TW201639879A - Insulin-like growth factor 2 (IGF2) signaling and modulation - Google Patents

Abstract

The present invention resides in the discovery that the specific interaction between insulin-like growth factor 2 (IGF2) and integrin is involved in integrin-mediated cellular signaling, such as enhanced proliferation of cells expressing integrin, especially integrin [alpha]v[beta]3. Thus, this invention provides for a novel method for inhibiting integrin signaling by using an inhibitor of IGF1-integrin binding. A method for identifying inhibitors of IGF1-integrin binding is also described. Further disclosed are polypeptides, nucleic acids, and corresponding compositions for inhibiting integrin signaling.

Description

Communication and regulation of insulin-like growth factor 2 (IGF2) Statement of Rights to the Research and Development Funded by the Federal Government

The present invention was completed by the National Institutes of Health with the authorization number CA13015 with the assistance of the government. The government has certain powers over the invention.

Related application

The present application claims priority to U.S. Provisional Patent Application No. 62/104,608, filed on Jan. 16, s.

The present invention relates to the signaling and regulation of insulin-like growth factor 2 (IGF2).

Insulin-like growth factor 1 (IGF1) and insulin-like growth factor 2 (IGF2) are two polypeptide endosomes with high structural similarity (75-kD). They also share a substantial degree of structure with human proinsulin Similarity.

The combinatorial protein is a family of cell adhesion receptors that regulate cell-extracellular matrix interactions with cell-cell interactions. Ligand binding affinity signaling (internal to external communication) from the intracellular regulatory complex protein has been proposed. Each of the combination proteins is a heterodimer containing alpha and beta subunits. Eighteen alpha and eight beta subunits have been identified that bind to form 24 combinatorial proteins.

Combination proteins have been reported to play an important role in cancer proliferation and aggressiveness. For example, a high concentration of the combination protein [alpha]v[beta]3 has been reported to be involved in the growth and/or progression of melanoma, neuroblastoma, breast cancer, colorectal cancer, ovarian cancer, and cervical cancer.

IGF1 has been implicated in cancer progression. One of the main functions of IGF1 is to inhibit apoptosis. IGF1 is resistant to chemotherapy and radiation therapy. IGF1 is shown to increase in breast, lung, prostate, and many other cancers. It is known that during IGF1 signaling, IGF1 binds to both the combination protein and its receptor IGF1R, and the combinatorial protein binding-deficient mutant of IGF1 is shown to be a dominant negative inhibitor of IGF1R-mediated IGF1 signaling.

A known role of IGF2 during pregnancy is growth promoting endocrine. In humans, the IGF2 gene is located on chromosome 11p15.5, and the gene accession number and amino acid sequence of the human IGF2 mRNA sequence are NM_000612 and NP_000603, respectively.

Both insulin receptor (IR) and type 1 insulin-like growth factor receptor (IGF1R) are members of the tyrosine kinase class of membrane receptors [2]. There are two splice variant isomers in IR; "B" isomer (IR-B) Only insulin is recognized, but the "A" isomer (IR-A) recognizes both insulin and insulin-like growth factor 2 (IGF2) [3]. Heterodimers are formed by half IR and half IGF1R, and these are well known heterozygous receptors [3, 4]. Ligands of IR or IGF1R that bind to the extracellular domain result in activation of the cytoplasmic domain tyrosine kinase. This leads to phosphorylation of the IR receptor (IRS) family of membrane proteins, and activation of PI3K, AKT and various downstream reaction networks [5]. IR-A is the major IR isomer and is found in a variety of cancers, including breast, large intestine and lung cancers. Abnormal autocrine or paracrine manifestations of ligands (especially IGF2) are common in many cancers [6], and the emergence of IR-A/IGF2 circuits represents an "addiction" to IR/IGF1R activation. . In cells with a high IR-A:IGF1R ratio, IGF2 produced by autocrine stimulation stimulates cell growth by IR-A stimulation. Blocking IGF2 or IR significantly inhibited growth, confirming the association of IR-A/IGF2 loops in cancer. In contrast, IR-B is mainly expressed in the liver, also in muscle, adipose tissue and kidney, and only binds to insulin. Since blocking IR-B will affect normal glucose metabolism in these tissues, blocking IR-B should be used with caution as a treatment.

Since IR-A is the therapeutic target of cancer, efforts are made to create IGF2 mutants that bind to IR-A instead of IR-B in order to achieve potential therapeutics by inhibiting tumorigenesis for the purpose of regulating cell signaling in cancer cells. benefit. Unfortunately, currently available kinase inhibitors for IR or IGF1R are unable to resolve IGF1R, IR-A or IR-B, whereas anti-IGF1R antibodies are unable to block IR-A or IR-B. When a dominant negative IGF1 mutant has been used as an inhibitor of IGF1R (IGF1 decoy), then an IGF2 mutant is produced. There is great interest (such as a dominant negative mutant similar to IGF1 decoy, which is defective in its ability to bind to a combinatorial protein, but still binds to IGF1R and IR-A) to further investigate IGF2 signaling and explore therapeutic uses for this type of inhibitor. Feasibility (for example, for the treatment of conditions involving inappropriate cell proliferation including various cancer formations).

The present invention provides novel methods and compositions for inhibiting IGF2 signaling in cells based on the discovery that IGF2 interacts with specific combinatorial protein molecules to participate in the regulation of IGF2 regulation. Thus, in one aspect, the invention relates to a method for inhibiting IGF2 signaling in a cell comprising the step of contacting a cell with an inhibitor of an effective amount of an IGF2-binding protein.

In certain embodiments, the combinatorial protein is αvβ3. In certain embodiments, the combinatorial protein is alpha 5 beta 1 or alpha 6 beta 4 . In certain embodiments, the inhibitor is an IGF2 mutant comprising a disubstituted R37E and R38E in an amino acid sequence of a wild-type IGF2 protein (eg, SEQ ID NO: 1). In certain embodiments, the inhibitor is the IGF2 mutant R24E/R37E/R38E, or the IGF2 mutant R34E/R37E/R38E, or the IGF2 mutant R24E/R34E/R37E/R38E. In certain embodiments, the cells are located within the body of the patient. In certain embodiments, the contacting step is performed orally. In certain embodiments, the contacting step is performed intravenously, subcutaneously, intraperitoneally, or intratumorally. For example, administration can be by using a pump similar to an insulin pump (a medication for therapeutic agents in a continuous subcutaneous infusion therapy). The device of the treatment device is carried out.

As recognized, excessive IGF2 signaling may result in undesired cellular responses, such as abnormal cell proliferation and inflammatory responses, which may instead cause or promote a variety of diseases and conditions, such as proliferative diseases, including, for example, melanoma, neuroblasts. Cancer of cell tumor, breast cancer, colon cancer, ovarian cancer and cervical cancer; inflammatory diseases such as joint diseases, rheumatoid arthritis and osteoarthritis; autoimmune diseases such as ankylosing spondylitis, autoimmune grapes Inflammation, multiple sclerosis, autoimmune diabetes, and rheumatoid arthritis; osteoporosis; angiogenesis (related to inflammation and cancer, and other diseases involved in hypervascularization); The various types of conditions of fibrous tissue formation (eg, fibrosis).

In a second aspect, the invention relates to a method for identifying an inhibitor of IGF2-binding protein binding. The method comprises the steps of: (1) contacting a combination protein with a polypeptide comprising a binding protein binding sequence of IGF2 under conditions which permit binding of the IGF2-combined protein in the presence of a test compound; and (2) detecting the polypeptide-combined protein The degree of binding, wherein the degree of binding decreases when compared to the extent of binding of the untested compound, indicating that the compound is an inhibitor of IGF2-binding protein binding.

In certain embodiments, the combinatorial protein is αvβ3. In other embodiments, the combinatorial protein is alpha 5 beta 1 or alpha 6 beta 4 . In certain embodiments, the polypeptide comprises the sequence of the human IGF2 protein (eg, SEQ ID NO: 2) C domain. In certain embodiments, the polypeptide comprises the full length of a human IGF2 protein (eg, SEQ ID NO: 1). In certain embodiments, the polypeptide is further A heterologous amino acid sequence, such as glutathione sulfur transferase (GST), is included. In certain embodiments, the polypeptide further comprises pegylation (polyethylene glycol (PEG) such as one or more residues (eg, one or more Arg residues at positions 24, 34, 37, and 38) A modification of the covalent attachment or combination of polymer chains which can be PEGylated or PEGylated by substitution with other amino acids such as Lys. PEGylation can be carried out on amino acids, including lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine . In addition, the N-terminal amine group and the C-terminal carboxylic acid may also be used (directly or via a functional group) as a site for PEGylation. In certain embodiments, the combinatorial protein is expressed on the cell surface.

In a third aspect, the invention relates to an isolated polypeptide comprising an amino acid sequence having the following characteristics: (1) and a sequence of a naturally occurring wild-type IGF2 protein (such as a wild-type human IGF2 protein) having at least 95% sequence Consistency; (2) at least two Arg residues at positions 37 and 38 of the wild-type human IGF2 protein contain substitutions; and (3) inhibition of IGF2-combined protein binding. The invention also relates to an isolated nucleic acid encoding the polypeptide, and a recombinant expression cassette comprising the nucleic acid or an isolated host cell comprising the recombinant expression cassette.

In certain embodiments, the combinatorial protein is αvβ3. In other embodiments, the combinatorial protein is alpha 5 beta 1 or alpha 6 beta 4 . In certain embodiments, at least three of the Arg residues at positions 24, 34, 37, and 38 are substituted. In certain embodiments, the Arg residues at positions 24, 34, 37, and 38 are substituted. In certain embodiments, each Arg residue is substituted with a Glu residue.

In a fourth aspect, the invention relates to a composition comprising: (A) a physiologically acceptable excipient, and (B) a polypeptide comprising an amino acid sequence of the following characteristics: (1) and naturally occurring The sequence of the wild-type IGF2 protein (especially the wild-type human IGF2 protein) has at least 95% sequence identity; (2) at least two Arg residues at positions 37 and 38 of the wild-type human IGF2 protein comprise a substitution; and (3) ) inhibits IGF2-binding protein binding. The invention also relates to a composition of a nucleic acid encoding a polypeptide having a pharmaceutically acceptable excipient as described above. Such compositions have various diseases and conditions for treating excessive IGF2 signaling resulting in undesired cell proliferation and inflammatory responses including, but not limited to, the conditions set forth above.

In certain embodiments, the polypeptide is the IGF2 mutant R24E/R37E/R38E, or the IGF2 mutant R34E/R37E/R38E, or the IGF2 mutant R24E/R34E/R37E/R38E. In certain embodiments, the polypeptide is PEGylated as described in other paragraphs.

In a fifth aspect, the invention relates to a kit for inhibiting IGF2 signaling comprising a composition of a polypeptide or nucleic acid having a pharmaceutically acceptable excipient as described above. Other types of instructions or user information are usually included in the kit. The kits of the present invention are useful for treating various diseases and conditions associated with excessive IGF2 signaling resulting in undesired cell proliferation and inflammatory responses including, but not limited to, the conditions set forth above.

Figure 1. Sequence and chain composition of IGF1, IGF2 and insulin [1]. IGF-specific C- and D-domains are gray and pink, respectively Color; insulin B and A chains in IGF1/2 and their equivalents are highlighted in yellow and blue, respectively. The important residues for IGF-1R or IR binding are red, while the reaction residues associated with IGFBP are green (the emphasized residue mutation results in at least a 90% decrease in binding; this substituted residue results in an italicized The affinity of the receptor and IGFBP has a higher impact). The amino acid sequences of IGF2, IGF1 and insulin are shown in Figure 1 (SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 4, respectively).

Figure 2 R36E/R37E of IGF1 inhibits cell viability and tumorigenesis, whereas WT IGF1 increases cell viability and tumorigenesis in mouse breast cancer Met-1 cells expressing WT IGF1 or R36E/R37E [10]. Above: Cell viability. Met-1 transfected cell lines were cultured in poly(polyethyl methacrylate) coated Petri dishes in DMEM for 48 h and cell viability was measured by MTS analysis (n=6). Bottom: Tumor formation in vivo. IGF1 (WT or mutant) stably secreted by Met-1 cells was injected into the breast fat pad of FVB mice (two mice per injection, 10 ⁵ cells each) without further screening. Tumor growth was monitored by measuring the size of the tumor using a caliper. Statistical analysis was performed on days 25-31 using t-test (n=10), *P<0.05.

Figure 3. IGF2 binds to the combinatorial protein [alpha]v[beta]3 and produces a combined protein binding defect mutant of IGF2. a) Soluble combinatorial protein αvβ3 binds to immobilized IGF2 in an ELISA-type binding assay. The wells of a 96-well microtiter plate are coated with IGF2 at increasing concentrations. The immobilized IGF2 was cultured in soluble recombinant αvβ3 (5 μg/ml) in Tyrode-HEPES buffer containing Mg ²⁺ . The bound αvβ3 line was measured with the anti-combined protein β3 mAb. b) CHO cells (β3-CHO cells) expressing αvβ3 are attached to IGF2, whereas those expressing αvβ1 (β1-CHO cells) are not. The wells of the 96-well microtiter plate were coated with IGF2 at increasing concentrations. 3-3-CHO cells or β1-CHO cells (10 ⁵ cells/well) were cultured in DMEM to be cultured with immobilized IGF2 and the bound cells were counted. c) and d) mutations in the predicted IGF2-combined protein binding surface inhibit the binding protein binding of IGF2. Based on the alignment of IGF1 and IGF2 (Fig. 1), several Arg residues of IGF2 were selected for mutagenesis. A combined mutation of several Arg residues effectively inhibits the binding protein binding of IGF2.

Figure 4. IGF2 mutants with combined protein binding defects are functionally defective and dominantly inactivated. a) Several combination protein-deficient IGF2 mutants are defective in increasing cell viability. As described, the survival rate of β3-CHO cells was measured by MTS analysis from polyHEMA-coated wells [10]. b) Excess IGF2 mutants inhibited cell viability by WT IGF2 (25 ng/ml).

Figure 5 IGF1 communication and related biological procedures.

Figure 6. IGF1 decoy affects the morphology and Oct-4 and Nanog expression in Met-1 mouse breast cancer cells. It has been reported that the performance of the IGF1 decoy inhibits tumorigenesis of Met-1 cells, while the performance of WT IGF1 is enhanced. a. Cell shape on the tissue culture plate. Met-1 cells expressing R36E/R37E (IGF1 decoy) have epithelial cell-like profiles different from those of cells expressing only WT IGF1. b. Western blot analysis of cell lysates. Met-1 cells expressing IGF1 decoy showed low Oct-4 and Nanog tables by Western blot analysis of cell lysates Now. Alpha tubulin is used as an internal reference. This means that the IGF1 decoy may inhibit reverse differentiation by endogenous IGF1 and/or IGF2.

Figure 7 Continuous infusion of IGF2 decoy using insulin pump technology. The insulin pump is a small device that is about the size of a small mobile phone that is worn outside. It delivers a precise insulin dose that is consistent with the body's needs. This technique can be used to infuse IGF1 or IGF2 decoys.

Figure 8 (A) Intentional map of micrograph analysis. The grid of BSACy5 is printed on an epoxy-coated glass cover slip (micro-touch printing). The gap is filled with streptavidin and biotinylated monoclonal antibodies against the membrane protein bait. The cells on this microbial wafer grow and the bait will be distributed in the serosa based on the antibody micrograph. Interaction with the second fluorescently labeled bait protein is explored by measuring the extent of the co-pattern. (B) HeLa cells transiently express GFP-IRS-3 and live cell images of anti-IR antibody functionalized coverslips. The IR-IRS-3 interaction is explored by a strong GFP IRS-3 co-pattern in the IR-rich microdomain.

Figure 9: Total reflection fluorescence (TIRF) images showing the co-assembly of β3 combination proteins by IGF-IR media. The HeLa cell line was transiently transfected with IGF-IR-RFP and β3 combinatorial protein GFP and grown on microbial wafers coated with anti-IGF-IR antibodies. Co-assembly of the β3 combinatorial protein GFP into the IGF-IR rich region indicates an interaction between the IGF-IR and β3 combination proteins.

The word "inhibition" as used herein means any detectable Negative effects of the target biological program detected, such as binding between IGF2 and the combination protein αvβ3, or its downstream procedures include IGF1 receptor (IGF1R) or insulin receptor type A (IR-A) phosphorylation, AKT and ERK1 /2 activation, and the possibility of cell proliferation, tumor formation and metastasis. Generally, when compared to the control group, the inhibition is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or higher of the IGF2-combined protein binding or the above A drop in any of the downstream parameters.

The term "nucleic acid" or "polynucleotide" means a single or double-stranded form of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and its polymer. Unless specifically limited, the term encompasses nucleic acids comprising known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to natural nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants (eg, degenerate codon substitutions), dual genes, homologous genes, SNPs and complementary sequences, as well as sequences explicitly indicated. In particular, degenerate codon substitution can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is substituted with a mixed base and/or a deoxyglycoside residue ( Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91 -98 (1994)). The term nucleic acid can be used interchangeably with genes, cDNA, and mRNA encoded by genes.

The term "gene" means a DNA fragment involved in the manufacture of a polypeptide chain. It may comprise a coding region between individual coding fragments (exons) Front and back (leader and tail) areas and insertion sequences (introns).

The term "amino acid" means naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that act in a manner similar to naturally occurring amino acids. The naturally occurring amino acids are encoded by the genetic code, and the amino acids are subsequently modified, such as hydroxyproline, gamma carboxy glutamic acid, and O phosphoryl phosphate. An amino acid analog means a compound having the same basic chemical structure as a naturally occurring amino acid, that is, an alpha carbon, a carboxyl group, an amine group, and an R group bonded to hydrogen, such as homoserine, nortylamamine. Acid, methionine sulfur oxide, methionine methyl sulfonium. Such analogs have a modified R-based acid (such as orthanoic acid) or a modified peptide backbone, but maintain the same basic structure as the naturally occurring amino acid. "Amino acid mimetic" means a chemical compound having a structure different from that of a general amino acid, but acts in a manner similar to a naturally occurring amino acid.

There are a number of known methods in the art that allow unnatural amino acid derivatives or analogs to be incorporated into a polypeptide chain in a position-specific manner, see, for example, WO 02/086075.

The amino acids herein may be represented by the commonly known three letter symbols or by the one-letter symbols suggested by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, a nucleotide can be represented by a generally accepted single word code.

"Conservatively modified variants" are applied to both amino acid and nucleic acid sequences. For a particular nucleic acid sequence, "conservatively modified variant" means a nucleic acid encoding the same or substantially the same amino acid sequence, or The nucleic acid of the same sequence does not encode an amino acid sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any particular protein. For example, the codons GCA, GCC, GCG, and GCU all encode alanine amino acids. Thus, for each position of the alanine specified by the codon, the codon can be exchanged for any corresponding codon that does not alter the encoded polypeptide. This nucleic acid variant is a "silent variation" which is one of the conservatively modified variants. Each nucleic acid sequence encoding a polypeptide herein also describes a silent variation of each of the possible nucleic acids. Those skilled in the art will appreciate that each codon in a nucleic acid (other than AUG, which is typically the only codon for methionine, and TGG, which is typically the only codon for tryptophan), can be modified to produce functionally identical Molecule. Thus, each silent variation of a nucleic acid encoding a polypeptide is implicit in each of the described sequences.

With regard to amino acid sequences, those skilled in the art will be aware of alterations, additions or deletions of individual substitutions or deletions of nucleic acids, peptides, polypeptides or protein sequences of a single amino acid or a small proportion of amino acid in the coding sequence ( The deletion or addition is a "conservatively modified variant" whose alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitutions provide functionally similar amino acids well known in the art. This conservative modification variant is, but not exclusive of, the polymorphic variants, interspecies homologs and dual genes of the invention.

The following 8 groups each comprise an amino acid which is conservatively substituted with each other: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) aspartame (N), glutamic acid (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L) , methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), sulphate (T); and 8) cysteine (C), methionine (M)

(See, for example, Creighton, Proteins, W. H. Freeman and Co., N. Y. (1984)).

The amino acids herein may be represented by the commonly known three letter symbols or by the one-letter symbols suggested by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, a nucleotide can be represented by a generally accepted single word code.

In the present specification, the amino acid residue is a relative position number based on the leftmost residue (numbered 1) of the unmodified wild type polypeptide sequence.

As used herein, it is described in the context that two or more polynucleotide or amino acid sequences are "identical" or "consistent" in proportion, meaning that when comparing and comparing maximal correspondences through a comparison window (comparison window) , or using one of the following sequence comparison algorithms or by manual alignment and visual measurement of a specified region, two or more sequences or subsequences are identical or the amino acid residues or nucleotides have the same specific ratio (eg, The core amino acid sequence responsive to IGF combinatorial protein binding is at least 80% identical to the reference sequence, preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, eg, the C domain sequence of the wild-type IGF2 protein). This sequence is called "substantially consistent." With regard to polynucleotide sequences, this definition also refers to the complementarity of the test sequences. Preferably, the identity occurs in a region of at least about 50 amino acids or nucleotides in length, or more preferably in the region of 75-100 amino acids or nucleotides in length.

For sequence comparison, typically a sequence is used as a reference sequence to compare the test sequence to it. When using the sequence comparison algorithm, the test and reference sequences are entered into the computer, if necessary, the subsequence coordinates are set, and then the sequence algorithm program parameters are set. You can use preset program parameters or set additional parameters. The sequence comparison algorithm then calculates the percentage of sequence identity of the test sequence relative to the reference sequence based on the program parameters. For sequence comparison of nucleic acids and proteins, the BLAST and BLAST 2.0 algorithms discussed below and preset parameters were used.

As used herein, a "comparison window" encompasses a segment of reference to any number of consecutive positions selected from the group consisting of from 20 to 600, typically from about 50 to 200, more typically from about 100 to 150, wherein in two sequences After ideal alignment, the sequences can be compared to a reference sequence of the same number of consecutive positions. Methods of algorithms for comparing sequences are well known in the art. Sequence ideal alignments for comparison can be performed, such as by Smith & Waterman's local homology algorithm, Adv. Appl. Math. 2: 482 (1981), by Needleman & Wunsch homology alignment calculus Method, J. Mol. Biol. 48: 443 (1970), by Pearson & Lipman's similarity search method, Proc. Nat'l. Acad. USA 85:2444 (1988), implemented by computerization of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or borrowed By manual alignment and visual inspection (see, for example, Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

Examples of algorithms suitable for determining percent and sequence similarity of sequence identity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., (1990) J. Mol. Biol. 215: 403-410 and Altschul, respectively. Et al. (1977) Nucleic Acids Res. 25: 3389-3402. Software for performing BLAST analyses is publicly available on the National Center for Biotechnology Information (ncbi.nlm.nih.gov). When compared to a letter of the same length in the database sequence, the algorithm includes identifying the high-sequence pair (HSP) by matching the short letter of length W in the query sequence, which matches or satisfies certain positive threshold scores. T. T means the adjacent letter score threshold (Altschul et al., supra). These initial pairs of adjacent letters are used as seeds to initiate a search to find longer HSPs containing the seeds. The hit letters are then extended at both ends along each sequence to maximize the cumulative alignment score as much as possible. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for matching residue pairs; must > 0) and N (penalty score for mismatched residues; must < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. When the following occurs, the letters extending in all directions are aborted: the accumulated alignment score falls to the number X of its maximum value; due to the accumulation of one or more negative residues Product, the cumulative score becomes 0 or lower; or the sequence reaches the terminal. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotides) uses a preset letter size (W) of 28, an expected value (E) of 10, M = 1, N = -2, and a comparison of the two strands. For amino acid sequences, the BLASTP program uses a default letter size (W) of 3, an expected value (E) of 10, and a BLOSUM62 score matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989). )).

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One similarity measure provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability of an accidental match between two nucleotide or amino acid sequences. For example, a nucleic acid is considered to be similar to a reference sequence if the minimum sum probability of the comparison test nucleotide to the reference nucleotide is less than about 0.2, more preferably less than about 0.01, and most preferably less than 0.001.

As described below, two nucleic acid sequences or polypeptides are substantially identical, meaning that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with an antibody raised against the polypeptide encoded by the second nucleic acid. Thus, the polypeptide is typically substantially identical to the second polypeptide, for example, the two peptides differ only in conservative substitutions. As described below, in addition, substantially identical two nucleic acid sequences means that two molecules or their complementary sequences are heterozygous for each other under stringent conditions. In addition, the fact that two nucleic acid sequences are substantially identical means that the same primer can be used to amplify the sequence.

"Polypeptide", "peptide" and "protein" are available in this article. Used interchangeably to mean a polymer of an amino acid residue. These three words for amino acid polymers, one of which naturally corresponds to the amino acid corresponding to the amino acid residue, and naturally occurring amino acid polymers and non-naturally occurring amino acids. Artificial chemical mimics of polymers. As used herein, the term encompasses any length of an amino acid chain, including full length proteins, wherein the amino acid residues are linked by a covalent peptide bond.

The term "effective amount" as used herein means the amount of therapeutic effect produced by the administered substance. Effects include prevention, correction or suppression of the symptoms of the disease/condition and progression of any detectable degree of associated complications. The exact amount will be judged according to the therapeutic purpose and by those skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding ( 1999); and Pickar, Dosage Calculations (1999)).

A "characteristic cassette" is a nucleic acid that is organized (recombined or synthesized) by allowing a particular polynucleotide sequence to be transcribed from a particular polynucleotide sequence in a host cell. The performance cassette can be part of a plastid, viral genome or nucleic acid fragment. Generally, the expression cassette includes a polynucleotide to be transcribed and is operably linked to a promoter.

As used herein, "polypeptide comprising an IGF2-combined protein binding region" means a polypeptide comprising a core amino acid sequence that generally corresponds to the amino acid sequence C domain of the wild-type IGF2 protein. The IGF2 amino acid sequence is shown in Figure 1 (SEQ ID NO: 1) and its C domain sequence is SRVSRRS (SEQ ID NO: 2). IGF1 and insulin amino acid sequence Also shown in Figure 1 (SEQ ID NO: 3 and SEQ ID NO: 4, respectively). The full length amino acid sequence of the pre-IGF2 protein is set forth in GenBank Accession No. NP_000603 or P01344 in the Swissprot protein library. The mature IGF2 protein amino acid sequence corresponds to the 25-91 segment of the pre-IGF2 protein sequence. This core amino acid sequence contains some variation, such as amino acid breaks, additions or substitutions, but the C domain sequence still maintains a general degree of sequence homology (eg, at least 80%, 85%, 90%, 95% or Higher sequence homology) and ability to bind to the combinatorial protein αvβ3. In addition to this core sequence in response to the ability of the polypeptide to bind to a combinatorial protein, the polypeptide may comprise one or more amino acid sequences of homologous origin (eg, an additional sequence of IGF2 from the same protein) or a heterologous origin (eg, from another Sequence of related proteins). Some examples of "polypeptides comprising an IGF2-combined protein binding site" include the C domain sequence of full-length wild-type IGF2. Alternatively, affinity or epitope calibration (such as GST calibration) can be included in the polypeptide to facilitate purification, isolation or immobilization of the polypeptide. The polypeptide may be further modified to enhance its ease of use, to improve stability and/or bioavailability by glycosylation, pegylation, or the like, or to incorporate one or more, as long as it retains the ability to bind to the combination protein αvβ3. A naturally occurring base acid (such as D-amino acid).

"Antibody" means a polypeptide substantially encoded by an immunoglobulin gene or a plurality of immunoglobulin genes or fragments thereof that specifically binds to and recognizes an analyte (antigen). The recognized immunoglobulin genes include the kappa, λ , α , γ , δ, ε, and mu constant regions, as well as a myriad of immunoglobulin variable region genes. Light chains are classified as either kappa or lambda . Heavy chains are classified as gamma , mu , alpha , delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively.

Exemplary immunoglobulin (antibody) structural units comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having a "light" (about 25 kD) and a "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids that is primarily used for antigen recognition. Terms variable light chain (V _L) and variable heavy chain (V _H) means the light and heavy chains, respectively.

The presence of antibodies, such as intact immunoglobulins or some well characterized fragments as produced by digestion with various peptidases. Thus, for example, pepsin digests the disulfide bond of an antibody in a hinge region to produce F(ab)' ₂ , a dimer of Fab, which itself is a light-incorporated _VH- C _H 1 by a disulfide bond. chain. Interrupting the disulfide bond in the hub under mild conditions reduces F(ab)' ₂ , thereby converting the F(ab)' ₂ dimer to a Fab' monomer. The Fab' monomer is essentially a Fab having a portion of a hinge region (see Paul (Ed.) Fundamental Immunology, Third Edition, Raven Press, NY (1993)). When various antibody fragments are defined in terms of digestion of intact antibodies, those skilled in the art will appreciate that the fragments can be synthesized de novo chemically or by utilizing recombinant DNA methods.

Further modification of antibodies by recombinant techniques is also well known in the art. For example, a chimeric antibody from an antigen binding region (variable region) of an antibody having an animal from one of the constant regions of other animal antibodies is bound. Generally, the antigen binding region is derived from a non-human animal and the constant region is derived from a human antibody. The presence of a human constant region reduces the likelihood that an antibody will be rejected by a human recipient as a foreign object. On the other hand, "humanized" antibodies Combines a smaller portion of a non-human antibody with a human component. Generally, humanized antibodies comprise highly variable regions, or complementarity determining regions (CDRs), of non-human antibodies grafted to the appropriate human antibody cassette region. The antigen binding site may be wild type or modified with one or more amino acid substitutions, such as modifications to be closer to human immunoglobulins. Chimeric and humanized antibodies can be accomplished using recombinant techniques well known in the art (see, for example, Jones et al. (1986) Nature 321 :522-525).

Thus, the term "antibody" as used herein also includes antibody fragments (such as single-chain Fv of chimeric or humanized antibodies) produced by modification of the entire antibody or by recombinant antibody synthesis using recombinant DNA methods.

I. Introduction

Efforts were made to establish dominant negative IGF2 mutants using strategies successfully used to establish dominant negative IGF1 mutants that retain the ability to bind IGFIR but attenuate the ability to bind to combinatorial proteins. IGF2 is structurally similar to IGF1, and the key IGF1 amino acid residues that bind the binding protein to IGF1 are retained in IGF2. The dominant-inactivated IGF2 mutant was investigated for its ability to inhibit IR-A along with IGF1R and to block the IR-A/IGF2 loop in cancer. In its preliminary study, the inventors have demonstrated that IGF2 binds to a combinatorial protein. They have also produced several IGF2 mutants that bind to defective proteins. These mutants have been shown to function in signaling and inhibit cell viability, i.e., dominant inactivation, increased by wild-type (WT) IGF2. Because these dominant negative IGF2 mutants or IGF2 decoys effectively inhibit IR-A (and IGF1R), they are used in cancer treatment as Therapeutic agents are useful.

II. Manufacture of polypeptides related to IGF2 A. General reorganization techniques

The present disclosure discloses general methods and techniques in the field of recombinant genetic material, including Sambrook and Russell, Molecular Cloning, A Laboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Ausubel et al. Eds., Current Protocols in Molecular Biology (1994).

For nucleic acids, the size is expressed in kilobases (kb) or base pairs (bp). These are estimates obtained from agarose or acrylamide gel electrophoresis, from sequencing nucleic acids, or from published DNA sequences. For proteins, the size is expressed in thousands of auricular (kDa) or amino acid residues. The protein size is estimated by gel electrophoresis, by sequencing the protein, by the resulting amino acid sequence, or by the published protein sequence.

Oligonucleotides which are not commercially available can be chemically synthesized, for example, according to the solid phase phosphite triester method, first proposed by Beaucage & Caruthers, Tetrahedron Lett. 22: 1859-1862 (1981), using an automatic pairing synthesizer As described in Van Devanter et. al., Nucleic Acids Res. 12: 6159-6168 (1984). Purification of the oligonucleotides is carried out using any art-recognized strategy, such as the native acrylamide gel electrophoresis or anion exchange HPLC described in Pearson & Reanier, J. Chrom. 255: 137-149 (1983). .

The sequence, polynucleotide encoding the IGF2 gene encodes a polypeptide comprising a combinatorial protein binding domain (i.e., C domain) of IGF2, and the double-stranded template strand is sequenced using, for example, Wallace et al., Gene 16:21-26 (1981). Synthetic oligonucleotides can be verified by termination or secondary selection.

Bw and IGF2 related polypeptide coding sequence

Polynucleotide sequences encoding wild-type IGF2 proteins have been identified, particularly wild-type human IGF2 proteins, and are available from commercial suppliers. For example, GenBank Accession Nos. of human IGF2 mRNA and protein sequences are NM_000612 and NP_000603, respectively.

Rapid advances in human genome research have led to the selection of human DNA sequence repositories for any gene segment with a certain proportion of sequence homology to a known nucleotide sequence, such as a human IGF previously defined. It becomes possible. Any DNA sequence thus identified can then be obtained by chemical synthesis and/or polymerase chain reaction (PCR) techniques, such as overlap extension. For short sequences, complete de novo synthesis is sufficient; and synthetic probes are used to further isolate full-length coding sequences from human cDNA or genome libraries to obtain larger genes.

Alternatively, a nucleic acid sequence encoding human IGF2 can be isolated from a human cDNA or genomic DNA library using standard selection techniques such as polymerase chain reaction (PCR), wherein homology-based primers are typically available from encoding IGF2. Nucleic acid. The most commonly used techniques for this purpose are described in the standard herein, such as Sambrook and Russell above.

Available in the market or can be configured to obtain human IGF A cDNA library of coding sequences. General methods for isolating mRNA, making cDNA by reverse transcription, binding cDNA to a recombinant vector, transfecting into a recombinant host for propagation, screening and colonization are well known (see, eg, Gubler and Hoffman, Gene, 25:263- 269 (1983); Ausubel et al., supra). The amplified nucleotide sequence segment is obtained by PCR, and this segment can be further used as a probe to isolate the full-length polynucleotide sequence encoded by the cDNA library IGF2. A general description of suitable procedures can be found in Sambrook and Russell as described above.

A similar procedure can be followed to obtain a full-length sequence encoding human IGF2 from a human genome. Human genomic libraries are commercially available or established according to various methods recognized in the art. In general, in order to establish a genomic library, DNA is first extracted from the tissue in which IGF2 may be found. The DNA is then mechanically sheared or enzymatically digested to produce fragments of about 12-20 kb in length. This fragment is then isolated from the undesired size of the polynucleotide fragment by gradient centrifugation and inserted into the vector of phage lambda. These vectors and phages are packaged in vitro. Recombinant phages were analyzed by plaque plaque assay as described in Benton and Davis, Science, 196: 180-182 (1977). Colony hybridization is performed as described in Grunstein et al., Proc. Natl. Acad. Sci. USA, 72: 3961-3965 (1975).

Based on sequence homology, degenerate oligonucleotides can be designed as primer sets and can be used under appropriate conditions (see, eg, White et al., PCR Protocols: Current Methods and Applications, 1993; Griffin and Griffin, PCR Technology, CRC Press). PCR under Inc. 1994) to amplify nucleosides from cDNA or genomic libraries A segment of the acid sequence. A full length nucleic acid encoding IGF2 is obtained using the amplified fragment as a probe.

In obtaining a nucleic acid sequence encoding IGF2, the coding sequence can be further modified by several known techniques, such as restriction enzymes, PCR, and PCR-related methods to generate coding sequences for IGF2-related polypeptides, including IGF2 mutants. (especially dominant negative type) and a polypeptide comprising a combinatorial protein binding sequence derived from IGF2. The polynucleotide sequence encoding the desired IGF2-related polypeptide can then be sub-selected into a vector, for example, a expression vector such that the recombinant polypeptide can be produced from the resulting construct. Further modifications to the coding sequence, such as nucleotide substitutions, can then be made to alter the properties of the polypeptide.

A variety of steps for generating mutations have been published and described in the art and can be readily used to modify polynucleotide sequences encoding polypeptides associated with IGF. See, e.g., Zhang et al., Proc. Natl. Acad. Sci. USA, 94: 4504-4509 (1997); and Stemmer, Nature, 370: 389-391 (1994). The programs can be used individually or in combination to produce sets of nucleic acids, and thus produce a plurality of encoded polypeptides. Kits for generating mutations, gene bank establishments, and other diverse production methods are commercially available.

Mutation methods that produce diversity include, for example, site-directed mutagenesis (Botstein and Shortle, Science, 229: 1193-1201 (1985)), mutagenesis using a template containing uracil (Kunkel, Proc. Natl. Acad. Sci. USA, 82: 488-492 (1985)), oligonucleotide site-directed mutagenesis (Zoller and Smith, Nucl. Acids Res., 10:6487-6500 (1982)), thiophosphorylation-modified DNA mutagenesis (Taylor et al., Nucl. Acids Res., 13:8749-8764 and 8765-8787 (1985)) and the use of nicked double-stranded DNA Mutagenesis (Kramer et al., Nucl. Acids Res., 12:9441-9456 (1984)).

Other possible methods for generating mutations include point-mismatched repair (Kramer et al., Cell, 38: 879-887 (1984)), mutagenesis using a defective host species (Carter et al., Nucl. Acids Res., 13 : 4431-4443 (1985)), disruptive mutagenesis (Eghtedarzadeh and Henikoff, Nucl. Acids Res., 14: 5115 (1986)), restriction screening and restriction purification (Wells et al., Phil. Trans.R) .Soc. Lond. A, 317: 415-423 (1986)), Mutagenesis by Whole Gene Synthesis (Nambiar et al., Science, 223: 1299-1301 (1984)), Double Strand Repair (Mandecki, Proc. Natl. Acad. Sci. USA, 83: 7177-7181 (1986)), mutagenesis by polynucleotide chain termination method (US Patent No. 5,965,408) and error-prone PCR (Leung et al., Biotechniques, 1:11-15 (1989)).

C. Nucleic acid modification of codon usage preferences in host organisms

The polynucleotide sequence encoding the polypeptide associated with IGF2 can be further altered to conform to the codon usage preferences of a particular host. For example, the codon usage preferences of bacterial cell varieties can be used to obtain polynucleotides encoding the recombinant polypeptides of the invention and including the codons of this variety. The frequency of codon usage expressed by the host cell can be maximized by the host cell The average frequency of codon usage preferences in gene expression is calculated (eg, the computing services provided by the Kazusa DNA Research Institute website in Japan). This analysis is preferably limited to genes that are highly expressed by the host cell.

Upon completion of the modification, the coding sequence is verified by sequencing and subsequently sub-selected to the appropriate expression vector for recombinant production of the polypeptide associated with the IGF.

D. Chemical synthesis of polypeptides associated with IGF2

The amino acid sequence of the combined protein binding site provided is derived from human IGF2, such as the C domain sequence of IGF2. Thus, polypeptides comprising such IGF2-combined protein binding sequences can also be chemically synthesized using conventional peptide synthesis or other procedures well known in the art.

It can be synthesized by solid phase peptide synthesis using a similar method to Merrifield et al., J. Am. Chem. Soc., 85: 2149-2156 (1963); Barany and Merrifield, Solid-Phase Peptide Synthesis, in The Peptides: Analysis, Synthesis, Biology Gross and Meienhofer (eds.), Academic Press, NY, vol. 2, pp. 3-284 (1980); and Stewart et al., Solid Phase Peptide Synthesis 2nd ed., Pierce Chem. Co. , The program described in Rockford, Ill. (1984) synthesizes a polypeptide. During the synthesis, the N-alpha-protected amino acid has a protective side chain which is gradually added to the growing polypeptide chain linked by its C-terminus and linked to a solid support, i.e., polystyrene beads. The peptide is synthesized by linking an amine group of an N-α-protected amino acid to an α-carboxy group of an N-α-protected amino acid, wherein the α-carboxy group is reacted with a reagent such as dicyclohexylene carbon Amine) is activated by reaction. Attachment of a free amine group to an activated carboxyl group results in the formation of a peptide bond. The most common use is an N-alpha-protecting group comprising Boc (acid labile) and Fmoc (base labile).

Those skilled in the art will appreciate that materials suitable as solid carriers include, but are not limited to, the following: halomethyl resins such as chloromethyl or bromomethyl resins; hydroxymethyl resins; phenolic resins such as 4-(alpha) -[2,4-Dimethoxyphenyl]-Fmoc-aminomethyl)phenoxy resin; tert-alkyloxycarbonyl-hydrazidated resin. Such resins are commercially available and are prepared by those skilled in the art.

Briefly, the C-terminal N-alpha-protected amino acid is first attached to a solid support. The N-α-protecting group is then removed. The deprotected alpha amine linkage is coupled to the activated alpha carboxyl group of the next N-alpha-protected amino acid. This procedure is repeated until the desired peptide is synthesized. The resulting peptide is then cleaved from the insoluble polymer carrier and the amino acid side chain deprotects. Longer peptides can be obtained by condensation of protected peptide fragments. Details of suitable chemicals, resins, protecting groups, protected amino acids, and reagents are well known in the art and will not be discussed in detail herein (see Atherton et al., Solid Phase Peptide Synthesis: A Practical Approach, IRL Press (1989) and Bodanszky, Peptide Chemistry, A Practical Textbook, 2nd Ed., Springer-Verlag (1993)).

III. Performance and purification of peptides associated with IGF2

After verifying the coding sequence, according to the code disclosed in this article The polynucleotide sequence of the peptide, the polypeptide of the invention associated with IGF2 can be produced using routine techniques of recombinant genes in the art.

A. Performance system

In order to obtain a high amount of the nucleic acid encoding the IGF2-related polypeptide of the present invention, a nucleotide encoding a typical coding polypeptide is subcultured into a expression vector containing a guide transcription, a transcription/translation terminator and a ribosome binding site. The powerful promoter of the point. Suitable bacterial promoters are known in the art and are described in Sambrook and Russell, supra, and Ausubel et al., supra. Bacterial expression systems for expressing polypeptides are available, for example, from E. coli, Bacillus sp., Salmonella, and Caulobacter. The kits used for this performance system are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are known in the art and are also commercially available. In one embodiment, the eukaryotic expression vector is an adenoviral vector, an adeno-associated viral vector, or a retroviral vector.

Promoters used to direct the expression of a heterologous nucleic acid depend on the particular application. The promoter is arbitrarily positioned relative to the heterologous transcription initiation site at the same distance from the transcription start site as in its natural environment. As is known in the art, some variation can be tolerated in this distance without loss of promoter function.

In addition to a promoter, a performance vector typically includes a transcription unit or a representational cassette comprising all of the additional elements required for expression of a polypeptide associated with IGF2 in a host cell. Thus, a typical performance cassette contains a promoter operably linked to a nucleic acid sequence encoding a polypeptide associated with IGF2, and efficient transcription, ribosome binding sites, and translation termination. The signal required for polyadenylation. A nucleic acid sequence encoding a polypeptide associated with IGF2 is typically ligated to a cleavable signal peptide sequence to facilitate secretion of the polypeptide by the transforming cell. This signal peptide includes juvenile hormone esterase from tissue plasminogen activator, insulin, and nerve growth factor, and Heliothis virescens. Additional elements of the cassette may include enhancers and, if the genomic DNA is used as a structural gene, an intron that functionally splicing the donor site and the acceptor site.

In addition to the promoter sequence, the performance cassette should also contain the downstream of the transcriptional termination region of the structural gene to provide efficient termination. The termination region may comprise the same gene as the promoter sequence or may comprise a different gene.

The specific expression vector used to transmit genetic messages into cells is not particularly absolute. Any conventional vector that is expressed in eukaryotic or prokaryotic cells can be used. Standard bacterial expression vectors include plastids such as pBR322-based plastids, pSKF, pET23D, and fusion expression systems such as GST and LacZ. Epitope calibration can also be added to recombinant proteins for conventional isolation methods such as c-myc.

Expression vectors containing regulatory elements from eukaryotic viruses are typically used in eukaryotic expression vectors such as the SV40 vector, the papilloma virus, and vectors derived from the Epstein-Barr virus. Other exemplary eukaryotic vectors include pMSG, pAV009/A ⁺ , pMTO10/A ⁺ , pMAMneo-5, baculovirus pDSVE, and the SV40 early promoter, the SV40 late promoter, the metallothionein promoter, the murine mammary tumor virus promoter, Any other vector that allows for protein expression under the guidance of a Ros sarcoma virus promoter, a polyhedrin promoter or other promoters that display efficient expression of eukaryotic cells.

Certain expression systems have indicators that provide gene amplification such as thymidine kinase, hygromycin B phosphotransferase, and dihydrofolate reductase. Alternatively, under the guidance of the polyhedrin promoter or other potent baculovirus promoters, high-yield expression systems that are not involved in gene amplification are also suitable for encoding (such as baculovirus vectors in insect cells) encoded by NRG. A polynucleotide sequence of a polypeptide.

Elements typically included in the expression vector also include a replication unit that acts in E. coli (a marker that encodes antibiotic resistance to allow for the presence of recombinant plastid bacteria), and a specific restriction enzyme in the non-essential region of the plastid. Cut to allow insertion of eukaryotic sequences. The specific antibiotic resistance selection gene is not absolute, and any of a number of drug resistance genes known in the art is appropriate. If necessary, the prokaryotic sequence is randomly selected such that it does not interfere with the replication of DNA in eukaryotic cells. Similar to the antibiotic resistance screening index, metabolic screening indicators based on known metabolic pathways can also be used as a method for screening transformed host cells.

When the periplasmic expression of a recombinant protein (such as a polypeptide associated with IGF2 of the invention) is desired, the expression vector further comprises a sequence encoding a secretion signal, such as an OppA (periplasmic oligopeptide binding protein) secretion signal or a modified version thereof ( Directly attached to the 5' end of the protein to be expressed). This signal sequence traverses the cell membrane into the periplasmic space to direct the production of recombinant proteins in the cytoplasm. The expression vector may further comprise a sequence encoding a signal peptidase 1 having an enzyme cleavage signal sequence capability when the recombinant protein enters the periplasmic space. For a detailed description of the periplasmic products of recombinant proteins, see, for example, Gray et al., Gene 39: 247-254 (1985), U.S. Patent Nos. 6,160,089 and 6,436,674.

Those skilled in the art will appreciate that a variety of conservative substitutions can be made to any wild type or mutant IGF2 or a polypeptide comprising a combination protein binding sequence of IGF2 to produce a modified polypeptide that still retains the ability to bind to a combinatorial protein. Does not trigger IGF2 downstream communication. In addition, modifications of the polynucleotide coding sequence may also permit a particular expression of the host's codon expression preferences without altering the resulting amino acid sequence.

B. Transfection method

Bacterial, mammalian, yeast, insect or plant cell lines expressing a large number of IGF2-related polypeptides are produced using standard transfection methods, which are then purified using standard techniques (see, eg, Colley et al., J. Biol. Chem. 264: 17619-17622 (1989); Guide to Protein Purification, in Methods in Enzymology, vol. 182 (Deutscher, ed., 1990)). Transduction of eukaryotic and prokaryotic cells according to standard techniques (see, eg, Morrison, J. Bact. 132: 349-351 (1977); Clark-Curtiss & Curtiss, Methods iw Enzymology 101: 347-362 (Wu et al., eds) , 1983).

Any known procedure for introducing a foreign nucleotide sequence into a host cell can be used. This includes the use of calcium phosphate transfection, condensed amines, protoplast fusion, electroporation, liposomes, microinjection, plasma carriers, viral vectors and any other known DNA, cDNA, synthetic DNA Or other foreign genetic material introduced into the host cell (see above) Sambrook and Russell). It only requires the use of specific genetic engineering procedures that are capable of successfully introducing at least one gene into a host cell to be capable of expressing a polypeptide associated with IGF2.

C. Recombination produces purified peptides associated with IGF2

When the expression of the recombinant IGF2-related polypeptide in the transfected host cell is confirmed (e.g., by immunoassay such as Western blot analysis), the host cell is then cultured at the appropriate scale for purification of the recombinant polypeptide.

1. Purification of peptides produced by bacterial recombination

Generally, after inducing a promoter, a polypeptide associated with the IGF2 of the present invention is produced by large-scale recombination of the transformed bacteria, and although expressed, the polypeptide forms an insoluble aggregate. There are several steps that apply to the purification of protein inclusion bodies. For example, purification of aggregate proteins (hereinafter referred to as inclusion bodies) generally involves extracting, isolating and/or purifying inclusion bodies by dissociation of bacterial cells, such as by culturing about 100-150 μg/ml of lysozyme and 0.1%. In the buffer of ethyl phenyl polyethylene glycol (Nonidet P40, nonionic detergent). The cell suspension can be ground using a Polytron pulverizer (Brinkman Instruments, Westbury, NY). Alternatively, the cells can be sonicated on ice. Those skilled in the art will be able to understand other methods of lysing bacteria as described by Ausubel et al., Sambrook and Russell, supra.

The cell suspension is generally centrifuged and the pellet containing the inclusion body is resuspended in the buffer, which does not dissolve but cleans the inclusion Body, such as 20 mM Tris-HCl (pH 7.2), 1 mM EDTA, 150 mM NaCl, and 2% Triton-X 100, a non-ionic detergent. It may be necessary to repeat the washing step to remove as much of the cell debris as possible. The remaining inclusion body flaky precipitate can be resuspended in a suitable buffer (eg, 20 mM sodium phosphate, pH 6.8, 150 mM NaCl). Those skilled in the art will be able to understand other suitable buffers.

Following the washing step, the inclusion bodies become soluble by the addition of a solvent that conforms to both strong hydrogen acceptors and strong hydrogen donors (or combinations of solvents each having such properties). The proteins forming the inclusion bodies can then be rejuvenated by dilution or dialysis in a compatible buffer. Suitable solvents include, but are not limited to, urea (about 4 M to about 8 M), formamide (at least about 80% by volume/volume), and hydrazine hydrochloride (about 4 M to about 8 M). Certain solvents can make aggregate-forming proteins soluble, such as SDS (sodium dodecyl sulfate) and 70% formic acid, which are not suitable for use because they may cause irreversible denaturation of proteins, accompanied by loss of immunity and/or activity. This program. While guanidine hydrochloride and similar agents are denaturing agents, this denaturation is not irreversible and renaturation occurs upon removal (eg, by dialysis) or dilution of the denatured agent to re-form the desired immunological and/or biologically active protein. After solubilization, the protein can be separated from other bacterial proteins by standard separation techniques. Further description of purification of recombinant polypeptides from bacterial inclusion bodies can be found, for example, in Patra et al., Protein Expression and Purification 18: 182-190 (2000).

In addition, recombinant polypeptides, such as those associated with IGF2, may be purified from bacterial periplasm. The recombinant protein is exported to the periplasm of the bacteria, and the periplasmic fragments of the bacteria can be isolated by cold osmotic shock, except for methods known to those skilled in the art (see Ausubel et al., supra). In order to separate the recombinant protein from the periplasm, the bacterial cells are centrifuged to form a pellet. The pellet was resuspended in a buffer containing 20% sucrose. Is the dissociation cell, 5mM MgSO _4, by centrifugation and the bacterial pellet was resuspended in ice, and maintained in an ice bath for about 10 minutes. The cell suspension was centrifuged and the supernatant was carefully poured out and stored. The recombinant protein present in the supernatant can be isolated from the host protein by standard separation techniques well known to those skilled in the art.

2. Standard protein separation technology for purification

When a recombinant polypeptide of the invention (such as an IGF2 mutant or a polypeptide comprising an IGF2-combined protein binding sequence) is expressed in a host form in a soluble form, purification can follow the standard protein purification procedure described below. This standard purification procedure is also applicable to the purification of chemically synthesized polypeptides associated with IGF2.

i. Solubility fractionation

If the protein mixture is complex, as an initial step, typically the initial salt fractionation can separate many undesired host cell proteins (or proteins derived from cell culture substrates) from the recombinant protein of interest (such as the polypeptide associated with IGF2 of the invention). A preferred salt is ammonium sulfate. Ammonium sulfate precipitates proteins by effectively reducing the amount of water in the protein mixture. The protein is then precipitated according to its solvency. The more hydrophobic the protein, the more likely it will precipitate. Concentration of ammonium sulfate. The general procedure is to add saturated ammonium sulfate to the protein solution such that the resulting ammonium sulfate concentration is between 20-30%. This will precipitate most of the hydrophobic protein. This precipitate is discarded (unless the protein of interest is hydrophobic) and ammonium sulfate is added to the supernatant to achieve a concentration known to precipitate the protein of interest. The precipitate is then solubilized in the buffer and, if necessary, the excess salt is removed by dialysis or diafiltration. Other methods depending on the solubility of the protein, such as cold ethanol precipitation, are well known to those skilled in the art and can be used to fractionate complex protein mixtures.

Ii. Size difference filtering

Based on the calculated molecular weight, larger and smaller protein sizes can be separated by ultrafiltration using membranes of different pore sizes (eg, Amicon or Millipore membranes). As a first step, the protein mixture is ultrafiltered through a membrane having a molecular weight critical pore size that is less than the molecular weight of the protein of interest (e.g., the polypeptide associated with IGF2). The ultrafiltered retentate is then ultrafiltered to a molecular weight that is greater than the molecular weight of the protein of interest. The recombinant protein will pass through the membrane to the filtrate. The filtrate can then be chromatographed as described below.

Iii. Column chromatography

A protein of interest, such as a polypeptide associated with IGF of the invention, can be isolated from other proteins depending on its size, surface net charge, hydrophobicity, or affinity for the ligand. In addition, antibodies against IGF2 fragments (such as binding protein binding sites) can be conjugated to the column matrix and associated with IGF2 The polypeptide is immunopurified. All such methods are well known in the art.

Those skilled in the art will appreciate that chromatographic techniques can be performed on any scale and using equipment from many different manufacturers, such as Pharmacia Biotech.

IV. Identification of IGF-binding protein binding inhibitors A. IGF combinatorial protein binding assay

In vitro assays can be used to detect IGF2-binding protein binding and to identify compounds that inhibit IGF2-binding protein binding. In general, such an assay can be carried out in the presence of IGF2 (such as human IGF2) and a combination of proteins (such as [alpha]v[beta]3), which are known to bind to each other under conditions that permit binding. For convenience, one of the binding partners may be immobilized on a solid support and/or labeled with a detectable moiety. A third molecule, such as an antibody that binds to one of the partners (which may include a detectable label) may also be used to facilitate detection.

In some cases, binding assays can be performed in a cell-free environment; however, in other cases, binding assays can be performed on the cell surface, typically using recombinant or endogenous recombination of cells to represent appropriate combinatorial protein molecules. Further details and some examples of such combined analysis can be found in the examples section of this specification.

To screen for compounds capable of inhibiting IGF2-binding protein binding, the above analysis was performed in the presence and absence of test compounds, followed by comparison of the extent of IGF2-combined protein binding. If the IGF2-combined protein binding is inhibited by at least 10% in the presence of the test compound, more preferably at least To the extent of 20%, 30%, 40% or 50%, or even higher, the test compound is considered an inhibitor of IGF2-binding protein binding and further testing can be accepted to confirm its ability to inhibit IGF2 signaling.

Binding assays also have been used to confirm that a polypeptide comprising a combinatorial protein binding sequence derived from IGF does specifically bind to a combinatorial protein. For example, a polypeptide comprising an IGF2 protein C domain but a non-full length IGF2 sequence can be recombinantly expressed, purified and placed in a binding assay with the combinatorial protein [alpha]v[beta]3, which is used in a control group to provide a reference full length wild-type IGF2 protein. If sufficient combined protein binding capacity is considered, a polypeptide comprising an IGF2-combined protein binding sequence can be used instead of the wild type full length IGF2 protein in binding assays for identifying inhibitors of IGF2-binding protein binding. Similarly, a polypeptide comprising a core sequence can be found to have a high degree of homology (e.g., 90%, 95% or higher) to the C domain sequence of the wild-type IGF2 protein and, if appropriate, can be substituted for the wild type. Binding analysis of full-length IGF2 protein in an inhibitor for recognition of IGF2-binding protein binding.

Inhibitors of IGF2-combination protein binding can have a variety of chemical and structural characteristics. For example, the inhibitor may be a non-functional IGF2 mutant that retains the ability to bind to a combined protein, an antibody that interferes with IGF2-binding protein binding to IGF2 or a combination protein, or any small molecule or large that simply blocks the interaction between IGF2 and the combinatorial protein. molecule. Essentially any chemical compound can be tested as an inhibitor of potential IGF2-combination protein binding. Most preferably, it is soluble in water or an organic (especially DMSO based) solution. Inhibitors can be identified by screening combinatorial libraries containing a large number of potentially potent compounds. As described herein, such a combinatorial chemical library can be one or more Multiple analyses are screened to identify library members (especially chemicals or sub-categories) that display the desired characteristic activity. Thus, the identified compound can be used as a "lead compound" or it can be used as a potential or actual therapy.

The preparation and screening of combinatorial chemical libraries are well known to those skilled in the art. This combinatorial chemical library includes, but is not limited to, a peptide library (see, e.g., US Patent 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493 (1991) and Houghton et al., Nature 354:84 -88 (1991)) and a sugar library (see, e.g., Liang et al., Science, 274: 1520-1522 (1996) and US Patent 5, 593, 853). Other chemicals used to create a library of chemical diversity can also be used. Such chemicals include, but are not limited to, peptoids (PCT Publication No. WO 91/19735), encoded peptides (PCT Publication WO 93/20242), random biological oligomers (PCT Publication No. WO 92/). 00091), benzodiazepines (US Pat. No. 5,288,514), diversomers such as betaine, benzodiazepines and dipeptides (Hobbs et al., Proc. USA 90: 6909-6913 (1993)), an inter-vinyl peptide (Hagihara et al., J. Amer. Chem. Soc. 114: 6568 (1992)), with a β-D-glucose scaffold Non-peptide peptidomimetics (Hirschmann et al., J. Amer. Chem. Soc. 114: 9217-9218 (1992)), organo-organic complexes of small compound libraries (Chen et al., J. Amer. Chem .Soc. 116:2661 (1994)), oligocarbamate (Cho et al., Science 261: 1303 (1993)), and/or peptidyl phosphonates (Campbell et al., J. Org. Chem. 59: 658 (1994)), nucleic acid libraries (see above Ausubel, Berger and Sambrook), peptides Nucleic acid libraries (see, e.g., US Patent 5,539,083), antibody libraries (see, e.g., Vaughn et al., Nature Biotechnology, 14(3): 309-314 (1996) and PCT/US96/10287), organic small molecule libraries (see, e.g., benzene) And diazepines, Baum C & EN, Jan 18, page 33 (1993); isoprenoids, US Patent 5, 569, 588; thiazolidinones and m-thiazinones, US Patent 5, 549, 974; pyrrolidines, US Patents 5,525,735 and 5,519,134; morphinyl compounds, US Patent 5,506,337; and benzodiazepines, US Patent 5,288,514).

B. IGF2 communication analysis

Inhibitors of IGF2-combination protein binding have their ability to inhibit IGF2 signaling, especially for cancer patients overexpressing one or more combinatorial protein molecules as anti-cancer therapies. The assay for confirming the inhibitory effect of such an inhibitor can be carried out in vitro or in vivo. In vitro assays typically involve exposing cultured cells to inhibitors and observing biological and biochemical changes in the subsequent cells. For example, upon exposure to 0.1-20μg / ml of the inhibitor by 0.5 to 48 hours, such as used or incorporated BrdU H ³ - thymidine glycosides direct cell count, tetrazolium salt 3- [4,5-dimethyl Thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethyl Oxyphenyl)-2-(4-sulfonic acid phenyl)-2H-tetrazolium (MTS) cell proliferation assay, chicken embryo chorioallantoic membrane (CAM) analysis, TUNNEL analysis, annexin V binding assay, etc. Proliferation/survival status of appropriate cells, such as those expressing the combination protein αvβ3. Further changes downstream, such as phosphorylation activation of IGF1R, IR-A, AKT or ERK1/2, can also be observed due to IGF2 signaling to provide evidence of inhibition of IGF2 signaling. Furthermore, the tumorigenicity of cancer cells is a useful parameter for observation and can be tested by methods such as colony formation analysis or soft agar analysis. A detailed description of certain example analyses can be found in the Examples section of this disclosure. When it is observed that the IGF communication drops by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or higher, as indicated by any of the above parameters, the inhibitory effect may be It was measured.

The effect of the IGF2-binding protein binding inhibitor of the present invention can also be confirmed by in vivo analysis. For example, an inhibitor of the IGF2 combinatorial protein can be injected into an animal that is immune to the immune system (such as a nude mouse, SCID mouse, or NOD/SCID mouse), thus allowing xenografting of the tumor. The method of injection can be in the form of intravenous, intraperitoneal or intratumoral. Tumor progression was compared to control group animals with similar tumors but no inhibitors, and then observed by a variety of methods, such as measuring tumor volume and scoring secondary damage resulting from metastasis. Some exemplary in vivo analyses are detailed in the Examples section of this disclosure. When the negative effects of tumor growth or metastasis in the test group have been established, inhibitory effects can be detected. Preferably, the negative impact is reduced by at least 10%; more preferably, by at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.

V. Drug composition and application

The invention also provides a pharmaceutical composition or physiological composition comprising an effective amount of a compound which inhibits binding of an IGF combination protein, such as a dominant negative IGF2 mutant R24E/R37E/R38E, R34E/R37E/R38E or R24E/R34E/R37E/ R38E or its encoding nucleic acid inhibits IGF2 signaling in both prophylactic and therapeutic applications. Such pharmaceutical or physiological compositions also include one or more pharmaceutically or physiologically acceptable excipients or carriers. The pharmaceutical compositions of the invention are suitable for use in a variety of drug delivery systems. Suitable formulations for use in the present invention are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985). For a review of drug delivery methods, see Langer, Science 249: 1527-1533 (1990).

The pharmaceutical composition of the present invention can be administered by various routes such as oral, subcutaneous, transdermal, intramuscular, intravenous or intraperitoneal. A preferred route for administering the pharmaceutical composition is a local delivery of from about 0.01 to 5000 mg, preferably from 5 to 500 mg, of the IGF2-combined protein binding inhibitor to a daily dose of 70 kg of adult per day to an organ suffering from exacerbation of IGF2 overexpression. Or tissue (eg intratumoral injection into the tumor). Suitable doses may be administered in a single daily dose or in multiple doses at appropriate intervals, for example two, three, four or more daily doses.

A pharmaceutical composition comprising an IGF2 combinatorial protein inhibitor is prepared using an inert and pharmaceutically acceptable carrier. The pharmaceutical carrier can be a solid or a liquid. Solid form preparations include, for example, powders, tablets, dispersed granules, capsules, sachets, and suppositories. The solid carrier can be one or more substances, which can also be used as a diluent, a flavoring agent, a solubilizing agent, a lubricant, a suspending agent, and a viscous agent. A mixture or tablet disintegrating agent; it may also be a coating material.

In the case of powders, the carrier will usually be a finely divided solid, such as an IGF2 dominant negative mutant polypeptide. In the case of a tablet, the active ingredient (inhibitor of IGF2-binding protein binding) is mixed with a carrier having an appropriate ratio of necessary binding properties and compressed into a desired shape and size.

In order to produce a pharmaceutical composition in the form of a suppository, a low melting wax such as a mixture of fatty acid glycerides and cocoa butter is first melted, and then the active ingredient is dispersed therein by, for example, stirring. The molten homogeneous mixture is then poured into a suitably sized mold and allowed to cool and solidify.

The powders and tablets preferably comprise from about 5% to about 70% by weight of the active ingredient of the IGF2-combined protein-bound inhibitor. Suitable carriers include, for example, magnesium carbonate, magnesium stearate, talc, lactose, granulated sugar, pectin, dextrin, starch, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter Wait.

The pharmaceutical composition may comprise a formulation of an active compound of an IGF2-binding protein binding inhibitor, the active compound being provided as a carrier with a coating material, wherein a capsule in which the inhibitor (with or without other carrier) is surrounded by the carrier is provided. The carrier is thus associated with the compound. The drug pack can also be prepared in a similar manner. Tablets, powders, packs and capsules can be used as solid dosage forms suitable for oral administration.

The liquid pharmaceutical composition includes, for example, a solution suitable for oral or parenteral administration, a suspension, an emulsifier suitable for oral administration. A sterile aqueous solution of the active ingredient (eg, a dominant negative IGF2 mutant polypeptide) or A sterile solution of the active ingredient in a solvent of water, buffered water, physiological saline, PBS, ethanol or propylene glycol is an example of a liquid composition suitable for parenteral administration. The composition may comprise pharmaceutically acceptable auxiliary substances, such as pH adjustment and buffering agents, tonicity adjusting agents, wetting agents, detergents, and the like, which are required to approximate the physiological environment.

The sterilizing solution can be prepared by dissolving the active ingredient (such as an IGF2-binding protein binding inhibitor) in a desired solvent system, and then passing the resulting solution through a membrane filter to sterilize or, alternatively, in a sterilizing environment The sterilized compound is dissolved in a pre-sterilized solvent. The resulting aqueous solution can be packaged or lyophilized for use, and the lyophilized formulation is combined with a sterile aqueous carrier prior to administration. The pH of the formulation is usually between 3 and 11, preferably from 5 to 9, and more preferably from 7 to 8.

The pharmaceutical composition comprising the IGF2-combined protein binding inhibitor can be administered for prophylactic and/or therapeutic treatment. In therapeutic applications, the composition is administered to a patient who has suffered a condition in which the IGF2 or a member of the combination protein family is overexpressed in an amount sufficient to prevent, treat, reverse or at least partially alleviate or prevent the condition and its complications. The condition. A quantity sufficient to accomplish this is defined as a "therapeutically effective dose." The use of this effective amount will depend on the severity of the disease or condition and the weight and general condition of the patient, but for a 70 kg patient, typically a range of inhibitors ranging from about 0.1 mg to about 2,000 mg per day, For a 70 kg patient, a dose of the inhibitor from about 5 mg to about 500 mg per day is more often used.

In a prophylactic application, a pharmaceutical composition comprising an IGF2-combined protein binding inhibitor is administered in an amount sufficient to delay or prevent the onset of symptoms. A patient who is at risk of developing or developing an undesired risk of over-expressing the disease or condition of the IGF2 or combination protein. This amount is defined as the "preventive effective dose." In this application, the precise amount of the inhibitor is again dependent on the patient's state of health and body weight, but for a 70 kg patient, it is usually in the range of about 0.1 mg to about 2,000 mg per day, for a 70 kg patient, More typically from about 5 mg to about 500 mg.

Single or multiple administrations of the composition can be performed with the dosage level and mode selected by the attending physician. Under any conditions, whether treated or prevented, the pharmaceutical preparation should provide an amount sufficient to effectively inhibit IGF2-binding protein binding by IGF2 signaling in the patient.

VI. Therapeutic applications using nucleic acids

A variety of diseases can be treated by a nucleic acid-related therapeutic method that introduces a polypeptide inhibitor that binds to a combination protein IGF2 into a cell such that the coding sequence is transcribed and a polypeptide inhibitor is produced in the cell. Suitable for treating diseases by this method, the method comprising broad-spectrum solid tumors whose survival and growth depend to some extent on the continuous communication of IGF2 or members of the combination protein family. For a discussion of genetic treatment and the application of gene therapy for acquired diseases, see Miller Nature 357: 455-460 (1992); and Mulligan Science 260: 926-932 (1993).

A. Vector for gene delivery

A polypeptide encoding an inhibitor of IGF2-binding protein binding (such as a dominant negative mutant R24E/R37E/R38E, R34E/R37E/R38E or The polynucleotide of R24E/R34E/R37E/R38E) can be incorporated into a vector for delivery to a cell or organelle. Examples of vectors useful for this purpose include the ability to direct nucleic acid expression in a target cell to express a plastid. In other examples, the vector is a viral vector system in which the polynucleotide is incorporated into a viral genome capable of transfecting a target cell. In a preferred embodiment, a polynucleotide encoding a polypeptide inhibitor is operably linked to a performance and control sequence that directs expression of the polypeptide in a desired host cell of interest. Thus, it can be achieved that the polypeptide inhibitor is expressed in a suitable environment in a target cell.

B. Gene delivery system

Viral vector systems for use in expressing an inhibitor of polypeptide IGF2-binding protein binding include, for example, naturally occurring or recombinant viral vector systems. Suitable viral vectors include replication competent, replication defective, and conditionally replicating viral vectors, depending on the particular application. For example, the viral vector can be obtained from human or bovine adenovirus, vaccinia virus, herpes virus, adeno-associated virus, mouse parvovirus (MVM), HIV, Sindbis virus, and retrovirus (including but not limited to Rous sarcoma virus) ) the genome, and MoMLV. Typically a gene of interest (e.g., a polypeptide inhibitor encoding a polypeptide of the invention) is inserted into the vector to allow for packaging of the genetic construct, usually with viral DNA, followed by infection of the susceptible host cell and expression of the desired gene.

As used herein, "gene delivery system" means any device used to deliver a nucleic acid of the invention to a target cell. In certain embodiments of the invention, the nucleic acid is conjugated to a ligand of a cellular receptor for transmission through a suitable linking moiety (Wu et al., J. Biol. Chem. 263: 14621-14624 (1988); WO 92/06180) promotes ingestion (eg, sputum sputum and endocytosis of endosomes). For example, a nucleic acid can be linked to a serum sialic acid-free serum mucin (asialo-oromucocid), which is a ligand for hepatocytes lacking a sialoglycoprotein receptor.

Similarly, a viral sheath for packaging a genetic construct comprising a nucleic acid of the invention can be modified by the addition of a receptor ligand or an antibody specific for the receptor to allow receptor-mediated endocytosis into a particular cell. (See, for example, WO 93/20221, WO 93/14188 and WO 94/06923). In certain embodiments of the invention, the DNA construct of the invention is linked to a viral protein, such as an adenoviral particle, to promote endocytosis (Curiel et al., Proc. Natl. Acad. Sci. USA 88: 8850 -8854 (1991)). In other embodiments, the molecular conjugation of the invention may include a microtubule inhibitor (WO/9406922), a synthetic peptide that mimics influenza virus hemagglutinin (Plank et al., J. Biol. Chem. 269: 12918- 12924 (1994)) and nuclear localization signals (such as SV40 T antigen) (WO93/19768).

Retroviral vectors can also be used to introduce the coding sequences of the polypeptide inhibitors of the invention into cells or organelles. Retroviral vectors are produced by genetic manipulation of retroviruses. The viral genome of the retrovirus is RNA. Upon infection, the genomic RNA is reverse transcribed into a copy of DNA that is integrated into the chromosomal DNA of the transduced cell with high stability and efficiency. The integrated DNA copy is called the provirus and is inherited by the daughter cells like other genes. Wild type retroviral genome and Proviral DNA has three genes: the gag, pol and env genes, which are accompanied by two long terminal repeat (LTR) sequences. The gag gene encodes an internal structure (nucleocapsid) protein; the pol gene encodes an RNA-directed DNA polymerase (reverse transcriptase); and the env gene encodes a sheath glycoprotein. 5' and 3' LTR act as transcription and polyadenylation of viral RNA. Adjacent to the 5'LTR is the sequence required for reverse transcription of the genomic (tRNA primer binding site) and is used to efficiently package viral RNA into particles (Ψ sites) (see Mulligan, In: Experimental Manipulation of Gene Expression, Inouye (ed), 155-173 (1983); Mann et al., Cell 33: 153-159 (1983); Cone and Mulligan, Proceedings of the National Academy of Sciences, USA, 81: 6349-6353 ( 1984)).

The design of retroviral vectors is well known to those of ordinary skill in the art. In short, if the sequence required for encapsidation (or packaging of retroviral RNA into an infectious virus) is lost from the viral genome, the result is a cis-acting effect that prevents nucleocapsid RNA encapsidation (cis Acting) defects. However, the resulting mutants still guide the synthesis of all viral proteins. The sequences from the retroviral genome described above have been disrupted, and as is well known in the art, cell lines containing mutant genomes are stably integrated into the chromosome and used to construct retroviral vectors. Preparation of retroviral vectors and their use are described in many publications, including, for example, European Patent Application EPA 0 178 220; U.S. Patent 4,405,712, Gilboa Biotechniques 4:504-512 (1986); Mann et al., Cell 33: 153-159 (1983); Cone and Mulligan Proc. Natl. Acad. Sci. USA 81: 6349-6353 (1984); Eglitis et al. Biotechniques 6: 608-614 (1988); Miller et al. Biotechniques 7: 981-990 (1989); Miller (1992) as described above; Mulligan (1993) and WO 92/07943, supra.

The retroviral vector particle is prepared by recombinantly inserting a desired nucleotide sequence into a retroviral vector, and packaging the vector with a retroviral capsid protein by using a packaging cell strain. The resulting retroviral vector particles are not replicable in the host cell, but can be integrated into the host cell genome as a proviral sequence containing the desired nucleotide sequence. As a result, the patient is able to produce, for example, a polypeptide or polynucleotide of the invention, and thus restore the cells to a normal phenotype.

The packaging cell line used to prepare the retroviral vector particles is a typical recombinant mammalian tissue culture cell line which produces the necessary viral structural proteins required for packaging, but is unable to produce an infectious virus. On the other hand, defective retroviral vectors are used which lack these structural genes but encode the remaining proteins necessary for packaging. In order to prepare a packaged strain, the infectious site of the desired retrovirus can be cultured and the packaging site is broken. Cells containing this organization will express all viral structural proteins, but the inserted DNA cannot be packaged. Alternatively, the packaging cell line can be prepared by transforming the cell line with one or more expression plastids encoding the appropriate core and sheath protein. In these cells, the gag, pol and env genes can be derived from the same or different retroviruses.

Many packaging cell lines suitable for the present invention are also useful in the prior art. Examples of such cells include Crip, GPE86, PA317, and PG13 (see Miller et al., J. Virol. 65: 2220-2224 (1991)). Examples of other packaging cell lines are in Cone and Mulligan Proceedings of the National Academy of Sciences, USA, 81: 6349-6353 (1984); Danos and Mulligan Proceedings of the National Academy of Sciences, USA, 85: 6460-6464 (1988). ; as described above by Eglitis et al. (1988) and Miller (1990) as described above.

A packaging cell line capable of producing retroviral vector particles having a chimeric sheath protein can be used. In addition, retroviral vectors can be packaged using amphotropic or heterophilic sheath proteins, such as those from PA317 and GPX packaging cell lines.

C. Pharmaceutical preparations

When used in a pharmaceutical use, the nucleic acid encoding the polypeptide of the IGF combination protein binding inhibitor is typically disposed in a suitable buffer, which may be any pharmaceutically acceptable buffer, such as phosphate buffered saline or sodium phosphate/sodium sulfate, Tris buffer, glycine buffer, sterile water, and other buffers known to those of ordinary skill, such as those described by Good et al. Biochemistry 5:467 (1966).

The composition may additionally include a stabilizer, an accelerator or other pharmaceutically acceptable carrier or vehicle. A pharmaceutically acceptable carrier can comprise a physiologically acceptable compound, for example, to stabilize the nucleic acid of the invention and What is the relevant carrier. Physiologically acceptable compounds can include, for example, carbohydrates (such as glucose, sucrose, or polyglucose), antioxidants (such as ascorbic acid or glutathione), chelating agents, low molecular weight proteins, or other stabilizers or excipients. 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 known to include, for example, phenol and ascorbic acid. For examples of carriers, stabilizers or adjuvants, see Remington&apos;s Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985).

D. Application of the formulation

Formulations containing a nucleic acid encoding a polypeptide inhibitor that binds between IGF2 and a combination protein can be delivered to any tissue or organ using any delivery method known to those of ordinary skill in the art. In certain embodiments of the invention, a nucleic acid encoding an inhibitor polypeptide is formulated for intravenous, intraperitoneal or intratumoral injection.

Formulations comprising a nucleic acid of the invention are typically administered to a cell. The cells can be provided as part of a tissue (such as an epithelial membrane), or as an isolated cell (such as tissue culture). The cells can be placed in vivo, ex vivo or in vitro.

The formulation can be introduced into the tissue of interest by in vivo or ex vivo by a variety of methods. In certain embodiments of the invention, the nucleic acids of the invention are introduced into cells by such methods as microinjection, calcium phosphate precipitation, liposome fusion, ultrasound, electroporation or biolistic. In other embodiments, the nucleic acid is directly ingested directly by the tissue of interest.

In certain embodiments of the invention, the nucleic acid of the invention is administered ex vivo to cells or tissues explanted from the patient and subsequently returned to the patient. Examples of in vitro administration of therapeutic gene constructs include Nolta et al., Proc Natl. Acad. Sci. USA 93(6): 2414-9 (1996); Koc et al., Seminars in Oncology 23 (1) ): 46-65 (1996); Raper et al., Annals of Surgery 223(2): 116-26 (1996); Dalesandro et al., J. Thorac. Cardi. Surg., 11(2): 416- 22 (1996); and Makarov et al., Proc. Natl. Acad. Sci. USA 93(1): 402-6 (1996).

The effective dose of the formulation will vary depending on a number of different factors, including the mode of administration, the target site, the physiological state of the patient, and other drug administration. Therefore, the therapeutic dose will need to be titrated to optimize safety and effectiveness. In determining the effective amount of the carrier to be administered, the physician should assess the particular nucleic acid used, the disease state being diagnosed; the age, weight and overall condition of the patient, circulating plasma concentrations, carrier toxicity, progression of the disease, and anti-carrier antibodies. produce. The size of the dose is also determined by the existence, nature, and extent of any adverse side-effects associated with administration of the particular carrier. For the practice of the invention, a typical dosage range is from about 10 ng to 1 g, 100 ng to 100 mg, 1 μg to 10 mg or 30 to 300 μg of DNA per patient. Typical dosages range from about 0.01 to about 50 mg per kilogram of body weight, preferably from about 0.1 and about 5 mg per kilogram of body weight or from about 10 ⁸ to 10 ¹⁰ or 10 ¹² particles per injection. Typically, for a typical 70 kg patient, the naked nucleic acid dose equivalent of the vector is from about 1 [mu]g to 100 [mu]g, and the dose of the vector comprising the retroviral particles is calculated to produce a binding between the inhibitory combination protein and IGF2 (such as human IGF2). An equal amount of nucleic acid of a polypeptide.

VII. Set

In accordance with the method of the present invention, the present invention also provides a kit for suppressing IGF2 communication. The kit typically includes a container containing a pharmaceutical composition having an effective amount of an inhibitor of IGF2-binding protein binding (such as a dominant negative mutant R24E/R37E/R38E, R34E/R37E/R38E or R24E/R34E/R37E/ R38E or a polynucleotide sequence encoding the polypeptide), and a message material containing instructions on how to dispense the drug composition, including the type of patient that can be treated (eg, a cancer patient with IGF2 or a combination protein overexpression), course of treatment (such as dose and frequency), and the route of administration.

Instance

The following examples are provided by way of illustration only and not by way of limitation. Those skilled in the art will readily appreciate that a variety of non-critical parameters can be altered or modified to produce substantially identical or similar results.

Example 1

IGF1 and IGF2 have similar signaling functions: IGF1 and IGF2 are polypeptide endosomes (75-kD), which have a high structural similarity to human proinsulin (Fig. 1). By acting in conjunction with IGF1R (receptor tyrosine kinase), IGF1R is ubiquitous in a variety of cell types. Since IGF1 and IGF2 are involved in cell growth, I sought IGF1R inhibition. Make potential measures to treat and prevent cancer. Ligand binding induces phosphorylation of specific tyrosine residues of IGF1R. The phosphotyrosines are then bound to a transfer molecule such as Shc and the insulin receptor matrix (IRS)-1. Phosphorylation of these proteins leads to activation of the PI3K and MAPK signaling pathways [11].

Development of dominant negative inhibitor IGF1 mutants: The combination proteins αvβ3 and α6β4 are overexpressed in a variety of human cancers and are associated with poor patient prognosis [12], but the role of these combination proteins in cancer has not yet been established. The present inventors have previously proposed the direct and specific binding of IGF1 to the reports of αvβ3 [7] and α6β4 [8]. Although R36E/R37E is still bound to IGF1R, the combination protein-deficient mutant of IGF1 (R36E/R37E) is defective in enhancing cell viability and inducing IGF signaling. Interestingly, WT IGF1 induced the formation of ternary complexes (αvβ3-IGF1-IGF1R and α6β4-IGF1-IGF1R), while R36E/R37E did not. It is pointed out that these combination proteins bind directly to IGF1, and the formation of subsequent ternary complexes is the key to IGF signaling [7,8].

A model of IGF1 signaling has been proposed in which IGF1 binds to IGF1R on the cell surface, and the combinatorial protein is recruited to the IGF1-IGF1R complex by direct binding to IGF1 to form an IGF1R-IGF1-combined protein ternary complex. If the formation of a ternary complex is critical in IGF signaling, the combination protein of IGF1 binds to a defective R36E/R37E mutant that is antagonized, while R36E/R37E binds stably to IGF1R and competes with WT IGF1 for binding to IGF1R. The inventors demonstrated that excess R36E/R37E inhibits the induction of WT IGF1 induction in vitro. worth it Note that R36E/R37E inhibits the anchorage-independent growth of cancer cells in vitro and tumorigenesis in vivo, while WT IGF1 is significantly enhanced (Fig. 2) [10]. R36E/R37E IGF1 has the potential to be a therapeutic agent in cancer ("IGF1 decoy"). It may be observed that excess IGF1 decoy inhibits the binding of WT IGF1 to the cell surface in cancer cells, presumably competing for IGF1 decoy and WT IGF1 to bind to IGF1R on the cell surface [10].

The insulin receptor (IR) is overexpressed in cancer cells: the insulin receptor (IR) is structurally very similar to IGF1R and is a member of the tyrosine kinase of the membrane receptor [2]. IR is usually not normally expressed in cancer cells, which regulate both metabolic and non-metabolic effects of insulin. When compared, the average IR content in cancerous breast tissue is 6 times higher than normal breast tissue [13]. Approximately 80% of breast cancer samples have much higher IR content than normal breast tissue, while approximately 20% of IR values are more than 10 times higher than normal breast tissue [13]. Functional studies indicate that IR responds to insulin in breast cancer more than in normal breast cells [14].

IR-A and IR-B have different functions: IR occurs in two isomers (IR-A and IR-B). The most important functional difference between the two isomers is the high affinity for IR-A for IGF2 (ligand binding to IR-A, but not to IR-B, Table 1). IR-A is mainly expressed in the prenatal period. It enhances the action of IGF2 during embryonic and fetal development. It is also prominent in adult tissues, especially in the brain. Conversely, IR-B is primarily expressed in highly differentiated adult tissues, including the liver, which enhances the metabolism of insulin. IR-A preferentially induces mitotic and anti-apoptotic signals, IR-B preferentially induces cell differentiation signals [15].

IR-A in cancer is overexpressed: IR splicing in cancer cells is altered, thus increasing the IR-A:IR-B ratio, which profoundly affects the response of cells to circulating insulin and IGF2. IR-A is the major IR isomer in various cancers, including breast, colon and lung cancer [16]. In particular, IR-A is the major IR isomer in breast cancer cell lines (64-100% of total IR) and in a range of breast cancer tissue samples (40-80%) [17]. In contrast, IR-A is 30-50% overall IR in normal breast cell and tissue samples [17]. This points out that IR-A plays an important role in cancer.

The autocrine IR-A/IGF2 loop plays an important role in cancer: IR-A is the IGF2 receptor [16]. This provides further insight into the overexpression of IR in cancer. It is worth noting that breast cancer cells produce IGF2 in an autocrine manner. In cells with a high ratio of IR-A:IGF1R, autocrine-produced IGF2 stimulates cell growth by IR-A stimulation. In these cells, blocking IGF2 or IR significantly inhibited growth, confirming the association of this autocrine loop (IR-A/IGF2 loop) in cancer [17]. IR-A binding to IGF2 is associated with stimulation growth and cell invasion [16], whereas IR-B, which does not bind IGF2, is associated with differentiation and metabolic signaling [13].

The heterodimer consists of half IR and half IGF1R (Table 1), and these lines are known to be heterozygous receptors [3, 4]. Like most cancers that exhibit both IR and IGF1R, they display multiple heterozygous receptor species rather than a single receptor type. In each condition, the kinase activity of the receptor results in phosphorylation of members of the IR matrix (IRS) family of proteins, and this results in PI3K, Activation of AKT and various downstream networks [5]. Abnormal autocrine or paracrine [6] and IR-A/IGF2 circuits of IGF2 may represent an "addiction" to IR/IGF1R activation in many cancers.

Dominant inactivated IGF2 (IGF2 decoy) has therapeutic utility: if it blocks IR-A without affecting IR-B, it will likely inhibit tumorigenesis effectively. Unfortunately, currently available kinase inhibitors for IR or IGF1R are unable to resolve IGF1R, IR-A or IR-B. Furthermore, anti-IGF1R antibodies are unable to block IR-A or IR-B. The inventors have established an IGF1 decoy that effectively inhibits IGF1-induced tumor cell survival and tumorigenesis in vivo [10]. However, the IGF1 decoy cannot inhibit IR-A or IR-B. To inhibit IR-A, an effective therapeutic target in cancer, IGF2 was utilized to bind to IR-A rather than IR-B (Table 1). The inventors used strategies successfully used for IGF1 decoys and other dominant negative growth factors (see below) to establish IGF2 decoys. IGF2 is structurally similar to IGF1, and the key IGF1 amino acid residues that bind the binding protein to IGF1 are retained in IGF2. The IGF2 decoy together with IGF1R inhibits IR-A and blocks the IR-A/IGF2 loop in cancer. In its preliminary study, the inventors found that IGF2 binds to the combinatorial protein αvβ3. The IGF2 mutation system of the combinatorial protein binding defect was generated using a strategy for IGF1 (Fig. 3). The IGF2 mutant was found to be defective in signaling function and it inhibited increased cell viability (dominant inactivation) by WT IGF2 (Fig. 4).

Dominant inactivation technique - a new platform for drug discovery: using docking simulations, the inventors identified several growth factors as new combinatorial protein ligands (FGF1 [18], IGF1 [7], and neuregulin 1 (neuregulin) -1, NRG1)) [19]. These growth factors bind directly to combinatorial proteins (such as αvβ3 and/or α6β4), and this interaction plays an important role in growth factor signaling. The combinatorial protein binding defective growth factor is produced by introducing a mutation at a predetermined combinatorial protein binding site. The combinatorial binding-deficient FGF1 mutant (R50E) is defective in the induction signal and the induced ternary complex (combined protein-FGF1-FGF1 receptor), which still binds to the FGF1 receptor [20]. In addition, R50E is dominantly inactive and inhibits signaling induced by WT growth factors and inhibits tumor growth [20] and angiogenesis [21]. U.S. Patent No. 8,168,591 (a composition and method related to anti-FGF agents) has published a dominant negative FGF1 mutant in 2012. The combinatorial protein binding-deficient NRG1 mutant (3KE) is defective in ErbB3 signaling, and the mutant still binds to Erbβ3 [19]. WT NRG1 induces a ternary complex (combined protein-NRG1-Erbβ3), while 3KE does not. 3KE is also a dominant negative mutant (inventors have not published results). Recently, the inventor proposed fractalkine The chemokine domain of (CX3CL1) (transmembrane chemokine) is reported to bind to the combination proteins αvβ3 and α4β1 [22]. CX3CL1 induces the formation of a ternary complex (combined protein, CX3CL1 and its specific receptor CX3CR1). In addition, the combination protein-deficient mutant of fractalkine is a dominant negative antagonist of CX3CR1 [22]. These findings indicate that combinatorial protein growth factor receptors interfere with each other by direct binding to growth factors and may be a common mechanism in many growth factors. This strategy is a new platform for drug discovery.

Mutants of human proteins can be used as therapeutic agents: there are precedents for the use of human protein mutants for human diseases. Human growth hormone (hGH) mutants have been used as antagonists of GH receptors in the treatment of acromegaly (Pevisol, trade name Somavert) [23]. Gly-120 of hGH was mutated to Arg (G120R), and this mutant was further modified by poly(ethylene glycol) (PEG)-5000 to extend half-life. Pevisolole prevents functional dimerization of hGH receptors by inhibiting stereoconfiguration changes in GHR dimers [23]. Pevisol is generally similar to the safety reported in clinical trials and can effectively reduce IGF1 in patients with acromegaly that are difficult to treat with conventional treatments [24]. Therefore, an IGF2 dominant negative mutant can be used as a therapeutic agent.

Innovation: IR-A is over-expressed in cancer and is the therapeutic target of cancer. Current treatment is not available for IR-A. Antibodies against IGF1R did not affect IR-A or IR-B, and antibodies against IR did not recognize IR-A and IR-B. Kinase inhibitors do not recognize IR-A, IR-B, and IGF1R. IGF2 binds to IR-A and IGF1R but does not bind to IR-B. In order to target IR-A, a dominant negative IGF2 mutant has been developed. The so-called "IGF2 decoy" has advantages over antibodies and kinase inhibitors. In addition to being a specific antagonist against IR-A, IGF2 decoys have better penetration rates for tumor tissue than IgG due to their smaller size. IGF2 decoys are possible therapies. To identify dominant negative IGF2, the same strategy that is effective for other growth factors such as FGF1, IGF1, and neuromodulin 1 is employed. The resulting study of several candidate IGF2 decoys is particularly useful for establishing the role of combinatorial proteins in IGF2 and IR-A signaling.

IGF2 uniquely identifies IR-A and IR-B. A dominant negative mutant of IGF2 against IR-A, which is overexpressed in cancer and plays an important role in cancer progression.

Rationale: Impeding IGF2 function to reduce IR-A communication and IGF1R communication, and as a reasonable way to inhibit cancer progression. Develop a dominant form of inactivation of IGF2 to achieve this goal. The dominant negative form of IGF1 is directed against IGF1R, but not in IR-A or non-IR-B [7-10]. The dominant negative pattern of IGF2 is directed against IR-A (rather than IR-B) and IGF1R and inhibits its activation. In its preliminary study, the inventors have established several candidates for dominant negative IGF2 mutants and have continued to characterize these IGF2 mutants in in vitro and in vivo experiments.

Study Design: In a preliminary study, the bacterial expression construct of IGF2 was generated by subcloning cDNA encoding IGF2 into the NdeI/XhoI site of PET28a. The protein is expressed as an insoluble inclusion body, and as described, the insoluble IGF2 is refolded [7]. The ability of IGF2 to bind to a combinatorial protein using a recombinant soluble combination protein as an ELISA-type binding assay As tested, and as described, the cell adhesion assay was performed using the recombinant recombinant protein CHO cell assay [7]. The results showed that CHO cells (β3-CHO cells) expressing αvβ3 strongly adhered to IGF2 in a dose-dependent manner, but none of those expressing αvβ1 (β1-CHO cells) ( FIG. 3 ). These findings indicate that αvβ3 interacts with IGF2. Recombinant IGF2 induced IGF1R phosphorylation in a dose-dependent manner (data not shown), which also indicated that IGF2 preparation was practical. Various mutations were introduced into IGF2, and several IGF2 mutants were identified as defective in the combination protein (Fig. 3c and d). The ability of these IGF2 mutants to induce signals was tested in β3-CHO cells and it was found that combinatorial protein binding-deficient mutants were defective in enhancing cell viability (Fig. 4a). It was observed that the excess combination protein binding-deficient IGF2 mutant inhibited cell viability by WT IGF2 (Fig. 4b). The conclusion is that the combinatorial protein binding-deficient mutant is not only defective in signaling function, but also dominantly inactivated.

1) Description of combinatorial protein binding defective IGF2 mutant: Mutants that bind to IGF1R [7] (commercially available) were further investigated using the soluble IGF1R in an ELISA type assay as previously described. As described, the ability of the combination protein-deficient mutant of WT IGF2 and IGF2 to induce ternary complex formation was tested [7], and it was confirmed that WT IGF2 induced a ternary complex, but the combination protein-binding defective mutant did not.

2) Ability to test IGF2 activation of IGF1R and IR: IGF2 mutants were tested for their ability to induce tyrosine phosphorylation of IR and IGF1R. MCF-7 cells were stimulated with IGF2 (WT and mutant) and used to phosphorylate-IR and phosphorylate IGF1R specific antibodies by Western blotting (commercially available) Analyze cell lysate. The combinatorial binding-deficient IGF2 mutant was verified to be defective in inducing IR and IGF1R phosphorylation, but the IGF2 mutant, which was not defective in combinatorial protein binding, retained the full signaling function. This is consistent with previous studies found that the combined protein-deficient IGF1 that is defective in IGF1R activation is defective [7].

3) Study of the role of combinatorial protein-IGF2 binding in IGF2/IR-A or IGF1R signaling: To study the ability of WT IGF2 to induce ternary complex formation (combined protein-IGF2-IGF1R or combinatorial protein-IGF2-IR-A). Cells (such as β3-CHO cells or MCF-7 cells) are cultured with WT IGF2, and the combination protein β3 or IGF1R (or IR) is immunopurified from cell lysates. As described, the purified material was then analyzed by Western blotting [7]. The combinatorial protein binds to a defective IGF2 mutant to reveal whether it induces ternary complex formation. WT IGF2 induces the formation of a ternary complex, but the mutant does not. These experiments show a new role for combinatorial proteins in IR signaling, and IGF2 mutants are an important tool for these studies.

4) Study of IGF2 mutant inhibition of WT IGF2-induced signaling: IGF2 mutants were tested for inhibition of WT IGF2-induced signaling. Culture and treat the non-transformed cells (such as NIH3T3 in a general tissue culture dish) or transformed cells (such as the above, such as MCF-7 in a plastic well groove coated with polyHEMA) by serum starvation. Reduce cell matrix interaction [10])). The cells are then stimulated with WT IGF2 and/or IGF2 mutants. Cell viability was measured using MTS analysis. Cell proliferation was analyzed using BrdU incorporation. When too much IGF2 mutant inhibits cell activity and proliferation induced by WT IGF2, it is as defined above Sexual inactivation [8-10]. The IGF2 mutant was tested for its ability to inhibit IR and IGF1R phosphorylation induced by WT IGF2 in transgenic and transduced cells as described above. As described above, cell lysates were analyzed using Western blotting methods for phosphorylated or non-phosphorylated IR and IGF1R specific antibodies [8-10].

5) In vivo testing of dominant negative IGF2 mutants: In vitro display of dominant negative IGF2 mutants were selected to test their ability to inhibit tumor growth in vivo. IGF2 (6His tag, WT and mutant) in a secreted vector (such as pSecTag) is stably expressed in cancer cells (such as MDA-MB231) and tested to confirm that IGF2 is secreted from the antibody using anti-His tag. The MDA-MB231 cell line was chosen for its high expression of IR-A. As described in IGF1, transfected cells are characterized by colony formation in soft agar in vitro, in response to serum starvation and chemotherapeutic (by MTS analysis) survival and in hyperplasia (by BrdU incorporation analysis) ) ability [10]. WT IGF2 enhances cell survival and proliferation, whereas dominant negative IGF2 inhibits it. WT IGF2 and dominant negative IGF2 were injected into mice as described, and their effects on tumorigenesis were observed [10]. Tumor tissue analysis indicated that IGF2 mutants inhibit tumorigenesis and angiogenesis by affecting IR-A signaling in tumor cells and microenvironments.

Statistical analysis and assay power analysis: Comparison of tumor growth curves across groups using standard repeated measurement of mixed models [25]. These models allow measurements of unequal spacing or subsequent unequal lengths, for example, if some mice develop an unsustainable tumor burden and sacrifice early. These models were developed to specifically test IGF2-treated mice and control groups, followed by testing for the effect of increasing dose on tumor growth rate to identify optimal dose concentrations. A qualitative analysis of the original breast cancer Met-1 orthograft has been found to detect a difference of 20% between the treatment and control groups of each group of 8 mice [26,27]. Usually 10 to 12 mice are used per group unless the data from the trial indicate better than 20%, under which 8 mice are used per group.

Direct binding of IGF1 to the combination protein and IGF1R (and leading to the formation of a ternary complex) has been reported to be the key to IGF1 signaling [7]. IGF1 and IGF2 are similar in structure and signaling (except for IGF2 binding to IR-A but IGF1). IGF2 requires direct combinatorial protein binding, and the resulting complex formation (combined protein-IGF2-IGF1R/IR-A) plays a key role in IGF2 signaling. Furthermore, the key Arg residues (Arg36 and Arg37) that bind to the IGF1 combinatorial protein are identical to those in IGF2. Some combinatorial protein binding defective IGF2 mutation systems are produced by mutating these residues in IGF2 with nearby Arg residues. It also shows that IGF2 mutants inhibit increased cell viability by WT IGF2. Therefore, these mutants are dominantly inactivated. These mutants are further characterized in that the above experiments identify which are the most potent antagonists of IR-A and IGF1R. However, IGF2 mutants inhibit the IR-A/IGF2 loop, which can serve as an important therapeutic target in cancer. IGF2 decoys can provide a greater therapeutic benefit for cancer patients.

IGF binding protein (IGFBP) may affect the half-life of the IGF2 decoy. Only a small fraction of all IGF2 (0.5 to 2% according to the literature) existed in free form, while the remainder was bound to IGFBP, of which approximately 80% bound to IGFBP3. It is possible to modify the binding position of IGF2 in IGFBP Inhibition of IGFBP binding to IGF2. The two amino acid residues of IGF2 (green dot in Figure 1) are key to IGFBP binding [1]. Additional mutations were introduced into the IGF2 decoy to determine if IGFBP binding in vivo affects the availability of the IGF2 decoy. It has been reported that IGF1 mutants that are unable to bind to IGFBP have a longer half-life than WT IGF1 (20 min for WT IGF1 and 20 h for IGF1 mutants that fail to bind to IGFBP) and are substantially more potent than WT IGF1 [28] . Therefore, similar mutations in IGF2 can improve the stability of IGF2 in vivo.

When IGF1R antibodies were used, IGF1R signaling in both normal and tumor IGF1R-positive cells was inhibited, and IGF1R in IGF1R-positive cells in the hypothalamic-pituitary axis involved in IGF1 feedback-induced inhibition of growth hormone secretion (GH) The communication was also suppressed. This results in a substantial increase in GH which stimulates the liver to increase IGF production and also causes insulin resistance in the insulin target tissue, which increases the glucose concentration, thereby causing an increase in insulin production. IGF1R tyrosine kinase inhibitors have a similar effect, but they also block the insulin receptor (review [29]). When the IGF2 decoy is used in vivo, it inhibits IGF1R and IR-A, which reduces the effect of enhancing insulin production on normal and cancer cells. However, since IR-B is not affected by IGF2 decoy, the effect of IGF2 decoy on normal cells is minimized. This was confirmed by observing the metabolism of IGF2 decoys in vitro and in vivo (eg, serum glucose and insulin concentrations).

Example 2 Development of a dominant negative IGF2 mutant (IGF2 decoy)

Purpose: IGF2 uniquely identifies IR-A and IR-B. IGF2 The dominant negative mutation system is designed to target IR-A, which is overexpressed in cancer and plays an important role in the progression of cancer.

Rationale: Blocking the IGF2 function is expected to reduce IR-A signaling and IR-B signaling without affecting IGF1, and can be used as a reasonable method to inhibit cancer progression. This goal was achieved by developing a dominant inactivation type of IGF2. The dominant negative form of IGF1 is directed against IGF1R but not IR-A or IR-B [9, 10, 30]. The dominant negative form of IGF2 targets IR-A (but not IR-B) and IGF1R and inhibits its activation. In the preliminary study, several candidate dominant negative IGF2 mutants have been established. The characteristics of these IGF2 mutants in vitro and in vivo are described in detail.

Method and material

Cell line: Since no transforming cells (NIH3T3) and several transformed cells (including human breast cancer MCF7, mouse breast cancer Met-1 and CHO cells) responded well to WT IGF1, they were previously used to study IGF1. Bait. These cell lines were used for this IGF2 decoy study. In addition, human breast cancer MDA-MB231 cells were used to study IGF2, which also exhibited IR-A in addition to IGF1R. HEK293 cells (commercially available from Clontech) that express the Tet-On transactivator can also be used to test inducible IGF2 decoy expression systems.

a) Characterization of combinatorial binding-deficient IGF2 mutants:

IGF1R binds. Use of soluble IGF1R as described previously to detect mutant binding to IGF1R by ELISA type analysis [30] Purchased on the surface). Briefly, if an IGF2 mutant can compete for binding to a fixed IGF1R in a 96-well plate in an ELISA-type competition binding assay, WT IGF2 will be biotinylated and tested. Most IGF2 mutants successfully competed with WT IGF2 for binding to IGF1R.

Tyrosine phosphorylation. It was examined whether the IGF2 mutant induced tyrosine phosphorylation of IR and IGF1R. Cells with IGF2 (WT and mutant) (such as MCF-7) were stimulated and cell lysates were analyzed by Western blot using antibodies specific for phospho-IR and phospho-IGF1R (commercially available). The combinatorial binding-deficient IGF2 mutant is defective in inducing IR and IGF1R phosphorylation, but the IGF2 mutant, which is not defective in combinatorial protein binding, retains the full signaling function.

b) Study the role of the combined protein IGF2 interacting in IGF2/IR-A or IGF1R communication:

IGF2 mutants were tested to investigate inhibition of signaling induced by WT IGF2. Culture and treat the non-transformed cells (such as NIH3T3 in a general tissue culture dish) or transformed cells (such as the above, such as MCF-7 in a plastic well groove coated with polyHEMA) by serum starvation. Reduce cell matrix interaction [10])). The cells are then stimulated with WT IGF2 and/or IGF2 mutants. Cell viability was measured using MTS analysis. Cell proliferation was analyzed using BrdU incorporation. When too much IGF2 mutant inhibits cell activity and proliferation induced by WT IGF2, it is a dominant inactivation as defined above [8, 9, 10]. Testing of IGF2 mutants inhibits IR induced by WT IGF2 in untransformed and transduced cells as described above And the ability of IGF1R to phosphorylate. Cell lysates were analyzed using Western blotting for antibodies specific for phosphorylated or non-phosphorylated IR and IGF1R [8, 9, 10]. Non-transformed HEK 293 cells that induced expression of IGF2 (WT and mutant) were made using the Tet-On system (data not shown). When initially induced to test for inducible IGF2 decoy induced by deoxycycline, further characterization of HEK293-transfected cells was expected due to the expected response of HEK293 cells to IGF2 decoy. As in NIH3T3 cells, the cell morphology and communication of the effect of the IGF2 decoy was examined.

result

IGF1 and IGF2 are similar in structure and signaling (except for IGF2 binding to IR-A but IGF1). Therefore, IGF2 is expected to require direct combinatorial protein binding, and the resulting complex formation (combined protein-IGF2-IGF1R/IR-A) plays a key role in IGF2 signaling. Furthermore, the key Arg residues (Arg36 and Arg37) that bind to the IGF1 combinatorial protein are identical to those in IGF2. During the preliminary study, some combinatorial protein-binding defective IGF2 mutation systems were generated by mutating these residues in IGF2 with nearby Arg residues. It shows that the IGF2 mutant inhibits increased cell viability by WT IGF2. Therefore, these mutants are dominantly inactivated. One goal of this study was to establish that IGF2 signaling through IGF1R and/or IR-A requires direct binding of protein binding, and IGF2 mutants that are defective in binding protein binding are dominantly inactive, like IGF1. Other purposes are to identify the most effective IGF2 decoys for a variety of practical applications.

discuss

One possible way to enhance the action of IGF2 mutants is to introduce pegylation into a predetermined combinatorial protein binding site. For this purpose, one of the K residues in the combinatorial protein binding site remains unchanged and all other K residues are mutated to R. The retained K residue is then crosslinked to the PEG. This strategy inhibits combinatorial protein binding and thus inhibits IGF signaling, resulting in a longer protein half-life.

Example 3 Study on the effect of IGF2 decoy on cancer stem cells and tumorigenesis in vivo initial research:

The previously produced dominant negative IGF1 affects the cancer phenotype. WT IGF1 is enhanced, while IGF1 decoy inhibits tumorigenesis, and tumor formation uses Met-1 mouse breast cancer cells, which are transfected with IGF1 decoy (Fig. 2). The IGF1 decoy is expected to inhibit IGF1R signaling in CSC and inhibit CSC-related phenotypes, including self-renewal capacity, enhanced epithelial-to-mesenchymal transition (EMT) potential, and increased resistance to therapeutic intervention ( Chemistry and radiation therapy). Whether the IGF1 decoy affects the type and performance of stem cell markers in Met-1 cells has been investigated. The IGF1 decoy induced an epithelial-like pattern (Fig. 6a) and decreased Oct-4 and Nanog expression in Met-1 cells transfected with IGF1 decoy (Fig. 6b). The IGF1 decoy inhibits the stem cell characteristics and dedifferentiation of CSC. Transfection of WT IGF1 does not effectively affect stem cells Characteristics and morphology, which may also be that Met-1 cells exhibit endogenous IGF1 or 2.

Fundamental

In a preliminary study, the IGF2 mutant was composed in a secretory vector in the triple negative breast cancer cell line MDA-MB231. These transfected cells did not grow well (data not shown). This observation strongly supports the theory that these are effective inhibitors of IGFR/IR-A communication. This may be a challenge in in vitro research. Alternatively, it may modulate the expression of IGF2 WT and mutants using a Tet-On inducible system [31] (Clontech Lab). HEK293 cells that induce expression of IGF2 (WT and mutant) have been produced.

HEK293 transfectants are useful for establishing dominant inactivation of IGF2 mutants in non-transformed cells. IGF2 expression was induced by the use of deoxytetracycline in the culture medium and tested for the ability of IGF2 mutants to inhibit IGF signaling and proliferation. Like transformed cells, MDA-MB231 human triple-negative breast cancer cells were selected for their high expression of IR-A. Met-1 mouse breast cancer cells were also selected to use homologous mice with intact immune systems. As described in IGF1, transfected cells are characterized by the formation of colonies in soft agar in vitro, in response to serum starvation and chemotherapy (by MTS analysis), and in hyperplasia (by BrdU incorporation analysis) Ability [10]. WT IGF2 enhances cell survival, proliferation, and tumorigenesis, whereas dominant negative IGF2 mutants (IGF2 decoy) inhibit it.

Research design a) Effect of mutant IGF2 on the expression of cancer stem cells (CSC)

1) Effect of IGF2 decoy on the expression of stem cell characteristics: Study the effect of IGF1 decoy on the phenotype of MDA-MB231 cells. Since homologous mice with intact immune systems can be used for in vivo tumorigenesis studies (Figure 2), Met-1 mouse breast cancer cell lines are derived from the widely used polyomavirus medium T (PyV-mT) transgenic mouse model. Breast cancer is produced [32], which induces the expression of IGF2 WT and mutants. First, cancer cells that induce secretion of WT IGF2 or IGF2 decoy have intact stem cell characteristics without further proliferation or colonization, tumorigenesis, morphology, and gene expression. IGF2, which is expressed in transfected cells, is induced and the cells are then fixed and stained with Oct-4 and Nanog for labeling with other stem cells. Secretion of IGF2 (6His tag) was detected by Western blotting using a medium using anti-His antibody. A laser scanning cytometer was used to quantitatively analyze the performance profile of stem cell characteristic markers in cells. As a control group, cells treated with WT IGF2 or cells not treated with IGF2 were used. WT IGF2 is enhanced, while IGF2 decoy inhibits the performance of stem cell signatures. Alternatively, qtPCR was used to observe the concentration of stem cell markers. These experiments demonstrate that the IGF2 decoy is directed against CSC.

2) The effect of IGF2 decoy on the CSC population. The side population (SP) methodology will be used as one of the tools for analysis in this study. It is shown to be activated in active ABCG2 efflux pumps in many types of stem cells and pump the cell-incorporated DNA binding dye Hoechst 33342 out of stem cells (SP). SP cells were detected using a flow cytometer. The IGF2 decoy was reduced, while the WT IGF1 enhanced the proportion of SP cells.

3) Effect of in vitro IGF2 decoy on chemotherapy. Master of CSC One of the characteristics is the resistance and rebound of the killing effect caused by chemotherapy. The functional characteristics of the CSC are tested along with the measurement of the CSC mark. Cell viability was compared between parental cells (non-CSC) and CSC in the presence of IGF2 decoy or WT IGF2. It was also tested whether the IGF2 decoy competitively inhibited the increased cell viability of WT IGF2 in the presence of a chemotherapeutic agent. WT IGF2 enhances chemotherapy while IGF2 decoy decreases. The performance of stem cell markers was observed during treatment.

4) Evaluation of tumor initiation ability by in vivo IGF2 decoy. Cells with a CD44 ⁺ /CD24 ^- expression profile have been widely accepted as cancer-like stem cells in breast cancer. Two methods were used to assess the efficacy of IGF2 decoy in preventing and/or inhibiting tumor-initiating ability of murine Met-1 cells (in syngeneic mouse models) or human MDA-MB-231 (in immunocompromised xenograft models). CD44 ⁺ /CD24 ^- cells were sorted and collected via the Aria III FACS system. The sorted cells (or mammary gland cells produced under serum-free conditions) were injected in situ into the mammary fat pad of the mouse. Tumor-inoculated mice received IGF2 decoy (experimental group), vehicle control group, and IGF2 mutant (experimental group) for observation of tumor formation in vivo. Since these cells are modified to contain a dual-reporter system (from the Stanford University Molecular Imaging Research Program (MIPS) Dr. Sanjiv Gambhir generous gift: Firefly luciferase 2 and enhanced GFP, L2G Therefore, non-invasive in vivo tumor formation observation (growth and metastasis) can be achieved vertically in the same animal. Tumor biopsies were collected after the experimental period and used for further analysis.

b) Effect of IGF2 decoy on tumorigenesis

A variety of in vivo tumor generation experiments were performed to analyze tumor pathology. As described, cancer cells that induced expression of IGF2 (WT or mutant) were injected into mice and tumor formation was observed [10]. The cancer cells are injected subcutaneously. The expression of IGF2 is induced in vitro by deoxytetracycline in the matrix, while in the body by drinking water. Analysis of tumor tissues revealed that IGF2 mutations inhibit tumorigenesis and angiogenesis by affecting IR-A signaling in tumor cells and microenvironments. In order to maintain the integrity of the host immune system, in addition to xenografts, a combination of Met-1 mouse breast cancer cells that induce secretion of IGF2 decoy and homologous mice (FVB mice) were also used. The induction of IGF2 decoy secretion begins when the tumor is inoculated, first confirming that the IGF2 decoy participates in tumorigenesis. When this is successful, the timing of IGF2 decoy induction (such as when the tumor system is detectable, when the tumor is fully developed) can be altered. This shows whether the IGF2 decoy inhibits the development of a complete tumor. By observing the extent of angiogenesis, it can be determined whether the IGF2 decoy inhibits angiogenesis. Transfected cells were treated with deoxytetracycline prior to inhibition of CSC population in cancer cells. The concentration of CSC in vitro was tested as described above. Tumor formation in pretreated cells in vivo was observed (no further oxytetracycline treatment). Pretreated cells showed reduced tumor production compared to untreated cells.

Statistical analysis and assay power analysis: Comparison of tumor growth curves across groups using standard repeated measurement of mixed models [24]. These models allow for unequal measurement spacing or length of subsequent studies, for example, if some mice develop an unsustainable tumor burden and sacrifice early. Develop these models to The IGF2-treated mice and control groups were specifically tested and then tested for the effect of increasing dose on tumor growth rate to identify the optimal dose concentration. A qualitative analysis of the in situ transplantation of mouse breast cancer Met-1 has been found to detect a difference of 20% between the treatment and control groups of 8 mice per group [26,27]. Therefore, typically 10 to 12 mice are used per group unless the data from the trial indicate better than 20%, under which 8 mice are used per group.

result

The IGF2 decoy is a potent antagonist of IGF1R and IR-A and affects the stem cell characteristics and proliferation of CSC. IGF2 decoys are more effective at inhibiting tumorigenesis than IGF1 decoys. By comparing the IGF2 decoy and the IGF1 decoy, it is possible to assess the effect of IR-A that inhibits the CSC phenotype. The IGF2 mutant can be further described in detail to identify which of IR-A and IGF1R is the most potent antagonist. The IGF2 decoy inhibits the IR-A/IGF2 loop (an important therapeutic target in cancer). The IGF2 decoy can therefore provide a greater benefit to cancer patients.

discuss a) Effect of IGF2 decoy on heterozygous receptors

Whether the IGF2 decoy effectively inhibits heterozygous receptors containing IR-B (Table 1), IRB/IR-A, and IR-B/IGF1R is not known at this time. To address this problem, individual heterozygous receptors (such as CHO cells) lacking IR on cells and detecting heterozygous receptors in response to IGF2 stimuli The specificity of the bait.

b) short half-life of the IGF2 decoy

1) IGFBP binding to defective IGF2 decoy: IGF binding protein (IGFBP) may affect the half-life of IGF2 decoy in vivo. Only a small fraction of the total IGF2 (0.5 to 2% according to the literature) was present in free form, while the remainder was bound to IGFBP, of which approximately 80% bound to IGFBP3. It is possible to inhibit IGFBP binding to IGF2 by modifying the binding site of IGF2 at IGFBP. The two amino acid residues of IGF2 (green in Figure 1) are key to IGFBP binding. Additional mutations were introduced into the IGF2 decoy (such as the E7R mutation) to test whether IGFBP binding in vivo affects the availability of the IGF2 decoy. It has been reported that IGF1E3R mutants that are unable to bind to IGFBP are substantially more potent than WT IGF1 [28]. Therefore, similar mutations in IGF2 may be more effective than IGF2.

2) Continuous infusion of IGF2 decoy: stabilization of the IGF2 decoy in the body may be difficult. To solve this problem, a small device (insulin pump) can be used for continuous infusion of insulin (this is programmable) to the patient (Figure 7). An osmotic pump can also be used for the same purpose.

3) PEGylation of IGF2 mutants (modified by biomolecules covalently conjugated to polyethylene glycol (PEG), a non-toxic, non-immune polymer): production with site-specific PEGylation IGF2 mutant. PEGylation has long been used to extend the half-life of biological agents. Site-directed PEGylation of WT IGF1 (in Lys-68) has been reported to have a longer half-life than the unpegylated version (20-30 min) (>140h). The PEGylation of IGF2 in vivo does not seriously affect efficacy [33]. Site-directed pegylation of Lys-68 by cleavage of two other Lys residues at positions 25 and 60 to Arg and removal of the N-terminal portion by hydrolysis to remove PEGylation of the N-terminal amine residue Achieved. IGF2 has only Lys residues at position 65. IGF2 (WT and mutant) were PEGylated, and the N-terminal PEG was then removed by hydrolysis (thrombin) to remove the N-terminal portion. Branched PEG (NHS-PEG) (molecular weight 40 Kd) (commercially available) was activated using branched N-hydroxysuccinylamine. As stated, the conditions for crosslinking are in buffer pH 8 to 10 and cultured at 4 to 22 ° C for up to 1 h [33]. The N-terminal His tag and PEG were digested overnight at 25 ° C by thrombin. The digested material is further purified by gel filtration and ion exchange chromatography.

4) IGF2 WT and mutants as Fc fusion proteins: Fc fusion has been widely used to stabilize proteins in vitro and in vivo. Two Fc-fusion proteins of the TNF receptor (Etanercept/Enbrel) mimic the inhibition of naturally occurring soluble TNF receptors and have a significantly extended half-life (70 to 132 hrs) in the bloodstream, and therefore organisms The effect is stronger and longer lasting than the naturally occurring soluble TNF receptor. The IGF2 decoy Fc-fusion protein (His tag) was synthesized in CHO cells and purified using NA-NTA affinity chromatography. To study the stability and utility of the IGF1 decoy Fc fusion protein. In the preliminary study, Fc-IGF1 was fully functional and Fc-IGF2 was also functional.

Example 4 Study of combined protein and IGF1R/IR- under IGF1/IGF2 stimulation A interaction Fundamental

In previous biochemical studies, it was shown that IGF1 induces the formation of ternary complexes on the cell surface (combined proteins αvβ3 and α6β4-IGF1-IGF1R). IGF2 also induces the formation of a ternary complex (combined protein αvβ3/α6β4-IGF2-IGF1R). Since IGF2 binds to IR-A, IGF2 is expected to induce the formation of the combinatorial protein-IGF2-IR-A complex. Two methods were used to detect the formation of ternary complexes: coprecipitation and imaging. It shows the association of the combination protein with IGF1R/IR-A and the combination protein by coprecipitation.

Research design a) Study the interaction between the combination protein and IGF1R/IR-A by co-immunoprecipitation

MDA-MB231 or other cancer cells that exhibit IR-A, IGF1R or hybrid receptors are used for this purpose. As used previously for IGF1, cells with WT or mutant IGF2 were stimulated and the combination protein β3 was immunoprecipitated from cell lysates. IGF1R or IR-A in purified material is detected by Western blotting using antibodies specific for IGF1R or IR. WT IGF2 induces the formation of the combined protein-IGF2-(IR-A/IGF1R) complex, but the IGF2 mutant does. This demonstrates that IGF1R/IR-A and combinatorial proteins interfere with each other in an IGF2-dependent manner. Due to human intervention and severe conditions, the co-immunoprecipitation technique has some limitations: it does not reflect the interaction of living cell contents, and the transmission of high frequencies is often difficult. Explain the pseudo-positive and quantitative [34]. Therefore, quantitative live cell imaging with better resolution in time and space is preferred.

b) Studying the interaction between the combination protein and IGF1R/IR-A by imaging and micropatterning

Micropattern-based quantification: Techniques for studying membrane protein into specific micropatterns in the plasma membrane to study protein-protein interactions have been published [35]. This concept is to rearrange the bait directly laterally in the viable cell plasma membrane by growing cells on the micropatterned surface by binding the partner to the bait (eg, IGF1R). The bait-prey interaction is read by quantifying the common redistribution of fluorescent prey (such as combinatorial proteins) (Figure 8). As noted, the creation of microstructured surfaces will be performed by microcontact printing [36]. Antibodies or ligands comprising biotinylated endocrine, toxin or purified protein can be applied to bait redistribution. Most importantly, this technology has been successfully used to analyze receptor tyrosine kinase (RTK) downstream signaling events including EGFR, IR, and IGF1R [36].

Localization of β3-EGFP and IGF1R-RFP: β3-GFP having a conformation of GFP at the C-terminus of the combination protein β3 was produced as described [37]. In a preliminary study, β3-GFP and IGF1R-RFP lines were expressed in HeLa cells and colocalization of β3 with IGF1R was studied (Fig. 9). The β3 combination protein is co-assembled into the IGF1R-rich region, indicating the interaction between the IGF1R and the β3 combination protein. Since this experiment was performed in the presence of serum, it is likely that the cells were exposed to WT IGF1/IGF2. The effect of dominant negative IGF2 (IGF2 decoy) was investigated using a serum-free matrix. WT IGF2 assists in the formation of a ternary complex (combined protein-IGF2-IGF1R) but the IGF2 decoy (combined protein binding lacks mutation) does not.

Interaction between the combination protein β3 and IR-A: it investigated whether the combination protein β3 interacts with IR-A in an IGF2-dependent manner. IR-A-GFP was produced by mutagenesis from IR-B-GFP (pEGF-N, available from Addgene) to remove 12 amino acid residues (since alternative splicing IR-A is shorter than IRB) 12 amino acids). The resulting IR-A cDNA was inserted into the RFP vector (pRFP-N). HeLa cells were transiently transfected with IR-ARFP and β3-combined protein-GFP and grown on microbial wafers coated with anti-IGF-IR antibodies. When WT IGF2 is present, the β3-combined protein-GFP is co-assembled into the IR-A-RFP-rich region. When the IGF2 decoy is present, the β3-combined protein-GFP is not co-assembled into the IR-A-RFP, indicating the interaction between the IR-A and β3-combined proteins, and this interaction requires IGF2 combinatorial protein interaction.

result

IGF2 induces the binding protein-IGF1R/IR-A interaction through the formation of a ternary complex. The IGF2 decoy is a reagent for solving this problem. WT IGF2 induces the formation of a ternary complex, but the combination protein binds to the defective IGF2 decoy. In a preliminary study, it was shown that the combinatorial protein β3-EGFP was present in the region rich in IGF1R-RFP (Fig. 9). This co-localization may be IGF1/IGF2-dependent (using IGF1 and IGF2 decoy detection). Photo-bleaching fluorescence recovery (FRAP) is used in more detail when establishing IGF1R/IR-A interaction with the combination protein in an IGF2-dependent manner Study this interaction. FRAP is an optical technique that quantifies the two-dimensional lateral diffusion of fluorescently labeled probes and is therefore a very useful tool for protein-bound biological research. The FRAP experiment confirmed whether the combination proteins β3 and IGFIR interacted dynamically.

discuss

When antibodies to IGF1R are used, IGF1R signaling is in both normal and tumor IGF1R positive cells, as well as in IGF1R positive cells in the hypothalamic-pituitary axis involved in IGF1 feedback inhibition of growth hormone (GH) secretion. inhibition. This will result in a substantial increase in GH, which stimulates the liver to increase IGF production and also causes insulin resistance in insulin target tissues to increase glucose concentration and thereby lead to increased insulin production. IGF1R tyrosine kinase inhibitors have a similar effect, but they also block the insulin receptor. When an IGF2 decoy is used in vivo, its inhibition reduces IGF1R and IR-A, which enhance the role of insulin production in normal and cancer cells. However, since IR-B is not affected by IGF2 decoy, the effect of IGF2 decoy on normal cells will be minimized. This can be addressed by observing the metabolism of the IGF2 decoy in vitro and in vivo (eg, serum glucose and insulin concentrations).

All patents, patent specifications, and other publications (including GenBank accession numbers) cited in this specification are incorporated by reference in their entirety for all purposes.

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Claims (32)

A method for inhibiting IGF2 signaling in a cell comprising the step of contacting the cell with an effective amount of an IGF2-combined protein binding inhibitor. The method of claim 1, wherein the combinatorial protein is αvβ3. The method of claim 1, wherein the combination protein is α5β1 or α6β4. The method of claim 1, wherein the inhibitor comprises two substituted IGF2 mutants, R37E and R38E. The method of claim 1, wherein the inhibitor is the IGF2 mutant R24E/R37E/R38E. The method of claim 1, wherein the inhibitor is the IGF2 mutant R34E/R37E/R38E. The method of claim 1, wherein the inhibitor is an IGF2 mutant R24E/R34E/R37E/R38E. The method of claim 1, wherein the cell line is in a patient. The method of claim 1, wherein the contacting step is performed by oral or intravenous, subcutaneous, intraperitoneal or intratumoral injection. A method for identifying an IGF2-binding protein binding inhibitor, the method comprising the steps of: (1) binding a combination protein to a combination protein comprising IGF2 in the presence of a test compound under conditions permitting binding of the IGF2-combined protein The polypeptide contact of the column; and (2) the degree of binding of the polypeptide-combined protein is detected, wherein a decrease in the degree of binding when compared to the degree of binding in the absence of the test compound indicates that the compound is an IGF2-combined protein binding inhibitor. The method of claim 10, wherein the combinatorial protein is αvβ3. The method of claim 10, wherein the combination protein is α5β1 or α6β4. The method of claim 11, wherein the polypeptide comprises a C domain sequence of a human IGF2 protein. The method of claim 11, wherein the polypeptide comprises the full length of the human IGF2 protein. The method of claim 11, wherein the polypeptide further comprises a heterologous amino acid sequence. The method of claim 15, wherein the heterologous amino acid sequence is glutathione S-transferase (GST). The method of claim 11, wherein the combined protein is expressed on the cell surface. An isolated polypeptide comprising an amino acid sequence, the amino acid sequence (1) having at least 95% sequence identity to a wild-type IGF2 protein sequence; (2) comprising at positions 37 and 38 of the wild-type human IGF2 protein Substitution of at least two Arg residues; and (3) inhibition of IGF2-binding protein binding. Such as the polypeptide of claim 18, wherein the combination egg White αvβ3. The polypeptide of claim 18, wherein the combination protein is α5β1 or α6β4. A polypeptide according to claim 18, wherein at least three of said Arg residues at positions 24, 34, 37 and 38 are substituted. The polypeptide of claim 18, wherein the Arg residues at positions 24, 34, 37 and 38 are substituted. A polypeptide according to claim 21 or 22, wherein each of said Arg residues is substituted with a Glu residue. A composition comprising the polypeptide of claim 18 and a pharmaceutically acceptable excipient. The composition of claim 24, wherein the polypeptide is an IGF2 mutant R24E/R37E/R38E. The composition of claim 24, wherein the polypeptide is an IGF2 mutant R34E/R37E/R38E. The composition of claim 24, wherein the polypeptide is an IGF2 mutant R24E/R34E/R37E/R38E. An isolated polynucleotide encoding a polypeptide as in claim 18 of the patent application. A recombinant expression cassette comprising a polynucleotide as in claim 28, which polynucleotide is arbitrarily linked to a promoter. An isolated host cell comprising a performance cassette as set forth in claim 29 of the scope of the patent application. a composition comprising, as claimed in claim 28 A polynucleotide or a performance cassette as described in claim 29 of the patent application and a pharmaceutically acceptable excipient. A kit for suppressing IGF communication, comprising the composition of claim 24 or 31.