Antiproliferative peptides and antibodies for their detection

This application is a divisional of U.S. patent application Ser. No. 11/181,331 filed Jul. 14, 2005, which claims priority to U.S. Provisional Application No. 60/587,919 filed Jul. 14, 2004, the contents of which are incorporated herein by reference in their entirety.

The present invention relates to peptides and peptidomimetics, which may be used therapeutically to inhibit abnormal cell proliferation. It is based at least in part, on the discovery of a class of developmental peptides or proteins (DPs) isolated from embryonic tissues which have been found to exhibit antiproliferative effects on a variety of damaged cells ranging from pre-cancerous to resistant cancer cells and virally infected cells. Developmental peptides and molecules that mimic the structure and function of these peptides are capable of inhibiting abnormal cell proliferation.

In addition, the present invention relates to specific antibodies generated against developmental peptides and their functional equivalents, their use to determine the peptide expression in various normal and pathologic tissues and biological fluids, and their use as biomarkers specifically but not limited to cancer. The present invention further investigates the use of these antibodies for isolating and characterizing additional developmental peptides exhibiting antiproliferative action that may share homology to developmental peptides described herein.

The present invention relates to developmental peptides and peptidomimetics which exert antiproliferative effects. The developmental peptides antiproliferative effects may be attributed to their ability to control the delicate balance between proliferative and antiproliferative factors in developing tissues within the embryo, and actually continuing such function throughout adulthood. Developmental peptides may further have a similar antiproliferative homeostatic role in the adult, preventing abnormal proliferation, such as it occurs in cancer.

The discovery is based on the theory that pregnancy can be viewed as a reversible form of cancer. In that context, embryonal cells are rapidly proliferating, migrating, and invading the maternal body. In contrast to cancer cells, the embryonal cells undergo differentiation, give rise to organ development and to a major shift from structure to function leading to the birth of an individual. In the case of cancer, altered cell proliferation leads in general to disrupted function and consequently to the demise of the individual, unless the cancer is aggressively controlled and eradicated. An additional aspect of pregnancy is that of maternal immune tolerance, which is a reversible phenomenon and conditional in general on the health of the conceptus. In contrast, frequently, the presence of cancer is associated with immune suppression and altered immune response.

As described in U.S. Pat. No. 5,648,340 entitled “Gestational agents for controlling cell proliferation” filed Jan. 17, 1995 and PCT Application No. PCT/US91/08046 filed Oct. 31, 1991, entitled “Proteins Purified from Mammalian Gestational Tissue which Controls Cell Proliferation” incorporated herein by reference, developmental peptide agents have been broadly identified to operate by controlling the development of the embryo such that proliferation, placental invasivity and differentiation may occur without substantially injuring the maternal host. U.S. Pat. No. 5,648,340 discloses the purification of protein extracts having a molecular weight less than 10,000 (and particularly less than 8,000 daltons) which have antiproliferative activity and other agents of less than 3,000 daltons, which oppositely exhibit proliferative activity. The protein preparations described therein are described as high molecular weight extracts effective against a wide variety of viruses, as well as isolation and sequencing of a low molecular weight seven amino acid set of peptides which exert antiproliferative effects. In U.S. application Ser. No. 10/117,728 filed Apr. 4, 2002 entitled “Gestational agents which modulate cell proliferation,” the profound and multi-targeted effects of high molecular weight developmental peptides on cancer cells proliferation is described. Developmental peptides inhibited tumor promoters and promoted tumor inhibitors acting on both cell cycle and cell cycle independent intracellular proteins, with minimal effect exerted on normal white blood cells. Overall effect of developmental peptides appeared to be exerted at the G0-G1 transition phase. On MCF7 cells (estrogen receptor positive breast cancer cells), P53 phosphorylation increased while pRb decreased, mdm2 separated from p53, and later p21 was induced. Cyclin D1 and E were blocked, MAPkinase temporarily dephosphorylated, Bcl2 was blocked while BAD increased.

The present invention relates to the further isolation and characterization of several developmental peptides as well as sequencing of such peptides, for example, from mammalian adult liver, and documenting their presence in a native form also within the porcine embryonal liver. In addition, generation of synthetic developmental peptides and peptidomimetics and testing of their activity against cancer cells and virally infected cells and the mechanism of developmental peptide action is further described. Further, the present invention relates to the use of developmental peptides and KLH as carrier for the generation of polyclonal antibody in chicken (IgY) and determining the expression of the proteins and peptides that contain a conserved seven to eleven amino acid sequence in a variety of embryonal and adult tissues.

The identification of developmental peptides in both fetal and adult tissues and their highly preferential expression in normal epithelium point to their important biological role in rapidly replicating cells where negative regulators are likely to have a significant role in assuring proper control of proliferation. Also their expression in highly proliferating and invasive cells as seen in the first trimester trophoblast further confirms its in vivo role in maintaining control of proliferation. On the other hand, their increased expression in tumors associated with an altered developmental peptide profile support the view that altered expression as a cause or consequence of malignant transformation and thereby, the use of developmental peptides as biomarkers, or therapeutics is envisaged. Developmental peptides appear to act through specific receptors that are expressed by abnormal and not normal cells. As such, as seen in the embryo, developmental peptides appear to act locally on the receptor target aimed in eliminating abnormal cells. As such, homeostasis in the body is maintained where normal cells secrete and abnormal cells respond to developmental peptides. One aspect of the present invention provides a method for identifying the developmental peptide receptor and creating a pharmacophore to examine developmental peptide/receptor interaction.

One aspect of the present invention provides for developmental peptides, peptidomimetics and methods of their use for inhibiting abnormal cell proliferation.

Thus, according to one aspect of the invention, a synthetic developmental peptide is provided, which binds to a developmental peptide receptor and inhibits abnormal cellular proliferation. The developmental peptide is preferably a seven to eleven amino acid peptide. In preferred embodiments, the developmental peptide comprises the sequence N-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID No.1). In other embodiments, the developmental peptide comprises the sequence of N-Val-Leu-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID No.2), N-Ile-Glu-Val-Leu-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID No.3), N-Ile-Asp-Val-Leu-Gly-LIys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID No.4), N-Ile-Arg-Val-Leu-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID No.5), N-Ile-Glu-Val-Thr-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID No.6), N-Ile-Asp-Val-Thr-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID No.7), or N-Ile-Arg-Val-Thr-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID No.8), Gly-Lys-Arg-Ile (SEQ ID No.9), or Lys-Gly-Thr. In further embodiments, the developmental peptide has the sequence Xaa_m-Gly-Lys-Arg-Ile-Xaa_n, wherein Xaa_m, and Xaa_n, each represent an amino acid and wherein m independently has a value from 0 to 20, preferably less than 10, and wherein n independently has a value from 0 to 20, preferably less than 10, or mimetics thereof. In further embodiments, the developmental peptide has the sequence Xaa_m-Lys-Gly-Thr-Xaa_n, wherein Xaa_m, and Xaa_n each represent an amino acid and wherein m independently has a value from 0 to 20, preferably less than 10, and wherein n independently has a value from 0 to 20, preferably less than 10, or mimetics thereof.

The present invention also provides for non-peptide or partial peptide mimetic of any of the aforementioned developmental peptides.

In another aspect, the present invention provides for a method of inhibiting abnormal cellular proliferation comprising administering the aforementioned developmental peptides or peptidomimetics in an amount sufficient to inhibit abnormal proliferation. In another embodiment, the cell is disposed within a living organism, preferably a mammal, more preferably a human.

In a further aspect of the invention, a compound that binds to developmental peptide receptors and inhibits abnormal cell proliferation is provided. The compound has the formula R₁-R₂-R₃-R₄-R₅-R₆-R₇—OH, wherein R₁ is Gly or a mimetic of Gly, R₂ is Lys or a mimetic of Lys, R₃ is Arg or a mimetic of Arg, R₄ is Ile or a mimetic of Ile, R₅ is Lys or a mimetic of Lys, R₆ is Gly or a mimetic of Gly and R₇ is Thr or a mimetic of Thr. In alternative embodiments, the compound may comprise the formula X-R₁-R₂-R₃-R₄-R₅-R₆-R₇—OH, wherein X may comprise two to four amino acid residues or mimetics of said residues, R₁ is Gly or a mimetic of Gly, R₂ is Lys or a mimetic of Lys, R₃ is Arg or a mimetic of Arg, R₄ is Ile or a mimetic of Ile, R₅ is Lys or a mimetic of Lys, R₆ is Gly or a mimetic of Gly and R₇ is Thr or a mimetic of Thr. For example, X may comprise the sequence Val-Leu, Ile-Glu-Val-Leu, Ile-Asp-Val-Leu, Ile-Arg-Val-Leu, Ile-Glu-Val-Thr, Ile-Asp-Val-Thr, Ile-Arg-Val-Thr, or mimetics thereof.

The present invention also provides for a methodology for isolating developmental peptides from adult and embryonal tissues that have selective antiproliferative effects on damaged cells, including, for example, cancerous cells and virally infected cells, as compared to normal cells. This methodology in one of its non-limiting embodiments allows for isolation and identification of developmental peptides containing a seven amino acid sequence N-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID No.1) present in both embryonal and adult mammalian tissue. More preferably, the developmental peptide is of the sequence N-Val-Leu-Gly-Lys-Arg-Ile-Lys-Gly-Thr-OH (SEQ ID No.2) that is present in both embryonal and adult mammalian liver. The developmental peptide may be isolated using chromatographic techniques or other isolation techniques known in the art.

In another embodiment, a method for identifying and analyzing developmental peptides and developmental peptide-like sequences within a protein database is provided. Preferably, the analysis is performed on Val-Leu-Gly-Lys-Arg-Ile-Lys-Gly-Thr (SEQ ID No.2) versus the human protein database.

A further embodiment of the present invention relates to the characterization and sequencing of a peptide that has significant antiproliferative effects on mammalian cancer cells. Preferably the developmental peptide has a sequence Xaa_m-Gly-Lys-Arg-Ile-Xaa_n, wherein Xaa_m and Xaa_n each represent an amino acid and wherein m independently has a value from 0 to 20, preferably less than 10, and wherein n independently has a value from 0 to 20, preferably less than 10, or mimetics thereof. In further embodiments, the developmental peptide has the sequence Xaa_m-Lys-Gly-Thr-Xaa_n, wherein Xaa_m and Xaa_n each represent an amino acid and wherein m independently has a value from 0 to 20, preferably less than 10, and wherein n independently has a value from 0 to 20, preferably less than 10, or mimetics thereof.

For example, the present invention relates to demonstrating the developmental peptide antiproliferative effect against various cancer cells including but not limited to breast and androgen receptor negative prostate cancer cells. In a further non-limiting embodiment, the present invention relates to demonstrating developmental peptide antiproliferative effect against various virally infected cells. While not wanting to be limited by theory, the antiproliferative effect may be exerted by blocking protein synthesis and creating mitochondrial collapse. The antiproliferative effect could also be exerted against leukemia and cancerous cells of the lung, liver, kidney, ovary, uterus, colon and the like.

Further, methods of inhibiting cancer cell proliferation comprising administering an effective amount of developmental peptides or peptidomimetic thereof to a subject in need of such treatment. As such, the proteins, peptides and peptidomimetics of the invention may be useful for the treatment of cancer and other proliferative disorders. In a further embodiment, a method of potentiating chemotherapeutic agents by administering a developmental peptide or mimetic thereof is provided. Methods of inhibiting viral replication and proliferation comprising administering an effective amount of developmental peptides or a peptidomimetic thereof to a subject in need of such treatment. In addition, administration of developmental peptides before and during pregnancy may help reduce/eliminate teratogenicity/toxicity of different compounds or exposures.