separated by three (3) amino acid residues (i.e., WX1X2X3W). Polypeptide no. 16 (SEQ ID NO: 11) belongs to the second class of polypeptides that do not include a recognizable core structure.
Evaluation of IL-23 mediated cell signaling activity need not be limited to measuring pSTAT3 and STAT3 levels. The inhibition of other components of the IL-23 signaling pathway (e.g., the Jak-STAT signaling cascade) may also be used to determine if a polypeptide is inhibiting IL-23 mediated cell signaling. For example, IL-23 mediated cell signaling can be assessed by determining translocation of STAT3 to the nucleus, activity of the IL-17A and IL-17F genes, or the differentiation of Th17 cells. These cell signaling assays are well known in the art and one of ordinary skill in the art would be able to optimize the assays for its application to the present invention.
It is noted that polypeptides that include the core structure may tolerate some variations and still possess its ability to bind IL-23R and inhibit IL-23 mediated cell signaling thereof. It is intended that the present invention encompasses variants that still possess the biological activity of binding to IL-23R and inhibits its subsequent cell signaling. Accordingly, polypeptides of the present invention retain their biological activity notwithstanding the position of the core structure (e.g., WX1X2X3W) within the length of the polypeptide.
In one embodiment, the present invention encompasses polypeptides that contain the core structure insofar as they inhibit binding and signaling of IL-23R. It is found that the location of the core structure may vary within the polypeptide without affecting the ability of the polypeptide to bind IL-23R and inhibit IL-23. For example, in some instances (e.g., peptide nos. 9, 10, 19, 22, 24, 25, 30, 32), the core structure is located nearer the amino terminus of the polypeptide. In other instances (e.g., peptide nos. 4, 12, 26, 28), the core structure is located nearer the carboxy terminus of the polypeptide. In yet other instances (e.g., peptide no. 7, 14, 18, 20, 21, 23, 29, 52, 60, 65, 66, 68, 70, 71, 72, 74, 77, 79), the core structure is centrally located within the polypeptide. The present invention encompasses all of these polypeptides containing the core structure, regardless its location.
In another embodiment, the present invention is directed to polypeptides that have an overall length of twelve (12) amino acid residues. In our initial screening, it was found that certain polypeptides having twelve (12) amino acid residues are effective at binding IL-23R and inhibiting IL-23. It is noted that polypeptides of varying amino acid lengths are functionally equivalent in the binding assays and Competitive ELISA described herein. In one embodiment, polypeptides that include a core structure and have an overall length of nine (9) amino acids bind to IL-23R and inhibit IL-23 (see, e.g., SEQ ID NO: 56 (peptide no. 23.1)). In one embodiment, polypeptides that include a core structure and have an overall length of sixteen (16) amino acids bind to IL-23R and inhibit IL-23 (see, e.g., SEQ ID NO: 54 (peptide no. 1.5) and SEQ ID NO: 57 (peptide no. 23.2), respectively). In one embodiment, polypeptides that include a core structure and have an overall length of eighteen (18) amino acids bind to IL-23R and inhibit IL-23 (see, e.g., SEQ ID NO: 55 (peptide no. 1.6) and SEQ ID NO: 58 (peptide no. 23.3)). Accordingly, the overall amino acid length of the polypeptide (having a core structure) may vary insofar as the polypeptide can function to inhibit binding of IL-23R and IL-23 cell signaling. Preferably, the overall amino acid length of the polypeptide covers from nine (9) to eighteen (18) amino acids. Preferably, the overall amino acid length of the polypeptide is twelve (12) amino acids.
Polypeptides of the present invention may be modified, for example, substitution, deletion, or addition of amino acids that have minimal influence on the inhibiting activity towards IL-23R binding and cell signaling thereof. In the present invention, it should be noted that substitution of either tryptophan (W) on each side of the core structure destroys the polypeptide's functionality (i.e., its ability to bind IL-23R and inhibit IL-23). Exemplary of such substitution includes polypeptides of SEQ ID NOs: 45-47. Insofar as the two (2) tryptophan (W) residues are present in the polypeptide (i.e., the core structure is present), the polypeptides are found to tolerate some amino acid changes throughout the other parts of the polypeptide sequence.
In one embodiment, the present invention provides polypeptides that include a change of amino acid residues in and outside the two (2) tryptophan core structure. One of skilled in the art would conveniently determine the optimal changes in the amino acid residues yet still possess the ability to inhibit binding to IL-23R and cell signaling thereof.
A “conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. In generally, the following groups of amino acid represent conservative changes: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, try, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.
The amino acids that are present inside the core structure (e.g., X1X2X3 within the WX1X2X3W motif) or flanking the core structure (e.g., outside the WX1X2X3W motif) include any of the standard twenty-two (22) amino acid residues in their L- or D-form. They may also be a non-standard amino acid notwithstanding the amino acids that are not found in proteins (for example, carnitine, GABA, and L-DOPA), or are not produced directly and in isolation by standard cellular machinery (for example, hydroxyproline and selenomethionine).
In the present invention, a predominant polypeptide group is that the two (2) tryptophan residues are separated by a few amino acid residues (e.g., WX1X2X3W, WX1X2X3, X4W, and the like). In one exemplary embodiment, the present invention provides a polypeptide having a core structure of WX1X2X3W, where X1 is W. In another exemplary embodiment, the present invention provides a polypeptide having a core structure of WX1X2X3W, where X2 is W. In yet another exemplary embodiment, the present invention provides a polypeptide having a core structure of WX1X2X3W, where X3 is an aromatic amino acid residue (i.e., tryptophan, tyrosine or phenylalanine). Preferably, X3 is tryptophan. However, it should be noted that when one of X1, X2 or X3 is tryptophan, the remainder of X1, X2 or X3 cannot be tryptophan.
A non-limiting exemplary list of the core structures that bind to IL-23R and inhibit IL-23 include: WVQYW (SEQ ID NO: 94), WYNYW (SEQ ID NO: 95), WMTYW (SEQ ID NO: 96), WATYW (SEQ ID NO: 97), WEMYW (SEQ ID NO: 98), WKDYW (SEQ ID NO: 99), WEHYW (SEQ ID NO: 100), WQNYW (SEQ ID NO: 101), WQDVW (SEQ ID NO: 102), WMQYW (SEQ ID NO: 103), WHAYW (SEQ ID NO: 104), WQTYW (SEQ ID NO: 105), WQSFW (SEQ ID NO: 106), WYAYW (SEQ ID NO: 107), WEDYW (SEQ ID NO: 108), WEDFW (SEQ ID NO: 109), WEDAW (SEQ ID NO: 110), WLQYW (SEQ ID NO: 111), WLNYW (SEQ ID NO: 112), WNLAW (SEQ ID NO: 113), WLNFW (SEQ ID NO: 114), WRWFW (SEQ ID NO: 115), WEDWW (SEQ ID NO: 116), WIDWW (SEQ ID NO: 117), WDDWW (SEQ ID NO: 118), WEEWW (SEQ ID NO: 119), WQDWW (SEQ ID NO: 120), WENWW (SEQ ID NO: 121), WQNWW (SEQ ID NO: 122), WEKWW (SEQ ID NO: 123), WKDWW (SEQ ID NO: 124), and WKKWW (SEQ ID NO: 125).
According to preferred aspects of the present invention the peptide is a cyclic peptide. In one preferred embodiment, the cyclic peptide is SEQ ID NO: 130 or SEQ ID NO: 131.
It is surprising to discover the core structure of our novel polypeptides that possess the ability to bind to IL-23R and effectively inhibit IL-23. Contrary to what would be expected, the amino acid sequences of the core structure of the present polypeptides do not correspond to any sequence within IL-23 or within IL-23R. The biological activity of the novel peptides suggests that the present polypeptides function equivalently to IL-23 subunit p19 but lack the ability to activate IL-23R signaling pathways. Despite the knowledge of the crystallographic structure of IL-23, the purported receptor interaction sites of IL-23 do not show any amino acid sequence that resemble or is homologous to the core structure (e.g., WX1X2X3W) of the present invention. (See, e.g., Lupardus, et al., J. Mol. Bio., 382:931-41, 2008). BLAST data further demonstrate that there is no significant similarity between the core structure (e.g., WX1X2X3W) and the linear amino acid sequence of p19. Given the dissimilarity in amino acid sequence, one of ordinary skill in the art would not have predicted or expected the present polypeptides to bind IL-23R.
The present inventors also surprised to discover that the present polypeptides are not limited (by and large) to any specific amino acid sequence provided that the polypeptide includes a core structure. The present polypeptides all exhibit the same biological properties (i.e., inhibit IL-23R binding and IL-23 cell signaling). This holds true whether amino acid changes are “conservative” or “drastic.”
One skilled in the art would recognize that there are several methods of determining whether an amino acid substitution is “conservative” or “drastic.” Amino acids are generally divided into families based on the properties of their side chains. Aspartate and glutamate are acidic. Lysine, arginine, and histidine are basic. Glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan are hydrophobic. Asparagine, glutamine, cysteine, serine, threonine, and tyrosine are neutral and hydrophilic. Phenylalanine, tryptophan, and tyrosine also are classified as aromatic. These classes typically convey variant properties on polypeptides according to the characteristics of the amino acid side chain.
The hydropathic index of amino acids is a method for assessing whether an amino acid change is “conservative” or “drastic.” Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5).
One standard method for assessing whether a change is “conservative” or “drastic” amino acids is based on hydrophilicity. The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5) and tryptophan (−3.4).
A “conservative” amino acid substitution involves substitution of a native amino acid residue with a normative residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position or substitution of one amino acid residue with another amino acid residue having the same or a similar hydropathic or hydrophilicity score. As used herein, a “drastic” amino acid substitution involves a substitution where there is a change in the polarity or charge of the amino acid residue at that position or substitution of one amino acid residue with another amino acid residue having a different hydropathic or hydrophilicity score.
It is generally believed that a conservative amino acid substitution is not expected to alter biological activity. Kyte, et al., J. Mol. Biol., 157: 105-131 (1982). Conversely, a drastic amino acid substitution is expected to alter biological activity. Contrary to what one of skill in the art would expect, the present inventors found functional equivalency between polypeptide sequences that include a core structure (e.g., WX1X2X3W), even when drastic amino acid changes are made.
The novel polypeptides of the present invention may be synthesized by methods known in the art. For example, polypeptides can be prepared using solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. These synthesis methods are well-known to those of skill in the art (see, for example, Merrifield, J. Am. Chem. Soc. 85:2149 (1963), Stewart et al., “Solid Phase Peptide Synthesis” (2nd Edition), (Pierce Chemical Co. 1984), Bayer and Rapp, Chem. Pept. Prot. 3:3 (1986), Atherton et al., Solid Phase Peptide Synthesis: A Practical Approach (IRL Press 1989), Fields and Colowick, “Solid-Phase Peptide Synthesis,” Methods in Enzymology Volume 289 (Academic Press 1997), and Lloyd-Williams et al., Chemical Approaches to the Synthesis of Peptides and Proteins (CRC Press, Inc. 1997)). Variations in total chemical synthesis strategies, such as “native chemical ligation” and “expressed protein ligation” are also standard (see, for example, Dawson et al., Science 266:776 (1994), Hackeng et al., Proc. Nat'l Acad. Sci. USA 94:7845 (1997)).
Polypeptides of the present invention can be purified or isolated after synthesis. Protein purification methods are well known in the art, and are described, for example in Deutscher, et al. (ed., 1990, In: Guide to Protein Purification, Harcourt Brace Jovanovich, San Diego).
In one aspect, the present invention is directed to a pharmaceutical composition comprising a novel polypeptide of the present invention, and a pharmaceutical acceptable carrier. The present composition is believed to reduce activation of cells implicated in the IL-23 mediated inflammatory response and thus are useful and have practical utility in preventing and treating IL-23 mediated diseases. Such composition may be administered to a mammal (e.g., human) to inhibit binding of IL23R (by IL-23) and cell signaling thereof. The present composition aids to prevent, ameliorate or treat the symptoms of IL-23 mediated inflammatory disorders. The present composition is useful in treating IL-23 mediated diseases including, but not limited to, psoriasis, rheumatoid arthritis, and inflammatory bowel diseases such as ulcerative colitis and Crohn's disease and the like.
In one aspect, the present invention provides a method of preparing a pharmaceutical composition that contains a novel polypeptide of the present invention and a pharmaceutically acceptable excipient.
Pharmaceutically acceptable excipients are known in the art (see, e.g., Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, Pa. (1984)), including methods of combining an active ingredient (e.g., novel polypeptide) with the pharmaceutical acceptable excipient. Formulation of the therapeutic agents (i.e., novel polypeptides) may be prepared by mixing with physiologically acceptable excipients, or stabilizers in the form of, e.g., lyophilized powders, slurries, aqueous solutions or suspensions (see, e.g., Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, N.Y.; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, N.Y.; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, N.Y.; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, N.Y.).
In another aspect, the present invention provides a method of treating IL-23 mediated immunological diseases by administering to a human a pharmaceutical composition comprising the novel polypeptides. The suitable route of administration may include injection or infusion by intravenous, intramuscular, or by sustained release systems. The pharmaceutical compositions containing novel peptides may additionally contain other active ingredients such as anti-infective agents, antibiotics, or anti-IL-23R antibodies and the like.
Effective amounts of the present composition for a particular patient may vary depending on factors such as the condition being treated, the overall health of the patient, the method route and dose of administration and the severity of side affects (see, e.g., Maynard, et al. (1996) A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, Fla.; Dent (2001) Good Laboratory and Good Clinical Practice, Urch Publ., London, UK). It will be apparent to those of ordinary skill in the art that the therapeutic effective amount of the inventive peptides of this invention will depend, inter alia, upon many factors such as the administration schedule, the unit dose of peptide administered, whether the peptide is administered in combination with other therapeutic agents, the immune status and health of the patient, the therapeutic activity of the peptide administered and the judgment of the treating physician. An effective dose protocol can be conveniently devised by one of ordinary skill in the art. (See, e.g., Yang, et al., New Engl. J. Med. 349:427-434, 2003; Herold, et al., New Engl. J. Med. 346:1692-1698, 2002; and Portielji, et al., Cancer Immunol. Immunother. 52:133-144, 2003).
The following examples are provided to further illustrate various preferred embodiments and techniques of the invention. It should be understood, however, that these examples do not limit the scope of the invention described in the claims. Many variations and modifications are intended to be encompassed within the spirit and scope of the invention.
IL-23R is composed of 629 amino acids, whose sequence (accession no. NM 144701.2) has been reported by Oppmann et al. (Immunity 13 (5), 715-725, 2000). To date, no crystallization structure has been reported for IL-23R; and its three dimensional structure remains unknown. Chemtob et al. relied on the linear amino acid sequence of IL-23R and created peptides corresponding to portions of the IL-23R. Using this rational drug design approach, these authors reported several IL-23R antagonists and an IL-23R agonist. The purported amino acid structure for the IL-23R antagonists includes LPDEVTCV, TEEEQQYL, TEEEQQYL, and MEESKQLQL. Because these synthetic polypeptides are based on the IL-23R, they were reported to act as an antagonist in blocking IL-23 binding to IL-23R and subsequent cell signaling.
In this study, we chose a different approach. Instead of a rational drug design based on a computer generated model, we used a random peptide approach using a Phage Display Screening. This novel approach was adopted to maximize the chances to identify novel polypeptides that bind to IL-23R (i.e., our screened polypeptides do not have corresponding IL-23R amino acid sequence; rather, our screen polypeptides are expected to mimic IL-23 and bind to IL-23R).
The present inventors have identified polypeptides that share common core structures. These polypeptides function to inhibit binding of IL-23 to IL-23R and to inhibit cell signaling of IL23R. These polypeptides also bind to IL-23R. It is surprising to note that our screened polypeptides do not resemble that of IL-23 when the amino acid sequence is considered. Beyer et al. and Lupardus et al. have reported the crystallized structure for IL-23. Comparison of the purported crystallized structure unexpectedly reveals little, if any, similarity between our screened polypeptides and portions of the IL-23.
In the present Phage Display Screening, the assay essentially involves four steps (See,
The details of the Phage Display Screening are described as follows:
A) Phage Display Library Expressing Random Peptide Sequences
A phage library displaying approximately 1×1011 pfu random 12-mer peptide sequences was prepared (obtained from New England BioLabs (Ipswich, Mass.) (Cat. No. E8110S)). The phage library was then used in our Phage Display Screening. To identify and select phages that express peptide sequences that were bound, we employed bio-panning. Two independent bio-panning assays were performed.
(i) Bio-Panning (Screening) Using a Soluble Recombinant Δ9 IL-23R (i.e., Δ9-Flag Screening)
In previous studies we had identified a soluble recombinant IL-23R lacking a domain (i.e., between the 349 amino acid residue to 629 amino acid residue) but possess an extra eight (8) amino acid residues (i.e., Δ9 protein) due to a messenger RNA splicing event. (See, Kan et al., Genes Immun. (7):631-9, 2008; Mancini G et al., Genes Immun. 2008 (6):566-9, 2008; and Yu et al., J. Immunol. 185(12):7302-8, 2010). We had further shown that this particular soluble IL-23R variant (also known as Δ9 protein) functions equivalently as the full-length IL-23R in terms of its IL-23 binding activity. (See, Yu et al., J. Immunol. 185(12):7302-8, 2010).
In the initial bio-panning assay, phages were screened using purified Flag-tagged fusion protein containing the soluble recombinant Δ9 which contains the entire extracellular domain of IL-23R (i.e., Δ9-Flag) (See
Data obtained using this Δ9-Flag screening revealed that this approach yielded a total of thirteen (13) polypeptides, indicating a need to improve efficiency. After sequence confirmation, many of the screened polypeptides were found to be the Flag sequence, in part because the anti-Flag antibody used in the screening process may also capture random peptides that resemble the Flag sequence.
A list of the novel polypeptides (i.e., six (6) polypeptides) (polypeptide nos. 52, 60, 61, 66, 75, and 79) (SEQ ID NOs: 1-6, respectively) screened using the Δ9-Flag screening approach is summarized in
(ii) Bio-Panning (Screening) Using a Soluble Recombinant IL-23R (Containing the Entire Extracellular Domain but Lacking the Transmembrane and Intracellular Domains) (i.e., IL-23R/Fc Chimera Screening)
We improved the Phage Display Screening Assay. To avoid potential interaction between Flag and anti-Flag, we chose to use a different capturing reagent.
To this end, we planned to prepare a soluble recombinant IL-23R lacking the signal peptide sequence (i.e., 1-23 amino acids). This soluble recombinant IL-23R has the protein portion between the 24 amino acid residue to 353 amino acid residue. In contrast to the full-length IL-23R (1-629 amino acids in length), this soluble recombinant IL-23R lacks the transmembrane domain and cytoplasm domain (i.e., between the 354 amino acid residue to 629 amino acid residue). The soluble recombinant IL-23R may be coupled to a human IgG1 Fc (i.e., between the 100 amino acid residue to 330 amino acid residue) and created an IL-23R/Fc chimera (i.e., IL-23R/Fc).
We obtained the IL-23R/Fc from a commercial source (i.e., R&D Systems, Minneapolis, Minn.; Cat. No. 1400-IR-050). To mediate capturing, we employed protein A sepharose to capture the Fc portion of the IL-23R/Fc. Such molecular device eliminates the binding between Flag and anti-Flag. (
Using this IL-23R/Fc chimera screening approach, we obtained a total of twenty-two (22) unique polypeptides that bound to IL-23R, demonstrating an efficient screening.
A list of the novel polypeptides (i.e., 22 polypeptides) (polypeptides nos. 1, 2, 3, 4, 5, 6, 7, 9, 10, 13, 14, 15, 18, 19, 20, 22, 23, 24, 25, 27, 29, 30 and 32) (SEQ ID NOs: 7-29, respectively) screened using the IL-23R/Fc chimera screening approach is summarized in
B) Elimination of Non-Specific Binding
To avoid non-specific binding, we performed a negative selection step. Either protein A sepharose or anti-Flag affinity gel was incubated with 1×1011 pfu of phage display library at room temperature for 15 minutes. The mixture was centrifuged at 5,000 rpm for 2 minutes to separate the phages that bound non-specifically from unbound phages. The phages in the supernatant were then screened using either the Δ9-Flag followed by anti-flag agarose or the soluble recombinant IL-23R (containing the entire extracellular domain, but lacking both the transmembrane and intracellular domains) followed by Fc-protein A sepharose to obtain phages displaying sequences that bind to IL-23R (as detailed above).
C) Detection of IL-23R Binding
(i) Using Δ9-Flag Followed by Anti-Flag Agarose
Phage-containing supernatant (300 μl) was incubated with anti-Flag affinity gel and Flag-tagged fusion protein containing the entire extracellular domain of IL-23R (Δ9-Flag) (
(ii) Using Soluble Recombinant IL-23R (Containing the Entire extracellular Domain, but Missing the Transmembrane and Intracellular Domains) Followed by Fc-Protein A Sepharose
The second independent screening was performed by incubating the phage-containing supernatant (300 μl) with protein A sepharose (20 μl) and recombinant human IL-23R/Fc (10 μg) (
D) Amplification of Binding Phages in E. coli
Phages that bound IL-23R were removed from the affinity gel or sepharose beads by adding 1 ml of glycine elution buffer (0.2 M glycine-HCl, pH 2.2, 0.1% BSA) to the resin and incubating the resin-buffer mixture for 10-minutes at room temperature. The resin was removed by centrifuging the mixture at 5,000 rpm for 2 minutes, which leaves the relevant phages in the supernatant. The eluted phages were neutralized by adding 150 μl of 1 M Tris-HCl, pH 9.1 to the supernatant.
Next, binding phages were amplified. Phages were introduced to an early-log 20 ml culture of E. coli ER2738. The mixture was incubated for 4.5 hours at 37° C. with shaking. Following incubation, the culture was centrifuged at 12,000 rpm for 10 minutes to remove cell debris and E. coli cells. To precipitate the phages, the upper 80% of the supernatant was incubated overnight at 4° C. with 3 ml of 20% polyethylene glycol (PEG)/2.5 M NaCl. The solution was centrifuged at 12,000 rpm for 30 minutes to collect the precipitated phages. The supernatant was discarded. The pellet was re-suspended in 1 ml of TBS. 200 μl of 20% PEG/2.5 M NaCl was added to the supernatant and the mixture was incubated on ice for 1 hour. The solution was centrifuged at 12,000 rpm for 10 minutes and the pellet re-suspended in 200 μA of TBS.
The phage concentration in the amplified pool was measured by the phage titer on LB/IPTG/Xgal plates. The phage titer was determined by counting blue plaques. Two additional rounds of bio-panning were performed as described above. After 3 rounds of bio-panning, the eluted phages were subjected for titer before phage amplification.
Individual plaques (i.e., blue plaques) were randomly selected and amplified by adding the plaques to 1 ml of E. coli ER2738. The mixture was incubated at 37° C. with shaking for 5 hours. The cultures were centrifuged to remove cell debris and the pellets discarded. 500 μl of the supernatant (i.e. LB containing amplified individual phage identified after 3 around of bio-panning) was apportioned for DNA sequencing. The remaining supernatant volume was used in a phage ELISA.
DNA from the phages that bound to either Δ9-Flag or to IL-23R/Fc chimera was sequenced to determine the amino acid sequence of the polypeptides that bound IL-23R.
In this study, phages were precipitated from the 500 μl of supernatant obtained following the phage display assay. 200 μl of 20% PEG/2.5 M NaCl was added to the 500 μl phage-containing supernatant (1:2.5 volume to volume ratio of PEG/NaCl to supernatant). The mixture was incubated for 20 minutes at room temperature. Phages were collected by centrifuging at 12,000 rpm for 10 minutes. The supernatant was discarded and the pellet re-suspended in 100 μl iodide buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 4 M sodium iodide). 250 μl ethanol was added and the solution incubated at room temperature for 20 minutes. The solution was centrifuged at 12,000 rpm for 10 minutes. The supernatant was discarded and the pellet was washed with 0.5 ml of cold 70% ethanol. After another centrifugation at 12,000 rpm for 10 minutes, the supernatant was discarded and the pellet, containing the phage DNA, was suspended in 20 μl TE buffer.
For DNA sequencing, we used Beckman Coulter GenomeLab DTCS Quick Start Kit (Cat. No. 60812) (Beckman Coulter, Fullerton, Calif.). The concentration of phage DNA was measured by the NanoDrop at OD280. (NanoDrop Technologies inc, Wilmington, Del.). Approximately 150 ng of phage DNA was used for each sequencing reaction. 1 pmol of −96 GIII sequencing primer (20-mer), (supplied with New England BioLabs Ph.D.™ 12 Phage Display Peptide Library Kit, Ipswich, Mass.) was used for each reaction. The thermal cycling program was 20 seconds at 96° C., 20 seconds at 50° C., and 4 minutes at 60° C., for 30 cycles. Ethanol precipitation was performed according to the Beckman Coulter kit insert. DNA sequencing was performed using Beckman Coulter CEQ 8000.
Δ9-Flag Screening:
With respect to the Δ9-Flag screening, eighty (80) bound phages were selected and subject to DNA sequencing. Among the eight (80) bound phages, the DNA sequencing study reveals thirteen (13) polypeptides, of which six (6) of the polypeptides have novel amino acid sequences.
Analysis of the polypeptide sequence reveals that these novel polypeptides contain at least one (1) tryptophan (W) residue. Interestingly, three (3) of the six (6) polypeptide sequences (i.e., polypeptide nos. 52, 60, and 66) included a WX1X2X3W core structure.
IL-23R/Fc Chimera Screening:
With respect to the IL-23R/Fc chimera screening, thirty-two (32) bound phages were selected and subjected to DNA sequencing. Among these bound phages, DNA sequencing reveals twenty-nine (29) polypeptides, of which twenty-two (22) of the polypeptides have novel amino acid sequences.
Analysis of the polypeptide sequence reveals that all twenty-two (22) of the phages expressing peptide sequences included at least one tryptophan (W) residue. Fifteen (15) of the twenty-two (22) polypeptide sequences (i.e., polypeptide nos. 1, 4, 7, 9, 10, 14, 15, 20, 22, 23, 25, 27, 29, 30 and 32) included a WX1X2X3W core structure.
Δ9-Flag and IL-23R/Fc Chimera Screening:
Of interest is the observation that one amino acid sequence obtained from the Δ9-Flag screening matches that of IL-23R/Fc chimera screening (i.e., polypeptide nos. 52, 65, 71, 74 and 77 from Δ9-Flag screening match the polypeptide no. 23 from IL-23R/Fc chimera screening). Accordingly, both Δ9-Flag screening and IL-23R/Fc chimera screening together yielded a total of twenty seven (27) novel polypeptides. A full list of all the sequences obtained using these two screening approaches is shown in
To confirm the binding of phages isolated using IL-23R/Fc conjugated to protein A sepharose to IL-23R, we performed a phage ELISA. IL-23R/Fc was used to select peptide sequences from the phage stock obtained in our bio-panning assay. Sixteen (16) isolated phages were tested for their ability to bind IL-23R in the ELISA. Twelve (12) different polypeptide sequences were expressed on these sixteen (16) phages. Two empty phages (i.e., numbers 8 and 11) were run as controls.
Bound phage was detected by adding 2 μg/ml of biotinylated anti-M13 antibody (Cat. No.: ab17269) (Abcam, Cambridge, Mass.). Streptavidin-horseradish peroxidase (HRP) was added to detect anti-M13 antibody bound to IL-23R bound phages. Peroxidase activity (representing the level of M13 captured onto plates) was measured by adding 100 μl of a tetramethylbenzidine (TMB) to each well. The color intensity was directly proportional to the amount of the bound M13 phage. Optical density was read at 450nm.
A total of twenty-seven (27) different polypeptide sequences were obtained from the Phage Display Screening assays. The screening with Δ9-Flag yielded six (6) novel polypeptide sequences. The screening with IL-23R/Fc chimera yielded twenty-two (22) novel polypeptide sequences. Altogether, the two approaches yielded a total of twenty seven (27) novel polypeptide sequences.
Interestingly, Δ9-Flag screening and the IL-23R/Fc chimera screening produced one (1) polypeptide sequence in common (i.e., polypeptide nos. 23 and 52), indicating that commonality of using the Δ9-Flag and IL-23R/Fc chimera approaches.
We also screened a minor portion of polypeptides (33.3%) containing a different core structure that is different from the WX1X2X3W.
A few of the selected polypeptides possess a single tryptophan (W). Because the presence of a single tryptophan is ubiquitous in many proteins, we believe that additional features within the polypeptides (e.g., neighboring amino acid residues) play a role in determining the polypeptide's ability to inhibit IL-23 binding to IL-23R.
Overall, the present study shows that WX1X2X3W is the major core structure of the polypeptides that bound to IL-23R and inhibit cell signaling thereof.
The ability to bind to IL-23R is a property of the screened polypeptides obtained from our Phage Display Screening, because the screening assay is based on the ability of the polypeptides to bind to IL-23R. To further assess whether the polypeptides are physically bound to IL-23R, we performed immunoprecipitation assays using the extracellular domain of IL-23R (i.e., Δ9).
(i) Generation and Expression of Polypeptide-Fc Fusion Proteins
Polypeptide-Fc fusion proteins were obtained by designing expression constructs (i.e., polypeptide-Fc expression constructs) that expressed the amino acid sequence of interest fused to Fc.
Forward (F) and reverse (R) oligonucleotides corresponding to the desired peptide sequences were designed as set forth in Table 1.
Equal amounts of forward and reverse oligonucleotides were annealed into double strand form by incubating at 95° C. for 10 minutes. After the mixture cooled down to room temperature, 1 μl of double strand oligonucleotide was ligated with linearized Fc expression construct (Cat. No. ppfc2-mg2ae1) (InvivoGen, San Diego, Calif.). The linearized vector was prepared by treating the DNA with NcoI and BglII restriction enzymes. The ligated DNA was then transformed into Top10 competent cells (Invitrogen, Carlsbad, Calif.). The sequence of the expression constructs were verified by DNA sequencing. Fusion constructs were expressed by transfecting HEK-293T cells with 2 μg of polypeptide-Fc expression constructs.
We performed the immunoprecipitation using six (6) polypeptides that included the core structure WX1X2X3W (polypeptide nos. 1, 4, 7, 10, 22 and 32) and one polypeptide that included a single tryptophan (W) (polypeptide no. 16).
(ii) In Vitro Binding Assay—Polypeptide Binding to IL-23 Receptor
We next tested if the synthetic polypeptides we generated could bind IL-23R by performing an in vitro binding assay involving Fc-fusion protein and protein A bead (i.e., immunoprecipitation assay with Δ9).
The immunoprecipitation assays confirmed that the polypeptides screened from the Phage Display Screening can bind to IL-23R. The majority of the polypeptides expressed the core structure WX1X2X3W, indicating that this core structure is essential in IL-23R binding.
Immuno-precipitation studies established that polypeptides including the core structure can bind to IL-23R. In this next series of studies, we tested if the polypeptides could inhibit binding of IL-23R in the presence of IL-23 (i.e., could prevent IL-23 from binding to IL-23R). To do so, we performed the Competitive ELISA for IL-23R binding.
Six (6) ELISA were performed. Polypeptide nos. 1 and 7 were examined by Competitive ELISA with IL-23. Both polypeptide no. 1 and polypeptide no. 7 include the core structure WX1X2X3W. Polypeptide no. 16 (which included only a single tryptophan) was run as a negative control. The ELISA data is summarized in Table 2.
We examined if our polypeptides could inhibit IL-23R mediated cell signaling. DB cells (CRL-2289, ATCC, Manassas, Va.) were used in a cell based assay to study the effect of synthetic peptides on IL-23R mediated cell signaling. DB cells are human B cell lymphoma cell lines, which express IL-23R on their surface.
Activation of STAT3 by phosphorylation (p-STAT3) was used as a measurable indicator for the IL-23R mediated cell signaling in the DB cells. STAT3 is a transcription factor activated by IL-23 and phosphorylated in response to IL-23 binding to its receptor. Thus, higher relative levels of p-STAT3 to total p-STAT exist in cells where IL-23 has bound IL-23R. Conversely, when IL-23 cannot bind to IL-23R, STAT3 is not phosphorylated and cells exhibit a lower relative level of p-STAT3 to total p-STAT.
To determine the inhibitory affect of our peptides, we tested cell lysates for the expression level of p-STAT3 in response to IL-23 in the absence or the presence of synthetic polypeptide nos. 1, 7 or 16 and compared the p-STAT3 levels to total STAT3 levels.
DB cells (3×106) were cultured in 1 ml of RPMI+10% FBS. The cells were pre-incubated with synthetic polypeptide (i.e., polypeptide nos. 1, 7, 16) (100 μM) for 30 minutes at 37° C. 30 ng/ml of IL-23 was added to the cells and incubated at 37° C. for an additional 20 minutes. Cells were washed with 1 ml of ice-cold PBS and lysed in 50 μl ProteoJET mammalian cell lysis reagent (Fermentas, Glen Burnie, Md.) with protease and phosphatase inhibitors (1 μl per 100 μl) (Sigma, St. Louis, Md.). Cell lysates were centrifuged at 12,000 rpm for 10 minutes. 10 μl of sample loading buffer (Bio-Rad, Berkeley, Calif.) was added to the supernatant. 35 μl of the mixture was run on 4-12% PAGE (Bio-Rad, Berkeley, Calif.). The proteins were transferred to an Immun-Blot PVDF membrane (Bio-Rad, Berkeley, Calif.) and then blocked in 5% milk/TPBT for 1 hour at room temperature. p-STAT3 was detected by probing the membrane with polyclonal rabbit anti-p-STAT3 antibodies (Cell Signaling Technology, Beverly, Mass.). After the membrane was stripped, STAT3 was detected using polyclonal rabbit anti-STAT3 antibodies (Cell Signaling Technology, Beverly, Mass.).
We observed a decrease in the level of p-STAT3 as well as the ratio of p-STAT3 to total p-STAT in samples treated with our polypeptides. This data indicates that the polypeptides inhibited the activity of IL-23R and cell signaling in response to IL-23.
We studied the dose response effect of our polypeptide (e.g., polypeptide no. 1). We examined IL-23R cell signaling activity using the level of p-STAT3 and the ratio of p-STAT3 to total STAT3 in DB cells.
DB cells were pre-incubated with varying amounts of polypeptide no. 1 (100 μM, 50 μM, 10 μM, 5 μM, 1 μM) for 30 minutes at 37° C. To stimulate IL-23R, 30 ng/ml of IL-23 was added to the cells and the cells incubated for 20 minutes at 37° C. Cell lysates were prepared to examine the expression levels of p-STAT3 and total STAT3 as previously described.
As shown in
These results show that peptide no. 1 inhibits the effect IL-23 in cells in a concentration dependent manner.
We extended our Competitive ELISA (
We chose peptide number 1, 9, 23 and 27 to generate the dose-response curve in our competitive ELISA. We varied the concentrations of synthetic peptides (100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.5625 μM or 0 μM) with fixed amount of IL-23 (50 ng/well) in the assay.
We next sought to determine the importance of the tryptophan (i.e., W) residues in the consensus sequences we identified. To do so, we altered one or both of the tryptophan residues in our peptide sequences. The synthetic peptides were used in the competitive ELISA. The corresponding peptide-Fc fusion proteins were also constructed and used in our in vitro binding assay to examine its binding to IL-23R.
We designed three alternative synthetic peptides based on the sequence of synthetic peptide no. 1. The peptide sequence of synthetic peptide no. 1 was modified at one or both of the tryptophan residues. This resulted in three (3) new synthetic peptide sequences designated as 1.1, 1.2 and 1.3 (collectively, “modified synthetic peptides”). The peptide sequences of synthetic peptide no. 1 and the modified synthetic peptides are depicted in
We designed forward (F) and reverse (R) oligonucleotide sequences corresponding to the desired peptide sequence for the construction of peptide-Fc fusion protein. The oligonucleotide sequences are set forth in Table 4.
Equal amounts of forward and reverse oligonucleotides were annealed into double strand form by incubating at 95° C. for 10 minutes. After the mixture cooled down to room temperature, 1 μl of double strand oligonucleotides was ligated with linearized Fc expression construct (Cat. No. ppfc2-mg2ae1) (InvivoGen, San Diego, Calif.). The linearized vector was prepared by treating the DNA with NcoI and BglII restriction enzymes. The ligated DNA was then transformed into Top10 competent cells (Invitrogen, Carlsbad, Calif.). The sequence of the Fc expression constructs were verified by DNA sequencing.
After the peptide-Fc fusion constructs were prepared, we tested if the fusion proteins could bind extra-cellular region of IL-23R (i.e. Δ9). We performed an in vitro binding assay (i.e., immunoprecipitation assay) wherein we examined if the modified synthetic peptides could bind to Δ9.
Fusion constructs were expressed by transfecting HEK-293T cells with 2 μg of Fc expression constructs. 1 ml of cell culture medium was incubated with 20 μl of protein A sepharose at room temperature for 1 hour to precipitate the Fc proteins. 1 ml of Δ9 containing culture medium was added to the mixture (i.e. Fc proteins and protein A sepharose) and was incubated overnight with mixing at 4° C. The mixture was centrifuged and the supernatant was removed. The remaining protein A resin was then washed 5 times with 1M1 PBS. The proteins in the precipitate were then denatured by addition of sample loading buffer (Bio-Rad, Berkeley, Calif.).
The denatured proteins were subjected to 4-12% PAGE (Bio-Rad) and transferred to Immun-Blot PVDF membrane (Bio-Rad). Membranes were blocked in 5% milk/TPBT at room temperature for 1 hour. Anti-Flag antibody (1:1,000) (Sigma, St. Louis, Mo.) and anti-mouse antibody (1:3,000) were used to detect the Δ9 and Fc proteins respectively.
We next performed a competitive ELISA (as shown in
Six (6) ELISAs were performed. Control assays were performed in which (i) no IL-23 or peptide was added; (ii) IL-23 was added and peptide was not; and, (iii) IL-23 was added and peptide no. 1 was added. In each competitive assay, 50 μg of human IL-23 and 50 μM of the applicable modified peptide (either polypeptide no. 1.1, 1.2 or 1.3) were added to the wells and allowed to compete for binding to IL-23R. The relative amount of IL-23 bound to the IL-23 receptor was determined by reading optical density at 450 nm.
These results indicate an important role of the tryptophan residues in the core structure WX1X2X3W in binding to IL-23R and inhibiting IL-23R activity.
Interleukin 23 (IL-23) belongs to the IL-12 family and is structurally similar to IL-12 in that the two cytokines share the p40 subunit. Mature IL-23 comprises of p19 and p40 subunits whereas IL-12 is composed of p35 and p40 subunits. IL-23 binds to a heterodimeric receptor (IL-23R), which is comprised of an IL-23α and an IL-12Rβ1 chain. IL-12Rβ1 is also a component of IL-12R complex. IL-12R comprises the IL-12Rβ1 and IL-12Rβ2 chains. It is desirable to specifically inhibit IL-23R while not inhibiting the activity associated with IL-12R.
To study the specificity of our synthetic peptides, we examined the effect of polypeptide no. 1 on IL-12R in a Competitive ELISA. Polypeptide no. 16 was run as a negative control.
Competitive ELISA were performed exactly the same as example 6 except the plate was coated with IL-12Rβ2 (2 μg/ml) or IL-23R (2 μg/ml). In addition, IL-12 (50 ng/well) and IL-23 (50 ng/well) was used in the plate coated with IL-12Rβ2 and IL-23R respectively. The rest of the experimental setup was the same as example 6.
These results indicate that the inhibitory effect of peptide no. 1 is specific for the IL-23R pathway.
We next performed Flow Cytometry to examine expression of IL-23R on two cell lines, Jurkat T cell and DB cell. Jurkat T cells and DB cells were stained with either isotype control antibodies (i.e., negative control) or anti-IL-23R antibodies.
In preparation for staining, cells were washed 2× with ice-cold PBS. 100 μl of blocking buffer (PBS+5% heat inactivated human serum) was added to re-suspend the cell pellet and the mixture was incubated for 30 minutes on ice. Antibodies (either control isotype antibody or biotinylated anti-IL-23R antibody) (0.5 μg/100 μl) were added and the mixture incubated for 30 minutes on ice followed by washing with ice-cold PBS for 3 times. The washed cells were re-suspended in 100 μl of blocking buffer containing 0.2 μl of streptavidin-PE (Cat. No. 554061BD) (Pharmingen, Phillipines) and were incubated on ice for 30 minutes followed by washing 1 ml PBS for 3 times. The cells were fixed in 1% of paraformaldehyde in PBS. The fixed cells were then acquired and analyzed in a Flow Cytometer.
These results indicate that IL-23R is expressed on DB cell surface but not on Jurkat T cell surface.
We examined the affinity of peptide no. 1 to bind to IL-23R on the cell surface. Jurkat T cells (i.e., IL-23R negative cell) and DB cells (i.e., IL-23R positive cell) were incubated with peptide no. 1-Fc fusion protein that had been biotinylated to permit detection. In total, eight assays were performed. Four assays were performed in each cell line: (i) staining with biotinylated Fc (i.e., control); (ii) staining with 0.1 μg of biotinylated synthetic peptide no. 1-Fc chimera (no. 1-Fc); (iii) staining with 0.5 μg of biotinylated no. 1-Fc; and, (iv) staining with 1 μg of biotinylated no. 1-Fc.
HEK-293T cells cultured in DMEM+10% FBS were transfected with 10 μg of Fc expression constructs using FuGENE® HD (Roche Cat. No. 04709705001). The cells were washed with PBS. The culture medium was replaced with serum-free CD293 media (Cat. No. 11913019) (Invitrogen, Carlsbad, Calif.) 16 hours after transfection. The cultured media were collected 72 hours after the transfection and concentrated by using Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-30 membrane (Cat. No. UFC903024) (Millipore, Billerica, Mass.). The Fc proteins were purified using the Protein A IgG purification kit (Cat. No. 44667) (Pierce, Rockford, Ill.). The quantity of purified Fc proteins was measured by OD280.
Fc proteins were biotinylated using the EZ-Link® Micro Sulfo-NHS-Biotinylation Kit (Cat. No. 21925) (Pierce, Rockford, Ill.). 50 μg of purified Fc proteins in 200 ul of phosphate-buffered saline (PBS pH=7.4) were mixed with 2 μl of sulfo-NHS-biotin solution and incubated on ice for two hours. Excess biotin was removed by centrifuging at 1,000 g for 2 minutes using the Zeba Desalt Spin Column provided in the kit. The collected flow-through solution contained biotinylated Fc protein.
We examined if the binding of peptide no. 1 to cell surface IL-23R is reduced when IL-23R levels are reduced. siRNA was administered to diminish the expression level of IL-23R.
We performed four assays: (1) control siRNA (i.e., siRNA that would not bind to IL-23R RNA) stained with control antibodies; (2) control siRNA stained with anti-IL-23R antibodies; (3) IL-23R siRNA (i.e., siRNA specific for IL-23R RNA) stained with control antibodies; and, (4) IL-23R siRNA stained with anti-IL-23R antibodies.
DB cells (5×106) were transfected with siRNA using Amaxa® Human T cell Nucleofector® Kit (Cat. No. VPA-1002) (Lonza, Allendale, N.J.). The control siRNA and human IL-23R siRNA were obtained from Thermo Scientific Dharmacon® (Cat. No. D-001810-01-05 (control) and L-007976-00 (IL-23R)) (Lafayette, Colo.). DB cells were precipitated by centrifugation at 200×g for 10 minutes. The cell pellet was re-suspended in 100 μl of Nucleofector® solution containing either control siRNA or IL23R siRNA. The cell and siRNA suspension were transferred into certified cuvette. The cuvette was inserted into the Nucleofector® Cuvette Holder and V-024 program was applied. 500 μl of the pre-equilibrated culture medium (RPMI+10% FBS) was gently added to the cuvette and was transferred into one well of a 12-well plate. The cells were incubated for 48 hours and were used in the Flow Cytometry experiment. The cells were stained with either isotype control antibodies or anti-IL23R antibodies as previously described in Example 15.
These data show that IL-23R levels were reduced by the addition of IL-23R siRNA.
We examined the effect of the IL-23R siRNA in DB cells and determined if it may affect the binding of polypeptide no. 1-Fc to DB cell IL-23 receptors.
We performed four assays: (1) control siRNA (i.e., siRNA not specific to IL-23R) stained with biotinylated Fc; (2) IL-23R siRNA stained with biotinylated Fc; (3) control siRNA stained with biotinylated polypeptide no. 1-Fc; and, (4) IL-23R siRNA stained with biotinylated polypeptide no. 1-Fc. Fc proteins were purified and biotinylated as set forth in Example 16.
These data show that the addition of IL-23R siRNA reduces the amount of polypeptide no. 1 that is binding to the cells. Thus, given that IL-23R siRNA reduces the levels of IL-23R on the cells, the data suggests that polypeptide no. 1 binds to the cell surface through IL-23R.
We examined the effect of peptide length on IL-23 inhibition. Polypeptides with a total amino acid length of 9, 16 or 18 amino acids were prepared by deleting or adding amino acids to sequences of polypeptide nos. 1 or 23. Despite a change in the overall length of the polypeptide, the core structure of the polypeptide was retained.
The modified polypeptides were used in a Competitive ELISA with IL-23, which was performed in accordance with the procedure set forth in Example 6.
(i) Core Structure WX1X2X3W-Polypeptide No. 1
We prepared three (3) polypeptides of various lengths based on the amino acid sequence of polypeptide no. 1.
(ii) Core Structure WX1X2WW-Polypeptide No. 23
We also prepared three (3) polypeptides of various lengths based on the amino acid sequence of polypeptide no. 23.
These data show that if the core structure is retained and the length of the polypeptide is twelve (12) amino acids or longer, no adverse affect on inhibitory activity was noticed.
We altered the number of amino acids between tryptophan residues (thereby destroying the core structure WX1X2X3W) and tested the role of tryptophan spacing on inhibition of IL-23 binding to its receptor.
The modified polypeptides were used in a Competitive ELISA with IL-23. The Competitive ELISA was performed in accordance with the procedure set forth in Example 6.
(i) Modified Sequences Based Upon Polypeptide No. 1
Based on the sequence of polypeptide no. 1, we prepared six (6) modified polypeptides (polypeptide nos. 1.7, 1.8, 1.9, 1.10, 1.11 and 1.12) in which the number of amino acid residues between tryptophan residues was changed (i.e., they no longer included the core structure WX1X2X3W). The
WGASSVQYRVQW
These data suggest that the spacing between tryptophan residues, and specifically the core structure WX1X2X3W, plays an influential role in the ability of a polypeptide to inhibit IL-23 binding to its receptor.
(ii) Modified Sequences Based Upon Polypeptide No. 9
Using the amino acid sequence of polypeptide no. 9, we designed five (5) additional peptides (i.e., polypeptide nos. 9.1, 9.2, 9.3, 9.4 and 9.5) in which the number of amino acid residues between tryptophan residues was changed (i.e., they no longer included the core structure WX1X2X3W).
These data suggest that the spacing between tryptophan residues, and specifically the core structure WX1X2X3W, plays an influential role in the ability of a polypeptide to inhibit IL-23 binding to its receptor.
We examined the role of the three (3) amino acid residues (i.e., X1X2X3) within the core structure WX1X2X3W on IL-23 inhibition. To do so, we substituted the amino acid residues by: (i) simultaneously substituting all three amino acid residues between the tryptophan (W) residues, or (ii) substituting (one at a time) of the three amino acid residues between the tryptophan (W) residues (i.e., substituting at one of X1, X2 or X3).
Using polypeptide no. 1 as a control, we prepared the modified polypeptides and studied the polypeptide modification (See,
(i) Simultaneous Substitution of All Three (3) Amino Acids
In this series of studies, we simultaneously substituted all three amino acid residues of polypeptide no. 1 while maintaining the remaining amino acid residues unchanged. We used two approaches: (i) drastic amino acid substitution change; and (ii) conservative amino acid substitution change.
In the drastic substitution change approach, the original amino acid residues were replaced by amino acids with different properties on the side chain. For example, an amino acid with a non-polar side chain was replaced with amino acid with polar uncharged side chain. An amino acid with aromatic side chain was changed to amino acid with aliphatic side chain. Polypeptide no. 1.13 was modified using the dramatic change approach.
In the conservative change approach, the original amino acid residues were replaced by amino acids with the same properties on the side chain. For example, amino acid with a non-polar side chain was replaced with another amino acid with non-polar side chain. An amino acid with aromatic side chain was changed to another amino acid with aromatic side chain. Polypeptide no. 1.14 was modified by the conservative change approach.
(ii) Gradual Substitution of Amino Acids
In this study, we changed the (3) three amino acid residues (i.e. VQY) of the polypeptide no. 1 in the core structure WX1X2X3W individually. The conservative change approach was applied in this study. Three (3) modified polypeptides based on the amino acid sequence of polypeptide no. 1 were generated (i.e., polypeptide nos. 1.12, 1.13 and 1.14).
We concluded that the amino acid residues X1X2X3 within the core structure WX1X2X3W play a limited, if any, role in a polypeptide's ability to inhibit IL-23 binding to its receptor. This observation is also consistent with the results obtained in our studies wherein we performed simultaneous substitution of all three (3) amino acid residues in the core structure WX1X2X3W.
We examined the role of tryptophan (W) on binding to IL-23R and inhibiting IL-23. We prepared modified polypeptides that included additional tryptophan (W) residues adjacent to, but outside of, the WX1X2X3W core structure. One (1) or two (2) tryptophan residues were added to either side of the WX1X2X3W core structure of polypeptide no. 1.
The modified polypeptides were used in a Competitive ELISA with IL-23. The Competitive ELISA was performed in accordance with the procedure set forth in Example 6.
Thus, it appears that additional tryptophan (W) residues adjacent to (but outside of) the WX1X2X3W core structure may attribute to solubility of the polypeptide (i.e., additional tryptophan (W) residues may adversely affect the solubility of the polypeptides).
We examined the contribution of the amino acids adjacent (on both sides) to the core structure WX1X2X3W of polypeptide no. 1 on the inhibitory properties of the polypeptides. We prepared several modified polypeptides based on the amino acid sequence of polypeptide no. 1. We maintained the core structure WX1X2X3W of the polypeptides, but changed the amino acid residues before (i.e., SGAS) or after (i.e., VQR) the WX1X2X3W core structure. (See,
The modified polypeptides were examined in a Competitive ELISA, which was performed in accordance with the procedure set forth in Example 6.
(i) Conservative Chance of Adjacent Amino Acids
Polypeptide nos. 1.19 and 1.20 were modified by the conservative change approach, in which the original amino acid residues were replaced by amino acids with the same properties on the side chain.
This suggests that it is the core structure WX1X2X3W and not the surrounding amino acid residues that are responsible for binding to IL-23R and inhibiting IL-23.
(ii) Drastic Chance of Adjacent Amino Acids
Polypeptide nos. 1.24 and 1.25 were prepared by modifying polypeptide no. 1 according to a drastic change approach in which the original amino acid residues were replaced by amino acids with different properties on the side chain.
Many polypeptides screened from our Phage Display Screening possess variations in amino acids adjacent to the WX1X2X3W core structure. Despite these amino acid variations, all exhibit the ability to bind to IL-23R as well as inhibiting IL-23R signaling. (See,
Competitive ELISA studies indicate that polypeptide no. 23 may have the strongest inhibitory effect of all the polypeptides selected in our Phage Display Screening. (See
But, unlike the majority of the polypeptides selected, polypeptide no. 23 possesses a tryptophan (W) at X3 instead of a tyrosine (Y). Further, polypeptide no. 23 contains the amino acid residue glutamate (E) at X1 and the amino acid residue aspartate (D) at X2. Among all the amino acids, aspartate (D) and glutamate (E) are the only two negatively charged residues.
In this series of studies, we examined the negatively charged amino acids inside the core structure on the inhibitory activity of polypeptide no. 23. To do so, we prepared several modified polypeptides based on the amino acid sequence of polypeptide no. 23. We modified polypeptide no. 23 by: (i) changing the negatively charged amino acid residues into different negatively charged residues (either D to E or E to D); (ii) changing the negatively charged amino acid residues into polar and uncharged residues; or (iii) changing the negatively charged amino acid residues to positively charged residues.
The modified polypeptides were studied in a Competitive ELISA with IL-23 which was performed in accordance with the procedure set forth in Example 6. Polypeptide no. 23 was run as a control.
(i) Changing the Negatively Charged Amino Acid Residues into Another Negatively Charged Residue
In this example, we changed the core structure of polypeptide no. 23 (i.e., WEDWW) into WDDWW (i.e., peptide no. 23.4) and WEEWW (i.e., peptide no. 23.5). The negative charge of the amino acids was preserved in these modified polypeptides.
(ii) Changing Negatively Charged Amino Acid Residues to Polar and/or Uncharged Amino Acid Residues
We determined the effect of changing the negatively charged amino acids in the WEDWW motif of polypeptide no. 23 into polar and uncharged amino acids. We prepared three (3) modified polypeptides by changing aspartate (D) and glutamate (E) into asparagine (N) and glutamine (Q), respectively.
(iii) Changing Negatively Charged Amino Acid Residues to Positively Charged Amino Acid Residues
Because the negatively charged residues in the WEDWW motif of peptide no. 23 could be substituted by the uncharged amino acids without major effect on its inhibitory activity, we examined whether these residues could be replaced by positively charged amino acids. We prepared three (3) modified polypeptides (i.e., polypeptide nos. 23.9, 23.10 and 23.11) in which either one or both of the negative amino acid residues were changed to positively charged lysine (K).
In sum, the results of this example (Example 22 parts (i), (ii) and (iii)) suggest that the charge of the amino acid residues in the core structure does not appear to be an important determinant of the ability of a polypeptide to inhibit IL-23 binding to its receptor.
We examined the importance of tryptophan (W) residue inside the core structure WX1X2X3W (i.e., the W at X3) of polypeptide no. 23. It is well recognized that tryptophan residue has an aromatic side chain. To examine the role of tryptophan at X3, we prepared three (3) modified polypeptides. In modified polypeptide nos. 23.12 and 23.13, we changed tryptophan (W) to tyrosine (Y) and phenylalanine (F), respectively. Tyrosine (Y) and phenylalanine (F) both include, like tryptophan (W), an aromatic side chain. In modified polypeptide no. 23.14, tryptophan (W) was changed to the non-polar, aliphatic amino acid residue alanine (A). Table 17 and
The modified polypeptides were examined in the Competitive ELISA, which was performed in accordance with the procedure set forth in Example 6.
In sum, it appears that polypeptides that include an aromatic amino acid at position X3 of the core structure WX1X2X3W, have a greater ability to inhibit IL-23 binding its receptor. This observation was consistent with our phage display screening result in which 30 out of 31 of the screened polypeptides (96.8%) contained an aromatic amino acid at position X3 of the core structure. (See,
The polypeptide no. 23 has the core structure of WX1X2X3W, wherein X1 is glutamate (E) X2 is aspartate (D) and X3 is tryptophan (W). Based on the observations made in Example 22 and Example 23, the X1 and X3 amino acid residues inside the core structure of polypeptide no. 23 may be glutamine (Q) and tyrosine (Y) respectively.
In this study, we continued to modify the polypeptide no. 23. We modified the polypeptide no. 23 with a new core structure having an amino acid sequence of “WQDYW.” Table 18 and
The modified polypeptide was examined in the Competitive ELISA, which was performed in accordance with the procedure set forth in Example 6. The IC50 value of polypeptides was calculated.
We chose the polypeptide no. 23.15 and used it to examine the effect of peptide length on IL-23 inhibition. Polypeptides having a total amino acid length of 5, 7 or 9 amino acids were prepared by deleting amino acids from the amino acid sequences of polypeptide no 23.15. Despite the varying length of the polypeptides, the core structure of the polypeptide remained WQDYW.
The modified polypeptides were used in a Competitive ELISA with IL-23, which was performed in accordance with the procedure set forth in Example 6.
These data show that reducing the total amino acid length of the polypeptide with the core structure WQDYW has adverse effect on inhibitory activity.
Cyclic peptide is a polypeptide on which the amino terminus and carboxyl terminus are themselves linked together with a peptide bond that forms a circular (i.e., cyclic) chain. Using the polypeptide no. 23.15, we generated the polypeptide no. 23.19, which is a cyclic peptide, by adding a cysteine residue on both side of polypeptide no. 23.15. A disulfide bond (i.e., a covalent bond) is formed by coupling the thiol group on the cysteine residue in a head-tail fashion to generate a cyclic peptide. Table 20 and
Cyclic Peptide Synthesis:
We obtained the crude peptide following the method of solid phase synthesis with TRT resin. We placed the sample in aqueous solution of 0.5 mg/ml concentration and adjust the pH to 8.5, dropping 10 equivalent of H2O2, then the cyclization reaction start and be retained for one hour. After the detection and the reaction being confirmed completed, we separated the sample by HPLC (solute: acetonitrile (ACN) with 0.1% TFA+H2O with 0.1% TFA, C18 column). The desired sample obtained will be freeze-dried and HPLC verified.
The cyclic polypeptide was examined in the Competitive ELISA, which was performed in accordance with the procedure set forth in Example 6. The IC50 value of polypeptides was calculated.
We examined the effect of peptide length relating to the cyclic polypeptide 23.19 on IL-23 inhibition. Cyclic Polypeptides with a total amino acid length of 7, 9, or 11 amino acids were prepared by deleting amino acids to sequences of polypeptide no 23.19. Despite varying the polypeptide length, the core structure of the cyclic polypeptide (“WQDYW”) was retained.
The modified polypeptides were used in a Competitive ELISA with IL-23, which was performed in accordance with the procedure set forth in Example 6.
These data show that reducing the polypeptide length of the cyclic polypeptide (containing the “WQDYW” core structure) has an adverse effect on the inhibitory activity.
Polypeptide no. 23.15-Fc fusion protein was obtained by designing expression constructs (i.e., polypeptide-Fc expression constructs) that expressed the amino acid sequence of interest fused to Fc.
Forward (F) and reverse (R) oligonucleotides corresponding to the desired peptide sequences were designed as set forth in Table 20.
Equal amounts of forward and reverse oligonucleotides were annealed into double strand form by incubating at 95° C. for 10 minutes. After the mixture cooled down to room temperature, 1 μl of double strand oligonucleotide was ligated with linearized Fc expression construct (Cat. No. ppfc2-mg2ae1) (InvivoGen, San Diego, Calif.). The linearized vector was prepared by treating the DNA with NcoI and BglII restriction enzymes. The ligated DNA was then transformed into Top10 competent cells (Invitrogen, Carlsbad, Calif.). The sequence of the expression constructs were verified by DNA sequencing. Fusion protein was expressed by transfecting HEK-293T cells with the polypeptide-Fc expression construct. We performed the purification using protein A-agarose bead to isolate #23.15-Fc fusion protein.
The isolated polypeptide no. 23.15-Fc fusion protein was used in a Competitive ELISA with IL-23, which was performed in accordance with the procedure set forth in Example 6. The IC50 value of polypeptides was calculated.
These data show that fusing the polypeptide no. 23.15 to Fc protein increased its inhibitory activity.
(i) Generation of Cell Based Assay—DB Cells—STAT3-Luc
In Example 13, we examined expression of IL-23R on the DB cells. In a flow cytometry assay, DB cells were stained with either isotype control antibodies or anti-IL-23R antibodies (
The Cignal STAT3 reporter (SA Biosciences, CCS-9028L), designed to measure the transcriptional activity of STAT3 homodimers, was transiently transfected into the DB cells. The stably transfected STAT3-Luc reporter clone was selected using 200 ng/ml of Hygromycin B.
The stably transfected STAT3-Luc reporter DB cells (0.5×106) were cultured in 100 μl of RPMI+10% FBS. IL-23 was added to the cells and incubated at 37° C. for 4 hours. Luciferase activity was measured using Dual-Glo luciferase assay system (Promega).
As depicted in
This result clearly demonstrates that the stably transfected DB-STAT-Luc reporter cells can be used as cell-based assay to measure the activity of IL-23R pathway upon IL-23 cytokine stimulation.
ii) Measurement of IC50 Values of Cyclic Polypeptide no. 23.19 and Polypeptide No. 23.15-Fc Fusion Protein
The isolated cyclic polypeptide no. 23.15-Fc fusion protein and cyclic polypeptide no. 23.19 were used in a cell-based assay to measure the IC50 values. The stably transfected STAT3-Luc reporter DB cells (0.5×106) were cultured in 100 μl of RPMI+10% FBS. The cells were pre-incubated with different concentration of isolated cyclic polypeptide no. 23.15-Fc fusion protein or polypeptide no. 23.19 for 30 minutes at 37° C. 50 ng/ml of IL-23 was added to the cells and incubated at 37° C. for an additional 4 hours. Luciferase activity was measured using Dual-Glo luciferase assay system (Promega). The IC50 values of isolated cyclic polypeptide no. 23.15-Fc fusion protein and cyclic polypeptide no. 23.19 are 2.5 μM and 2.5 μM respectively. The IC50 values obtained from this cell-based assay are comparable to the IC50 values obtained from the cell-free assay.
This result clearly demonstrates that both isolated cyclic polypeptide no. 23.15-Fc fusion protein and cyclic polypeptide no. 23.19 are active in the cell-based assay to inhibit the IL-23 signaling.
In this study, we tested if the cyclic polypeptide no. 23.15, cyclic polypeptide no. 23.19 and the isolated polypeptide 23.15-Fc fusion protein could inhibit binding of mouse IL-23 to mouse IL-23R. Mouse IL-23 (p19 Gene Accession #: NM—031252; p40 Gene Accession #: NM—008352). Mouse IL-23R (Gene Accession #: NM—144548). To do so, we performed the Competitive ELISA.
The isolated polypeptide no. 23.15-Fc fusion protein, cyclic polypeptide no. 23.19 and polypeptide no. 23.15 were used in a Competitive ELISA with IL-23, which was performed in accordance with the procedure depicted in
This result clearly demonstrates that both isolated polypeptide no. 23.15-Fc fusion protein, cyclic polypeptide no. 23.19 and polypeptide no. 23.15 are active in the cell-free assay to inhibit the mouse IL-23 cytokine binding to mouse IL-23R, and that the polypeptide no. 23.15-Fc fusion protein exhibits the strongest inhibitory activity.
In all the above-mentioned examples, polypeptide and isolated polypeptide no. 23.15-Fc fusion protein not only bind to IL-23R but also block the IL-23 cytokine binding to IL-23R. This observation suggests that the polypeptides containing the core structure WX1X2X3W and IL-23 cytokine bind to the same region of IL-23R.
In this study, we examined which regions of IL-23R proteins were required for the binding to IL-23 and polypeptide no. 23.15-fc fusion protein. To do so, we generated a series of IL-23R deletion proteins as illustrated in the
Culture media from 293T cells transfected with different FLAG-tagged IL-23R deletion constructs were incubated with 200 ng of either IL-23 or polypeptide no. 23.15-fc fusion protein, then immunoprecipitated with anti-FLAG M2 affinity gel. The immunoprecipitate was subjected to Western blotting and polypeptide no. 23.15-Fc fusion protein or IL-23 was visualized with either anti-mouse IgG or anti-hIL-12/23p40 respectively. IL-23 or polypeptide no. 23.15-fc fusion protein was detected in the precipitate when 1-348, 1-318 or 1-250 protein was present in the precipitation reaction (
These experiments confirmed that IL-23 and polypeptide no. 23.15-fc fusion protein bind to the same region of IL-23R.
In Example 11 (above), the results clearly indicate an important role of the tryptophan (W) residues in the core structure WX1X2X3W in binding to IL-23R and inhibiting IL-23R activity. In Example 30, the experiments confirmed that IL-23 and polypeptide no. 23.15-fc fusion protein bind to the same region of IL-23R. However, we could not identify the sequence of core structure (i.e. WX1X2X3W) on p19 subunit of IL-23 cytokine (See,
Given the importance of “W” residues on the inhibitory activity of peptides, we examined the role of the five (5) tryptophan residues found on the p19 subunit of IL-23 (See,
To do so, we performed site-directed mutagenesis to change the “W” residue present on the IL-23 p19 subunit into “G” residue separately one at a time. We created a total of six (6) expression constructs; namely, WT (Wild type—no mutation), W1 (convert W1 into G), W2 (convert W2 into G), W3 (convert W3 into G), W4 (convert W4 into G) and W5 (convert W5 into G) of the p19 subunit of IL-23. The expression construct of p40 subunit of IL-23 was also made.
In order to produce IL-23 cytokines (contains p19 and p40 subunits), expression constructs of p40 and corresponding p19 were co-transfected into the 293T cells. Conditional media were collected 48 hours after transfection. We prepared a total of six (6) different conditional media containing corresponding IL-23 cytokines (WT, W1, W2, W3, W4 and W5). We then examined the expression level of cytokines and their binding to IL-23R by ELISA.
i) Measurement of Expression Levels of IL-23 Cytokines
Expression levels of IL-23 cytokines in the conditional media were measured by Human IL-23 ELISA Ready-SET-Go!® Set (eBioscience).
To prepare the ELISA, microtiter plates were coated with anti-p19 antibody and incubated overnight at 4° C. The plates were blocked for 1 hour at room temperature using the blocking buffer provided in the ELISA kit. After blocking and washing with PBST for three (3) times, recombinant IL-23 or 100 μA of conditional media were added to each well and the plate was incubated overnight at 4° C.
Bound IL-23 cytokines were detected by adding biotinylated anti-p40 antibody. Streptavidin-horseradish peroxidase (HRP) was added to detect anti-p40 antibody bound to IL-23 cytokines Peroxidase activity (representing the level of IL-23 cytokine captured onto plates) was measured by adding 100 μl of a tetramethylbenzidine (TMB) to each well. The color intensity was directly proportional to the amount of the bound IL-23. Optical density was read at 450nm.
ii) Measurement of IL-23 Cytokines Binding to IL-23R
In
Eight (8) ELISAs were performed. Binding of wild type (WT) or mutated IL-23 cytokines (W1, W2, W3, W4 and W5) to IL-23R was examined by ELISA illustrated in
iii) Measurement of IL-23 Cytokines Binding to IL-23R by Competition Assay
As depicted in
Eight (8) competitive ELISAs were performed. Wild type (WT) or mutated (W1, W2, W3, W4 and W5) IL-23 cytokines in the conditional media were used as competitor in this assay. No signal was detected when there was no biotinlyated IL-23 in the well. In the absence of conditional media, biotinlyated IL-23 cytokine bound effectively to the immobilized IL-23R-Fc. In contrast, biotinlyated IL-23 cytokine failed to bind to IL-23R in the wells of conditional media containing WT, W1, W3 or W4 IL-23 cytokine Only half of the biotinlyated IL-23 cytokine bound to its receptor in the well of conditional medium containing W2 IL-23. Biotinlyated IL-23 cytokine in the well of conditional medium containing W5 IL-23 bound as effectively as biotinlyated IL-23 cytokine in the well with no competitor. The ELISA data is summarized in
Altogether, these data show that W1, W3 and W4 of p19 subunits of IL-23 cytokine are not essential to IL-23 binding to IL-23R whereas W2 of p19 subunit of IL-23 cytokine plays a limited role on receptor binding. Intriguingly, W5 of p19 subunit of IL-23 cytokine is indispensable to the receptor binding.
In Example 32 (above), we showed the indispensable role of the tryptophan residue (W5) in p19 subunit of IL-23 cytokine to bind to IL-23R. We further confirmed the critical role of the W5 residues in the p19 subunit of IL-23 on the receptor binding using cell-based assay developed in the Example 29. To do so, we purified the WT and W5 IL-23 cytokines.
In order to produce IL-23 cytokines (contains p19 and p40 subunits), expression constructs of p40 and corresponding p19 (WT or W5) were co-transfected into the 293T cells. Conditional media were collected 48 hours after transfection. We prepared two (2) different conditional media containing corresponding IL-23 cytokines (WT and W5). The WT and W5 p19 subunits of IL-23 cytokine were tagged with 6×His at the C-terminus. The IL-23 cytokines in the conditional media were purified by using HisTALON Gravity Column (Clontech). The concentration of the purified IL-23 cytokines was measured by absorbance at 280 nm using NanoDrop (Thermo Scientific). The purity of the isolate IL-23 cytokines was examined using SDS PAGE followed by Coomassie Blue staining (See,
In Example 29 (above), we detailed the development of stably transfected STAT3-Luc reporter clone. IL-23 cytokine stimulates the STAT3 activity in a dose-dependent manner in this cell-based assay. The stably transfected STAT3-Luc reporter DB cells (0.5×106 cells) were cultured in 100 μl of RPMI+10% FBS. Recombinant IL-23 (Humanzyme) or isolated IL-23 (WT or W5) was added to the cells and incubated at 37° C. for 4 hours. Luciferase activity was measured using Dual-Glo luciferase assay system (Promega).
As shown in
In contrast, when we added the isolated W5 IL-23 cytokine at different concentrations (50,000, 5,000, 500 and 50 ng/ml) to the stable cells, none of them showed significant activation of the STAT3 reporter system, indicating that the W5 IL-23 cytokine lacks the ability to stimulate the STAT3 activity.
Altogether, these data show that W5 of p19 subunit of IL-23 cytokine is indispensable to the receptor binding.
Experimental Methods and Protocols
1. Phage Display—Negative Selection Assay to Remove Phages that Non-Specifically Bind to Protein A Sepharose
20 μA of Protein A sepharose (GE Healthcare, Waukesha, Wis.) or anti-Flag affinity gel (Sigma, St. Louis, Mo.) was washed with 1 ml of TBST (TBS+0.1% Tween) and re-suspended in 1 ml of blocking buffer (TBST+3% BSA+0.02% sodium azide). The resin was mixed at 4° C. for 1 hour and was washed four times (4×) with 1 ml of 0.1% TBST. 1×1011 pfu of phage display 12-mer peptide library (Cat. No. E8110S) (New England BioLabs, Ipswich, Mass.) was added to the washed resin and the mixture was incubated at room temperature for 15 minutes. The mixture was centrifuged at 5,000 rpm for 2 minutes to precipitate the bead-bound phages. The non-specifically bound phages were removed. The supernatant containing unbound phages were used in assays to isolate IL-23R binding phages.
2. Selection of Phages Displaying Sequences that Bind to IL-23R
300 μl of the phage-containing supernatant from the negative selection assay was incubated overnight at 4° C. with mixing with 20 μl of protein A sepharose and 10 μg of rhIL23R-Fc (Cat. No. 1400-IR-050) (R&D Systems, Minneapolis, Minn.) or Flag-tagged Δ9 and anti-Flag affinity gel. The mixture was centrifuged at 5,000 rpm for 2 minutes to precipitate the bead-bound phages (i.e., phages displaying sequences that bound IL-23R). The supernatant was removed and the resin was washed ten times (10×) with 1 ml of 0.1% TBST. Phages were eluted from the sepharose beads by adding 1 ml of elution buffer (0.2 M glycine-HCl, pH 2.2, 0.1% BSA) to the resin and incubating the resin-buffer mixture for 10-minutes at room temperature. The beads were separated from the phages by centrifuging the elution mixture at 5,000 rpm for 2 minutes leaving the phages in the supernatant. The supernatant was added to 150 μl of 1 M Tris-HCl, pH 9.1 to neutralize the eluted phages.
3. Amplification of IL-23R Binding Phages
The eluted phages were introduced to an early-log 20 ml culture of E. coli (i.e., ER2738). The mixture was incubated for 4.5 hours at 37° C. with shaking. Following incubation, the culture was centrifuged at 12,000 rpm for 10 minutes to remove cell debris and E. coli cells. To precipitate the phages, the upper 80% of the supernatant was incubated overnight at 4° C. with 3 ml of 20% polyethylene glycol (PEG)/2.5 M NaCl. The solution was centrifuged at 12,000 rpm for 30 minutes to collect the precipitated phages. The supernatant was discarded. The pellet was re-suspended in 1 ml of TBS. 200 μA of 20% PEG/2.5 M NaCl was added to the supernatant and the mixture was incubated on ice for 1 hour. The solution was centrifuged at 12,000 rpm for 10 minutes and the pellet re-suspended in 200 μA of TBS.
The phage concentration in the amplified pool was measured by the phage titer on LB/IPTG/Xgal plates. The phage titer was determined by counting blue plaques. Two additional rounds of bio-panning were performed as described above. After 3 rounds of bio-panning, the eluted phages were enumerated by titration before phage amplification.
4. Isolation of Single M13 Plaque for DNA Sequencing and Phage ELISA
Individual plaques (i.e., blue plaques) were randomly selected and amplified by adding the plaques to 1 ml of E. coli ER2738. The mixture was incubated at 37° C. with shaking for 5 hours. The cultures were centrifuged to remove cell debris and the pellets discarded. 500 μl of the supernatant (i.e. LB containing amplified individual phage identified after 3 around of bio-panning) was apportioned for DNA sequencing. The remaining supernatant volume was used in a phage ELISA.
5. DNA Extraction and Sequencing
DNA from the phages that bound to either Δ9-Flag or to IL-23R/Fc chimera was sequenced to determine the amino acid sequence of the polypeptides that bound IL-23R.
Individually amplified phages were precipitated from the 500 μl of supernatant obtained following the phage display assay. 200 μl of 20% PEG/2.5 M NaCl was added to the 500 μl phage-containing supernatant (1:2.5 volume to volume ratio of PEG/NaCl to supernatant). The mixture was incubated for 20 minutes at room temperature. Phages were collected by centrifuging at 12,000 rpm for 10 minutes. The supernatant was discarded and the pellet re-suspended in 100 μA iodide buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 4 M sodium iodide). 250 μA ethanol was added and the solution incubated at room temperature for 20 minutes. The solution was centrifuged at 12,000 rpm for 10 minutes. The supernatant was discarded and the pellet was washed with 0.5 ml of cold 70% ethanol. After another centrifugation at 12,000 rpm for 10 minutes, the supernatant was discarded and the pellet, containing the phage DNA, was suspended in 20 μl TE buffer.
For DNA sequencing, we used Beckman Coulter GenomeLab DTCS Quick Start Kit (Cat. No. 60812) (Beckman Coulter, Fullerton, Calif.). The concentration of phage DNA was measured by the NanoDrop at OD280. (NanoDrop Technologies inc, Wilmington, Del.). Approximately 150 ng of phage DNA was used for each sequencing reaction. 1 pmol of −96 GIII sequencing primer (20-mer), (supplied with New England BioLabs Ph.D.™ 12 Phage Display Peptide Library Kit, Ipswich, Mass.) was used for each reaction. The thermal cycling program was 20 seconds at 96° C., 20 seconds at 50° C., and 4 minutes at 60° C., for 30 cycles. Ethanol precipitation was performed according to the Beckman Coulter kit insert. DNA sequencing was performed using Beckman Coulter CEQ 8000.
6. Phage ELISA
Microtiter plates were coated with 2 μg/ml of recombinant human IL-23R/Fc and incubated overnight at 4° C. The plates were blocked for 2 hours using 10% FBS/TBST at room temperature. 10 μl of 10×PBS was added to each well. 90 μl of M13 phage-containing LB media was also added. To permit phage binding to the IL-23R, the plate was incubated overnight at 4° C.
Bound phage was detected by adding 2 μg/ml of biotinylated anti-M13 antibody (Cat. No.: ab17269) (Abcam, Cambridge, Mass.). Streptavidin-horseradish peroxidase (HRP) was added to detect anti-M13 antibody bound to IL-23R bound phages. Peroxidase activity (representing the level of M13 captured onto plates) was measured by adding 100 μl of a tetramethylbenzidine (TMB) to each well. The color intensity was directly proportional to the amount of the bound M13 phage. Optical density was read at 450nm.
7. Immuno-Precipitation Assays
1 ml of cell culture media that included polypeptide-Fc fusion protein was incubated with 20 μl of protein A sepharose at room temperature for 1 hour. 1 ml of Δ9 containing culture medium was added to the mixture (i.e., Fc proteins and protein A sepharose) which was incubated at 4° C. overnight with mixing. The mixture was centrifuged, the supernatant removed and the remaining protein A resin washed 5 times with 1 ml PBS to form a precipitate. The precipitated proteins were denatured by addition of sample loading buffer (Bio-Rad, Berkeley, Calif.) and run on 4-12% PAGE and transferred to Immun-Blot PVDF membrane. Membranes were blocked in 5% milk/TPBT at room temperature for 1 hour. Anti-Flag antibody (1:1,000) (Sigma, St. Louis, Mo.) and anti-mouse antibody (1:3,000) were used to detect the Δ9 and Fc proteins, respectively.
8. Competitive ELISA
Recombinant human IL-23R-Fc (2 μg/ml) was coated onto microtiter plates as a capture reagent. IL-23 and polypeptides were added to compete for binding to the immobilized IL-23R-Fc. 50 μg human IL-23 (Cat. No. HZ-1048) (Humanzyme, Chicago, Ill.) in the presence or absence of 100 μM peptides was added to the well and incubated at 4° C. overnight. IL-23 that bound to IL-23R-Fc was detected using biotinylated anti-p40 antibody (1:250). The bound anti-p40 antibody was detected using streptavidin-horseradish peroxidase (HRP) (1:500) and 100 μl/well of a tetramethylbenzidine (TMB) substrate was added to measure peroxidase activity. The color was measured at optical density (OD) 450nm. The color intensity was directly proportional to the amount of the bound IL-23 protein.
9. Generation of Peptide IL-23R/Fc Fusion Expression Constructs
Polypeptide-Fc fusion proteins were obtained by designing expression constructs (i.e., polypeptide-Fc expression constructs) that expressed the amino acid sequence of interest fused to Fc.
Forward (F) and reverse (R) oligonucleotides corresponding to the desired peptide sequences were designed as set forth in Table 18.
The oligonucleotides were re-suspended in TE buffer at 200 μM concentration. Equal amount of forward and reverse oligonucleotides were annealed into double strand form by incubated at 95° C. for 10 minutes. After the mixture cooled down to room temperature, 1 μl of double strand oligonucleotides was used as an insert to ligate with linearized Fc expression construct (Cat. No. ppfc2-mg2ae1) (InvivoGen, San Diego, Calif.). The linearized vector was prepared by treating the DNA with NcoI and BglII restriction enzymes. The ligated DNA was then transformed into Top10 competent cells (Invitrogen, Carlsbad, Calif.). All the Fc expression constructs were verified by DNA sequencing. The ppfc2-mg2ae1 is a plasmid used to construct peptide-Fc Fusion proteins by fusing a sequence encoding a corresponding peptide sequence to the Fc region of an immunoglobulin. These peptide-Fc fusion proteins contain Fc region of mouse IgG2a. The Fc region comprises the CH2 and CH3 domains of the murine IgG2a heavy chain and the hinge region. The hinge serves as a flexible spacer between peptide sequence and the Fc region, allowing each part of the molecule to function independently. Additionally, the plasmid features the IL2 signal sequence for the generation of peptide-Fc fusion proteins secreted into the culture medium.
10. Purification of Fc Proteins
HEK-293T cells cultured in DMEM+10% FBS were transfected with 10 μg of Fc expression constructs using FuGENE® HD (Roche Cat. No. 04709705001). The cells were washed with PBS and the culture medium was replaced by serum free CD293 media (Cat. No. 11913019) (Invitrogen, Carlsbad, Calif.) 16 hours after transfection. The cultured media were collected 72 hours after the transfection and concentrated by using Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-30 membrane (Cat. No. UFC903024) (Millipore, Billerica, Mass.). The Fc proteins were purified using the Protein A IgG purification kit (Cat. No. 44667) (Pierce, Rockford, Ill.). The quantity of purified Fc proteins was measured by OD280.
11. Biotinylation of Purified Fc Proteins
Fc proteins were biotinylated using the EZ-Link® Micro Sulfo-NHS-Biotinylation Kit (Cat. No. 21925) (Pierce, Rockford, Ill.). 50 μg of purified Fc proteins in 200 μl of phosphate-buffered saline (PBS pH=7.4) was mixed with 2 μl of sulfo-NHS-biotin solution and incubated on ice for two hours. Excess biotin was removed by centrifuging at 1,000 g for 2 minutes using the Zeba Desalt Spin Column provided in the kit. The collected flow-through solution contained biotinylated Fc protein.
12. Cell Surface Staining for the Flow Cytometry
The cells were first washed twice with ice-cold PBS. 100 μl of blocking buffer (PBS+5% heat inactivated human serum) was added to re-suspend the cell pellet. The mixture was incubated on ice for 30 minutes followed by the addition of staining reagents, such as control isotype-biotinylated goat IgG, biotinylated anti-IL23R, biotinylated Fc protein and biotinylated peptide-Fc fusion proteins. After 30 minutes of incubation on ice, the cells were washed thrice with ice-cold PBS. The washed cells were re-suspended in 100 l of blocking buffer containing 0.2 μl of detection reagent (Streptavidin-PE, BD Pharmingen, Cat. No. 554061) and were incubated on ice for 30 minutes followed by washing thrice with PBS. The cells were fixed in 1% (v/v) paraformaldehyde/PBS. The fixed cells were then acquired and analyzed.
13. Cell Based Assay—Luciferase Assay
DB cells express IL-23R on the cell surface. In addition, IL-23 stimulated the DB cells through the activation of STAT3 by phosphorylation (p-STAT3) (Example 7 (
The Cignal STAT3 reporter (SA Biosciences, CCS-9028L) is designed to measure the transcriptional activity of STAT3 homodimers. STAT3 is activated through phosphorylation in response to various cytokines and growth factors including IL-23. The activation of STAT3 results in formation of STAT3 homodimers, which interact with the sis-inducible element (SIE), a promoter sequence, thereby inducing transcription activity of target genes. The STAT3 reporter is a luciferase construct under the control of multiple SIE. The STAT3-responsive luciferase construct encodes the firefly luciferase reporter gene under the control of a minimal CMV promoter and tandem repeats of the SIE transcriptional response element. The number of response elements and the intervening sequence between these response elements has been experimentally optimized to maximize the signal to noise ratio. This construct monitors both increases and decreases in the transcriptional activity of STAT3 homodimers, and hence the activity of the STAT3 signaling pathway and IL-23R pathway.
The Cignal STAT3 reporter (SA Biosciences, CCS-9028L), which is designed to measure the transcriptional activity of STAT3 homodimers, was transiently transfected into the DB cells. The stably transfected STAT3-Luc reporter clone was selected using 200 ng/ml of Hygromycin B. The activity of IL-23R pathway can be measured by the transcriptional activity of STAT3 homodimers. Phosphorylation of STAT3 induces the formation of homodimer, which results in translocation into nucleus. STAT3 homodimer in the nucleus binds to DNA and activates transcription. Therefore, the Luciferase activity is directly proportional to the activity of IL-23R pathway.
The DB cells stably transfected STAT3-Luc reporter (0.5×106) were cultured in 100 μl of RPMI+10% FBS. IL-23 was added to the cells and incubated at 37° C. for 4 hours. Luciferase activity was measured using Dual-Glo luciferase assay system (Promega).
14. Cell Based Assay—IL-23/IL-23R Signaling
3×106 DB cells were cultured in 1 ml of RMPI+10% FBS. The cells were pre-incubated with peptides at 4° C. for 30 minutes followed by the addition of 30 ng/ml of IL23 cytokine. After the stimulation for 20 minutes, the cells were washed with ice-cold PBS and lysed in ProteoJET mammalian cell lysis reagent (Fermentas) with protease and phosphatase inhibitors (Sigma). Lysates were centrifuged and supernatants were prepared for SDS-PAGE by addition of sample loading buffer (Bio-Rad). Lysates were subjected to 4-12% PAGE (Bio-Rad) and transferred to Immun-Blot PVDF membrane (Bio-Rad) per manufacturer's recommendations. Membranes were blocked in 5% milk/TPBT at room temperature for 1 hour. Membranes were first probed with antibodies against p-STAT3 (cell signaling technology), and then stripped and reprobed for STAT3 (cell signaling technology).
15. Site-Directed Mutagenesis—Generation of p19 Mutants
Expression construct of wild-type p19 subunit of IL-23 cytokine (WT) was generated by PCR using Pfx DNA polymerase (Invitrogen). The following primers were used to amplify wild-type p19 subunit of IL-23 cytokine from PBMCs cDNA (WT F: 5′ GCCACCATGCTGGGGAGCAGAGCT 3′ (SEQ ID NO: 136)) and (WT R: 5′TCAGTGATGATGGTGGTGATGTCCGCTGCCGGGACTCAGGGTTGCTGC 3′ (SEQ ID NO: 137)). The amplified PCR product was treated with Taq polymerase to add 3′-A overhang to each end of PCR. The gel-purified product was then subcloning into mammalian expression plasmid using the pcDNA3.3 TOPO TA Cloning kit from Invitrogen. The correct expression construct was subjected to validation by sequencing.
Expression constructs of p19 mutants (W1, W2, W3, W4 and W5) were constructed by site-directed mutagenesis using overlapping PCR. The expression construct of wild-type p19 subunit of IL-23 cytokine was used as PCR template. The following primers were used to mutate the corresponding Tryptophan residue “W” into Glycine residue “G”. Generation of p19 mutants was performed by PCR overlap extension. Two fragments, fragment 1: using WT F and W1 R, W2 R, W3 R, W4 R or W5 R; and fragment 2: using WT R and W1 F, W2 F, W3 F, W4 F or W5 F, were amplified using these primer pairs.
Two amplified fragments (1 and 2) for corresponding Tryptophan mutant were then joined together by overlap extension. The final combined fragment was subcloned into pcDNA3.3 TOPO expression vector. The correct expression constructs were subjected to validation by sequencing.
All publications and patents cited in this specification are herein incorporated by reference in their entirety. Although the invention has been described in connection with specific preferred embodiments and working examples, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments and examples. Various modifications and variations of the described composition, method, and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention.
The present application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 61/520,710 filed Jun. 14, 2011, the disclosure of which is hereby incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 61520710 | Jun 2011 | US |
