WO2012019192A2 - Cell-permeable molecules as growth factor receptor antagonists - Google Patents

Abstract

The invention provides growth factor receptor inhibitory molecules and methods of identifying such molecules, as well as methods of making such molecules and methods of using such molecules, for example in the treatment of cancer such as breast, colorectal, pancreatic, or lung cancer.

Description

CELL-PERMEABLE MOLECULES AS

GROWTH FACTOR RECEPTOR ANTAGONISTS Cross References to Other Applications

[0001] This application claims priority from U.S. Provisional Application 61/371,589, filed August 6, 2010, and U.S. Provisional Application 61/454,209, filed March 18, 2011. The disclosures of those applications are incorporated by reference herein in their entirety. Government Interest

[0002] This invention was made with U.S. government support under Grant No. DK 38761, awarded by the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health. The U.S. government may have certain rights in the invention. Field of the Invention

[0003] This invention relates generally to the field of modulating protein activity and, in particular, to modulating an activity of a growth factor receptor such as a receptor tyrosine kinase. Background of the Invention

[0004] The epidermal growth factor receptor (EGFR, also known as ErbBl and HER-1) is a member of the ErbB family of transmembrane receptor tyrosine kinases (RTKs). There are several ligands that bind one or more receptors in the ErbB family. The most common ligand for EGFR is the epidermal growth factor (EGF). Upon ligand binding, a conformational change allows for either homodimerization or heterodimerization and subsequent cross- phosphorylation at the intracellular C-terminal tail. The phosphorylated receptor provides a docking site for several adaptor proteins and enzymes which propagate the signals that eventually lead to a phenotypic outcome such as cell proliferation, growth, differentiation or apoptosis. The nature of the signal and outcome depends on the identity of the signaling receptors and ligand and the levels and availability of the various adaptor proteins and downstream effector enzymes.

[0005] EGFR is overexpressed in a majority of epithelial solid tumors and its

overexpression is often correlated with poor clinical outcome and therapeutic resistance. Additionally, deletions, insertions and point mutations that lead to a constitutively active form of EGFR having enhanced kinase activity are found in cancers, such as brain and lung cancers, respectively. Currently, there are several FDA approved drugs that target EGFR. For example, Erbitux® (cetuximab, Imclone Therapeutics, NY), a monoclonal antibody (mAb), binds to the extracellular ligand-binding domain of EGFR and blocks EGFR’s ligand binding. Herceptin® (trastuzumab, Genentech), another mAb, targets an EGFR family member, ErbB2. The tyrosine kinase inhibitors (TKIs), Iressa® (gefitinib, AstraZeneca, currently withdrawn from U.S. market) and Tarceva® (erlotinib, OSI pharmaceuticals), bind to and inhibit the active site of the intracellular EGFR kinase domain. However, a novel approach is necessary for EGFR targeted therapies because of limitations of the current therapies. For example, EGFR mAb therapies can cause severe allergic reactions. Further, the demonstrated effectiveness of Erbitux® is tumor shrinkage, not lengthened lifespan. There are also significant side effects associated with EGFR TKI treatment, such as bleeding and clotting problems that can lead to heart attack or stroke. TKI therapy also is often not very effective. In fact, Iressa® was relabeled to be given only to patients who have shown prior progress due to treatment with Iressa®, since no significant difference between Iressa® and placebo treatment was shown in clinical trials. In the majority of cases, the TKI and mAb therapies are prescribed in conjunction with other chemotherapies.

[0006] For the foregoing reasons, it is apparent that a need persists in the art for anti-cancer therapies that effectively target cancerous cells while presenting a minimized risk of deleterious effect on healthy cells of an animal such as a mammal, e.g., a human. Summary of the Invention

[0007] The disclosure satisfies at least one of the aforementioned needs by providing materials and methods for treating a variety of cancers using therapeutic peptides capable of modulating signal transduction mediated by a growth factor receptor, such as a receptor tyrosine kinase, thereby leading to effective treatment of cancer cells while exhibiting a greater degree of selectivity in deleteriously harming cancer cells versus healthy cells than is found with current anti-cancer treatment methodologies.

[0008] The invention provides an isolated peptide. In some embodiments, the isolated peptide comprises an amino acid sequence that is 15 or more amino acids in length (e.g., 15- 30 amino acids) and is derived from a juxtamembrane domain of a growth factor receptor, wherein the peptide inhibits access to the fully active conformation of the growth factor receptor. In some embodiments, the growth factor receptor is a receptor tyrosine kinase. In some embodiments, the peptide further comprises a cellular entry facilitator. Examples of cellular entry facilitators may include a TAT domain. In some embodiments, the peptide may comprise a sequence selected from the group consisting of SEQ ID NOS: 2-9, 13-25, and 28-35. In some embodiments, the peptide may comprise a sequence as set forth in SEQ ID NO: 2. In some embodiments, the peptide may comprise a sequence as set forth in SEQ ID NO: 3. In some embodiments, the peptide may comprise a sequence as set forth in SEQ ID NO: 32. In some embodiments, the peptide may be derived from the juxtamembrane domain of a member of the Epidermal Growth Factor Receptor family (e.g., EGFR or ErbB or HER-1, ErbB2 or HER-2, ErbB3, ErbB4), c-Met, Fibroblast Growth Factor Receptor (e.g., FGFR1, FGFR2), Platelet-derived Growth Factor Receptor (e.g., PDGFRA (PGFRA), PDGFRB (PGFRB)), Insulin Growth Factor Receptor (INSR), Insulin-like Growth Factor Receptor (e.g., IGF1R), or Vascular Endothelial Growth Factor Receptor (VEGFR (VGFR)) family of receptor tyrosine kinases. In certain embodiments, the peptide is derived from c- Met. In certain embodiments, the peptide is derived from Epidermal Growth Factor

Receptor. In some embodiments, the peptides according to the disclosure may be labeled using any conventionally known label, including fluorescent labels such as fluorescein and related compounds such as fluorescein isothiocyanate (FITC) and 5-carboxy-fluorescein (FAM).

[0009] In some embodiments, the peptide comprises one or more amino acid changes (e.g., substitutions, inserts and deletions) compared to said juxtamembrane domain. In certain embodiments, the one or more changes stabilize said peptide. In certain embodiments, the one or more changes induce the formation of a disulfide bonded bridge in the peptide. [0010] In some embodiments, the peptide comprises one or more modifications compared to said juxtamembrane domain. In certain embodiments, the one or more modifications comprise an aliphatic or other chemically inserted linker or bridge. Examples of aliphatic or chemically inserted linkers or bridges may include peptide linkers and cross-linking agents.

[0011] The invention further provides non-peptide mimetics of the juxtamembrane peptides discovered through functional screening, e.g., high throughput screening, with FAM- or FITC-labeled juxtamembrane peptide probes.

[0012] The invention also provides a kit. In some embodiments, the kit comprises a peptide comprising an amino acid sequence that is 15 or more amino acids in length and is derived from a juxtamembrane domain of a growth factor receptor, wherein the peptide inhibits access to the fully active conformation of the growth factor receptor, and a protocol for administration of the peptide to a subject in need thereof. In certain embodiments, the peptide further comprises a cellular entry facilitator.

[0013] The invention also provides a method for treating a condition in a subject in need thereof. In some embodiments, the method comprises administering a therapeutically effective amount of a peptide comprising an amino acid sequence that is 15 or more amino acids in length and is derived from a juxtamembrane domain of a growth factor receptor, wherein the peptide inhibits access to the fully active conformation of the growth factor receptor, to the subject. In certain embodiments, the peptide further comprises a cellular entry facilitator. In certain embodiments, the condition may be cancer. Examples of cancer include breast cancer, colorectal cancer, pancreatic cancer, and lung cancer. In alternative embodiments, the condition may be selected from the group consisting of malaria, chronic myeloproliferative disorders, and fibrotic diseases.

[0014] The invention also provides a method for preventing the development of cancer comprising administering a prophylactically effective amount of a peptide comprising an amino acid sequence that is 15 or more amino acids in length and is derived from a juxtamembrane domain of a growth factor receptor, wherein the peptide inhibits access to the fully active conformation of the growth factor receptor, to a subject at risk of developing cancer. In certain embodiments, the peptide further comprises a cellular entry facilitator. It is expected that prophylactically effective amounts of the therapeutic peptides will typically be within an order of magnitude of the dosing amounts effective for treatment, and it is recognized in the art that such dosing will be determined by the clinician after consideration of a variety of well-known factors (e.g., patient weight, age, general medical condition, degree of development of disease where relevant, type of disease where relevant, and the like).

[0015] The invention also provides a method of inhibiting an RTK in a cell, comprising contacting the cell with a molecule that binds to a juxtamembrane domain of the RTK and prevents multimerization of the RTK, thereby inhibiting the RTK in the cell. In certain embodiments, the molecule is an isolated peptide comprising an amino acid sequence that is 15 or more amino acids in length and is derived from a juxtamembrane domain of a receptor tyrosine kinase (RTK). In certain embodiments, the peptide further comprises a cellular entry facilitator. In certain embodiments, the cell is in the body.

[0016] The invention also provides a method of inhibiting multimerization of an RTK. In some embodiments, the method comprises contacting the RTK with a peptide comprising an amino acid sequence that is 15 or more amino acids in length and is derived from a juxtamembrane domain of a growth factor receptor, wherein the peptide inhibits access to the fully active conformation of the growth factor receptor. In certain embodiments, the peptide further comprises a cellular entry facilitator.

[0017] This invention also provides combination therapy methods in which the peptide of the invention can be administered concurrently with, prior to, or subsequent to, the administration of another therapeutic, such as another anti-cancer therapeutic, including, without limitation, a chemotherapeutic agent and a targeted therapy agent (e.g., an agent that targets an oncoprotein).

[0018] The invention also provides a method of identifying a molecule that inhibits a receptor tyrosine kinase (RTK). In some embodiments, the method comprises contacting a cell expressing the RTK with the molecule, and measuring the activity of the RTK in the cell, wherein a decrease in the activity as compared to the activity of the RTK in a control cell that has not been contacted with the molecule indicates that the molecule inhibits the RTK. In certain embodiments, the RTK is an EGFR. In certain embodiments, a receptor tyrosine kinase activity being inhibited is receptor tyrosine kinase-mediated signal transduction across the cell membrane.

[0019] The invention also provides a method for identifying a receptor tyrosine kinase (RTK) inhibitory molecule. In some embodiments, the method comprises incubating an RTK with a peptide comprising an amino acid sequence that is 15 or more amino acids in length and is derived from a juxtamembrane domain of a growth factor receptor, wherein the peptide inhibits access to the fully active conformation of the growth factor receptor, in the presence of a candidate RTK inhibitory molecule, and detecting the binding of the RTK to the peptide, wherein a decrease in the binding as compared to binding in the absence of the candidate molecule indicates that the candidate molecule inhibits the RTK. In certain embodiments, the peptide further comprises a cellular entry facilitator. In certain

embodiments, a receptor tyrosine kinase activity being inhibited is receptor tyrosine kinase- mediated signal transduction across the cell membrane. In certain embodiments, the candidate molecule is added prior to, concurrently with, or subsequent to, mixing the RTK and the peptide.

[0020] In some embodiments of the methods of this invention, the inhibition of the targeted growth factor receptor such as an RTK can be monitored through a surrogate biomarker. For example, the activation of EGFR correlates positively with the level of phospho-Erk and/or phosphor-Akt in a cell. Thus, EGFR activity can be measured by the level of cellular phospho-Erk and/or phosphor-Atk, and inhibition of EGFR can be indicated by decreased cellular phospho-Erk and/or phosphor-Akt levels.

[0021] Other features and advantages of the present invention will be better understood by reference to the following detailed description, including the drawing,the examples, and claims. Brief Description of the Drawings

[0022] FIGS. 1A and 1B are graphs showing cell viability assay results obtained by exposing MDA-MB-231 cells to varying concentrations of (A) TAT-EGFR (SEQ ID NO:3; circle), TAT peptide-RRRRTLRRLLQER (SEQ ID NO:8; square), RRRR-EGFR (SEQ ID NO:31, RRRRRRRHIVRKRTLRRLLQER; up arrow;“RRRR” disclosed as SEQ ID NO:38), or TAT peptide-RRRHIVRKRTLRRL (SEQ ID NO:32; down arrow), or (B) TAT- EGFR, TAT peptide-TLRRLLQER (SEQ ID NO:6; down arrow), or TAT peptide- VRKRTLRRLLQER (SEQ ID NO:5; square). Percent cell viability is plotted as a function of the log molar concentration of peptide.

[0023] FIGS. 2A-C are fluorescence microscopy images showing the distribution of (A) FAM-TAT-EGFR peptide in MDA-MB-231 cells after the cells had been incubated with the peptide for approximately 20 minutes, and (B) FAM-EGFR peptide (SEQ ID NO:3 without the TAT sequence) and (C) FAM-TAT peptide (SEQ ID NO:11) in MDA-MB-231 cells after the cells had been incubated with the respective peptide for several hours. Peptides were dosed at 1 μM. FAM is 5-carboxyfluorescein succinimidyl ester.

[0024] FIG. 2D is a graph showing the activity of the FAM- conjugated peptides (FAM- TAT-EGFR, FAM-EGFR and FAM-TAT) in a cell viability assay. MDA-MB-231 cells were serum starved and treated with the respective peptide for 24 hours.

[0025] FIGS. 3A-D are graphs showing results of cell viability assays and tumor size. Fig. 3A: MDA-MB-231 cells (breast cancer) or DLD-1 cells (colorectal cells) were treated with the TAT-EGFR peptide or the TAT peptide in serum-starved media (containing 0.1% BSA; “SS”) or in media containing 2.5% serum. FIG. 3B: The indicated cell types (A-549 (non- small cell lung cancer), Hep2G (hepatic cancer), DLD-1 (colorectal cancer), MDA-MB-435 (breast cancer), MDA-MB-231 (breast cancer), SK-BR-3 (breast cancer), and BT-474 (breast cancer)) were treated with the TAT-EGFR peptide or vehicle in serum-starved media (containing 0.1% BSA) or in media containing 2.5% FBS for 36 hours. The IC₅₀ values of the TAT-EGFR peptide in the various treatment conditions are shown in Table 4. FIG. 3C is a graph showing results of cell viability assays. MDA-MB-231 breast cancer cells and non- cancerous HMEC-689 and HMEC-636 cells were treated for 24 hours with the TAT-EGFR peptide. FIG.3D: MDA-MB-231 cells were dosed with the TAT-EGFR peptide alone (IC₅₀ of 7.7 μM) or in combination with 1 mg/ml Erbitux® (IC₅₀ of 3.8 μM). The viability is calculated by dividing the luminescent signal of each sample by that of the control cells. The TAT control peptide did not display any activity up to 200 μM in all cell lines tested.

[0026] FIGS. 4A-C are bar graphs showing results of soft agar assays. FIG. 4A: MDA- MB-231 were plated in agar containing media with 5% serum and treated twice weekly with 25 μM or 40 μM TAT-EGFR peptide, 40 μM TAT peptide, or vehicle (“control”). FIG.4B is a bar graph showing the effects of the TAT-EGFR peptide on colony formation. MDA- MB-231 breast cancer cells, DLD-1 colorectal cancer cells, A-549 lung cancer cells, and MIA-PaCa2 pancreatic cancer cells were treated with 10 μM, 20 μM, or 40 μM TAT-EGFR, 20 μM TAT peptide, or a vehicle control. Colonies were allowed to grow for two weeks. FIG.4C: MDA-MB-231 cells were plated as described above and treated with 10 μM TAT- EGFR peptide, 10 μM TAT-EGFR peptide with 0.5 mg/ml Erbitux®, 0.5 mg/ml Erbitux® alone, or vehicle. Colonies were counted manually after two weeks. Significance in Student's unpaired t-test relative to control value: P<0.05, 99% confidence interval. FIG.4C shows that in the Erbitux® synergy study, the combination group and the TAT-EGFR alone group are not significantly different from each other but the combination group is different from controls whereas the TAT-EGFR dose alone is not.

[0027] FIG. 5 is a graph with a table showing the anti-tumor effect of the EGFR peptide. MDA-MB-231 xenografts were grown in NCR/nude female athymic mice. Mice were injected twice weekly with 1 mg of TAT-EGFR or TAT peptide in 200 μl of PBS, or with PBS alone, subcutaneously proximal to the tumor site. Data shown are from a study designated Study I. Tumor size was measured using a caliper twice weekly and tumor volume was calculated by 0.5*length*width*height. The fold growth was calculated by dividing the size at each time point by the initial size of the tumor. Mice were sacrificed if the tumor reached a volume > 2000 mm³ or a dimension of 2 cm, or if tumor ulceration occurred. The graph on top shows a tumor growth curve plotted as a function of time in mice injected as described above. The table below indicates the number of mice that were within the endpoints of the study protocol.

[0028] FIG. 6 is a graph with a table showing the effect of the TAT-EGFR peptide on tumor growth in mice. Data are from a study designated Study II. The description for this figure is the same as that for FIG.5.

[0029] FIGS. 7A-B are graphs showing survival of mice treated with the TAT-EGFR peptide, measured by the number of days of survival without reaching an endpoint of weight loss or tumor size >2000 mm³. Circles: Mice treated with the TAT-EGFR peptide; Squares: Mice treated with saline; and Up arrows: Mice treated with the TAT peptide. Data from Study I are shown in FIG. 7A; data from Study II are shown in FIG.7B.

[0030] FIGS. 8A-C are graphs showing an anti-tumor effect of the TAT-EGFR peptide. MDA-MB-231 xenografts were grown in NCR/nude female athymic mice. Mice were injected with (1) 200 μl of 5 mg/ml TAT-EFR in saline, (2) a combination of 200 μl of 2 mg/ml Erbitux® in saline followed by 200 μl of 5 mg/ml TAT-EGFR peptide in saline, (3) 200 μl of 2 mg/ml Erbitux® in saline, or (4) saline alone as a control. Tumor size was measured using a caliper twice weekly and tumor volume was calculated by

0.5*length*width*height. FIG.8A provides data from Study III. FIG.8B provides data from Study IV, which involved mice injected peritoneally with the above dosages of the TAT-EGFR peptide, the TAT peptide, or saline. The graphs on top show the amount by which the tumors grew on each treatment day relative to the day that the treatment was initiated. Tables below indicate the number of mice that were within the endpoints of the study protocol. The fold tumor growth was calculated by dividing the size at each time point by the initial size of the tumor. Circles: Mice treated with TAT-EGFR peptide; Diamonds: Mice treated with TAT-EGFR peptide and Erbitux® (Etux); Down arrow: Mice treated with Erbitux® (Etux); and Squares: Mice treated with saline. FIG.8C provides data from Study IV showing tumor size over the course of treatment. Circles: Mice treated with TAT-EGFR peptide; squares: mice treated with the TAT peptide; triangles: mice treated with saline. Asterisks (*P 0.0325) indicate that the average size of the TAT-EGFR treated tumors is statistically different from the sizes of the saline- and TAT-treated tumors at that time point, using a one-tailed, nonparametric Mann-Whitney tests (95% confidence interval).

[0031] FIGS. 9A-B are graphs showing survival of mice treated with the TAT-EGFR peptide, measured by the number of days of survival without reaching an endpoint of weight loss or tumor size >2000 mm³. Circles: Mice treated with the TAT-EGFR peptide; Squares: Mice treated with saline; Up arrow: Mice treated with Erbitux®; Down arrow: mice treated with the TAT peptide; Diamond: Mice treated with TAT-EGFR in combination with

Erbitux®. Data from Study III are shown in FIG.9A; data from Study IV are shown in FIG. 9B. [0032] FIGS. 10A-B are images showing in vivo images of the distribution of the FAM- TAT-EGFR peptide. FIG.10A shows the distribution (1) before injection (control; left panel), (2) just after injection (center panel), and (3) 5.5 hours after injection (right panel, mouse on right). The image of the mouse on the left in the right panel was taken 5 hours after a subcutaneous injection with the FAM-TAT-EGFR peptide. FIG.10B shows images of FAM-TAT-EGFR peptide distribution, taken approximately every 2 minutes, from 20 minutes post injection until approximately 1 hour post injection. Note that the mouse was turned between different image acquisitions to show differential distribution.

[0033] FIGS. 11A-B are graphs showing results of cell viability assays. MDA-MB-231 cells (FIG. 11A) and A-549 cells (FIG.11B) were simultaneously treated with hepatocyte growth factor (HGF) and with a TAT-conjugated peptide derived from a c-Met

juxtamembrane domain sequence. The c-Met juxtamembrane peptide sequence is

KKRKQIKDLGSELVRYDARV (SEQ ID NO:2).

[0034] FIG. 12 is a graph showing the activity of an ErbB2 peptide in SKBR3 breast cancer cells. SKBR3 cells were plated in 96-well plates, serum-starved for 2 hours, and treated with the TAT-EGFR peptide for 48 hours. Cell viability was detected using a colorimetric tetrazolium cell viability dye.

[0035] FIGS. 13A-B are graphs showing Biacore^™ SPR binding isotherms for various concentrations of the EGFR peptide (FIG. 13A) and a peptide derived using the same logic in the juxtamembrane region of the insulin receptor (INSR,

RKRQPDGPLGPLYASSNPEYLSASDV; SEQ ID NO:24) (FIG.13B) binding to immobilized PIP2. Both peptides bind to PIP2. These data demonstrate that another JXM peptide is derived from a unique, active, specific region of the JXM domain.

[0036] FIGS. 14A-D are graphs depicting the mode of cell death induced by TAT-EGFR (645-662). MDA-MB-231 breast cancer cells were serum starved overnight then treated with 0 (control), 6, or 12 μM TAT-EGFR for 18 hours in FIGS.14A-B or 0 (control) or 10 μM TAT-EGFR for 30 minutes or 3 hours in FIGS.14C-D. Cells were stained with Annexin-V and propidium iodide (PI). Staining was as follows: unstained viable cells (lower left quadrant; propidium iodide staining of non-viable cells (upper left quadrant); Annexin-V plus propidium iodide staining of fully apoptotic and necrotic cells (upper right quadrant); annexin-V staining of apoptotic cells (lower right quadrant). The numbers in each quadrant represent the percentage of events/cells gated in each quadrant. In FIGS.14B and 14D, The results of four separate experiments were quantified and compared. A two-tailed, unpaired t test was used to determine whether the mean value for each treatment condition was significantly different from control with a 95% confidence interval (* signifies P=0.035; ** P<0.004).

[0037] FIGS. 15A-D are Western blot images for cleaved caspase-3 and a graph showing cell viability. FIG.15A shows data from MDA-MB-231 cells that were serum starved overnight, treated with the TAT-EGFR peptide for increasing lengths of time, harvested, lysed, and then analyzed by SDS-PAGE Western blot for the presence of cleaved caspase-3. FIGS.15B-C show data from MDA-MB-231 cells that were serum starved overnight, treated with 0 (control), 3, 6, or 12 μM TAT-EGFR (645-662) for 18 hours (FIG.15B) or with 0 (control) or 10 μM TAT-EGFR (645-662) for 15 minutes, 30 minutes, 3 hours, or 18 hours (separate control shown due to the longer duration of serum starvation) (FIG.15C), lysed, Western blotted, and then probed for the presence of cleaved caspase-3. Numbers at the bottom of the images represent the quantification of each band normalized to the respective control. FIG. 15D shows data from serum starved MDA-MB-231 cells that were treated overnight with 6.25, 12.5, or 25.0 μM of TAT-EGFR (645-662) for 0.5, 1, 3, or 24 hours and assayed for cell viability (Promega CELLTITER-GLO assay). A two-tailed, unpaired t test was used to determine if the mean value for each treatment condition was significantly different from control with a 95% confidence interval (* signifies P<0.003). The means for each concentration were compared at each time point and the significance is represented above the bar lines. The symbols above the 24 hours data points are comparisons between the data at each time point and the data at 24 hours (same concentrations were compared). The means were not significantly different between 0.5, 1 and 3 hours at the same concentration.

[0038] FIGS. 16A-B are images of Western blots demonstrating the ability of the TAT- EGFR peptide to bind to EGFR in cells. SK-N-MC cells lacking endogenous EGFR expression were used to exogenously express an EGFR intracellular domain (ICD), an ICD lacking JXM residues 645-662 (ΔJMA), or an ICD lacking the entire JXM domain (ΔJM). FIG.16A shows levels of binding of the TAT-EGFR or TAT peptides to the exogenously expressed EGFR proteins. FIG. 16B shows that free TAT-EGFR can compete for binding to EGFR proteins, reducing the binding of EGFR to TAT-EGFR-conjugated beads. IP, immunoprecipitation; SA, streptavidin; WB, Western blot; WCL, whole cell lysate.

[0039] FIG. 17 shows a graph and table demonstrating the effect of introducing a disulfide bridge in the EGFR peptide. MDA-MB-231 cells were serum-starved overnight, treated with varying concentrations of the TAT-EGFR or bridged TAT-EGFR peptide, and tested for cell viability.

[0040] FIGS. 18A-B are graphs showing intraperitoneal study IV Kaplan Meier Survival analyses and body weight monitoring. FIG. 18A shows the average body weight for each treatment group plotted over time. Plots and statistics were generated using PRISM 5.0 (GraphPad Software, Inc., USA) and error bars represent the standard error of the mean. Results are representative of two independent studies. FIG.18B shows a Kaplan Meier survival curve depicting the number of mice within endpoints, as defined by tumor size cutoff, tumor ulceration, and body conditioning scoring, at each time point. The inset in FIG.18B shows the median survival (the number of days at which the fraction of mice within endpoints is equal to 50%), plotted for each treatment group. The survival curves for the TAT and saline groups were compared to the survival curve for the TAT-EGFR (645- 662) group and the p-value was derived using the log-rank (Mantel-Cox) test. Asterisks (*) designate a significant difference with the indicated p-values.

[0041] FIGS. 19A-B are images and a graph showing data from MDA-MB-231 (human breast cancer) xenograft tumors grown in the subcutaneous flank region of nude mice.

Treatments with the indicated agents were commenced when tumors reached a size > 100 mm³. FIG.19A shows tumor slices from mice treated twice a week with the TAT-EGFR (645-662) peptide (40 mg/kg; 7 μmol/kg), TAT peptide (20 mg/kg; 7 μmol/kg), or vehicle (saline), intraperitoneally. H&E stained tumor slices shown are from mice treated intraperitoneally for 17 days with TAT-EGFR or saline and for 21 days with TAT. FIG.19B shows averages ± S.D. of quantified amounts of viable tumor and necrotic/dead tissue from batches of H&E images (TAT, N=6; Saline, N= 8; TAT-EGFR, N=14) of tumors from mice treated for 14 to 52 days. [0042] FIGS. 20A-E are images and a graph showing phospho-Erk and phospho-Akt levels in tumor slices. Frozen tumor sections were stained for phospho-Akt (S473) in FIG.20A or phospho-Erk in FIG.20C and counterstained with DAPI. Representative stained tumor sections are shown with the area in the box enlarged in the images below each section. Large scale bars = 500 μm and small scale bars = 50 μm. The intensity of the outer ~50 μM of the tumor slices was quantified and the average intensity of >8 measurements was plotted for each treatment group in FIGS. 20B and 20D. FIG.20E is a Western blot image of phospho- Erk present in the extracts of tumors from the treated animals. Each lane in FIG.20E represents one tumor from an individual mouse. FIG. 20F is a graph showing the statistical analysis of the Western blot data from FIG.20E. Each treatment group was compared statistically (*P≤0.03).

[0043] FIGS. 21A-B are graphs and a Western image showing that intraperitoneal treatment with TAT-EGFR reduces Erk signaling in MIA-PaCa-2 (pancreatic cancer) xenograft tumors. Nude mice bearing subcutaneous, MIA-PaCa-2 xenographic tumors were injected with the TAT-EGFR (645-662) peptide (40 mg/kg; 7 μmol/kg), TAT peptide (20 mg/kg; 7 μmol/kg), or vehicle (saline), intraperitoneally. Mice were treated twice a week and the tumor sizes were measured. In FIG.21A, the average for each group is plotted (left panel) and the individual growth curves for each treatment group are shown (inset). FIG. 21B shows data from mice with a tumor size of ~ 100 mm³ that were injected with TAT- EGFR (40 mg/kg; 7 μmol/kg) or saline. The tumor was extracted 30 minutes later and a cross-sectional area was lysed and analyzed for phospho-Erk. Dotted lines are at identical X and Y locations in each plot to differentiate that there are more mice in the TAT-645-662 treatment group in the lower right quadrant (representing lower size at later time points), compared to controls. The effect is not significant on average, as shown in FIG.21A.

[0044] FIGS. 22A-B are a Western blot image and a graph showing that TAT-EGFR (645- 662) affects downstream EGFR signaling. MDA-MB-231 cells were treated with TAT- EGFR (1.2, 2.55.010 and 20 μM), an EGFR specific tyrosine kinase inhibitor (2.0 μM TKI), TAT (20 μM), or vehicle for 30 minutes, followed by EGF treatment (10 ng/mL) for 10 minutes. Cell lysates were collected and analyzed by Western blot for phospho-Akt, total Akt, phospho-Erk (p44/p42) and total Erk (FIG.22A). Blots were stripped and re-probed with α-tubulin. The Erk and Akt data were analyzed and plotted as mean values from three experiments with the error bars representing the standard deviation from the mean (FIG. 22B). The significant differences (*P <0.04) were assessed between each treatment condition and the EGF-treated control.

[0045] FIG. 23 is a Western blot image showing that phospho-Akt S473 decreases with increasing amounts of TAT-EGFR (645-662) in MIA-PaCa-2 cells. MIA-PaCa-2 cells were treated with the indicated concentrations of TAT-EGFR (645-662) or TKI for 30 minutes, followed by EGF for 10 minutes. The presence of phospho-Akt (S473) and phospho-Erk was analyzed by Western blot. Detailed Description

[0046] This invention is based on our discovery that a peptide comprising an amino acid sequence from a juxtamembrane domain of a growth factor receptor (e.g., a receptor tyrosine kinase such as EGFR and c-Met) binds to the juxtamembrane domain of the growth factor receptor, thereby preventing access to the fully active conformation of the receptor (for example, by inhibiting dimerization or multimerization) and consequent activation of downstream signaling events. The fully active conformation of a receptor refers to a state of the receptor in which the receptor is capable of triggering downstream signaling events, such as a state in which the receptor is capable of performing its physiological enzymatic activity. For example, a fully active conformation of EGFR is an asymmetrical dimer, where the juxtamembrane A (JMA) region (amino acids 645-662) adopts an anti-parallel helical dimer conformation and the juxtamembrane B (JMB) region (amino acids 663-682) binds the C- lobe of the opposite monomer, forming a latch (Jura et al., Cell 137: 1293-1307 (2009)).

[0047] In our invention, the approach to disease treatment, e.g., cancer treatment by targeting growth factor receptors (e.g., the receptor tyrosine kinases discussed herein), takes advantage of the dimerization mechanism of the receptors. For example, in the case of EGFR, prior to activation by ligand binding, EGFR exists in an auto-inhibited state, where the dimerization face is buried. Upon ligand binding, EGFR undergoes a conformational change that exposes the dimerization face. Our work demonstrates that targeting the juxtamembrane domain of EGFR and other receptor tyrosine kinases mitigates these kinases’ signaling. Without being bound by theory, we believe that the peptides of this invention inhibit EGFR signaling by preventing access to the fully active conformation and subsequent downstream signaling (for example, by preventing dimerization). Additional data in our laboratory indicate that the JXM region is also involved in interactions with PIP2. The experimental evidence supports use of a peptide or small molecule inhibitor(s) designed to bind to the JXM domain of the EGFR as an agent to interfere with access to the fully active conformation of an RTK. In some embodiments of the invention, a peptide designed to bind to the JXM domain of the EGFR can be used to identify a small molecule inhibitor that binds to the JXM domain of the EGFR.

[0048] This invention offers advantages over the current approaches to RTK targeting. The current approaches are aimed at an RTK’s nucleotide and ligand binding sites. By targeting an RTK region that is mechanistically distinct from ligand and nucleotide binding, we expect that compensatory upregulation of other RTK family members will be less of an issue because the peptide will reduce the signaling potential of RTK homodimers as well as heterodimers. Further, non-cancerous cells will not be significantly affected by treatment with the peptide therapeutics disclosed herein. For example, ErbB protein levels are lower in non-cancerous cells and ErbB proteins are regulated by localization by epithelial barriers that are depolarized in cancerous cells. GROWTH FACTOR RECEPTORS

[0049] The inhibitory molecules of this invention can be used to inhibit a variety of mammalian growth factor receptors (such as RTKs), which include, but are not limited to, HER-1 (ErbB-1), HER-2 (ErbB-2/c-neu), HER-3 (ErbB-3), and HER-4 (ErbB-4), c-Met, fibroblast growth factor receptors (e.g., FGFR-1, FGFR-2, FGFR-3, and FGFR-4), insulin- like growth factor receptors (e.g., IGF1R), vascular endothelial growth factor receptors (e.g., VEGFR-1 and VEGFR-2), and platelet-derived growth factor receptors (e.g., PDGFRA). These growth factor receptors can be of any mammalian origin, including, without limitation, rodent origin (e.g., mouse and rat) and primate origin (e.g., human). INHIBITORY MOLECULES

[0050] The inhibitory molecules of this invention can be peptides derived from a juxtamembrane domain of a target growth factor receptor or from a juxtamembrane domain of a growth factor receptor closely related to the target receptor. In some embodiments, the peptide includes about 10-30 amino acid residues. In some embodiments, a portion (e.g., about 15-30 residues of the peptide is identical or substantially homologous to an amino acid sequence in the juxtamembrane domain of a growth factor receptor. A juxtamembrane domain is immediately C-terminal to the alpha-helical region of the transmembrane domain of the receptor.“Substantially homologous” means that at least about 75% (e.g., at least 80%, 85%, 90%, 95% or 99%) of the amino acid residues match over the defined length of two peptide sequences. Sequence homology can be identified by using standard software available in sequence databases, such as the BLAST program from the website of the National Center for Biotechnology Information or the ClustalW multiple sequence alignment tool from the European Bioinformatics Institute of the European Molecular Biology

Laboratory.

[0051] In some embodiments, the peptides of this invention can be derived from a growth factor receptor JXM domain by including the first amino acid immediately C-terminal to the alpha-helical transmembrane domain in the receptor. This amino acid and the following two or three amino acids are typically positively charged. The peptide sequence can be extended to include the following amino acids in the JXM domain, resulting in peptides of 15 to 30 amino acids.

[0052] In some embodiments, the inhibitory peptides do not contain any phosphorylatable tyrosine residues; where an inhibitory peptide is derived from a receptor juxtamembrane domain having a phosphorylatable tyrosine, that molecule will lack the phosphorylatable tyrosine, whether through substitution of another amino acid (naturally occurring or not) or through deletion of the tyrosine residue. In some embodiments, the inhibitory peptides will be helical or partially helical in structure. In some embodiments, the inhibitory peptides may contain modifications to induce a helical confirmation. In some embodiments, the inhibitory peptides may contain modifications to stabilize a helical conformation. Modifications can include, for example, naturally-occuring modifications (e.g., post-translational modifications) or modifications introduced, for example, by site-directed mutagenesis. For example, the inhibitory peptide can be internally“stapled” at its i, i+4 positions (one helical turn) or i, i+8 positions by an aliphatic bridge. An aliphatic bridge is a carbon bridge created between two unnatural amino acid side chains (e.g., (S)-N-Fmoc-2-(4’-pentenyl)alanine) that can be synthesized with, e.g., on-bead chemistry using Grubbs-I Catalyst (e.g., benzylidene- bis(tricyclohexylphosphine) Dichlorouthenium). An inhibitory peptide can also be“stapled” by, e.g., a disulfide bond between cysteines or modified cysteines, or by a lactam bridge (an amid bond between lysine and glutamate or derivatives thereof).

[0053] Sequences from juxtamembrane domains of some exemplary human growth factor receptors are provided below in Table 1. Peptides comprising all or a portion of such a juxtamembrane domain sequence, or variants of such peptides, can be used to inhibit the multimerization (e.g., dimerization and trimerization) and hence, activation, of the cognate growth factor or a growth factor closely related thereto. A number of such inhibitory peptides are disclosed herein (see, e.g., Tables 2 and 3, infra; and SEQ ID NOS:2-9, 12-25, and 29-32). Peptides substantially homologous to them or otherwise have similar structures are within the scope of this invention. In some embodiments, inhibitory peptides include peptides that are at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98, 99, 99.5 or 99.9 % identical to an inhibitory peptide derived from an RTK juxtamembrane domain, such as any of the inhibitory peptides disclosed herein. Inhibitory peptides also include peptides encoded by nucleic acids that hybridize under stringent conditions of 0.015 M sodium chloride, 0.0015 M sodium citrate at 65-68°C or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at 42°C to nucleic acids encoding any inhibitory peptide of about 15-30 amino acids that is derived from a RTK juxtamembrane domain. Further, the disclosure comprehends nucleic acids encoding any of the inhibitory peptides, as well as vectors comprising any such nucleic acid, a host cell comprising any such vector, methods of making an inhibitory peptide according to the disclosure, as well as methods of preventing or treating diseases or conditions characterized by RTK activity levels that are too high, such as cancer. Table 1 Juxtamembrane Domain Sequences from Families of Growth Factor Receptors

[0054] The peptides of this invention may contain naturally occurring amino acids and/or chemical analogs thereof. Amino acids can be in the D- or L- form. The peptide can be linear or cyclic, or have both structures. Amino acid mutations (e.g., substitution, deletion, and insertion) and chemical modifications, e.g., to the side chains and to the bonds formed between residues, can be introduced to the peptide to modify its stability, solubility, potency, immunogenicity, and other relevant pharmacological characteristics. For example, the peptide may contain an intramolecular disulfide bond to increase the molecule’s stability. Inhibitory molecules of this invention may also be conjugated covalently or non-covalently to other molecules that help improve the peptides’ pharmacokinetic characteristics such as half life and/or have additional therapeutic effects of their own. Examples of such molecules may include, but are not limited to, antibodies, antibody fragments (e.g., Fc domains), PEG molecules, albumin, and so on. In various embodiments, the peptides of this invention may have natural or unnatural glycosylation patterns, and/or may be PEGylated, prenylated, or stearylated peptides, or have similar modifications. In some embodiments, the peptides of the invention may be chemically modified to stabilize labile peptide bonds or form aliphatic or other covalent bridges (e.g., a lactam bridge) that stabilize the structure and/or protect the peptide from degradation and/or increase bioavailability or cell permeability.

[0055] In some embodiments, the peptide comprises a moiety that facilitates its entry into a target cell. Examples of such moieties include, without limitation, a TAT peptide (SEQ ID NO:1 or SEQ ID NO:11) or variants thereof. Other exemplary cell entry facilitators include, but are not limited to, any of the poly-arginine sequences (e.g., five to nine arginines at the N- or C-terminus (SEQ ID NO:39)) (as found in, for example, SEQ ID NO:4), the pVec sequence (LLIILRRRIRKQAHAHSK; SEQ ID NO:26), a penetration sequence such as an antennapedia peptide (e.g., RQIKIWFQNRRMKWKK (SEQ ID NO:27);

RVIRVWFQNKRCKDKK (SEQ ID NO:40)), and a polylysine sequence. The cell entry facilitator can be chemically conjugated to the targeting peptide, or can be fused to the targeting peptide via recombinant technology. Other methods of enabling cell penetration of the inhibitory peptide includes, without limitation, lipidification, PEGylation, and serum albumin (e.g., human serum albumin) conjugation

[0056] The peptides of this invention may be made from natural sources, produced recombinantly or manufactured synthetically.

[0057] In some embodiments, inhibitory molecules of this invention can be small molecules identified by using a peptide derived from a juxtamembrane domain of a target RTK or from a juxtamembrane domain of an RTK closely related to the target RTK. In some embodiments, a small molecule inhibitor can be identified by incubating an RTK with a peptide derived from a juxtamembrane domain of an RTK in the presence of a candidate RTK inhibitory molecule and detecting the binding of the RTK to the peptide. In certain embodiments, a decrease in the binding of the peptide as compared to binding in the absence of the candidate molecule indicates that the candidate molecule inhibits the RTK. In certain embodiments, the candidate molecule is added prior to, concurrently with, or subsequent to mixing the RTK and the peptide. Further, the disclosure comprehends methods of making inhibitory small molecules according to the disclosure, as well as methods of preventing or treating diseases or conditions characterized by RTK activity levels that are too high, such as cancer. THERAPEUTIC USES

[0058] The inhibitory molecules of this invention exhibit such high selectivity for target cells that therapeutically effective doses can be administered to a subject such as a human patient without causing deleterious consequences to healthy cells in that subject. Indeed, no toxicity due to treatment was observed in mice treated with inhibitory peptides of the invention (see, e.g., Example 7). Thus, the disclosure provides a new class of RTK inhibitors that exhibit promising efficacy in inhibiting RTK activity (e.g., tyrosine kinase activity) and in treating a variety of cancers while also providing better selectivity than current classes of RTK inhibitors (e.g., small-molecule inhibitors of the cytosolic kinase domain of RTKs). Examples of cancers that can be treated with the methods and compositions of this invention include, but are not limited to, breast cancer, colorectal cancer, lung cancer (e.g., non-small- cell lung cancer), pancreatic cancer, and prostate cancer.

[0059] The inhibitory molecules of this invention may be used to treat any other diseases where inhibition of a growth factor receptor such as an RTK is beneficial. In some embodiments, the inhibitory molecules are effective in treating inflammatory conditions such as those seen in malaria, fibrotic diseases, and chronic myeloproliferative disorders. Table 2 below provides examples of diseases and conditions that can be treated or prevented with the various inhibitory peptides of this invention. The listed diseases and/or conditions are associated with overactivation of RTKs family and that overactivation is amenable to down regulation with the compositions and methods of this invention.

[0060] The invention provides pharmaceutical compositions comprising a therapeutically effective amount of one or more peptides of the invention and optionally one or more pharmaceutically acceptable carriers such as physiologically compatible buffers. The compositions can be administered in any way that a skilled person in the art deemed appropriate for the disease/condition being treated. Examples of routes of administration include, without limitation, intravenous, intramuscular, intramedullary, intradermal, intraperitoneal, subcutaneous, oral, intranasal, and topical.

[0061] The pharmaceutical composition may contain formulation materials for modifying or maintaining, e.g., the pH, osmolarity, viscosity, clarity, color, isotonicity, sterility, stability, rate of dissolution or release, adsorption or penetration, of the composition.

Suitable formulation materials include, without limitations, amino acids, antimicrobials, antioxidants, buffers, chelating agents, solvents, stability enhancing agents, tonicity enhancing agents, etc. The pharmaceutical composition may be formulated to contain injectable microspheres, bio-degradable particles, polymeric compounds (e.g., polylactic acid, polyglycolic acid), beads, or liposomes that allow for desired mode of delivery of the peptide. Other suitable means for the introduction of the desired molecule include implantable drug delivery devices. Once formulated, the pharmaceutical composition may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or dehydrated (e.g., lyophilized) powder.

[0062] The pharmaceutical composition may be administered to a subject (e.g., a human patient) in dosages (e.g., single or multiple doses or continuous infusion) effective to achieve the desired effect. The dosages should be titrated to the individual patient. An effective dose may be in the range of from about 0.1 μg to about 40 mg per kilogram body weight per day. In some embodiments, a dose is administered to achieve peak plasma concentrations of 0.002 mg/ml to 30 mg/ml for the pharmacologically active ingredient.

[0063] The pharmaceutical composition may be administered concurrently or noncurrently with another therapeutic agent to achieve maximum therapeutic effects in the patient. For example, in treating cancer, the patient may be given a pharmaceutical composition of this invention in combination with a chemotherapeutic agent (e.g., 5-fluorouracil, docetaxel, paclitaxel, platinum-based therapies, and irinotecan) or another targeted therapy agent (e.g., therapies targeting RTKs (e.g., cetuximab) or other molecules involved in cancer pathways such as Mek, B-RAF, Erk, Akt, and Myc, and monoclonal antibody therapies targeting the same or other growth factor receptors or stromal cells (i.e., FAP)). SCREENING METHODS

[0064] The invention provides methods of identifying additional RTK inhibitors. In some embodiments, the methods entail contacting a cell expressing the RTK (e.g., EGFR) with said molecule; and measuring the activity of the RTK in said cell, wherein a decrease in said activity as compared to the activity of the RTK in a control cell that has not been contacted with the molecule indicates that the molecule inhibited the RTK. In other embodiments, the methods entail incubating an RTK with a peptide of this invention in the presence of a candidate RTK inhibitory molecule; and detecting the binding of the RTK to the peptide, wherein a decrease in the binding as compared to binding in the absence of the candidate molecule indicates that the candidate molecule inhibits the RTK. In these methods, the candidate molecule may be added prior to, concurrently with, or subsequent to, mixing the RTK and the peptide. Methods of measuring RTK activity and detecting binding to RTK are well known in the art. The methods can be used, for example, in screening small molecule combinatorial libraries and peptide libraries in a high-throughput manner. By way of example, the peptide of this invention can be immobilized on a 96-well plate, and ELISA assays can be performed to detect binding of the receptor to the immobilized peptide in the presence of a test compound. A reduction in receptor detection would indicate that the test compound is able to compete with the peptide in binding to the receptor and thus the test compound would be a candidate for inhibiting the receptor.

[0065] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Exemplary methods and materials are further described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art. Throughout this specification and claims, the word“comprise,” or variations such as“comprises” or“comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. The materials, methods, and examples are illustrative only and not intended to be limiting. EXAMPLES

[0066] The following examples illustrate embodiments of the invention. In these examples, unless otherwise indicated,“TAT-EGFR” or“TAT-EGFR(1-18)” refers to a peptide having a TAT peptide (SEQ ID NO:1 or SEQ ID NO:11)– a cell entry facilitator– conjugated at its C-terminal end to an 18-residue amino acid sequence

(RRRHIVRKRTLRRLLQER; SEQ ID NO: 28) identical to residues 645-662 of a human EGFR sequence (e.g., SEQ ID NO: 37, below). The sequence of TAT-EGFR is SEQ ID NO:3. [0067] LEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNNCEV

VLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYA LAVLSNYDANKTGLKELPMRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSDFLSN MSMDFQNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQCSGRCRGKSPSDC CHNQCAAGCTGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYS FGATCVKKCPRNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIG IGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEIT GFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDV IISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGP EPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRG PDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTG PGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMRRRHIVRKRTLRRLLQEREL VEPLTPSGEAPNQALLRILKETEFKKIKVLGSGAFGTVYKGLWIPEGEKVKIPVAIKEL REATSPKANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLITQLMPFGCLLDYVRE HKDNIGSQYLLNWCVQIAKGMNYLEDRRLVHRDLAARNVLVKTPQHVKITDFGLA KLLGAEEKEYHAEGGKVPIKWMALESILHRIYTHQSDVWSYGVTVWELMTFGSKPY DGIPASEISSILEKGERLPQPPICTIDVYMIMVKCWMIDADSRPKFRELIIEFSKMARDP QRYLVIQGDERMHLPSPTDSNFYRALMDEEDMDDVVDADEYLIPQQGFFSSPSTSRT PLLSSLSATSNNSTVACIDRNGLQSCPIKEDSFLQRYSSDPTGALTEDSIDDTFLPVPEY INQSVPKRPAGSVQNPVYHNQPLNPAPSRDPHYQDPHSTAVGNPEYLNTVQPTCVNS TFDSPAHWAQKGSHQISLDNPDYQQDFFPKEAKPNGIFKGSTAENAEYLRVAPQSSE FIGA (SEQ ID NO: 37) EXAMPLE 1

[0068] Studies of the juxtamembrane region of the Epidermal Growth Factor Receptor (i.e., JXM EGFR region) were initiated with various peptides derived from the EGFR JXM domain conjugated to the TAT sequence, which facilitated cellular entry (FIGS.2A-D). The peptides were assayed for their effect on cell viability in MDA-MB-231 cells (human breast cancer cells). The TAT-conjugated 18-mer (TAT-EGFR peptide; SEQ ID NO: 3) displayed an IC₅₀ value of about 12 μM in this assay (Table 3 and FIGS.1A and 1B). In order to optimize peptide length, the peptide sequence was shortened in increments of 5 amino acids (FIG.1A). Of all the shortened versions of the peptide assayed (SEQ ID NOs:5-9), none of them displayed higher activity than the TAT-EGFR peptide (FIGS.1A and 1B). Further, the TAT sequence was changed to extend the poly-arginine sequence at the N-terminus of the EGFR 18-mer, because poly-arginine sequences are known to facilitate cellular entry. The poly-R-EGFR peptide (SEQ ID NO: 4) displayed about 4-fold lower activity than the TAT- EGFR peptide. A TAT-conjugated scrambled sequence (“TAT-scrambled,” SEQ ID NO: 12) was used as a control.

[0069] Several cancer cell types (metastatic colorectal, metastatic and non-metastatic breast and metastatic non-small cell lung) that overexpress EGFR were treated with the TAT-EGFR peptide, and cell viability was assayed at the end of a 24-48 hour period. In each case, the peptide caused a reduction in cell viability, with IC₅₀ values ranging from 9 to 35 μM, depending on the cell type and the amount of serum present in the cell culture medium during treatment (FIGS.3A and 3B, and Table 3). In breast cancer cells, the peptide reduced cell viability synergistically with 0.5-1.0 mg/ml Erbitux® (cetuximab) (FIG.3D).

[0070] To evaluate the activity of the EGFR peptide in normal cells, the IC₅₀ profile was compared between MDA-MB-231 breast cancer cells and their non-cancerous counterpart HMEC (human mammary epithelial) cells (FIG.3C). Two strains of HMEC cells (HMEC- 689 and HMEC-636) were used. The HMECs were plated in HMEC growth media fully supplemented with nutrients and hormones. MDA-MB-231 cells were plated in full media (formulated for this cell type) overnight. The media then was exchanged for serum-starved media (buffered media with minimal nutrients) or HMEC growth media overnight. All cells were treated for 24 hours with increasing concentrations of the EGFR peptide, and IC₅₀s were calculated from the percent of viable cells in each culture relative to the vehicle-treated control. The EGFR peptide was slightly less active in MDA-MB-231 cells treated in serum starved media (with an IC₅₀ value of 12 μM), compared to MDA-MB-231 cells treated in HMEC media (which had an IC₅₀ value of 9 μM). However, the ability of the EGFR peptide to reduce viability of HMEC cells was markedly reduced, with an IC₅₀ of 23 μM in strain 636 and an IC₅₀ of 31 μM in strain 689. These values are 2.6-fold and 3.4-fold higher (indicating a decrease in activity), respectively, than in MDA-MB-231 cells. These results demonstrate that the EGFR peptide specifically inhibited the growth of breast cancer cells over normal breast cells.

[0071] Another study demonstrating the effect of the JXM-derived peptides of the invention on cell viability involved the exposure of SKBR3 breast cancer cells to TAT- EGFR or to a peptide derived from an ErbB2 (HER-2) JXM (SEQ ID NO: 13) linked to a TAT peptide (RKKRRQRRRG) (SEQ ID NO: 11), or to saline (FIG.12). Both the ErbB1 and ErbB2 JXM peptides induced a decrease in viability of this second line of breast cancer cells as compared to saline (FIG.12). Accordingly, the data support the use of a range of peptides derived from the juxtamembrane region of Receptor Tyrosine Kinase proteins, as disclosed herein, as RTK antagonists in the treatment or prevention of such diseases as cancer.

[0072] Further evidence of the functionality of the peptides disclosed herein was obtained from a binding study in which an EGFR JXM peptide and an insulin receptor (INSR) JXM peptide were exposed to immobilized Phosphatidylinositol (4,5)-Bisphosphate (PIP2). Both EGFR and INSR bind PIP2. As revealed in FIG.13, the Biacore SPR binding isotherms of each of these peptides to immobilized PIP2 demonstrated functional binding activity for each peptide, consistent with the role of such peptides in antagonizing an activity of an RTK in methods of treating or preventing such diseases as cancer. EXAMPLE 2

[0073] The ability of the peptide to enter cells was examined using fluorescence microscopy. Peptides were labeled with fluorescein (FAM) and dosed in MDA-MB-231 cells just prior to imaging. The FAM-TAT-EGFR peptide entered cells within 10-15 minutes (FIG.2A). The FAM-EGFR (no TAT-conjugation, FIG.2B) and FAM-TAT (no EGFR sequence, FIG.2C) peptides were not able to enter cells, as monitored for several hours or after an overnight treatment. These data confirmed that a cellular entry facilitator sequence (e.g., the TAT sequence) was necessary for cellular entry. These peptides were also assayed in the cell viability screen. The non-TAT-conjugated EGFR peptide and TAT peptide did not have an effect on cell viability, while the fluorescent TAT-EGFR peptide displayed similar activity to its non-fluorescent counterpart (FIG.2D). Overall, the peptides were monitored from just after dosing for several hours to overnight. The TAT and non-TAT EGFR peptide did not enter cells, even with incubation for several hours to overnight. The TAT-EGFR peptide entered cells within 10 to 15 minutes, remained there for several hours, and was excreted after about 12 hours (overnight).

[0074] As an initial experiment to analyze the kinetics of in vivo distribution of the TAT- EGFR peptide, the distribution and kinetics of the fluorescein-tagged peptide in the mouse after intraperitoneal (i.e., IP) or subcutaneous injection was analyzed using in vivo fluorescent imaging. FIGS.10A and 10B show a representative image sequence for a fluorescein-TAT-EGFR peptide intraperitoneal injection followed over the course of 2 hours. Images from the TAT-conjugated peptide and sub-cutaneous injections are similar.

Conclusions from these experiments are that the peptide(s) remain in the bloodstream for a minimum of 2-3 hours and are excreted within approximately 5 hours. EXAMPLE 3

[0075] To demonstrate a dose-dependent effect of EGFR peptides on colony formation, MDA-MB-231 (breast cancer) cells, DLD-1 (colorectal cancer) cells, A-549 (non-small cell lung cancer) cells, and MIA-PaCa2 (pancreatic cancer) cells were suspended in soft agar (100,000 cells) containing 5% serum and treated with 10 μM, 20 μM, or 40 μM of the TAT- EGFR (TAT 645-662) peptide, 20 μM of the TAT peptide, or a vehicle control (FIG.4C). Colonies were allowed to grow for 2 weeks with periodic dosing to maintain fresh dosing media and hydration of the agar. TAT-EGFR demonstrated the ability to kill each type of cancer cell in soft-agar assays (FIGS.4A-C). In these assays, the TAT-EGFR peptide reduced colony formation by about 50% in MDA-MB-231 breast cancer cells dosed at 25 μM or 40 μM TAT-EGFR with 5% serum (FIG.4A). TAT-EGFR peptide also reduced colony formation in DLD-1 colorectal cancer cells, A-549 lung cancer cells, and MIA-PaCa- 2 pancreatic cancer cells (FIG.4B). The peptide showed a slightly synergistic effect on colony formation of MDA-MB-231 cells when dosed in combination with 0.5 mg/ml Erbitux® (FIG.4C). EXAMPLE 4

[0076] To assess the mode of cell death induced by peptide treatment in MDA-MB-231 cells, apoptosis assays using FITC-Annexin-V with propidium iodide staining were performed and analyzed by flow cytometry. Serum starved MDA-MB-231 breast cancer cells were treated with 0 (control), 6, or 12 μM TAT-EGFR (645-662) for 18 hours (FIGS. 14A-B) or 0 (control) or 10 μM TAT-EGFR (645-662) for 30 minutes or 3 hours (FIGS. 14C-D) and stained with Annexin-V and propidium iodide (PI). Treatment with 6 μM TAT- EGFR increased the percentage of apoptotic cells (Annexin-V staining only) from 5.2±2.0 (S.D.)% in the untreated control to 7.7±1.0 (S.D.)%. Treatment with 12 μM TAT-EGFR increased the percentage of apoptotic cells (Annexin-V staining only) to 11.5 ± 2.8(S.D.)%. The total percentage of non-viable cells (Annexin-V and/or PI positive) increased from 15.0± 4.3(S.D.)% in the control to 25.8± 2.9 (S.D.)% with 6 μM TAT-EGFR and 44.1± 5.4(S.D.)% with 12 μM TAT-EGFR (FIGS.14A-B). The effect of treatment with the TAT-EGFR peptide was seen within the first 30 minutes of treatment, and did not change significantly after 3 hours (FIGS.14C-D).

[0077] Apoptosis was also measured by caspase-3 cleavage. MDA-MB-231 breast cancer cells were serum-starved overnight then treated with 10 μM TAT-EGFR for increasing lengths of time. The cells were then harvested, lysed, and analyzed by SDS-PAGE Western blotting for the presence of cleaved caspase-3. The amount of cleaved caspase-3 markedly increased after 18 hours of treatment (FIG.15A), indicating apoptosis of the cells. These results were confirmed in a second experiment wherein MDA-MB-231 cells were serum starved overnight then treated with 0 (control), 3.1, 6.3 or 12.0 μM TAT-EGFR (645-662) for 18 hours (FIG.15B) or with 0 (control) or 10 μM TAT-EGFR (645-662) for 30 minutes, 3 hours or 18 hours (FIG.15C). Increasing both the amount and length of incubation with TAT-EGFR resulted in increased amounts of cleaved caspase-3. When serum starved MDA- MB-231 cells were treated overnight with 6.25, 12.5 or 25.0 μM of TAT-EGFR (645-662) for 0.5, 1, 3 or 24 hours and assayed for cell viability, the percentage of viable cells relative to the untreated control decreased with increasing concentrations of TAT-EGFR (FIG.15D). EXAMPLE 5

[0078] In order to determine if TAT-EGFR interacts with the EGFR protein in cells, a binding assay was designed in which the intracellular domain (ICD) of EGFR (645-992), an EGFR ICD lacking the EGFR JXM region from 645-662, or the entire juxtamembrane region was exogenously expressed in SK-N-MC (Ewing’s sarcoma) cells lacking endogenous EGFR expression (FIGS.16A-B). These cells were treated with biotinylated versions of TAT- EGFR or TAT peptides for two hours. The cells were then washed rigorously, lysed, and the lysates incubated with streptavidin-coated beads overnight. Finally, the beads were washed, boiled, and analyzed by Western blot for the presence of EGFR. Cells treated with TAT- EGFR at 0.5 μM showed a strong increase (10- to 12-fold over the TAT-treated control) of bound EGFR in cells expressing the intracellular domain of EGFR (FIG.16A). Binding between TAT-EGFR and the EGFR ICD was diminished with expression of an EGFR ICD construct that lacked either the entire or a part of the JXM domain (FIG.16A). These results indicate that the interaction between TAT-EGFR and the EGFR ICD is specific and takes place at the JXM domain.

[0079] In a separate assay, the biotinylated TAT-EGFR and TAT peptides were conjugated to streptavidin beads and incubated overnight with SK-N-MC lysates from cells expressing either the ICD or the ICD lacking the JXM domain. The beads were then washed, boiled, and analyzed for the presence of bound EGFR. TAT-EGFR peptide beads pulled down the most EGFR ICD compared to a blank control (FIG.16B). Although the TAT peptide also pulled down the EGFR ICD, this was 4-fold lower than with TAT-EGFR. When free TAT- EGFR (3 or 10 μM) was added to the bead-lysate mixture, the free peptide competed for binding to EGFR ICD, reducing the binding of EGFR ICD to the peptide-conjugated beads (FIG.16B). As in the experiments above, binding between TAT-EGFR and the EGFR ICD was diminished with expression of an EGFR ICD construct lacking a JXM domain. EXAMPLE 6

[0080] To improve the stability of the EGFR peptide, two mutations were introduced in TAT-EGFR: R656C and Q660C (TAT-EGFR R656C/Q660C, SEQ ID NO: 32). These mutations were predicted to induce formation of a disulfide bridge in the peptide, thereby stabilizing the peptide. Additional mutations also were made to disrupt the helicity, and, therefore, conformation and/or stability, of the EGFR peptide.

[0081] A helical region is centered around the LRRLL (SEQ ID NO:44) sequence in the wild-type EGFR protein, where residues RR and LL have been thought to interact in the helical dimer. To test the effects of the R656C/Q660C mutations, MDA-MB-231 cells were serum-starved overnight and treated for 24 hours with increasing concentrations from 0.78 μM to 200 μM of TAT-EGFR, TAT-EGFR(R656C/Q660C) (TAT peptide- RRRHIVRKRTLCRLLCER (SEQ ID NO: 36) (the mutations are in boldface)), or other TAT-EGFR mutated peptides shown in Table 5 with a mutated helical region (RRLL (SEQ ID NO:45) was mutated two amino acids at a time, RR to GG, LL to GG, and LL to PP, to destabilize the helix). Cell viability was measured using the Promega CellTiter One assay and expressed as percentage of the assay value for untreated cells. Dose response curves were generated and fitted using GraphPad PRISM 5.0 (GraphPad Software, Inc., USA) to determine IC₅₀ values from the normalized curves. The IC50 values were generated using the log inhibitor-normalized response variable slope function (Y=100/(1+10^((LogIC50- X)*HillSlope)). Values are shown in Table 5 with standard deviation values from at least three independent experiments. The IC₅₀ of TAT-EGFR was 14±4 μM, compared to an IC₅₀ of 2.1 μM with TAT-EGFR(R656C/Q660C), which is stapled at the C’s to increase helicity (FIG.17). IC₅₀ values of TAT-EGFR peptides with other mutations were higher than 14±4 μM. The results demonstrated that the EGFR peptide was made significantly more potent with stabilization by the disulfide bridge. Each of the destabilizing mutations decreased the activity of the peptide due to the decrease in helicity as shown by circular dichroism.

EXAMPLE 7

[0082] In two separate MDA-MB-231 xenograft studies, subcutaneous treatment with the TAT-EGFR peptide (50 mg/kg) delivered twice a week proximal to, or in the vicinity of, a tumor site in NCR/nude female athymic mice significantly reduced late-stage tumor growth (FIGS.5 and 6) and increased lifespan at the endpoints of the study (FIGS.7A-B) compared to a saline control group. No significant weight loss occurred with peptide treatment. No signs of kidney or liver pathologies were observed with peptide treatment. An intraperitoneal mode of administration was tested in two subsequent MDA-MB-231 xenograft studies (FIGS.8A, 8B, and 8C). Mice were dosed twice a week with the TAT-EGFR peptide (50 mg/kg) intraperitoneally. TAT-EGFR treatment significantly reduced late-stage tumor growth (FIGS.8A and 8B), reduced overall tumor size (FIG.8C), and increased lifespan (according to study endpoints, FIGS.9 (percentage plot) and 18B (Kaplan-Meier plot)) compared to saline and TAT peptide control groups. No significant weight loss occurred with peptide treatment (FIG.18A).

[0083] In another study, MDA-MB-231 xenograft tumors were grown in the subcutaneous flank region of nude mice. When tumors reached a size > 100 mm³, the mice were injected intraperitoneally twice a week with the TAT-EGFR (645-662) peptide (40 mg/kg; 7 μmol/kg), TAT peptide (20 mg/kg; 7 μmol/kg), or vehicle (saline). Tumor slices from mice treated for 17 days with TAT-EGFR or saline and for 21 days with TAT were H&E stained and quantified for the amount of viable tumor and necrotic/dead tissue. The percentage of viable tissue in tumor slices from mice treated with TAT-EGFR was lower than the tumor slices from mice treated with TAT peptide or saline (FIGS.19A-B).

[0084] In another study, MDA-MB-231 xenograft tumors were grown in the subcutaneous flank region of nude mice that were treated twice a week with the TAT-EGFR (645-662) peptide (40 mg/kg; 7 μmol/kg), TAT peptide (20 mg/kg; 7 μmol/kg), or vehicle (saline), intraperitoneally for four days, once per day. On the last day, the mice were injected 30 minutes prior to tumor extraction. The frozen tumor sections were stained for phospho-Akt (S473) or phospho-Erk and counterstained with DAPI (FIGS. 20A and 20C, respectively), and at least eight intensity measurements for approximately the outer 50 μm of each section was quantified and averaged (FIGS.20B and 20D). Phospho-Akt levels were significantly lower between the TAT-EGFR sample and the TAT sample (p = 0.006), and between the TAT-EGFR sample and the saline sample (p = 0.02). But phospho-ERK levels were not significantly lower with TAT-EGFR overall, but were lower in some tumor regions. Tumor extracts were also lysed and analyzed by Western blot for each mouse (FIG. 20E), and the Western blot data was quantified and analyzed statistically (FIG. 20F). FIG.20F shows that phospho-Erk levels were significantly decreased in extracts from TAT-EGFR-treated tumors, as compared to extracts from saline- or TAT-treated tumors.

[0085] Data from MDA-MB-231 cells also confirmed the results seen in mice. MDA-MB- 231 cells were treated with TAT-EGFR (645-662) (1.2, 2.5, 5.0, 10, and 20 μM), an EGFR specific tyrosine kinase inhibitor (2.0 μM TKI), TAT (20 μM), or vehicle for 30 minutes, followed by EGF treatment (10 ng/mL) for 10 minutes. Phospho-Erk and phospho-Akt levels decreased with increasing amounts of TAT-EGFR (645-662) (FIGS.22A-B).

[0086] In addition to mice with MDA-MB-231 xenograft tumors, mice with MIA-PaCa-2 (pancreatic cancer) xenograft tumors also were tested for levels of Erk. In this study, nude mice bearing subcutaneous, MIA-PaCa-2 xenographic tumors were injected intraperitoneally twice a week with the TAT-EGFR peptide (40 mg/kg; 7 μmol/kg), TAT peptide (20 mg/kg; 7 μmol/kg), or vehicle (saline). Tumor sizes were measured (FIG.21A). Mice with a tumor size of ~ 100 mm³ were injected with TAT-EGFR (40 mg/kg; 7 μmol/kg) or saline, once a day for four days. After injection on the fifth day, the tumor was extracted 30 minutes later, and a cross-sectional area was lysed and analyzed for phospho-Erk (FIG.21B). Phospho-Erk levels were lower in the samples from mice treated with TAT-EGFR as compared to saline, although Erk levels were relatively constant.

[0087] MIA-PaCa-2 results in mice also were confirmed with cellular data. MIA-PaCa-2 cells were treated with increasing concentrations of TAT-EGFR (645-662) or TKI for 30 minutes, followed by EGF for 10 minutes. Phospho-Akt and phospho-Erk levels decreased with increasing concentrations of TAT-EGFR (645-662) (FIG. 23). EXAMPLE 8

[0088] In order to assess whether peptide treatment would elicit an immune response, non- immunocompromised mice (strain 129) were injected intraperitoneally with the TAT-EGFR peptide, TAT peptide, or saline. The temperatures of the mice were taken prior to injection and 30 minutes following injection (Table 6). At day 7, a slight (<1°F) increase in temperature was recorded 30 minutes after treatment with the TAT-EGFR peptide, which did not occur in the saline- or TAT peptide-treated mice. At days 0 and 3, there were no significant effects on body temperature increase with TAT-EGFR peptide relative to saline or TAT peptide treatment.

EXAMPLE 9

[0089] An alignment of the JXM in the ErbB family of receptors showed a high amount of homology within the JXM region, indicating that this region had functional significance (Table 7). There have been reports that the JXM region is required for activation of EGFR. There is also a significant degree of homology between the JXM regions of other families of receptors, consistent with the functional importance of this region. In some cases, homology ranges from 5 to 35% across various families. For example, c-Met and ErbB4 display 35% homology in the 20-amino-acid JXM region. The homology indicates that the same principle can be applied to designing inhibitors of the JXM regions of other growth factor receptors. Other receptor tyrosine kinases are involved in cancer or resistance to cancer therapies, such as IGF1R, c-MET, VEGFR, PDGFR and c-KIT (Table 1). Hence, peptides with sequences designed on the basis of the juxtamembrane region of other growth factor receptors can be synthesized and functionality confirmed by assay for activity in killing cancer cells in vitro and/or in an in vivo xenograft model. One such peptide that has been tested was modeled after the RTK c-Met. This peptide (SEQ ID NO: 2) has been demonstrated to function in accordance with the expectations disclosed herein. Hence, peptides with sequences homologous to other growth factor receptor tyrosine kinases, that are also overexpressed, e.g., in cancer, such as VEGFR, can be synthesized and expected functionality confirmed by assay for activity in killing cancer cells in vitro and/or in an in vivo xenograft model.

[0090] To demonstrate that targeting the JXM region is broadly applicable to growth factor receptor families with similar mechanisms of activation (ligand binding-induced dimerization and cross-phosphorylation), a TAT-conjugated JXM peptide modeled after the c-Met sequence was subjected to a cytotoxicity assay. Some of the peptide sequences that could be TAT-conjugated and assayed in these models are listed in Table 7. The activities of the c- Met peptide (SEQ ID NO: 2) in MDA-MB-231 and A-549 cells are shown in FIG.11. Cells were simultaneously treated with hepatocellular growth factor (HGF), the c-Met ligand. The TAT-conjugated c-Met peptide reduced cell viability in both cell lines by about 25% at a dose of 25 μM. The effect on cell viability was lower relative to the TAT-EGFR peptide; however, it should be noted that the sequence was not optimized (e.g., the sequence length and position with the c-MET JXM region was not varied). [0091] Variations on the subject matter of the disclosure provided herein will be apparent to those of skill in the art upon review of the present disclosure, and such variations are within the scope of the invention.

Claims

What is Claimed is:

1. An isolated peptide comprising an amino acid sequence that is 15 or more amino acids in length and is derived from a juxtamembrane domain of a growth factor receptor, wherein the peptide inhibits access to the fully active conformation of the growth factor receptor.

2. The peptide of claim 1, wherein the growth factor receptor is a receptor tyrosine kinase.

3. The peptide according to claim 1, further comprising a cellular entry facilitator.

4. The peptide according to claim 1, wherein the cellular entry facilitator comprises a TAT domain.

5. The peptide according to claim 1, wherein the peptide comprises a sequence selected from the group consisting of SEQ ID NOS: 2-9, 13-25, and 28-35.

6. The peptide according to claim 1, wherein the peptide is derived from the juxtamembrane domain of a member of the Epidermal Growth Factor Receptor family, c- Met, Fibroblast Growth Factor Receptor, Platelet-derived Growth Factor Receptor, Insulin- like Growth Factor Receptor, or Vascular Endothelial Growth Factor Receptor family of receptor tyrosine kinases.

7. The peptide according to claim 1, wherein the peptide is derived from c-Met.

8. The peptide according to claim 1, wherein the peptide is derived from Epidermal Growth Factor Receptor.

9. The peptide according to claim 1, wherein the peptide comprises one or more amino acid changes compared to said juxtamembrane domain.

10. The peptide according claim 9, wherein the one or more amino acid changes stabilize said peptide.

11. The peptide according to claim 9, wherein the one or more amino acid changes induce the formation of a disulfide bonded bridge in the peptide.

12. The peptide according to claim 1, wherein the peptide comprises one or more modifications compared to said juxtamembrane domain.

13. The peptide according to claim 12, wherein the one or more modifications comprise an aliphatic or lactam bridge.

14. The peptide according to claim 1, wherein the sequence is set forth in SEQ ID NO: 32.

15. The peptide according to claim 1, wherein the sequence is set forth in SEQ ID NO: 2.

16. The peptide according to claim 1, wherein the sequence is set forth in SEQ ID NO: 3.

17. A kit comprising a peptide according to claim 1 and a protocol for administration of the peptide to a subject in need thereof.

18. A method for treating a condition in a subject in need thereof, comprising administering a therapeutically effective amount of the peptide according to claim 1 to the subject.

19. The method according to claim 18, wherein the condition is cancer.

20. The method according to claim 19, wherein the cancer is selected from the group consisting of breast cancer, colorectal cancer, pancreatic cancer, and lung cancer.

21. The method according to claim 18, wherein the condition is selected from the group consisting of malaria, chronic myeloproliferative disorders, and fibrotic diseases.

22. The method according to claim 19, further comprising administering to the subject another anti-cancer therapeutic.

23. A method of inhibiting an RTK in a cell, comprising contacting the cell with a molecule that binds to a juxtamembrane domain of the RTK and prevents multimerization of the RTK, thereby inhibiting the RTK in the cell.

24. The method of claim 23, wherein the molecule is an isolated peptide comprising an amino acid sequence that is 15 or more amino acids in length and is derived from a juxtamembrane domain of a receptor tyrosine kinase (RTK).

25. The method of claim 23, wherein the cell is in the body.

26. A method of inhibiting multimerization of an RTK, comprising contacting the RTK with the peptide of claim 1.

27. A method of identifying a molecule that inhibits a receptor tyrosine kinase (RTK), comprising:

contacting a cell expressing the RTK with said molecule; and measuring the activity of the RTK in said cell, wherein a decrease in said activity as compared to the activity of the RTK in a control cell that has not been contacted with the molecule indicates that the molecule inhibits the RTK.

28. The method according to claim 27, wherein the RTK is an EGFR.

29. A method for identifying a receptor tyrosine kinase (RTK) inhibitory molecule, comprising

incubating an RTK with a peptide according to claim 1 in the presence of a candidate RTK inhibitory molecule; and

detecting the binding of the RTK to the peptide, wherein a decrease in the binding as compared to binding in the absence of the candidate molecule indicates that the candidate molecule inhibits the RTK.

30. The method according to claim 29, wherein the candidate molecule is added prior to, concurrently with, or subsequent to, mixing the RTK and the peptide.