RELATED APPLICATION

The present application gains priority from U.S. Provisional Patent Applications No.

61/535,998 filed 18 September 2011, incorporated by reference herein in its entirety and for all purposes.

SEQUENCE LISTING

The present application incorporates-by-reference nucleotide and/or amino acid sequences which are present in the file named "20120905_121-72-PCT_SequenceListing ST25.txt", which is 5 kilobytes in size, and which was created on 5 September 2012 in the IBM-PC machine format, having an operating system compatibility with MS-Windows, and is submitted herewith.

FIELD AND BACKGROUND OF THE INVENTION

The invention, in some embodiments, relates to the field of chemistry and pharmacology, and more specifically, but not exclusively, to a B-cell targeting peptide useful for selective targeting to B-cell leukemic cells. The invention, in some embodiments, relates to the field of cancer treatment, and more specifically, but not exclusively, to compositions comprising a peptide, and their use in the treatment and diagnosis of cancers such as B-cell leukemias. In particular, provided herein is a method of selectively directing an agent to a B- cell leukemic cell in a subject by administering to the subject a composition comprising the agent and peptide that targets a B-cell leukemia cell.

Cancer is a term used for diseases in which abnormal cells divide without control and are able to invade other tissues. Cancer cells can spread to other parts of the body (metastasis) through the blood and lymph systems.

Leukemia is a cancer that starts in blood-forming tissue such as the bone marrow and is characterized by an abnormal proliferation of blood cells, usually leukocytes. Leukemia is divided into four categories: myelogenous (myeloid) or lymphocytic (lymphoid), each of which can be acute or chronic.

Most forms of leukemia are treated by conventional therapy including chemotherapy, radiation therapy and in some cases, bone marrow transplant. Currently known chemotherapeutic agents lack cancer cell specificity, causing a wide range of adverse side-effects, including nausea, vomiting, anemia, and hair loss, their use limited due to concentration-dependent toxicity in normal cells. Furthermore, multiple drug resistance is frequently induced in cancer cells upon repeated exposure to chemotherapeutic agents. The therapeutic index of chemotherapeutic agents may be greatly improved if they could specifically target cancer cells and tissue.

SUMMARY OF THE INVENTION

Some embodiments of the invention provide compositions including B-cell leukemia targeting peptides and methods of using the targeting peptides in detecting, diagnosing, treating and monitoring treatment of cancer or cancer cells in vivo and in vitro. In some embodiments, the peptide is useful for, inter alia, selectively delivering therapeutic and or diagnostic agents to B-cell leukemic cells. In some embodiments, the peptide is a component in a composition whereby, for example, the agent is coupled to the peptide; the agent is complexed with the peptide and a carrier, or a peptide-decorated carrier encapsulates the agent.

Aspects of some embodiments of the invention relate to a peptide or a peptidomimetic thereof configured for selective binding to B-cell leukemic cells, in some embodiments allowing the peptide to act as a B-cell Leukemia targeting ligand. In some embodiments of the invention, the target cancer cell selectively internalizes the peptide. In some embodiments of the invention, the target cancer cell selectively internalizes a composition comprising the peptide.

Aspects of some embodiments of the invention relate to methods and compositions for the treatment and diagnosis or detection of cancer, such as B-cell leukemia, using targeted agent delivery by a composition comprising a peptide including for example peptide-agent conjugates, where the peptide moiety is configured for selective binding to, and in some embodiments, internalization by, B-cell leukemic cells.

Thus, according to an aspect of some embodiments of the invention there is provided a peptide, wherein the peptide comprises a subsequence of at least 5 consecutive amino acids selected from the sequence Thr-Ala-Thr-Ala-Ser-Gln-Ser (SEQ ID NO:l)

In some embodiments, the peptide is a heptapeptide comprising the sequence Thr- Ala-Thr-Ala-Ser-Gln-Ser (SEQ ID NO:l). In some embodiments, the peptide is a hexapeptide comprising a sequence selected from the group consisting of Thr-Ala-Thr-Ala-Ser-Gln (SEQ ID NO:2) and Ala-Thr-Ala-Ser- Gln-Ser (SEQ ID NO:3).

In some embodiments, the peptide is a pentapeptide comprising a sequence selected from the group consisting of Thr-Ala-Ser-Gln-Ser (SEQ ID NO:4), Thr-Ala-Thr-Ala-Ser (SEQ ID NO:5), and Ala-Thr-Ala-Ser-Gln (SEQ ID NO:6).

According to an aspect of some embodiments of the invention there is also provided a peptide or peptidomimetic containing the amino acid sequence set forth in any one of SEQ ID NO: 1-6, or a conservative substitute or peptidomimetic of one of these sequences. In some embodiments provided herein is a cyclic peptide or peptidomimetic comprising SEQ ID NO: l . In some embodiments the cyclic peptide is 7, 8, 9, 10, 11, 12, 13, 14, 15, or up to 35 amino acid residues long. In some embodiments the peptide according to SEQ ID NO: l is synthesized with terminal cysteine residues and cyclic by oxidation.

In some embodiments the peptide comprises an amino acid sequence set forth in one of SEQ ID NOS: 16-18. In some embodiments the peptide comprises an amino acid sequence set forth in SEQ ID NO: 16. In some embodiments the peptide comprises an amino acid sequence set forth in SEQ ID NO: 17. In some embodiments the peptide comprises an amino acid sequence set forth in one of SEQ ID NO: 18. According to an aspect of some embodiments of the invention there is also provided a peptide or peptidomimetic containing the amino acid sequence set forth in any one of SEQ ID NO: 16- 18, or a conservative substitute or peptidomimetic of one of these sequences.

In some embodiments provided herein is a cyclic peptide or peptidomimetic comprising SEQ ID NO: 16. In some embodiments the cyclic peptide is 7, 8, 9, 10, 11, 12, 13, 14, 15, or up to 35 amino acid residues long. In some embodiments the peptide comprising SEQ ID NO: 16 is synthesized with terminal cysteine residues and cyclicized by oxidation.

In some embodiments provided herein is a cyclic peptide or peptidomimetic comprising SEQ ID NO: 17. In some embodiments the cyclic peptide is 7, 8, 9, 10, 11, 12, 13, 14, 15, or up to 35 amino acid residues long. In some embodiments the peptide according to SEQ ID NO: 17 is synthesized with terminal cysteine residues and cyclicized by oxidation.

In some embodiments provided herein is a cyclic peptide or peptidomimetic comprising SEQ ID NO: 18. In some embodiments the cyclic peptide is 7, 8, 9, 10, 11, 12, 13, 14, 15, or up to 35 amino acid residues long. In some embodiments the peptide according to SEQ ID NO: 18 is synthesized with terminal cysteine residues and cyclicized by oxidation. According to an aspect of some embodiments of the invention there is also provided a peptide which includes one or two amino acid substitutions. Therefore, provided is a peptide comprising at least 7 amino acid residues having the sequence: X1-X2-X3-X4-X5-X6-X7 (SEQ ID NO: 7), wherein at least five of the at least 7 amino acid residues set forth in SEQ ID NO:7 are selected from the group of amino acid residues consisting of XI = T; X2 = A; X3 = T; X4 = A; X5 = S; X6 = Q and X7 = S. In some embodiments, the peptide comprises at least 6 consecutive amino acids selected from the group of at least 7 amino acid residues.

According to an aspect of some embodiments of the invention there is further provided a conjugate peptide-agent conjugate, wherein the peptide comprises at least 6 amino acid residues having a sequence selected from the group of sequences X 1-X2-X3-X4-X5-X6 (SEQ ID NO:8) and X2-X3-X4-X5-X6-X7 (SEQ ID NO:9), wherein at least five of the at least 6 amino acid residues are selected from the group of amino acid residues consisting of XI = T; X2 = A; X3 = T; X4 = A; X5 = S; X6 = Q and X7 = S.

In some embodiments the composition further comprises an agent, for example a therapeutic agent or a diagnostic agent. In some embodiments the peptide is directly linked to the agent. In some embodiments the peptide is linked to the agent via a linker.

According to an aspect of some embodiments of the invention provided is a peptide agent conjugate, wherein the peptide comprises at least five consecutive amino acids present in the sequence Thr-Ala-Thr-Ala-Ser-Gln-Ser set forth in SEQ ID NO: l . In some embodiments, the peptide-agent conjugate is prepared through chemical synthesis such as solid phase peptide synthesis.

In some embodiments, the agent is selected from an active agent and a diagnostic agent. In some embodiments, the agent comprises an active agent. In some embodiments, the active agent is selected from the group consisting of a toxin, an antibody, an antigen, a biological material, a chemical material, a drug, an enzyme, a hormone, a nucleic acid, a peptide, a protein, and a radioisotope.

In some embodiments, the agent comprises a diagnostic agent, such as a probe or a tracer. In some embodiments the diagnostic agent includes, but is not limited to, fluorescent, metallic, enzymatic and radioactive markers such as biotin, gold, ferritin, alkaline phosphatase, β-galactosidase, peroxidase, urease, fluorescein or derivative thereof, rhodamine or derivative thereof, tritium, ¹⁴ C , iodination, a luminescent label, a PET-active substance or a SPECT-active substance.

In some embodiments, the agent comprises a non-soluble anchoring agent, such as a bead or particle. According to an aspect of some embodiments of the invention provided is a composition comprising the peptide according to the teachings herein or a peptidomimetic thereof; and a carrier. In some embodiments, the composition comprises a peptide comprising an amino acid sequence set forth in any one of SEQ ID NOS: l-6. In preferred embodiments, the composition comprises a peptide comprising an amino acid sequence set forth in SEQ ID NOS: l . In some embodiments, the composition comprises a peptide comprising an amino acid sequence set forth in any one of SEQ ID NOS: 16-18.

In some embodiments the carrier is a pharmaceutically acceptable carrier. Such carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil or injectable organic esters.

In some embodiments, a pharmaceutically acceptable carrier contains physiologically acceptable compounds that, for example, stabilize or facilitate absorption of the peptide and/or agent. Such physiologically acceptable compounds include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. One skilled in the art knows that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the route of administration of the composition.

In some embodiments the carrier is a macromolecular structure such as a micelle, a liposome a microcarrier a nanocarrier or a dendrimer

According to an aspect of some embodiments of the invention there is also provided the use of a composition comprising the peptide according to the teachings herein, in diagnosis of cancer, in some embodiments B-cell leukemia. In some embodiments the peptide comprises a sequence set forth in any one of SEQ ID NOS: 1-6. In preferred embodiments the peptide comprises a sequence set forth in SEQ ID NO: l . In some embodiments the peptide is covalently linked to a diagnostic agent. In some embodiments the peptide is non-covalently bound to a diagnostic agent.

According to an aspect of some embodiments of the invention there is also provided the use of a composition comprising the peptide according to the teachings herein, in treatment of cancer, in some embodiments B-cell leukemia. In some embodiments the peptide comprises a sequence set forth in any one of SEQ ID NOS: 1-6. In preferred embodiments the peptide comprises a sequence set forth in SEQ ID NO: l . In some embodiments the peptide comprises a sequence set forth in any one of SEQ ID NOS: 16- 18. In some embodiments the peptide is covalently linked to a therapeutic agent. In some embodiments the peptide is non- covalently bound to a therapeutic agent. In some embodiments the peptide is a cyclic peptide.

According to an aspect of some embodiments of the invention, there is also provided the use of a composition comprising the peptide according to the teachings herein, for detection of the presence of cancer cells in a patient following cancer therapy. In some embodiments the peptide comprises a sequence set forth in any one of SEQ ID NOS: l-6. In preferred embodiments the peptide comprises a sequence set forth in SEQ ID NO: l . In some embodiments the peptide comprises a sequence set forth in any one of SEQ ID NOS: 16-18. In some embodiments the peptide is covalently linked to a diagnostic agent. In some embodiments the peptide is non-covalently bound to a diagnostic agent. In some embodiments the peptide is a cyclic peptide.

According to an aspect of some embodiments of the invention there is also provided the use of a composition comprising the peptide according to the teachings herein, in the manufacture of a medicament, such as a medicament for the treatment and/or diagnosis and/or detection of cancer, in some embodiments B-cell leukemia. In some embodiments the peptide comprises a sequence set forth in any one of SEQ ID NOS: l-6. In preferred embodiments the peptide comprises a sequence set forth in SEQ ID NO: l . In some embodiments the peptide comprises a sequence set forth in any one of SEQ ID NOS: 16-18. In some embodiments the peptide is covalently linked to a diagnostic agent. In some embodiments the peptide is non-covalently bound to a diagnostic agent. In some embodiments the peptide is a cyclic peptide.

According to an aspect of some embodiments of the invention the composition comprises the peptide according to the teachings herein, in a pharmaceutically acceptable carrier. In some embodiments, the composition is useful in the treatment and/or diagnosis and/or detection of cancer, in some embodiments B-cell leukemia. In some embodiments the peptide comprises a sequence set forth in any one of SEQ ID NOS: l-6. In preferred embodiments the peptide comprises a sequence set forth in SEQ ID NO: l . In some embodiments the peptide comprises a sequence set forth in any one of SEQ ID NOS: 16-18.In some embodiments the peptide is covalently linked to a therapeutic or diagnostic agent. In some embodiments the peptide is non-covalently bound to a therapeutic or diagnostic agent. In some embodiments the peptide is a cyclic peptide. In some embodiments the carrier comprises a dendrimer, a micelle, a liposome or a nanoparticle.

According to an aspect of some embodiments of the invention there is also provided a method of treatment and/or diagnosis and/or detection of cancer, comprising administering a pharmaceutical composition comprising the peptide set forth in any one of SEQ ID NOS: 1-6 or SEQ ID NOS 16-18 to an animal subject in need thereof, such as an animal in need of treatment or diagnosis of cancer, in some embodiments B-cell leukemia. In some embodiments, the animal is a human animal. In some embodiments, the animal is a non- human animal. In some embodiments the peptide comprises a sequence set forth in any one of SEQ ID NOS: 1-6. In preferred embodiments the peptide comprises a sequence set forth in SEQ ID NO: l . In some embodiments the peptide comprises a sequence set forth in any one of SEQ ID NOS: 16-18.

According to an aspect of some embodiments of the invention there is also provided a method for obtaining information useful in the diagnosis and/or detection of cancer comprising: providing an amount of a composition comprising the peptide according to the teachings herein; ex vivo combining the composition with tissue isolated from a subject; and determining the degree of binding of the peptide to the tissue, whereby the information is obtained by the degree of binding of the peptide to the tissue. In some embodiments, the composition further comprises a diagnostic agent configured for assisting in the determining of the degree of binding of the peptide. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal.

According to an aspect of some embodiments of the invention there is also provided a method for treating a cancer in a subject in need thereof, comprising: providing an amount of a composition comprising the peptide according to the teachings herein; ex vivo combining the composition with tissue isolated from the body of a subject; and whereby the combining reduces the occurrence and/or vitality of cancer cells (especially leukemic cells) in the tissue. In some embodiments, at least some of the tissue is returned to the body of the subject. In some embodiments, the composition further comprises a therapeutic agent configured for reducing the vitality of the cancer cells, for example, a therapeutic agent that is toxic to cancer cells (especially leukemic cells). In some embodiments, the composition further comprises an anchoring agent, for example an insoluble bead associated with the peptide, so that the combining of the composition with the tissue leads to binding of cancer cells to the anchoring agent through the peptide, thereby effecting the reducing of the occurrence of cancer cells in the tissue. In some embodiments, the method is used for the treatment and/or diagnosis and/or detection of cancer, in some embodiments B-cell leukemia. In some embodiments, the isolated tissue is blood. In some embodiments, subsequent to combining the composition and the isolated tissue, the tissue is prepared for return to the animal. In some embodiments the peptide comprises a sequence set forth in any one of SEQ ID NOS: 1-6. In preferred embodiments the peptide comprises a sequence set forth in SEQ ID NO: l . In some embodiments the peptide comprises a sequence set forth in any one of SEQ ID NOS: 16-18.

According to an aspect of the invention, provided herein is a method of detecting presence of a B-cell leukemic cell in a subject, comprising

a) providing a tissue sample from the subject;

b) contacting the tissue sample with a composition comprising a peptide conjugated to a diagnostic agent, wherein the peptide comprises up to 35 amino acids in length and an amino acid sequence selected from any one of SEQ ID NOS: l-6, and wherein the polypeptide is capable of binding to a B-cell leukemia cell; and

c) detecting a level of binding of the peptide conjugated to a diagnostic agent in the tissue sample as compared to a negative control,

wherein an increase in binding of the peptide conjugated to a diagnostic agent as compared to a negative control indicates the presence of a B-leukemic tumor cell in the tissue sample.

In some embodiments, the contacting is performed ex vivo. In some embodiments, the contacting is performed in vitro. As is clear to a person having ordinary skill in the art, such embodiments do not require the presence of the subject during the contacting.

In some embodiments the tissue sample is a blood sample or a bone marrow sample. According to an aspect of some embodiments of the invention, provided herein is a method of detecting the presence or absence of a B-cell leukemic cell in a subject, comprising:

a) administering a composition comprising a peptide conjugated to a diagnostic moiety to a subject, wherein the peptide comprises up to 35 amino acids in length and an amino acid sequence selected from any one of SEQ ID NOS: l-6, and wherein the polypeptide is capable of binding to a B-cell leukemic cell; and

b) detecting a level of binding of the peptide conjugated to a detectable molecule in the subject as compared to a negative control,

wherein an increase in binding of the polypeptide conjugated to a detectable molecule as compared to a negative control indicates the presence of a tumor cell in the subject.

In some embodiments the peptide comprises SEQ ID NO: l or a peptidomimetic thereof. In some embodiments the peptide is a cyclic peptide or peptidomimetic thereof. In certain embodiments, the diagnostic agent comprises fluorescent, metallic, enzymatic and radioactive markers such as biotin, gold, ferritin, alkaline phosphatase, β-galactosidase, peroxidase, urease, fluorescein or derivative thereof, rhodamine or derivative thereof, tritium, ¹⁴ C , iodination, a luminescent label, a PET-active substance or a SPECT-active substance. In another aspect of some embodiments of the invention, provided herein is a kit for detecting presence or absence of a tumor cell in a subject, including a composition comprising a peptide conjugated to a detectable molecule to a subject, wherein the peptide comprises up to 35 amino acids in length and an amino acid sequence selected from any one of SEQ ID NO: 1-6, and wherein the peptide is capable of binding to a B-cell leukemia cell. In some embodiments the peptide comprises SEQ ID NO: l . In some embodiments the peptide comprises a sequence set forth in any one of SEQ ID NOS:16-18. In some embodiments the peptide is a cyclic peptide or peptidomimetic thereof. In certain embodiments, the diagnostic agent comprises fluorescent, metallic, enzymatic and radioactive markers such as biotin, gold, ferritin, alkaline phosphatase, β-galactosidase, peroxidase, urease, fluorescein or derivative thereof, rhodamine or derivative thereof, tritium, ¹⁴ C, iodination, a luminescent label, a PET-active substance or a SPECT-active substance. In some embodiments, the detectable molecule is fluorescein or a fluorescein derivative. In some embodiments, the detectable molecule is rhodamine or a rhodamine derivative. The presence of a tumor cell in a subject is determined by identifying cells from the subject, which exhibit binding or internalization of the peptide conjugate. In some embodiments, the kit further comprises instructions for use.

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 the invention pertains. In case of conflict, the specification, including definitions, takes precedence.

As used herein, the terms "comprising", "including", "having" and grammatical variants thereof are to be taken as specifying the stated features, integers, steps or components but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof. These terms encompass the terms "consisting of and "consisting essentially o .

As used herein, the indefinite articles "a" and "an" mean "at least one" or "one or more" unless the context clearly dictates otherwise.

As used herein, when a numerical value is preceded by the term "about" the term "about" is intended to indicate +/-10%.

DESCRIPTION OF SOME EMBODIMENTS OF THE INVENTION

Some embodiments of the teachings herein relate to the field of chemistry and pharmacology, and more specifically, but not exclusively, to chemical entities including a peptide part useful for selective binding to B-cell leukemic cells. Some embodiments of the teachings herein relate to chemical entities comprising peptide-agent conjugates and their use in the treatment, diagnosis or detection of cancer, particularly B-cell leukemias.

In some embodiments, the peptide is selected according to its ability to bind with a high degree of specificity to unique, complementary receptors expressed on the surface of specific populations of target cells, particularly B-cells. In some embodiments, the peptide is selectively internalized by the target cancer cell. The peptide-agent conjugate is constructed to specifically target such cells where the peptide part constitutes a cell-targeting part of the conjugate.

The principles, uses and implementations of the teachings herein may be better understood with reference to the accompanying description. Upon perusal of the description present herein, one skilled in the art is able to implement the invention without undue effort or experimentation.

Before explaining at least one embodiment in detail, it is to be understood that the invention is not necessarily limited in its application to the details of construction and the arrangement of the components and/or methods set forth herein. The invention is capable of other embodiments or of being practiced or carried out in various ways. The phraseology and terminology employed herein are for descriptive purpose and should not be regarded as limiting.

The present inventor has found a peptide having the sequence Thr-Ala-Thr-Ala-Ser- Gln-Ser, as well as parts thereof, exhibits binding with a high degree of specificity to receptors expressed on the surface of B-cells in B-cell leukemia. Such a peptide can be combined with an agent, e.g., chemically-bonded directly or through a linker molecule to form a conjugate that is a peptide-agent conjugate constructed to target cells where the peptide part constitutes a cell-targeting part of the conjugate.

In some embodiments, the peptide comprises a subsequence of at least 5 amino acids selected from the sequence Thr-Ala-Thr-Ala-Ser-Gln-Ser.

In some embodiments, the peptide is a heptapeptide comprising the sequence Thr- Ala-Thr-Ala-Ser-Gln-Ser.

In some embodiments, the peptide is a hexapeptide comprising a sequence selected from the group consisting of Thr-Ala-Thr-Ala-Ser-Gln and Ala-Thr-Ala-Ser-Gln-Ser.

In some embodiments, the peptide is a pentapeptide comprising a sequence selected from the group consisting of Thr-Ala-Ser-Gln-Ser, Thr-Ala-Thr-Ala-Ser, and Ala-Thr-Ala- Ser-Gln. In some embodiments the peptide comprises a sequence set forth in any one of SEQ ID NOS: 16-18.

In some embodiments, the peptide comprises at least 7 amino acid residues having the sequence: X1-X2-X3-X4-X5-X6-X7, wherein at least five of the at least 7 amino acid residues are selected from the group of amino acid residues consisting of XI = T; X2 = A; X3 = T; X4 = A; X5 = S; X6 = Q and; X7 = S. In some embodiments, the peptide comprises at least 6 amino acids selected from the group of at least 7 amino acid residues.

In some embodiments, the peptide comprises at least 6 amino acid residues having a sequence selected from the group of sequences X1-X2-X3-X4-X5-X6 and X2-X3-X4-X5- X6-X7, wherein at least five of the at least 6 amino acid residues are selected from the group of amino acid residues consisting of XI = T; X2 = A; X3 = T; X4 = A; X5 = S; X6 = Q and X7 = S.

Further provided herein is a conjugate, which includes a peptide and an agent, wherein the peptide comprises a subsequence of at least 5 consecutive amino acids selected from the sequence Thr-Ala-Thr-Ala-Ser-Gln-Ser. As used herein, the term "agent" broadly refers to a physical, chemical, or biological material that can be linked to a peptide of the teachings herein and generally imparts a biologically useful function to the targeting peptide. An agent can be any natural or non-natural material including a biological material, such as a cell or phage; an organic chemical, such as a small molecule; a radionuclide; a nucleotide or oligonucleotide; a polypeptide; or a peptide or peptidomimetic. Depending on the embodiments, agents useful for implementing the teachings herein, for example in diagnostic and therapeutic methods include, without limitation, therapeutic agents; cancer chemotherapeutic agents, cytotoxic agents, pro-apoptotic agents, anti-lymphangiogenic agents, detectable labels and imaging agents; and tags or other insoluble supports. Depending on the embodiment, agents useful in implementing the teachings herein further include, for example, phage and other viruses, cells, liposomes, polymeric matrices, non-polymeric matrices or particles such as gold particles, microdevices and nanodevices, and nano-scale semiconductor materials. These and other agent known in the art can be components of a conjugate of the teachings herein, as disclosed herein.

In some embodiments, the agent comprises, for example, a toxin, an antibody, an antigen, a biological material, a chemical material, a drug, an enzyme, a hormone, a nucleic acid, a peptide, a protein. In some embodiments the agent comprises a macromolecular structure such as, for example, a micelle or a liposome. In some embodiments, the active agent comprises a chemotherapeutic agent, such that the conjugate comprises, for example, the peptide as a cell-targeting portion and the chemotherapeutic agent as an apoptosis-inducing portion. In some embodiments, the chemotherapeutic agent comprises a toxin, such as a plant toxin or a bacterial toxin. In some embodiments, the toxin is selected from the group consisting of ricin toxin, Pseudomonas exotoxin, pokeweed antiviral protein, saporin, gelonin, cyclohexamide, anisomycin and diphtheria toxin.

In embodiments where the agent is a chemotherapeutic agent, the peptide-agent conjugate is configured such that upon administration of the conjugate, the peptide binds to the specific receptor expressed on the surface of the cancer cells, whereupon the chemotherapeutic agent exerts an effect ultimately leading to death of the target cell. For example, in some exemplary embodiments, a peptide derived from Myelin Basic Protein, referred to here as (MBP) is chemically coupled to the anti-cancer drug doxorubicin (Dox). The resulting conjugate, MBP-Dox, is then cultured with cells expressing a cell surface antibody specific for the MBP peptide. During culture, MBP-Dox binds to the cells through the MBP receptor, the MBP-Dox is internalized into the cell and then the chemotherapeutic drug affects the cell, leading to cell death.

In some embodiments, the agent is a diagnostic agent. In some embodiments, the diagnostic agent is a probe or a tracer. In some embodiments, the diagnostic agent is a detectable agent, such as a fluorescent agent (e.g., fluorescein), an enzyme or a radioactive agent (e.g., includes a radioisotope). In some embodiments, the diagnostic agent is a chemical or biological signaling agent. For example, in some exemplary embodiments, the fluorescent molecule FITC is conjugated to a peptide specific for a selected target antibody. The peptide- FITC conjugate is added to a mixture of antibodies, including the target antibody, preferably in an assay format similar to the common Fluorescent Immunoassay test. The conjugate binds only to the target antibody, and this reaction can be used to measure the amount of target antibody in the mixture.

In some embodiments, the agent comprises a non-soluble anchoring agent, for example, a particle such as a glass, silica or polymer bead, to which the peptide is bonded, for example through the C-terminus with a diamine linker, as is known in the art of solid phase peptide synthesis. Such chemical entities are useful for making a filter selective for capturing cells to which the peptide has a high affinity, for example for extracting afflicted cells from a sample of blood. A conjugate is made using any suitable method or technique, for example, suitable synthetic methods with which a person having ordinary skill in the art of synthetic organic chemistry or peptide synthesis is familiar.

Typically, a suitable peptide sequence is first identified using libraries of peptides of varying sequence. Such libraries include phage peptide display libraries or chemical libraries according to techniques known in the art of molecular biology and chemistry.

In some embodiments, typically where the agent is a peptide or a protein, the conjugate is made as a single unit, for example, using a suitable organism such as a genetically modified cell such as a yeast or bacterial cell, or using a peptide-synthesis technique such as solid-phase peptide synthesis.

In some embodiments, the peptide and the agent are provided separately, each synthesized according to a suitable method, and combined to form the chemical agent, for example, chemically bonded together directly or through a linker. In such embodiments, the peptide is typically made for example, using a suitable organism such as a genetically modified yeast or bacteria, or using a peptide-synthesis technique such as solid-phase peptide synthesis.

After synthesis and if desired, the conjugate be purified by any method known in the art, such as, for example, high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography and the like.

Peptides and Peptidomimetics

Some embodiments of the teachings herein provide peptides exhibiting selective binding to B-cell leukemic cells. The term "selective binding" means that the peptides disclosed herein bind to the target cells with greater affinity than they binds to normal mammalian cells under specified conditions. According to one embodiment, the peptides of the teachings herein bind the target B-cell leukemic cells with an affinity greater by at least one order of magnitude than their binding affinity to other mammalian cells under specified conditions.

Provided herein is an isolated peptide containing the amino acid sequence set forth in SEQ ID NO: l, or a peptidomimetic thereof. The peptide or peptidomimetic disclosed herein can be, for example, cyclic or otherwise conformationally constrained and can have a variety of lengths, for example, a length of 40 residues, a length of 35 residues, a length of 30 residues, a length of 25 residues, a length of 15 residues, a length of 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 residues. As used herein, the term "residue" refers to amino acids or analogs thereof. In some preferred embodiments the peptide is cyclic, for example, by synthesizing a peptide with two cysteine residues (e.g, at the C and N termini) and forming a cyclicizing disulfide bond, through the cysteine side chains.

In one embodiment, provided herein is a peptide which contains the amino acid sequence set forth in any one of SEQ ID NO: 1-6, or a peptidomimetic based on the same amino acid sequence and has targeting, or homing, activity, toward B-cell leukemic cells. In a preferred embodiment, provided herein is a peptide which contains the amino acid sequence set forth in SEQ ID NO: l, or a peptidomimetic based on the same amino acid sequence and has targeting, or homing, activity, toward B-cell leukemic cells.

As used herein, the term "peptide" is used broadly to mean peptides, proteins, fragments of proteins and the like. One skilled in the art will recognize that the peptides disclosed herein may be synthesized as peptide mimetics. A peptide mimetic or "peptidomimetic", is a molecule that mimics the biological activity of a peptide but is not completely peptidic in nature. The term "peptidomimetic," as used herein, means a peptide- like molecule that has the activity of the peptide upon which it is structurally based. Such peptidomimetics include chemically-modified peptides, peptide-like molecules containing non-naturally occurring amino acids, and peptides and have an activity such as selective targeting activity of the peptide upon which the peptidomimetic is derived. A peptidomimetic can include amino acid analogs and can be a peptide-like molecule which contains, for example, an amide bond isostere such as a retro-inverso modification; reduced amide bond; methylenethioether or methylenesulfoxide bond; methylene ether bond; ethylene bond; thioamide bond; trans-olefm or fluoroolefin bond; 1,5-disubstituted tetrazole ring; ketomethylene or fluoroketomethylene bond or another amide isostere. One skilled in the art understands that these and other peptidomimetics are encompassed within the meaning of the term "peptidomimetic" as used herein.

Accordingly, provided herein is a peptide containing the amino acid sequence set forth in any one of SEQ ID NO: 1-6, or a conservative substitute or peptidomimetic of one of these sequences. In a preferred embodiment, the peptide contains the amino acid sequence set forth in SEQ ID NO: l, or a conservative substitute or peptidomimetic of the sequence. Further provided herein is a peptide containing the amino acid sequence set forth in any one of SEQ ID NO: 16- 18, or a conservative substitute or peptidomimetic of one of these sequences. As used herein, a "conservative substitute" is an amino acid sequence in which a first amino acid is replaced by a second amino acid or amino acid analog having at least one similar biochemical property, which can be, for example, similar size, charge, hydrophobicity or hydrogen-bonding capacity. For example, a first hydrophobic amino acid alanine can be conservatively substituted with a second (non-identical) hydrophobic amino acid such as alanine (Ala), valine (Val), leucine (Leu), or isoleucine (He), or an analog thereof. Similarly, a first amino acid having an uncharged side chain (i.e., serine, threonine and glutamine) can be conservatively substituted with a second (non-identical) amino acid such as serine (Ser), threonine (Thr), asparagine (Asn) and glutamine (Gin), or an analog thereof.

Further provided is a cyclic peptide comprising a peptide comprising the amino acid sequence set forth in any one of SEQ ID NO: 1-6 or SEQ ID NOS 16-18. In preferred embodiments the cyclic peptide comprises SEQ ID NO: l and has 9-39 amino acids, 12-35 amino acids or 15-25 amino acids. Methods for preparing a cyclic peptide have been established and are well known by those skilled in the art, see for example Davies, 2003. J Peptide Sci. 9:471.

Some embodiments of the teachings herein further provide a chimeric protein containing a peptide or peptidomimetic set forth in any one of SEQ ID NO: 1-6, fused to a heterologous protein, for example a therapeutic or diagnostic protein or peptidomimetic thereof. In one embodiment, the teachings herein provides a chimeric protein containing set forth in any one of SEQ ID NO: 1-6 fused to a heterologous peptide or protein.

The term "heterologous protein," as used herein in refers to a protein derived from a source other than the gene encoding the peptide according to the teachings herein or upon which the peptidomimetic is derived, and can be for example a mammalian, prokaryotic or plant protein. Accordingly, further provided herein is a bifunctional moiety which contains a peptide set forth in any one of SEQ ID NOS: 1-6 that selectively targets a B-cell leukemia cell, fused to a second peptide having a separate activity, for example a cytotoxic activity. In one embodiment, the heterologous peptide or protein has a therapeutic activity, for example a pro-apoptotic protein or peptide or a bacterial or plant toxin.

Further disclosed herein are isolated nucleic acid molecules that encode the B-cell targeting peptides and the chimeric proteins. Thus, in yet another aspect, an isolated nucleic acid molecules provided comprising (a) a nucleic acid molecule which codes for a peptide comprising an amino acid sequence of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or functionally equivalent fragments thereof, (b) degenerates of (a); and (c) complements of (a) and (b). In preferred embodiments the nucleic acid molecule includes a nucleic acid sequence set forth in any one of SEQ ID NO: 10, SEQ ID NO: l 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18,. As used herein a "nucleic acid molecule" refers to an oligonucleotide of about 15 to about 105 nucleotides in length.

Synthesis of Peptide and Peptide- Agent Conjugate

Peptides and peptide conjugates disclosed herein are synthesized according to methods known in the art, including, but not limited to recombinant methods as well as synthetic methods. In some embodiments, the targeting peptides according to the teachings herein are synthesized by synthetic methods. Peptides can be synthesized by a large variety of well-known techniques, such as solid-phase methods (FMOC-, BOC-, and other protection schemes, various resin types), solution methods (FMOC, BOC and other variants) and combinations of these. Automated devices for the purpose are available commercially, as are also routine synthesis and purification services. In certain embodiments, one or more protecting groups are used during synthesis, as are known in the art, such as FMOC, BOC, and trityl groups and other protecting groups. Protecting groups are often used for protecting amino, carboxyl, hydroxyl, guanyl and -SH groups, and for any reactive groups/functions.

The peptide agent conjugates disclosed herein may be prepared as fusion proteins or by other suitable recombinant DNA methods known in the art. Such an approach for preparing the peptides according to the teachings herein is preferred especially when the effector units and/or other optional units are peptides or proteins.

The peptide agent conjugates disclosed herein may be synthesized using chemical synthesis methods known in the art. Using solid phase chemistry, for example, one could attach a therapeutic agent or diagnostic agent directly to the amino or carboxylic group of the last amino acid in the sequence (depending on the direction of synthesis). Alternatively, combinatorial chemical methods known in the art can be used to couple the peptide to a therapeutic agent or to a diagnostic agent to generate a peptide conjugate.

Use of peptide/ peptide-agent conjugate

Some embodiments of the peptide provided herein was shown to specifically target B- cell leukemic cells. Leukemia cells are named after the specific blood cell that becomes cancerous, the lymphoid cells or the myeloid cells. There are four main types of leukemia in adults: acute lymphocytic leukemia (ALL); chronic lymphocytic leukemia (CLL); acute myeloid leukemia (AML); and chronic myeloid leukemia (CML). Other, less common types of leukemia, are generally subcategories of one of the four main categories, for example, prolymphocytic leukemia (PLL) and hairy cell leukemia (HCL), both of which are chronic B- cell leukemias. According to some embodiments, there is provided the use of a peptide according to the teachings herein, in the manufacture of a medicament, in some embodiments for the treatment of cancer. In some embodiments the medicament comprises a peptide set forth in any one of SEQ ID NOS: l-6 and a therapeutic agent. In some embodiments the peptide is coupled to the agent per se or via a linker. In some embodiments the medicament comprises a peptide set forth in any one of SEQ ID NOS: l-6 and a dendrimer, and further comprises a therapeutic agent

According to some embodiments, there is provided the use of a peptide according to the teachings herein, for treatment of a pathology, in some embodiments for the treatment of cancer.

According to some embodiments, there is provided a method for the treatment of a pathology, in some embodiments cancer, comprising administering a pharmaceutically effective amount of a composition comprising a peptide according to the teachings herein to an animal subject in need thereof. In some embodiments the subject is a mammal, preferably a human. In some embodiments, the subject is a non-human mammal.

In some embodiments, the cancer is B-cell leukemia.

Pharmaceutical compositions

A peptide or peptide conjugate according to the teachings herein may be administered to a subject per se or as a component of a pharmaceutical composition.

Pharmaceutical compositions and medicaments comprising a peptide and/or conjugate according to the teachings herein may be manufactured by any suitable method or combination of methods as known in the art such as are described in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference. Such methods include conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing. Pharmaceutical compositions and medicaments may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the proteins into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

For topical administration a composition comprising a peptide and/or conjugate according to the teachings herein may be formulated as solutions, gels, ointments, creams, suspensions, etc. as are well-known in the art. Systemic formulations include those designed for administration by injection, e.g., subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal, inhalation, oral or pulmonary administration.

For injection, a composition comprising a peptide and/or conjugate according to the teachings herein may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. The solution may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Alternatively, a composition may be in powder form for constitution with a suitable vehicle, e. g., sterile pyrogen- free water, before use.

For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For oral administration, a composition comprising a peptide and/or conjugate according to the teachings herein is readily formulated by combining the peptide or conjugate with pharmaceutically acceptable carriers well known in the art. Such carriers enable the composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. For oral solid formulations such as, for example, powders, capsules and tablets, suitable excipients include fillers such as sugars, e. g. lactose, sucrose, mannitol and sorbitol; cellulose preparations such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP); granulating agents; and binding agents. If desired, disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

If desired, solid dosage forms may be sugar-coated or enteric-coated using standard techniques.

For oral liquid preparations such as, for example, suspensions, elixirs and solutions, suitable carriers, excipients or diluents include water, glycols, oils, alcohols, etc. Additionally, flavoring agents, preservatives, coloring agents and the like may be added.

For buccal administration, a composition comprising a peptide and/or conjugate according to the teachings herein may take the form of tablets, lozenges, etc. formulated in conventional manner.

For administration by inhalation, some embodiments of the composition are conveniently delivered in the form of an aerosol spray from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the peptide and a suitable powder base such as lactose or starch.

The peptides and/or conjugates may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e. g., containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, a composition comprising a a peptide and/or conjugate according to the teachings herein may also be formulated as a depot preparation.

Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, a composition comprising a peptide and/or conjugate according to the teachings herein may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

Alternatively, other pharmaceutical delivery systems may be employed. Liposomes and emulsions are well known examples of delivery vehicles that may be used to deliver peptides and/or conjugates according to the teachings herein. Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity. Additionally, the peptides and/or conjugates may be delivered using a sustained-release system, such as semipermeable matrices of solid polymers containing the therapeutic agent. Various of sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the peptide or conjugate for a few weeks up to over 100 days.

The conjugates according to the teachings herein, especially the peptide part, may include charged side chains or termini, and may be included in any of the above-described formulations as the free acids or bases, as esters or as pharmaceutically acceptable salts. Pharmaceutically acceptable salts are those salts which substantially retain the biologic activity of the free acid or base and which are prepared, for example by reaction with inorganic acids. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than are the corresponding free forms. In some embodiments a peptide according to the teachings herein is used to decorate a carrier, including for example, a micelle, a liposome, a lipid particle, a microcarrier, a nanocarrier, a dendrimer, or a dendrimer. The peptide may be covalently or non-covalently associated with the carrier. In some embodiments the carrier and the targeting peptide are covalently bound. In some embodiments the carrier and the targeting peptide are non- covalently associated. In some embodiments the carrier and the targeting peptide are covalently bound via a surface modification of the carrier with a functional group or linker. A linker includes, for example a synthetic polymer, a natural polymer or a semi synthetic polymer (comprising natural and synthetic elements). In some embodiments the linker comprises a PEG moiety. In some embodiments the linker is peptide of about 2 to 16 amino acids or a C2-C16 hydrocarbon. In some embodiments the linker comprises a nucleic acid, a peptidyl moiety or a saccharide. In some embodiments the linker is a cleavable linker for example an enzyme cleavable linker cleavable in the presence of for example cathepsin B or thrombin.

The linker that links the peptide to the carrier may be incorporated into the carrier composition ab initio or may be combined with the prepared carrier.

Without wishing to be bound to theory, in some embodiments the peptide-decorated carrier is able to carry a therapeutic or diagnostic agent to a target cell, i.e. a B-cell leukemic cell.

In some preferred embodiments the carrier is a dendrimer. Multifunctional dendrimers are described, inter alia, in US 2010/0105857, to the Inventor and Assignee of the present application, and is incorporated herein by reference in its entirety.

In some embodiments the peptide is adsorbed onto a surface. In some embodiments, the surface is of a carrier particle (for example, a nanoparticle) made with either an inorganic or organic core. Examples of such nanoparticles include, but are not limited to, nanocrystalline particles, nanoparticles made by the polymerization of organic chains, activated carbon particles and protein-ceramic nanoplates.

Adsorption of the peptide and/or an agent to a surface for the purpose of delivery of the adsorbed molecules to cells is well known in the art. Preferably, the material comprising the adsorbent surface is biodegradable. Adsorption of the peptide and/or an agent to a surface may occur through non-covalent interactions, including ionic and/or hydrophobic interactions. In general, the features of carriers such as nanoparticles, such as surface charge, particle size and molecular weight, depend upon polymerization conditions, monomer concentration and the presence of stabilizers during the polymerization process. The surface of carrier particles may be modified, for example, with a surface coating, to allow or enhance adsorption of the peptide and/or agent.

Complexes

Peptides or peptide conjugates may be administered in the form of a microcarrier or nanocarrier complex. Accordingly, in some embodiments the teachings herein provide compositions comprising a peptide-micro carrier complex and a peptide nanocarrier complex.

Microcarriers useful in implementing such embodiments are less than about 150, 120 or 100 micrometers (μιη) in size, preferably about 10-60 μιη, and are preferably water insoluble. Nanocarriers useful in implementing the teachings herein are less than about 1 micrometer (um) in size, preferably about 10-1000 nm, and are water insoluble. Microcarriers and nanocarriers are preferably biocompatible and may be biodegradable A wide variety of biodegradable and nonbiodegradable materials acceptable for use as microcarriers and nanocarriers are known in the art.

Solid biodegradable microcarriers may be manufactured from biodegradable polymers including, but not limited to: biodegradable polyesters, such as poly(lactic acid), poly(glycolic acid), and copolymers (including block copolymers) thereof, as well as block copolymers of poly(lactic acid), poly(ethylene glycol), and poly (lactic-co-glycolic acid).

Nonbiodegradable materials suitable for manufacturing microcarriers are also known, including, but not limited to polystyrene, polypropylene, polyethylene, silica, ceramic, polyacrylamide, dextran, hydroxyapatite, latex, gold, and ferromagnetic or paramagnetic materials.

Suitable functional groups useful for coupling the peptide to an agent and or a carrier include: N-hydroxysuccinimide esters, isothiocyanates, and sulfonylchlorides, which form stable covalent bonds with amines, including amines in proteins and amine-modified nucleic acids; Iodoacetamides and maleimides, which form covalent bonds with thiol-functions, as in proteins; Carboxyl functions and various derivatives, including N-hydroxybenztriazole esters, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl, and aromatic esters, and acyl imidazoles; Alkylhalides, including iodoacetamides and chloroacetamides; Hydroxyl groups, which can be converted into esters, ethers, and aldehydes; Aldehydes and ketones and various derivatives, including hydrazones, oximes, and semicarbozones; Isocyanates (react with amines); Thiol groups, which may form disulfide bonds and react with alkylhalides (such as iodoacetamide); Alkenes, which can undergo a Michael addition with thiols, e.g., maleimide reactions with thiols; Phosphoramidites, which can be used for direct labeling of nucleosides, nucleotides, and oligonucleotides; Primary amines that may be coupled to variety of groups including carboxyl, aldehydes, ketones, and acid chlorides, among others.

Agents

The agents disclosed herein are, for example, therapeutic agents or diagnostic agents.

Therapeutic agents include agents that are cytotoxic such as toxins, immunomodulators including cytokines, lymphokines, chemokines, growth factors; hormones; hormone antagonists; enzymes; nucleic acid molecules including antisense and siR A; photoactive therapeutic agents, anti-angiogenic agents; and pro-apoptotic agents. The therapeutic agent may be delivered by coupling (i.e. covalently) to the peptide or may be administered in unconjugated form.

Examples of therapeutic agents useful in treating B-cell leukemias include chemotherapeutic drugs including conventional chemotherapeutic reagents such as alkylating agents, anti-metabolites, plant alkaloids including taxol, antibiotics and compounds including cisplatin, CDDP, methotrexate, vincristine, adriamycin, bleomycin, and hydroxyurea.

In a preferred embodiment, the therapeutic agent is a chemotherapeutic selected from the group consisting of nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas, gemcitabine, triazenes, folic acid analogs, anthracyclines, taxanes, nucleotide analogs, antibiotics, enzyme inhibitors, platinum based agents, vinca alkaloids, substituted ureas, methyl hydrazine derivatives, adrenocortical suppressants, hormone antagonists, endostatin, taxols, camptothecins, doxorubicins and their analogs, antimetabolites, alkylating agents, antimitotics, anti-angiogenic agents, tyrosine kinase inhibitors, mTOR inhibitors, pro- apoptotic agents, methotrexate, and combinations thereof. In some preferred embodiments the agent is doxorubicin. In some preferred embodiments the agent is doxorubicin. In some preferred embodiments the agent is chlorambucil. In some preferred embodiments the agent is Melphalan.

In another preferred embodiment, the therapeutic agent is a toxin selected from the group consisting of ricin, abrin, alpha toxin, saporin, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, Pseudomonas endotoxin and combinations thereof.

In another preferred embodiment, the therapeutic agent is an immunomodulator selected from the group consisting of a cytokine, a stem cell growth factor, a lymphotoxin, a hematopoietic factor, a colony stimulating factor, an interferon, erythropoietin, thrombopoietin and combinations thereof. In other preferred embodiments, the therapeutic agent is a radioisotope including radionuclides that substantially decay with Auger-emitting particles. Alpha and beta-particle- emitting radioisotopes are contemplated. Non-limiting examples of radioisotopes include ^m In, ¹⁷⁷ Lu, ²¹² B, ⁶² Cu, ⁶⁷ Cu, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, ³² P, ³³ P. Decay energies of useful beta-particle- emitting radioisotopes are preferably less than 1,000 keV, less than 100 keV, and preferably about 30-70 keV. Decay energies of useful alpha-particle-emitting radionuclides are preferably less than 10,000 keV, less than 8,000 keV, and preferably about 4,000-7,000 keV. A peptide radioisotope conjugate is synthesized using methods known in the art, for example a method as described in Wicki et al. (Clin Cancer Res 2007. 13(12):3696).

In other preferred embodiments the therapeutic agent is an enzyme selected from the group consisting of RNAase, DNAase, malate dehydrogenase, staphylococcal nuclease, delta- V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6- phosphate dehydrogenase, glucoamylase and acetylcholinesterase.

In other preferred embodiments the therapeutic agent comprises a nucleic acid molecule such as an antisense molecule, a siNA, a siRNA or a miRNA molecule. An "siRNA" molecule or an "RNAi molecule" refers to a nucleic acid that forms a double stranded RNA (dsRNA), which dsRNA has the ability to reduce or inhibit expression of a target gene. In one embodiment, an siRNA comprises a sense strand (passenger strand) that has substantial or complete identity to a target gene and forms a double stranded siRNA with a complementary antisense strand. Typically, the siRNA is at least about 15-50 nucleotides in length (e.g., each complementary sequence of the double stranded siRNA is 15-50 nucleotides in length, and the double stranded siRNA is about 15-50 base pairs in length, preferably about 18-30 base nucleotides, preferably about 18-25 or about 19-23 nucleotides in length, e.g., 19, 20, 21, 22, or 23 nucleotides in length.

Therapeutic uses

The peptides, peptide conjugates and compositions comprising the peptide according to the teachings herein will generally be used in an amount effective to achieve the intended purpose.

For use to treat or prevent a pathology, a composition comprising the peptide according to the teachings herein, is administered or applied in a therapeutically effective amount. A therapeutically effective amount is an amount effective to ameliorate or prevent the symptoms, or prolong the survival of, the patient being treated. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

For systemic administration, a therapeutically effective dose can be estimated initially from in vitro assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.

Initial dosages can also be estimated from in vivo data, e.g., animal models, using techniques that are well known in the art. One having ordinary skill in the art could readily optimize administration to humans based on animal data.

In cases of local administration or selective uptake, the effective local concentration of the chemical agent may not be related to plasma concentration. A person having ordinary skill in the art is able to optimize therapeutically effective local dosages without undue experimentation.

The amount of composition comprising a peptide according to the teachings herein administered will, of course, depend on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.

The therapy may be repeated intermittently while symptoms detectable or even when not detectable.

According to some embodiments, there is provided the use of the composition of any of the embodiments described above, in a method for the diagnosis of a pathology, in some embodiments, cancer.

According to some embodiments, there is provided the use of the composition according to the teachings herein in a method for the detection of the presence of cancer cells in a patient following cancer therapy. Such methods may be used, for example, for follow-up of a patient having undergone cancer therapy, and/or to monitor the efficacy of the treatment.

According to some embodiments, there is provided the composition according to the teachings herein, for the diagnosis of cancer.

According to some embodiments, there is provided a method for the diagnosis of a pathology, in some embodiments cancer, comprising administering a composition according to the teachings herein to a subject. In some embodiments the subject is a human animal. In some embodiments, the subject is a non-human animal. According to some embodiments, a peptide conjugate according to the teachings herein, is used to detect the presence of cancer in a subject, both before and after receiving a cancer therapy. In some embodiments, the cancer is B cell leukemia.

In some embodiments, the diagnostic agent comprises a diagnostic agent such as a fluorescent entity (e.g., fluorescein), an enzyme or a radioactive entity including a radioisotope to facilitate the detection of binding of the peptide conjugate to a cell.

According to some embodiments, there is provided an ex vivo method of treatment or diagnosis, comprising providing an amount of a composition comprising a peptide having an amino acid sequence set forth in any one of SEQ ID NO: 1-6 as described above, and ex vivo combining the composition with tissue isolated from an animal. In some embodiments, the method is useful for the treatment and/or diagnosis of cancer. In some such embodiments, subsequent to combining the composition with the isolated tissue, the tissue is prepared for returning to the animal from which it was isolated.

According to some embodiments, diagnosis or treatment comprises first isolating tissue from an animal. In some embodiments, the tissue is a blood sample. In some embodiments, the tissue is bone marrow. In some embodiments, the tissue is a lymph node

For example, in some embodiments, the composition includes an agent that is a non- soluble anchor such as glass beads, and an amount of the composition is packed in a vessel with a fluid inlet and a fluid outlet to constitute a filter. A sample of blood is passed into the vessel through the fluid inlet where cells having a high-affinity to the peptide (e.g. pathological cells) bind to the peptide in the composition. The blood recovered from the fluid outlet is substantially devoid of the pathological cells. The blood recovered from the fluid outlet can optionally be returned to the organism from which taken, in some embodiments after the addition of nutrients, salts, fluids and the like.

Exemplary embodiments of the teachings herein are discussed hereinbelow with reference to specific materials, methods and examples. The material, methods and examples discussed herein are illustrative and not intended to be limiting. In some embodiments, methods and materials similar or equivalent to those described herein are used in the practice or testing of embodiments of the teachings herein. It is to be understood that the invention is not necessarily limited in its application to the details of construction and the arrangement of the components and/or methods set forth in the following description and/or illustrated in the drawings. The invention is capable of other embodiments or of being practiced or carried out in various ways. EXPERIMENTAL

Example 1 : Identification and isolation of peptide from phage display library

B-cell leukemia cell -specific peptides were isolated using a phage display library first absorbed against a series of normal non-lymphoid tissue cells and then absorbed against a mixture of freshly obtained normal lymphocytes from several healthy humans. Finally, the "absorbed' phage library was subsequently screened for clones that bound to or were internalized by the cells from 3 CLL patients.

In further details, a phage display library was exposed to a culture of each type of normal cells. Non-binding phage were selected and screening was repeated. This cycle was repeated 5 times. Non-binding phage were then exposed to freshly obtained CLL cells from 3 patients and the screening repeated. This time binding and internalized phage were isolated. Phage clones binding to, or internalized into (10 each), CLL cells were randomly picked and after purification, the nucleic acid and amino acid sequences of the peptides were determined.. Ten peptides were sequenced from clones isolated from cell internalized phage and their sequences are shown in Table 1 , hereinbelow .

Table 1 Amino acid and nucleic acid sequences identified from isolated phage library

Four peptide species were found to selectively bind to the CLL cells. However amongst the 10 clones sequenced, the sequence Thr-Ala-Thr-Ala-Ser-Gln-Ser (SEQ ID NO: l) was repeated 6 times.

The isolated peptides were identified from a phage display library whereby a repertoire of peptides displayed on the surface of phage particles is screened. The peptide having an amino acid sequence set forth in SEQ ID NO: l was selected and shown to bind to, and internalize, B-cell leukemic cells while having no effect on normal cells. Table 2: Sequence ID numbers for amino acid and nucleic acid sequences disclosed herein

Patients and normal donors

Specimens were obtained from patients with chronic lymphocytic leukemia (CLL), as well as from normal donors. Informed consent was obtained from all patients prior to the study according to the standard regulations.

Cell preparation

The peripheral blood (PB) or bone marrow (BM) cells were subjected to density cut centrifugation over Ficoll-Hypaque. The mononuclear fractions were washed twice and used directly.

Isolation of phage binding to B-cell leukemic cells

This work was performed using the Ph.D-7 phage library, purchased from New England Biolabs with a diversity of approximately 10 ^u .

Initial selection of phage specific for B-cell leukemia used cells from normal individuals. The cells were incubated with the library 10 ¹¹ in PBS/0.1% milk at 4°C for 90 minutes. Unbound phage were collected and bound phage were discarded. The following screening was performed using cells from CLL patients. The phage that were bound to CLL cells were acid eluted by glycine (0.1 M, pH2.2) followed by cell lysis (30 mM Tris pH8.0, 1 mM EDTA) to elute the tightly bound phage. This second fraction was amplified by reinfection and growth in E. Coli. Amplified phage was purified by PEG precipitation and used for another round of binding. After growth on agar plates using antibiotic selection, individual colonies were randomly picked, amplified, and PEG purified. Binding to CLL cells was confirmed by a cell-based ELISA.

Peptide synthesis

Phage DNA was extracted using standard methods. Each clone was amplified using primers from the phage vector and each clone was amplified, purified and sequenced according to the manufacturer's instructions. Peptides were synthesized either in house or through a commercial vendor. Cytochemistry

Fresh cells from CLL patients were incubated with fluorescein-conjugated peptide (synthesis outsourced to a commercial vendor) at 1 μΜ for 30 minutes unless otherwise stated. After washing, cells were fixed with 3.7% paraformaldehyde and observed using a fluorescent microscope. Fluorescence was observed in B-cell leukemic cells but not in normal human cells.

Example 2: Testing the effect of a peptide conjugate on leukemic mouse cells in vitro

A human B-cell leukemic cell line is cultured in appropriate medium for 24 and 48 hrs together with free drug (e.g., Chlorambucil or Melphalan or Doxorubicin) or drug conjugated to a targeting peptide of the amino acid sequence set forth in SEQ ID NO: 1. Some cells are cultured in medium alone as control. At the end of the culture period, cells are centrifuged, medium is carefully removed and replaced with fresh medium plus XTT reagent and cells are re-incubated for a further 4 hrs. At that time the optical density in the wells is measured with an ELISA reader at both 480 and 680nm. The difference between these measurements is used to calculate the percent Growth Inhibition in test wells compared to control cells exposed to medium alone.

The negative effect of drug conjugated to targeting peptide on the homeostasis of leukemic cells is shown to be significantly greater than the effect of either free drug or medium alone.

Example 3 : Effect of peptide conjugate on human leukemic cells in vitro

A peptide-agent conjugate is prepared, for example, as in Example 2. Bone marrow and peripheral blood samples are obtained from human subjects diagnosed with multiple myeloma (group A), chronic lymphocytic leukemia (group B), and normal, healthy subjects (control group).

Aliquots of cell suspensions from leukemic and normal subjects are each added to 2 sets of microculture plates. To a first set of plates a composition comprising the peptide conjugate is added; to a second set of plates carrier is added alone.

After 3 days of culture, cell viability is tested using a standard MTT assay, and apoptosis studied using Annexin V assay.

The conjugate is found to exhibit a significant cytotoxic effect against leukemic cells of groups A and B in vitro, with no cytotoxic effect on normal cells. No cytotoxic effect is seen in plates treated with carrier alone.

Example 4: Effect of peptide conjugate on leukemic mice in vivo

A peptide-agent conjugate is prepared, for example, as in Example 2.

Nu/nu or SCID mice bearing human leukemic cells (for example from a patient with

Chronic Lymphocytic Leukemia) and control mice (mice not inoculated with leukemic cells) are treated i.v. with the pharmaceutical composition comprising a peptide disclosed herein and a therapeutic agent using a previously defined protocol (for example, one or more times a week for several weeks, depending on the specific experimental conditions used) and animals are then assessed for the presence of CLL cells and other overt signs of disease. A significant reduction in tumor burden and other signs of disease, as well as increased survival rate, is seen in mice treated with the pharmaceutical composition.

Example 5 : Effect of peptide -toxin conjugate on leukemic cells

A peptide-toxin conjugate (PTC) is prepared by coupling the peptide disclosed herein with a toxin, preferably ricin for use in delivering the toxin specifically to B-cell leukemic cells. A similar method is disclosed in Firer et al., Leuk Lymphoma. 2003 44(4):681-9, whereby ligand toxin conjugates (LTC) comprising the DNP hapten conjugated to ricin A toxin (DNP-RTA) were shown to specifically and effectively kill anti-DNP secreting murine hybridoma (U7.6) cells but not other hybridoma cells (1B12), a murine erythroleukemia cell line (Friend's Leukemia) or normal mouse spleen cells.

Likewise the PTC treatment kills B-cell leukemic cells from cell lines and cells isolated from leukemia patients. These data show that PTC therapy is an effective strategy for specific destruction of tumor cells such as B-cell leukemic cells. It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements. Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the scope of the appended claims.

Citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the invention.

Section headings are used herein to ease understanding of the specification and should not be construed as necessarily limiting.