Patent Application
20090215686
Hepatocyte growth factor (HGF, also known as scatter factor) and its receptor Met, a receptor tyrosine kinase (RTK), mediate a network of signaling pathways that control cell proliferation, survival, and motility (Birchmeier et al., 2003). Proper signaling of HGF-Met is essential for normal embryonic development and organ formation (Bladt et al., 1995; Schmidt et al., 1995). In the adult, HGF-Met signaling is involved in angiogenesis, wound healing, and liver regeneration (Borowiak et al., 2004; Huh et al., 2004; Zhang et al., 2003). Besides these normal physiological functions, aberrant activation of HGF/Met signaling has been closely associated with tumor growth, invasion, and metastasis. For example, Met activation by over-expression of the receptor is found in more than 50% of solid tumors and its hyper-activation is generally associated with poor prognosis (http://www.vai.org/met/). Thus, anti-cancer therapy based on Met antagonists has emerged as a prominent and rational goal of pharmaceutical research and development.
HGF is synthesized as an inactive, 90 kDa single chain precursor that is cleaved to form the mature growth factor consisting of two chains linked by a disulfide bond (Bottaro et al., 1991; Gherardi et al., 1989; Mars et al., 1993; Nakamura et al., 1989). The N-terminal α-chain (69 kDa) contains an N-terminal domain (N) followed by four repeats of the kringle domain (K1-K4); the C-terminal β-chain (34 kDa) contains a serine protease-homology (SPH) domain devoid of protease activity (
NK1 binds Met and is described either as a receptor antagonist or agonist in vitro depending on the context of assay formats and cell types (Lokker and Godowski, 1993; Lokker et al., 1994; Montesano et al., 1998). In vivo studies in transgenic mice, however, have clearly established that NK1 is a potent Met activator (Jakubczak et al., 1998). Despite the fact that the binding affinity of NK1 for Met is 5-20 fold weaker than that of HGF, the ability of NK1 to induce Met dimerization is readily observed and consistent with its agonistic activity in transgenic mice and in cell based assays (Catlow et al., 2003; Jakubczak et al., 1998; Lyon et al., 2004; Rubin et al., 2001; Schwall et al., 1996); and it has been clarified that the agonist activity of NK1 is dependent on the presence of glycosaminoglycans such as heparan sulfate (Catlow et al., 2003; Lyon et al., 2004; Rubin et al., 2001; Schwall et al., 1996). Whereas the detailed interactions between HGF and Met remain poorly characterized, mutagenesis data have pointed out that the fragment corresponding to NK1 is responsible for the high-affinity binding of HGF to Met (Holmes et al., 2007; Lokker and Godowski, 1993; Lokker et al., 1994). Recent mutagenesis and crystallographic data, however, also indicate that the C-terminal β-chain also binds directly to Met and is essential for the agonist activity of the full-length HGF (Stamos et al., 2004). Amino acid sequences for NK1 in various species are available. The human NK1 amino acid sequence is provided in the sequence listing at SEQ ID NO:1. The mouse NK1 amino acid sequence is provided in the sequence listing at SEQ ID NO:2. The PDB code for the mouse and human NK1 is 2QJ4 and 2QJ2, respectively.
Met, the receptor for HGF, was originally identified in a chemically transformed cell line as an oncogene produced by chromosomal rearrangement (Cooper et al., 1984). The 170 kD receptor encoded by the c-Met proto-oncogene contains a large extracellular domain (ectodomain), a transmembrane domain, and an intracellular tyrosine kinase domain (
Met is thought to be activated by HGF through ligand-induced receptor dimerization and there is evidence that HGF can form a complex with 2:2 stoichiometry with a fragment of the Met ectodomain corresponding to the sema and cystine-rich domain (Gherardi et al., 2006). Furthermore, there is evidence that Met on the cell surface is readily cross-linked into dimers or higher order oligomers upon treatment with HGF (Gherardi et al., 2006; Kong-Beltran et al., 2004). Activation of Met was shown to be mediated through receptor dimerization as first demonstrated by the fusion protein of the Met kinase domain with a TRP leucine zipper dimerization motif (Park et al., 1986). However, the mechanism of HGF-induced Met dimerization remains unclear as the isolated full-length Met ectodomain in complex with HGF is predominately monomeric in solution (Gherardi et al., 2003). Recent data shows that the Met sema domain is sufficient for the binding of HGF (Gherardi et al., 2003), and is required for receptor dimerization and activation (Kong-Beltran et al., 2004; Michieli et al., 2004).
Because aberrant activation of the HGF-Met signaling is closely correlated with tumor proliferation, progression, invasion, and metastasis (Birchmeier et al., 2003), targeting Met activation has become an intense area of anti-cancer therapeutical research. Current methods of Met inhibition include small molecules targeting the Met intracellular kinase domain (Christensen et al., 2003), antibodies against HGF or Met (Cao et al., 2001; Petrelli et al., 2006), decoy receptors using the Met extracellular domains (Kong-Beltran et al., 2004; Michieli et al., 2004), and HGF based Met antagonists like single chain HGF derivatives (Matsumoto and Nakamura, 2003; Mazzone et al., 2004). While these methods demonstrate various degrees of Met inhibition, they have displayed limitations with respect to their applications e.g. limited bioavailability and specificity of small molecule inhibitors, the large size and costly production of antibody and decoy Met receptors, protein stability and mixed agonist/antagonist properties of single chain HGF derivatives.
Although receptor dimerization is a general and accepted paradigm for activation of RTKs (Weiss and Schlessinger, 1998), diverse structural mechanisms of ligand-induced receptor dimerization have been revealed by crystallographic studies of ligand-receptor complexes of RTKs (Schlessinger, 2002). Dimerization of the receptors for vascular endothelial growth factor (VEGF) and nerve growth factor (NGF) is mediated by preformed dimeric ligands (Wiesmann et al., 1997; Wiesmann et al., 1999). In FGFR, heparin drives dimerization of FGF ligand and the receptor (Pellegrini et al., 2000; Schlessinger et al., 2000). In contrast, dimerization of EGFR (epidermal growth factor receptor) is through the receptor itself, independent of direct ligand-ligand interactions (Cho and Leahy, 2002; Ferguson et al., 2003; Garrett et al., 2002; Ogiso et al., 2002). The EGF binding site in the receptor is distinct from the receptor dimer interface but the binding of EGF induces a conformational change of the receptor to expose the dimerization loop, thus enabling the receptor to dimerize (Schlessinger, 2002).
The present invention includes NK1-based polypeptides comprising the amino acids of any of SEQ ID NO: 8-53, 58, or 59 or SEQ ID NO: 8-53, 58, or 59 with one or more conservative amino acid substitutions, wherein the polypeptide modulates Met activity as compared to wild type NK1; and derivatives thereof. Further, the present invention includes an NK1-based polypeptide comprising the amino acids of any of SEQ ID NO: 8-13, 20-36, 58, and 59 wherein the polypeptide is a Met antagonist; and derivatives thereof. Also, the present invention includes an NK1-based polypeptide comprising amino acids of any of SEQ ID NO: 54 or 55, or SEQ ID NO: 54 or 55 with one or more conservative amino acid substitutions; and derivatives thereof.
In another embodiment, the present inventions include: (1) an NK1-based polypeptide comprising amino acids of SEQ ID NO: 1 wherein the amino acid at one of wild type NK1 amino acid positions 77, 82, 83, 85, 122, 123, 124, 126, 127, 134, 137, 139, 140, 141, and 142 is substituted with an amino acid not normally found at that position in SEQ ID NO: 1, and wherein the polypeptide decreases Met activity as compared to wild type NK1, and derivatives thereof, (2) an NK1-based polypeptide comprising amino acids of SEQ ID NO: 1 wherein the amino acid at two of wild type NK1 amino acid positions 77, 82, 83, 85, 122, 123, 124, 126, 134, 137, 139, 141 and 142 is substituted with an amino acid not normally found at that position in SEQ ID NO: 1, and wherein the polypeptide decreases Met activity as compared to wild type NK1, and derivatives thereof, and (3) an NK1-based polypeptide comprising amino acids of SEQ ID NO: 1 wherein the amino acid at three of wild type NK1 amino acid positions 77, 83, 85, 122, 123, 124, 126, 127, 134, 137, 139, 140, 141, and 142 is substituted with an amino acid not normally found at that position in SEQ ID NO: 1, and wherein the polypeptide decreases Met activity as compared to wild type NK1, and derivatives thereof.
Also included is a composition comprising any of the above-described polypeptides and a pharmaceutically acceptable diluent or carrier.
A method of the present invention includes reducing Met activity in a cell comprising introducing the above-described polypeptides to the cell, e.g., a tumor cell. Another method includes increasing Met activity in a cell by introducing to the cell an NK1-based polypeptide comprising amino acids of any of SEQ ID NO: 54 or 55, or SEQ ID NO: 54 or 55 with one or more conservative amino acid substitutions; and a derivative thereof.
A further method includes designing an antagonist by converting a growth factor from a receptor tyrosine kinase agonist to an antagonist, wherein the native receptor dimerizes or oligomerizes to become active, comprising the steps: identifying a region of said growth factor that promotes said dimerization or oligomerization of said receptor; providing growth factor mutants in which at least one amino acid in said region of said growth factor that promotes said dimerization or oligomerization has been substituted with another amino acid; screening said mutants to determine receptor dimerization or oligomerization activity and to determine the binding affinity of the mutants to the receptor, and selecting one or more mutants that have a decreased ability as compared to wild type growth factor to promote dimerization or oligomerization of the receptor but retain the ability to bind the receptor. The receptor tyrosine kinase of interest may be, for example, Met, epidermal growth factor receptor (EGFR), nerve growth factor receptor (NGFR), vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR), stem cell factor receptor, and macrophage-stimulating protein receptor (RON). With this method, a proximity assay may be used to determine receptor dimerization or oligomerization activity or to determine the binding affinity of said mutants to the receptor.
The present inventions also include a polynucleotide coding for any one of the above-described polypeptides or derivatives; an expression vector comprising such polynucleotide or derivative operably linked to a promoter; and a host cell carrying the vector.
An additional method includes treating a patient in need of control of cell growth, cell proliferation, cell survival, or cell motility, said method comprising administering to said patient a therapeutically effective amount of one of the polypeptides or derivatives described above. Also, one method includes treating a patient in need of anti-cancer therapy, said method comprising administering to said patient a therapeutically effective amount of one of the above-described polypeptides or derivatives which acts as a Met antagonist.
A kit is also provided herein and includes a therapeutic dose of bioactive agent for use in treating a patient, comprising: a container; and a volume of material stored within the container in substantially sterile form; wherein the material comprises any one of the above-described polypeptides or a derivative thereof.
A further method of the present invention includes treating an abnormal cellular proliferation condition associated with a region of tissue in a living patient, comprising: delivering a therapeutically effective amount of a material to the region of tissue; inhibiting the abnormal cellular proliferation condition with the therapeutic amount of the material; wherein the material comprises any one of the above-described polypeptides or derivatives which acts as a Met antagonist. Alternatively, with this method, the region of tissue comprises a tumor, and the abnormal cellular proliferation condition comprises cancer, and further; the therapeutic amount of the material is delivered to the tumor; and proliferation of the cancer is inhibited with the therapeutic dose of the material.
The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings, certain embodiment(s) which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown. Drawings are not necessary to scale. Certain features of the invention may be exaggerated in scale or shown in schematic form in the interest of clarity and conciseness.
This application claims the benefit of the following provisional applications: Ser. No. 60/893,032 filed Mar. 5, 2007, entitled A MECHANISTIC BASIS FOR CONVERTING A GROWTH-FACTOR RECEPTOR AGONIST TO AN ANTAGONIST; Ser. No. 60/969,637 filed Sep. 2, 2007, entitled NK-1 BASED POLYPEPTIDES AND RELATED METHODS; and Ser. No. 60/981,952 filed Oct. 23, 2007, entitled NK-1 BASED POLYPEPTIDES AND RELATED METHODS, the entire contents of which are incorporated herein in their entirety.
Before the subject invention is described further, it is to be understood that the invention is not limited to the particular embodiments of the invention described below, as variations of the particular embodiments may be made and still fall within the scope of the appended claims. It is also to be understood that the terminology employed is for the purpose of describing particular embodiments, and is not intended to be limiting. Instead, the scope of the present invention will be established by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
All references, patents, patent publications, articles, and databases, referred to in this application are incorporated herein by reference in their entirety, as if each were specifically and individually incorporated herein by reference. Such patents, patent publications, articles, and databases are incorporated for the purpose of describing and disclosing the subject components of the invention that are described in those patents, patent publications, articles, and databases, which components might be used in connection with the presently described invention.
The information provided below is not admitted to be prior art to the present invention, but is provided solely to assist the understanding of the reader.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, embodiments, and advantages of the invention will be apparent from the description and drawings, and from the claims. The preferred embodiments of the present invention may be understood more readily by reference to the following detailed description of the specific embodiments and the Examples and Sequence Listing included hereafter.
For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the subsections that follow.
The text file filed concurrently with this application, titled “VAN67P392.txt” contains material identified as SEQ ID NO: 1-59, which material is incorporated herein by reference. This text file was created on Mar. 5, 2008, and is 109,775 bytes.
Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization described below are those well known and commonly employed in the art. Standard techniques are used for nucleic acid and peptide synthesis. Generally, enzymatic reactions and purification steps are performed according to the manufacturer's specifications. The techniques and procedures are generally performed according to conventional methods in the art and various general references (see generally, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., which is incorporated herein by reference), which are provided throughout this document. Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods, devices and materials are now described.
In this specification and the appended claims, the singular forms “a,” “an” and “the” include plural reference unless the context clearly dictates otherwise.
The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogues and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, alpha-carboxyglutamate, and O-phosphoserine. Amino acid analogues refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an alpha carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group (e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium). Such analogues have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
The term “cell” is used in its usual biological sense, and does not refer to an entire multicellular organism. The cell can, for example, be in vitro, e.g., in cell culture, or present in a multicellular organism, including, e.g., birds, plants and mammals such as humans, cows, sheep, apes, monkeys, swine, dogs, and cats. The cell can be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalian or plant cell).
A polypeptide that “modulates” Met activity either activates (promotes, enhances, increases) or inhibits (suppresses, blocks, decreases) Met activity and such modulation is identified by in vitro or in vivo assays of this activity or downstream activities, some of which assays are described hereinbelow.
“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogues or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogues include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses variants thereof (e.g., degenerate codon substitutions that encode the same amino acids) and complementary sequences, as well as the sequence explicitly indicated. The term “nucleic acid” is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.
The term “operably linked” refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences.
“Patient” refers to a mammal, preferably a human, in need of treatment for a condition, disorder or disease.
The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. These terms also include amino acid polymers in which one or more amino acid residue is an analogue or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
By “treatment” or “treating” it will be understood that this refers to any administration of a polypeptide intended to alleviate the severity of a disease being treated, to provide relief from the symptoms of the disease or to prevent or slow down the development of the disease in an individual with a disease condition or at risk of developing the disease condition.
By “therapeutically effective amount” is meant a dose that produces effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); and Pickar, Dosage Calculations (1999)).
The term “tumor cell” refers to a cancerous, pre-cancerous or transformed cell, either in vivo, ex vivo, and in tissue culture, that has spontaneous or induced phenotypic changes that do not necessarily involve the uptake of new genetic material. Although transformation can arise from infection with a transforming virus and incorporation of new genomic nucleic acid, or uptake of exogenous nucleic acid, it can also arise spontaneously or following exposure to a carcinogen, thereby mutating an endogenous gene. A “tumor” includes at least one tumor cell.
Here disclosed is a mechanistic basis for designing Met antagonists based on NK1, a natural variant of HGF containing the N-terminal and the first kringle domain of HGF. Through detailed biochemical and structural analyses, it was determined that both mouse and human NK1 induce Met dimerization via a conserved NK1-NK1 dimer interface. Both the mouse and human NK1 are able to bind and induce Met dimerization, and novel crystal structures of these two proteins reveal a conserved NK1-NK1 dimer interface. Mutations in the NK1-NK1 dimer interface abolish its ability to promote Met dimerization but retain full Met-binding activity. Importantly, by introducing mutations in the NK1 dimer interface it was found that the Met binding capability of NK1 can be separated from its ability to promote Met dimerization and activation. These NK1 dimer-interface mutants retain full Met binding ability, but inhibit HGF-mediated Met activation. Further, the disclosed NK1 mutants decrease or reduce Met activity (i.e., they act as Met antagonists) by inhibiting HGF-mediated cell scattering, proliferation, branching and invasion.
While not being bound by any particular theory, one plausible mechanism for NK1 to induce Met dimerization is that dimerization of Met is mediated by the NK1-NK1 dimer interface, in a manner similar to receptor/ligand complexes of VEGF and NGF (
The above mechanism of Met activation by HGF via NK1 dimerization provides a rational basis for designing an NK1-based Met antagonist by selectively disrupting the ability of NK1 to dimerize (
The NK1 mutants shown in
Additional mutants of the NK1 dimer interface are included in Table 1 (SEQ ID NO. 20-36). Mutations in the N domain (N), the linker region (L), kringle 1 (K1), and in combinations thereof (C) are shown in Table 1. These NK1-based polypeptides are Met antagonists, having reduced Met activity, as demonstrated by a MDCK colony scatter assay (See, Example 7 below).
With each mutant in Table 1, the residue has been changed to an alanine, however, it is contemplated that any substituted amino acid not normally found at the position of the altered residue would be acceptable. Such NK1 antagonists of Met are N77X; F82X:T83X:K85X; N77X:F82X:T83X:K85X; K122X; R126X; D123X:Y124X; D123X:N127X; D123X:Y124X:N127X; V140X:T142X; T139X:S141X:T142X; K137X:T139X:S141X:I142X; K137X:T139X:V140X:I142X; F82X:T83X:K85X:D123X:N127X; D123X:K137X; D123X:K137X:T139X:V140X:I142X; D123X:K137X:T139X:S141X:I142X; D123X:N127X:K137X:T139X:V140X:I142×, where “X” is any substituted amino acid not normally found at the indicated position (SEQ ID NO: 37-53).
One exception is the V140R mutant (SEQ ID NO: 54), which actually has an increased ability to promote Met dimerization over the wild type NK1 (
The NK1 based Met antagonists described here have several distinct advantages to known methods of Met inhibition. First, NK1 is a naturally circulated HGF variant that targets the extracellular domain of Met. Second, NK1 is a much smaller protein than an antibody, decoy Met receptor, or single chain HGF and NK4. Third, because of its small size, NK1 can be easily produced in large quantities and high purity. Fourth, the potency of the disclosed NK1-based antagonists is currently within one order of magnitude of HGF and approaching the potency of small molecules or antibodies. Finally, the disclosed antagonists are based on endogenous proteins with small variations, while antibodies are exogenous and may be antigenic.
The present invention includes methods for modulating Met activity in a Met expressing cell as compared to the Met activity from wild type NK1. As described hereinabove, most of the disclosed NK1 mutants are antagonists of Met activity, e.g., the polypeptides of amino acid sequences 8-11. That is, these polypeptides successfully bind Met in competition with wild type NK1, fail to induce Met dimerization, and inhibit Met activity (as shown in the Examples herein). Similarly, the method for modulating Met activity in a Met expressing cell also includes use of an NK1 mutant, or derivative thereof, that is an agonist of Met activity, e.g., the polypeptide of amino acid SEQ ID NO: 54. The method of modulating Met activity includes introducing one of the novel NK1 polypeptides, or a derivative thereof, into a Met-expressing cell. For example, as described below, in one embodiment a polynucleotide encoding one of the novel NK1 polypeptides, or a derivative thereof, is incorporated into a recombinant replicable vector that is used to replicate the polynucleotide in the Met-expressing cell.
Conservative Amino Acid Substitutions of the NK1 Mutants:
In one aspect, the present invention also include a polypeptide having an amino acid sequence of any of SEQ ID NO: 8-59, which polypeptide includes one or more “conservative amino acid substitutions”. Such polypeptide may differ from the original sequence such that it has 90%, 95%, or 98% identity with the amino acid sequence of SEQ ID NO: 8-59. It is well known in the art that the amino acids within the same conservative group can typically substitute for one another without substantially affecting the function of a protein.
Conservative substitution tables providing functionally similar amino acids are well known in the art (see for example Table 2). For example, one exemplary guideline to select conservative substitutions includes (original residue followed by exemplary substitution): ala/gly or ser; arg/lys; asn/gln or his; asp/glu; cys/ser; gln/asn; gly/asp; gly/ala or pro; his/asn or gln; ile/leu or val; leu/ile or val; lys/arg or gln or glu; met/leu or tyr or ile; phe/met or leu or tyr; ser/thr; thr/ser; trp/tyr; tyr/trp or phe; val/ile or leu. An alternative exemplary guideline uses the following six groups, each containing amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (I); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); (see also, e.g., Creighton, Proteins, W. H. Freeman and Company (1984); Schultz and Schimer, Principles of Protein Structure, Springer-Verlag (1979)).
One of skill in the art will appreciate that the above-identified substitutions are not the only possible conservative substitutions. For example, for some purposes, one may regard all charged amino acids as conservative substitutions for each other whether they are positive or negative. In addition, individual substitutions, deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence can also be considered “conservative amino acid substitutions” as long as the polypeptide retains its desired properties, i.e., with a conservative substitution in an NK1-based polypeptide antagonist of Met, the polypeptide will bind to Met and inhibit Met activity.
For a detailed description of protein chemistry and structure, see Schulz, G E et al., Principles of Protein Structure, Springer-Verlag, New York, 1978, and Creighton, T. E., Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, 1983, which are hereby incorporated by reference. The types of substitutions which may be made in the protein molecule may be based on analysis of the frequencies of amino acid changes between a homologous protein of different species.
Conservative substitution groups are set forth in Table 2 based on shared properties.
Various conservative substitutions are envisioned to be within the scope of the invention. For instance, it would be within the level of skill in the art to perform amino acid substitutions using known protocols of recombinant gene technology including PCR, gene cloning, site-directed mutagenesis of cDNA, transfection of host cells, and in-vitro transcription. The variants can then be screened for functional activity. Most acceptable deletions, insertions and substitutions according to the present invention are those which do not produce radical changes in the characteristics of the protein in terms of its desired Met binding and its inhibition of Met activity. However, when it is difficult to predict the exact effect of the substitution, deletion or insertion in advance of doing so, one skilled in the art will appreciate that the effect can be evaluated by routine screening assays such as those described here, without requiring undue experimentation.
Derivatives of NK1
A “derivative” of any one of the novel NK1 polypeptides includes additional chemical moieties not normally a part of the protein. Covalent modifications of the protein are included within the scope of this invention. Such modifications may be introduced into the molecule by reacting targeted amino acid residues with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. Such chemically modified and derivatized moieties may improve the protein's solubility, absorption, biological half life, binding affinity, and the like. These changes may eliminate or attenuate undesirable side effects of the protein in vivo. Moieties capable of mediating such effects are disclosed, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton Pa. (Gennaro 18th ed. 1990).
Cysteinyl residues most commonly are reacted with *alpha.-haloacetates (and corresponding amines) to give carboxymethyl or carboxyarmdomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, *alpha.-bromo-.beta.-(5-imidozoyl-)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.
Histidyl residues are derivatized by reaction with diethylprocarbonate (pH 5.5-7.0) which agent is relatively specific for the histidyl side chain. p-bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.
Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents reverses the charge of the lysinyl residues. Other suitable reagents for derivatizing .alpha.-amino-containing residues include imidoesters such as methylpicolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea, 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.
Arginyl residues are modified by reaction with one or several conventional reagents, including phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Such derivatization requires that the reaction be performed in alkaline conditions because of the high pK.sub.a of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine .epsilon.-amino group.
Modification of tyrosyl residues has permits introduction of spectral labels into a protein or peptide. This is accomplished by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizol and tetranitromethane are used to create O-acetyl tyrosyl species and 3-nitro derivatives, respectively.
Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R′—N—C—N—R′) such as 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide.
Aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions. Conversely, glutaminyl and asparaginyl residues may be deamidated to the corresponding glutamyl and aspartyl residues. Deamidation can be performed under mildly acidic conditions. Either form of these residues falls within the scope of this invention.
Derivatization with bifunctional agents is useful for cross-linking the polypeptide to a water-insoluble support matrix or other macromolecular carrier. Commonly used cross-linking agents include 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane.
Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization.
Other chemical modifications include hydroxylation of proline and lysine, phosphoiylation of the hydroxyl groups of seryl or threonyl residues, methylation of the .alpha.-amino groups of lysine, arginine and histidine side chains (T. E. Creighton, Proteins: Structure and Molecule Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminal amine, and, in some instances, amidation of the C-terminal carboxyl group.
Pharmaceutical Compositions and Kits:
In another embodiment, the present invention is a pharmaceutical composition of one of the disclosed NK1-based polypeptides, the NK1-based polypeptides with conservative amino acid substitutions, and derivatives thereof. In said composition, the polypeptide is dissolved in a pharmaceutically acceptable carrier or diluent, preferably an aqueous carrier. A variety of aqueous carriers or diluents can be used. These solutions are preferably sterile and generally free of undesirable matter. These compositions may be sterilized by conventional, well known sterilization techniques.
Pharmaceutically acceptable carriers can contain a physiologically acceptable compound that acts, e.g., to stabilize the composition or to increase or decrease the absorption of the agent and/or pharmaceutical composition. Physiologically acceptable compounds can include, for example, carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of the adjunctive and cancer agents, or excipients or other stabilizers and/or buffers. Detergents can also used to stabilize the composition or to increase or decrease the absorption of the pharmaceutical composition.
Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives which are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known and include, for example, phenol and ascorbic acid. One skilled in the art would appreciate that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound depends, for example, on the route of administration of the adjunctive anti-cancer agent and on the particular physio-chemical characteristics of the adjunctive anti-cancer agent.
The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of active agent in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs.
The pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration. For example, unit dosage forms suitable for oral administration include, but are not limited to, powder, tablets, pills, capsules and lozenges. It is recognized that polypeptides, when administered orally, may be protected from digestion. This is typically accomplished either by complexing the molecules with a composition to render them resistant to acidic and enzymatic hydrolysis, or by packaging the molecules in an appropriately resistant carrier, such as a liposome or a protection barrier. Means of protecting agents from digestion are well known in the art.
Also within the scope described herein is a kit for providing a therapeutically effective amount of a bioactive agent for use in treating a patient, comprising a container, a volume of material stored within the container in substantially sterile form; wherein the material comprises the pharmaceutical of one of the present NK1-based polypeptides, the NK1-based polypeptides with conservative amino acid substitutions, or derivatives thereof. The kit can further contain at least one additional reagent.
Polynucleotides, Vectors, and Host Cells:
A polynucleotide of the present invention is one which encodes a polypeptide of the invention as defined above. This includes DNA and RNA polynucleotides. A polynucleotide of the invention may be single or double stranded. Generally, a polynucleotide according to the present invention is provided as an isolate, in isolated and/or purified form, or free or substantially free of material with which it is naturally associated, such as free or substantially free of nucleic acid flanking the gene in the human genome, except possibly one or more regulatory sequence(s) for expression.
Sequences encoding all or part of the polypeptides of the invention and/or its regulatory elements can be readily prepared by the skilled person using the information and references contained herein and techniques known in the art (for example, see Sambrook, Fritsch and Maniatis, “Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989, and Ausubel et al, Short Protocols in Molecular Biology, John Wiley and Sons, 1992). These techniques include the use of site directed mutagenesis of nucleic acid encoding NK1. A polypeptide according to the present invention may be isolated and/or purified (e.g. using an antibody) for instance after production by expression from encoding nucleic acid (for which see below). Polypeptides according to the present invention may also be generated wholly or partly by chemical synthesis, for example in a step-wise manner. The isolated and/or purified polypeptide may be used in formulation of a composition, which may include at least one additional component, for example a pharmaceutical composition including a pharmaceutically acceptable excipient, vehicle or carrier. A composition including a polypeptide according to the invention may be used in prophylactic and/or therapeutic treatment.
Polynucleotides of the invention can be incorporated into a recombinant replicable vector. The vector may be used to replicate the nucleic acid in a compatible host cell. Thus in a further embodiment, the invention provides a method of making polynucleotides of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector. The vector may be recovered from the host cell. Suitable host cells are described below in connection with expression vectors.
Preferably, a polynucleotide of the invention in a vector is operably linked to a control sequence which is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector.
Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral e.g. ‘phage phagemid or baculoviral, cosmids, YACs, BACs, or PACs as appropriate. Vectors include gene therapy vectors, for example vectors based on adenovirus, adeno-associated virus, retrovirus (such as HIV or MLV) or alpha virus vectors.
The vectors may be provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter. The vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian vector. Vectors may be used in vitro, for example for the production of RNA or used to transfect or transform a host cell. The vector may also be adapted to be used in vivo, for example in methods of gene therapy. Systems for cloning and expression of a polypeptide in a variety of different host cells are well known.
Suitable host cells include bacteria, eukaryotic cells such as mammalian and yeast, and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, COS cells and many others.
Promoters and other expression regulation signals may be selected to be compatible with the host cell for which the expression vector is designed. For example, yeast promoters include S. cerevisiae GAL4 and ADH promoters, S. pombe nmt1 and adh promoter. Mammalian promoters include the metallothionein promoter which is can be included in response to heavy metals such as cadmium. Viral promoters such as the SV40 large T antigen promoter or adenovirus promoters may also be used. All these promoters are readily available in the art.
The vectors may include other sequences such as promoters or enhancers to drive the expression of the inserted nucleic acid, nucleic acid sequences so that the polypeptide is produced as a fusion and/or nucleic acid encoding secretion signals so that the polypeptide produced in the host cell is secreted from the cell.
Vectors for production of polypeptides of the invention of for use in gene therapy include vectors which carry a mini-gene sequence of the invention.
Vectors may be introduced into a suitable host cell as described above to provide for expression of a polypeptide of the invention. Thus, in a further aspect the invention provides a process for preparing polypeptides according to the invention which comprises cultivating a host cell carrying an expression vector as described above under conditions to provide for expression by the vector of a coding sequence encoding the polypeptides, and recovering the expressed polypeptides. Polypeptides may also be expressed in in-vitro systems, such as reticulocyte lysate.
A further embodiment of the invention provides host cells carrying the vectors for the replication and expression of polynucleotides of the invention. The cells will be chosen to be compatible with the said vector and may for example be bacterial, yeast, insect or mammalian.
The introduction of vectors into a host cell may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells (which may include cells actually transformed although more likely the cells will be descendants of the transformed cells) under conditions for expression of the gene, so that the encoded polypeptide is produced. If the polypeptide is expressed coupled to an appropriate signal leader peptide it may be secreted from the cell into the culture medium. Following production by expression, a polypeptide may be isolated and/or purified from the host cell and/or culture medium, as the case may be, and subsequently used as desired, e.g. in the formulation of a composition which may include one or more additional components, such as a pharmaceutical composition which includes one or more pharmaceutically acceptable excipients, vehicles or carriers.
A further aspect of the present invention provides a host cell containing nucleic acid as disclosed herein. The polynucleotides and vectors of the invention may be integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences which promote recombination with the genome, in accordance with standard techniques. The nucleic acid may be on an extra-chromosomal vector within the cell.
Methods of Treatment:
The polypeptides and polynucleotides of the present invention can be used in a method for treating Met-related conditions or diseases. Such treatment will be directed to treating a patient in need of controlling cell growth, cell proliferation, cell survival, or cell motility.
Polypeptides may be administered in any suitable form, for example in a pharmaceutical composition such as water, saline, dextrose, glycerol, ethanol and the like. Compositions may be formulated for injection, for example for direct injection to the site of intended treatment or intravenous injection.
Suitable doses or amounts of polypeptides will ultimately be at the discretion of the physician taking account of the nature of the condition to be treated and the condition of the patient. In general, dosage ranges will be 1 Âμg to 1 mg per kg body weight. The polypeptides may be administered by any suitable route, e.g. by i.v. or i.p injection, or directly to the site of treatment.
The polypeptides of the invention are useful for and can be delivered alone or as pharmaceutical compositions by any means known in the art, e.g., systemically, regionally, or, locally; by intraarterial, intratumoral, intravenous (IV), parenteral, intrapleural cavity, topical, oral, or local administration, as subcutaneous, intra-tracheal (e.g., by aerosol) or transmucosal (e.g., buccal, bladder, vaginal, uterine, rectal, nasal mucosa), intratumoral (e.g., transdermal application or local injection). Particularly preferred modes of administration include intraarterial injections, especially when it is desired to have a “regional effect,” e.g., to focus on a specific organ (e.g., brain, liver, spleen, lungs). For example, intra-hepatic artery injection is preferred if the anti-tumor regional effect is desired in the liver; or, intra-carotid artery injection, where it is desired to deliver a composition to the brain (e.g., for treatment of brain tumors), a carotid artery or an-artery of the carotid system of arteries (e.g., occipital artery, auricular artery, temporal artery, cerebral artery, maxillary artery, etc.).
Methods of Converting an Agonist to an Antagonist:
The ability to design specific mutations in the NK1 dimer interface that selectively disrupts NK1's ability to induce Met dimerization but retains its Met binding activity provides a mechanistic basis for designing Met antagonists for therapeutic applications. In order to serve as an RTK agonist, growth factors like HGF must have at least two functions, namely receptor binding and receptor activation. Thus, a mutated growth factor with selective disruption of its receptor activation ability but not its receptor binding may function as an RTK antagonist. Since activation of RTK is proposed to be mediated through a conserved mechanism of receptor dimerization or oligomerization, the disclosed method for design of NK1-based Met antagonists also provides a new concept for designing antagonists of other ligand-activated tyrosine kinase receptors. That is, the ability to separate Met binding of NK1 from its Met activation function has implications for antagonist design of other growth factor-activated tyrosine kinase receptors, i.e., by selectively abolishing the receptor activation ability but not the receptor binding of the growth factor. The methods disclosed herein can be used for designing an RTK antagonist (i.e., converting an RTK agonist to an RTK antagonist) for any RTK that is activated by receptor dimerization or oligomerization.
The family of ligand-activated receptor tyrosine kinases has a wide spread role in tumorigenesis and metastasis (Blume-Jensen and Hunter, 2001). The disclosed mechanistic-based design of Met antagonists also has important implications for other growth factor/tyrosine kinase receptor systems. RTKs active in such growth factor-RTK systems include epidermal growth factor receptor (EGFR), nerve growth factor receptor (NGFR), vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR), stem cell factor receptor, and macrophage-stimulating protein receptor (RON).
Like Met, VEGF, NGF and EGF receptors are activated through growth factor-induced receptor dimerization. To activate their receptors, NGF and VEGF contain separate receptor binding and dimerization surfaces (Wiesmann et al., 1997; Wiesmann et al., 1999). Based on the work disclosed herein with NK1 and Met, one can design VEGF-based and NGF-based antagonists by separating their receptor binding activity from their receptor dimerization activity. In the case of EGF, ligand binding induces conformational changes of the receptor that mediates direct receptor/receptor dimerization (Cho and Leahy, 2002; Ferguson et al., 2003; Garrett et al., 2002; Ogiso et al., 2002). A mutated EGF may induce a distinct conformational change of the receptor that is incompatible with receptor dimerization and activation. Given the relative small size of VEGF and EGF, such growth factor-based antagonists provide an exciting alternative to the current antibody-based approach.
Thus, the present invention also includes a method for designing an antagonist by converting a growth factor from a receptor tyrosine kinase agonist to an antagonist, wherein the native receptor dimerizes or oligomerizes to become active. The experimental design and methods utilized and set forth in the Examples below provide one means for performing such method. Steps for carrying out this method include: (1) identifying the region of the growth factor that promotes dimerization or oligomerization of the receptor (e.g., through crystallographic analysis); (2) providing mutants wherein at least one amino acid in the region of the growth factor that promotes said dimerization or oligomerization has been substituted with another amino acid; (3) screening these mutants to determine receptor dimerization or oligomerization activity and to determine the binding affinity of the mutants to the receptor; and (4) selecting the mutants that have a decreased ability to promote dimerization or oligomerization of the receptor but retain the ability to bind the receptor. More specifically, as described in the Examples, a proximity assay such as an AlphaScreen assay (Perkin Elmer, Waltham, Mass.), may be used to screen for and determine receptor dimerization or oligomerization activity or to determine the binding affinity of said mutants to the receptor. Further, as shown in the Examples, the binding affinity of the mutants to the receptor can be assayed through competition experiments with the wild type ligand.
Having now generally described the invention, the same will be more readily understood through reference to the following examples, which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified.
Production of NK1 Proteins
The human HGF NK1 (residues 28-209) was expressed as a 6×His-thioredoxin fusion protein from the expression vector pET-Duet1 (Novagen). The fusion protein contains a His6-Tag (MEHHHHHHMS) at the N terminus and a thrombin protease site between thioredoxin and NK1. Protein was expressed in the E. coli strain Rosetta/gami(DE) (Novagen) to promote disulfide bond formation. Bacterial cells transformed with the expression plasmid were grown in LB broth to an OD550 of ˜0.5 to 1.0 and induced with 100 μM isopropyl β-D-1-thiogalactopyranoside (IPTG) and 0.1% lactose for overnight at 22° C. Cells were harvested, resuspended in 200 ml extract buffer (495 mM NaCl, 25 mM Tris-HCl, pH 8.0, 5 mM imidazole, and 1 mg/ml lysozyme) per 12 liters of cells, and sonicated. The lysate was centrifuged at 20,000 rpm for 30 min, and the supernatant was loaded on to a 50 ml Ni-NTA agarose column. The column was washed with 500 ml extract buffer and eluted with buffer containing 25 mM Tris-HCl, pH8.0, and 500 mM imidazole. The eluted protein was cleaved overnight with thrombin at a protease/protein ratio of 1:1000 in the cold room and applied to a 5 ml HiTrap heparin HP column (Amersham Biosciences). The column was eluted with a gradient of 0.1 to 1.2 M NaCl, 25 mM Tris-HCl pH 8.0, where the 6×His-thioredoxin tag was in the flow through and NK1 was eluted at 800-1000 mM NaCl. Protein used for crystallization was further purified with gel filtration and cat ion exchange columns. Mouse NK1 and human NK1 mutants were purified with the same protocols.
Crystallization, Data Collection, and Structure Determination
For crystallization, proteins were concentrated to approximately 10 mg/ml in 25 mM Tris-HCl pH 8.0 and 100 mM NaCl. Crystals were grown in hanging drops containing equal volumes of protein and well solution. Crystallization conditions for human HGF/NK1 were 50 mM ammonium sulfate, 26-29% PEG 4000 (w/w), 100 mM Tris-HCl pH 8.0, 0.5 mM 0-octyl glucoside (βOG), and 5% ethylene glycol while crystallization conditions for mouse NK1 were 50 mM ammonium sulfate, 26-32% PEG 1000, 50 mM Tris-HCl pH 8.0, and 5% ethylene glycol. Crystals grew over weeks to months to a size of several hundred microns.
Data were collected with a MAR 225 CCD detector at 5-ID (DND-CAT) of the Advance Photon Source (APS) located at Argonne National Laboratory. Crystals were flash frozen in liquid nitrogen after a quick wash in cryoprotectant solution containing 50 mM ammonium sulfate, 29% PEG 4000 or 1000 (w/w) (human or mouse NK1 respectively), 100 mM Tris-HCl pH 8.0, 0.5 or 0 mM βOG, 100 mM NaCl, and 15% ethylene glycol. Data was integrated and scaled with HKL2000 (Otwinowski and Minor, 1997). The human NK1 crystallized in space group P21, a=49.40 Å, b=51.81 Å, c=73.17 Å, and β=107.87°, and the mouse NK1 in P63 with a=b=86.49 Å, c=104.45 Å. The structure was solved by molecular replacement using the PDB coordinates 1NK1. Molecular replacement and model refinement were performed with CNS, where twin fraction was incorporated for the refinement for the mouse structure (Brunger et al., 1998). Manual model building was done with the program 0 (Jones et al., 1991).
Production of Biotinylated NK1
The biotinylated NK1 was produced by fusing the 20 amino acid biotin acceptor peptide sequence from the pDW464 plasmid (Duffy et al., 1998) to the N-terminus of NK1 in the pET-Duet1 vector containing the His6-thioredoxin-NK1 fusion protein. This plasmid was coexpressed with the E. coli biotin holoenzyme synthetase (BirA) from a pACYC-Duet1 vector (Novagen). The biotinylated NK1 was purified and digested with thrombin as for native NK1, and was further purified with a monomeric avidin column (Pierce).
Production of the Met Extracellular Domain
The Met protein (residues 25-567, containing the sema domain and the cysteine-rich domain) was expressed as a C-terminal hexahistidine tag fusion protein from Lec 3.2.8.1 cells (Gherardi et al., 2003). Cells were grown in a 1:1 mixture of alpha-MEM and DMEM media with 2.5% FBS. Media was harvested every 5 days and concentrated prior to loading on a 50 ml nickel column (Qiagen) equilibrated with 25 mM Tris-HCl pH 7.5, 25 mM imidazole, 150 mM NaCl, and 10% glycerol. The column was washed with equilibrated buffer above until the absorbance at 280 nm reached baseline and then the protein was eluted with 175 mM imidazole, 25 mM Tris-HCl pH 7.5, and 10% glycerol. The eluted protein was dialyzed with 50 mM citrate pH 6.0, 100 mM NaCl, and 10% glycerol, and treated with endoglycosidase Hf (2.5 units per 1 μg of Met) were added at room temperature for 24 hours. Deglycosylated Met was loaded onto a 5 ml HiTrap blue HP column (Amersham Biosciences) and the column was washed with 5 to 10 column volumes of 20 mM Tris-HCl pH 8.0 and 200 mM NaCl. Met was eluted with 20 mM Tris-HCl pH 8.0 and 800 mM NaCl. Prior to use in AlphaScreen assays the protein was concentrated and the buffer exchanged with 25 mM Tris-HCl pH 8.0 and 100 mM NaCl in Amicon ultra centrifugal concentrators.
AlphaScreen Assays for NK1-Met Binding and Met Dimerization
The binding of NK1 to Met was determined by AlphaScreen assays using a hexahistidine detection kit from Perkin-Elmer. The experiment was performed using 10 nM biotin-NK1 and 6his-Met in the presence of 5 μg/ml donor and acceptor beads in a buffer containing 50 mM MOPS, pH7.4, 50 mM NaF, 0.05 mM CHAPS, and 0.1 mg/ml bovine serum albumin. Competition experiments were performed with 0.01 to 2.5 μM untagged ligand in the presence of 1 μM heparin and IC50 values for NK1-Met interaction were determined from a nonlinear least square fit of the data. All experiments were duplicated or triplicated with standard errors typically less than 10% of the measurements. The Met dimerization assays were prepared using 40 nM of 6his-Met in the presence of 5 μg/ml nickel chelate donor and acceptor beads (Perkin-Elmer). The effect of Met ligands and heparin on Met dimerization was determined using 1 μM untagged ligand and 1 μM heparin.
MDCK Cell Scatter Assay
HGF, wild type and mutated NK1, and heparin were used in the assay at final concentrations 60 ng/mL, 1 M, and 2 μM, respectively. The NK1 wt and mutants, heparin, or DMEM containing 5% FBS, 1% penicillin/streptomycin, and 1% L-glutamine were added to the appropriate wells of a 96-well plate. MDCK cells were diluted in the above media to 0.83×105 cell/mL and seeded into the 96-well plate at 0.083×105 cell/well. The cells were mixed by gently rocking the plate and incubated 10 min at 37° C., 5% CO2. After the incubation, HGF was added to the appropriate wells of the plate and incubated at 37° C., 5% CO2 overnight. The cells were fixed and stained using 0.5% crystal violet in 50% ethanol at room temperature for 10 min, washed with water, and allowed to dry prior to taking photos.
uPA-Plasmingen Assay
MDCK cells were seeded in a 96-well plate at 1500 cells per well and incubated overnight at 37° C., 5% CO2 in DMEM containing 10% FBS and 1% penicillin/streptomycin. HGF, NK1 wt and mutant, and heparin were used in the assay at final concentrations of 60 ng/mL, 1 μM, and 2 μM, respectively. NK1 wt or mutant and heparin were added to the cells and incubated 15 min at 37° C., 5% CO2. HGF was then added to the appropriate wells and incubated 24 hr at 37° C., 5% CO2. The plates were processed by first washing wells twice with phenol-red free DMEM, then adding 200 uL of reaction buffer, containing 50% (v/v) 0.05 units/mL plasminogen (Roche) in phenol-red free DMEM, 40% (v/v) 50 mM Tris buffer (pH 8.2), and 10% (v/v) 3 mM Chromozyme PL (Roche) in 100 mM glycine solution, to each well. The plates were incubated 4 hr at 37° C., 5% CO2, after which time the absorbance of each well was read using an automated spectrophotometric plate reader at 405 nm.
Thymidine Incorporation Assays
The highly proliferative sub-clone of MDCK cells were distributed into 96-well plates at 2×103 cells per well with DMEM supplemented with 10% FBS for 24 h. The cells were starved in DMEM without FBS for 24 h and treated with Heparin alone or Heparin in combination with wild type NK1 or various NK1 mutants for 1 h. Then the cells were further incubated with or without HGF/SF for 12 h. One microcurie of [3H]thymidine (Amersham) was added 4 h before analysis. Cells were washed with PBS and [3H]thymidine incorporation was measured by precipitation of whole cells with chilled 10% trichloroacetic acid, solubilization of precipitates with lysis buffer (0.02 M NaOH/0.1% SDS), suspension in 150 μl of scintillation mixture (Packard Bioscience) and measurement by a liquid scintillation counter (MicroBeta TriLux, PerkinElmer).
Branching Morphogenesis Assays
Branching morphogenesis in 3D Matrigel matrix was analyzed as follows: cells at a density of 50,000 cells/ml in DMEM supplemented with 10% FBS medium were mixed with an equal volume of Matrigel (Becton Dickinson), plated at 100 μl per well on a 96-well culture plate, and incubated for 30 min at 37° C. with 5% CO2 to allow gel formation. Cells were treated with Heparin alone or Heparin in combination with wild type NK1 or various NK1 mutants for 1 h. Growth medium with or without 100 ng/ml of human HGF/SF was introduced into each well. The cells were photographed at different time points.
To understand the detailed biochemical mechanisms of Met binding and activation by NK1, mouse and human NK1, the human NK1 mutant R134G, and the first 567 residues of the Met extracellular domain, which contains the sema and cystine-rich domains, were expressed and purified (
To determine the functional activity of the above purified proteins, the direct interactions of Met with the biotinylated NK1 were measured using AlphaScreen assays as illustrated in
To determine the binding affinities of the various NK1s for Met, competition experiments were performed using unlabeled NK1 proteins (
To probe the mechanism of Met activation by NK1, we designed a Met dimerization assay based on AlphaScreen technology (
The AlphaScreen is a proximity assay which can generate signals arising from Met dimerization or oligomerization. To determine the nature of the Met dimer/oligomer, we performed dynamic light scattering analysis to determine the hydraulic diameter of the Met/NK1 complex. In this assay, we used NK1 as our positive control since it has been shown to form a dimer in a heparin dependent manner (Chirgadze et al, 1998). In the absence of heparin, dynamic light scattering revealed that NK1 forms a mono-dispersed monomer with a hydraulic diameter of 54 Å (
To investigate the molecular mechanism by which NK1 induces Met dimerization, the crystal structure of mouse NK1 at a 2.4 Å resolution and a high resolution structure of human NK1 (1.8 Å) were determined for structural comparison. The statistics for the crystallographic data and refined structures are summarized in Table 3.
The head-to-tail NK1 dimer is formed by the intertwined packing between the N-domain and the C-terminal kringle domain of the two monomers that bury 2200 A2 of solvent accessible area. The interactions between the two monomers are summarized in
The mouse NK1 is 90.0% identical in sequence to the human NK1 (
To determine the role of the NK1 dimer configuration in the binding and activation of Met, four key residues (Y124, K85, D123, and N127) that form the dimer interface were mutated. These mutations were made in the human R134G background to facilitate purification. Since the dimer interface is far away from the heparin binding site located in the N domain (
Also, the Met binding activity of these NK1 mutants was measured by the same competition experiments as first shown in
To determine whether the above NK1 dimerization mutants can function as Met antagonists, we first performed protease assays for the urokinase-type plasminogen activator (uPA) since it is induced by HGF-mediated Met activation in MDCK cells and various cancer cell lines (Xie et al., 2005). In MDCK cells, uPA activity is consistently elevated by 4-5 folds by treatment with HGF (
Since Met activation by HGF also induces cell proliferation, we used thymidine incorporation to measure HGF-stimulated DNA synthesis. As shown in
HGF-mediated Met activation also affects cell morphological behavior e.g. cell scattering, branching, and invasion, thus we tested whether the NK1 mutants can inhibit the above HGF-mediated effects. As shown in
Materials and Methods.
Mutagenesis and Cloning—Mutagenesis of the full length and NK 1 fragments was performed in a site directed manner, using PCR-based techniques. Selection of mutant clones from unmutated parental DNA strands was achieved via methylation sensitive restriction digestion with DpnI. Mutants were confirmed by DNA sequencing before being sub-cloned into either pA71d or pPIC9K (Invitrogen) for expression in mammalian cells or in the yeast P. pastoris, respectively.
Transfections—Wild type and mutant full length HGF/SF proteins were expressed transiently in the mouse neuroblastoma Neuro2A cells using Lipofectamine 2000 (Invitrogen). Concentration of expressed proteins within the supernatants was measured using a sandwich enzyme-linked immunoassay (R&D Systems, Cat # DY294). Stable transfectants in the mouse NS0 myeloma cell line were selected using Hygromycin B at 0.75 mg/ml. Clones expressing the protein of interest were identified by a slot blot assay using a polyclonal sheep anti-human HGF/SF antibody (1W53) and expanded for protein production.
Protein purification—Expression and purification of the NK1 mutants were carried out as described by others (Chiragiadze, D Y, Nat Struct, Biol. 6, 72-9 (1999); Lietha, D, Embo J., 20, 5543-55 (2001). Purification of the wild type and mutant full length HGF/SF proteins was carried from the supernatants of the relevant transfectants using Heparin-Sepharose (Cat # 17-0998-01; Amersham Biosciences) followed by Mono-S (Cat # 17-0547-01; Amersham Biosciences) chromatography. Proteins were stored in 0.05M MES, 1 M NaCl pH 6.0.
Surface plasmon resonance—SPR was carried out as described in Holmes (J. Mol. Bio. 367, 395-408 (2007)) except that PBS-EP rather than HBS-EP was used as the running buffer and 5 M NaCl was used for chip regeneration. Briefly, a CM5 chip was coated with MET567 by amine coupling chemistry. Ligand was then flowed through the cell at various concentrations to allow calculation of the dissociation equilibrium (KD).
Small-angle X-ray scattering (SAXS) data collection and processing—Synchrotron X-ray scattering data were collected at the EMBL X33 beamline (DESY, Hamburg) 28 using a MAR345 image plate detector. The scattering patterns of all samples were measured at several solute concentrations ranging from 0.5 to 7.0 mg/ml. At a sample-detector distance of 2.7 m, the range of momentum transfer 0.1<<5 nm-1 was covered (s=4π sin(θ)/λ, where 2θ is the scattering angle and λ=0.15 nm is the X-ray wavelength). The primary data processing and evaluation of the overall structural parameters were performed using standard procedures by the program package PRIMUS 29. The molecular masses (MM) of the solutes were evaluated by scaling against reference solutions of bovine serum albumin. Particle shapes at low resolution were reconstructed by a multiphase bead modeling program MONSA (Svergun, Biophys J, 76, 2879-86 (1999). Two distinct phases were used to highlight the individual components (NK1 and MET567) in the complex. The modeling of the monomeric complex was constructed by simultaneous fitting of three scattering curves (free MET567, free LC and the 1:1 complex). In the case of the dimeric NK1-MET567 complex, the scattering curves from wild type NK1 and the complex were fitted and the modeling was constrained by P2 symmetry. Multiple MONSA runs yielded superimposable solutions and typical models are presented below.
Met Binding Assay—This was carried out using a competition assay using NUNC F96 Maxisorp 96-well plates (Cat # 442404) coated overnight with 50 ml/well of a 100 nM of a construct corresponding to the full length ectodomain (MET928) 21. Wells were blocked using a Reagent diluent (R&D Systems, Cat # DY995) before being incubated with mixtures of HGF/SF mutant proteins and 1×10-10M biotinylated wild type full length HGF/SF. Bound, biotinylated HGF/SF was detected using horseradish peroxidase-conjugated Streptavidin (DAKO Cat # P0397).
Western Blots—Western blots of ERK activation were performed as in Holmes (2007). Briefly, MK cells were treated with ligand for 5 mins, lysed and the level of ERK activation was measured by probing blots with antibodies against total and phosphorylated ERK1/2.
Cell motility assays—Madin-Darby canine kidney (MDCK) cells were used to seed 6-well tissue culture plates (Falcon) at a density of 2,500 cells per well. Cells were then incubated in DMEM (Gibco)+5% FCS (Gibco) at 37° C./5% CO2 for 48 hours and next treated with test proteins in DMEM+5% FCS and incubated overnight. Photographs were taken using a Hamamatsu camera with a phase contrast lens at 100 times magnification.
DNA synthesis assay—DNA synthesis assays were carried out as in Holmes (2007). Briefly, mouse keratinocyte (MK) cells were grown to confluence and then treated with the various ligands in the presence of 3[H]-methylthymidine. Cells were then lysed and mixed with scintillant to measure levels of incorporation of the radoactive isotype into DNA.
Results.
Several crystal structures of NK1 have consistently yielded a dimer arranged in a head to tail orientation (Chirgadze, D. Y. Nat Struct Biol, 6, 72-9 (1999); Lietha, D., Embo J., 20, 5543-55 (2001); Ultsch, M., Structure, 6, 1383-93 (1998); Watanabe, K, J. Mol. Bio, 319, 283-88 (2002). The dimer interface buries in excess of 2,000 A2 and consists of a central area in which the two inter-domain linkers (K122, D123, Y124, I125, R126 and N127) make extensive contacts and two adjacent areas in which residues between the N and K1 domains of different protomers make a number of further contacts.
An extensive number of individual or cluster alanine mutations have been introduced in the N and K1 domains and the inter-domain linker (
Wild type and mutant full length HGF/SF proteins were produced initially in Neuro2a cells by transient transfection and characterised for expression level, MET binding and MET activation. Eight mutants were not expressed well and were not characterised extensively. Data from the remaining 29 mutants are shown in
NK1 versions of the above mutants were expressed in the yeast P. pastoris and purified to homogeneity. The single linker mutants LC (Y 124A) and LE (N127A) bound MET with affinities indistinguishable from wild type NK1 in surface plasmon resonance experiments (1.83×10-7M for NK1, 1.49×10-7 M for the LC mutant and 0.83×10-7 M for the LE mutant) (
The stoichiometry of complexes formed by NK 1 mutants at the dimer interface with MET567, a fragment of MET ectodomain carrying the ligand-binding-propeller domain (Gherardi, E. et al., 100, 12039-44 (2003)), was investigated by small angle X-ray scattering (SAXS).
Dmax, nm
Rg, MM and Dmax are, respectively, radius of gyration, molecular mass and maximum size calculated from the scattering data. χ is the overall discrepancy between the experimental data and computed curves from the ab initio models.
The ability of the NK 1 mutants to induce migration of MDCK cells or Erk1/2 phosphorylation and DNA synthesis in MK cells was studied next. The LC mutant displayed limited activity on MDCK cells at the highest concentration tested (
Finally, the NK1 mutants were assayed for receptor antagonistic activity.
Point mutations were introduced at the crystallographic dimer interface following the hypothesis that such mutations would disrupt dimerization, and hence activity, without affecting receptor binding. The single linker mutations Y124A and N127A or the double mutation V140A:I142A in the K1 domain had the phenotype hypothesized. The single linker mutation D123A and complex mutations at the dimer interface, however, also affected receptor binding. This result was unexpected and is discussed first.
The evidence that opposite faces of the NK1 protomer are responsible for receptor binding and dimer formation has a strong foundation in crystallographic (Chirgadze, D. Y. Nat Struct Biol, 6, 72-9 (1999); Lietha, D., Embo J., 20, 5543-55 (2001); Ultsch, M., Structure, 6, 1383-93 (1998); Watanabe, K, J. Mol. Bio, 319, 283-88 (2002) and mutagenesis studies (Hartmann, G [published erratum appears in Curr Biol 1998 Oct. 8; 8(20):R739], Curr Biol, 8, 125-34 (1998); Lokker, N. A., Protein Eng, 7, 895-903 (1994)). Without being bound by any particular theory, it is believed that the interdomain linker is flexible (Watanabe, K, J. Mol. Bio, 319, 283-88 (2002); Gherardi, E. et al. Proc Natl Acad Sci USA 103, 4046-51 (2006)) and the first 3 amino acids (K 122, D123, Y 124) form a 3/10 helix. Conceivably, the linker mutation D123A may disrupt the 3/10 helix and thus the rotation and orientation of the N and K1 domains affecting indirectly, receptor binding.
While the foregoing specification has been described with regard to certain preferred embodiments, and many details have been set forth for the purpose of illustration, it will be apparent to those skilled in the art that the invention may be subject to various modifications and additional embodiments, and that certain of the details described herein can be varied considerably without departing from the spirit and scope of the invention. Such modifications, equivalent variations and additional embodiments are also intended to fall within the scope of the appended claims.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
| Number | Date | Country | |
|---|---|---|---|
| 60893032 | Mar 2007 | US | |
| 60969637 | Sep 2007 | US | |
| 60981952 | Oct 2007 | US |
