PEPTIDES DERIVED FROM MAGNETOTACTIC BACTERIA AND USE THEREOF

FIELD OF INVENTION

[1] The present invention is directed to; inter alia, peptides derived from magnetotactic bacteria, compositions comprising same and methods of use thereof.

BACKGROUND OF THE INVENTION

[2] Biomineralization is a process that can be found in all kingdoms of life. This process usually helps the organisms to harden a soft tissue by creating complex structures for their biological functions. It was shown that biominerals are under highly biological control that involves proteins as nucleation sites and/or as structural skeletons.

[3] In general, proteins play an active and important role in controlling metal Biomineralization, both inhibiting or promoting the nucleation and/or crystal growth through a combination of electrostatic and stereochemical interactions, as well as geometrical matching. In particular, proteins may locally alter the system supersaturation with respect to a particular mineral phase by concentrating ions in these regions close to charged patches on the protein surface, following the so called ionotropic effect. A number of studies suggested that proteins may act as a template for crystal growth, lowering the free energy needed for nucleation. One possible reason for protein-mineral interaction is that the distance between charged amino acids is similar to the distance between atoms in the crystalline structure.

[4] Magnetotactic bacteria (MTB) use iron biomineralization to create nanomagnetic crystals in a specialized organelle, the magnetosome, which is organized into a chain to help them to navigate through the geomagnetic field. The magnetosomes are magnetite (Fe ₃ 0 ₄ ) or gregite (Fe ₃ S0 ₄ ) nanoparticles with size range of 30-120 nm surrounded by bilayer membrane. Magnetite and gregite synthesis is dependent on the environment oxygen level where as the diversity in their size and shape depends on the bacteria species.

[5] Magnetosome formation is divided into three steps of: membrane invagination, iron uptake and biomineralization of magnetite particle. Each step involves specific proteins that can be found only in MTB and are located in specific magnetosomegenomic cluster. MamAB, the major operon existing in all MTB contains the core magnetosome-forming genes. In the Magnetospirillum genus, three additional operons exist: mamXY, mamCDFG and mms6. Deletion of these operons causes defects in both the biomineralization process and crystal maturation.

[6] MamC (mmsl3) is the most abundant magnetosome-associated protein. MamC is located in the mamCDFG operon and was suggested to be involved in iron Biomineralization. MamC is a highly conserved 12.4 kDa protein existing in most magnetotactic a-proteobcteria. Since MamC is tightly bound to the magnetosome membrane, it was also used as an anchor for magnetosome functionalization.

[7] Magnetic nanoparticles typically comprise nanocrystals made of oxides (or to a lesser extent of sulfides) of the elements in the fourth row of the periodic table (i.e. Cr, Mn, Fe, Co, and Ni). The ability to produce such magnetic nanoparticles is inevitable not only for the general understanding of magnetic properties in a nanometer regime but also for manifold technical applications ranging from magnetic resonance imaging, drug delivery, catalysts, and biosensing to nanoelectronics, semiconductor materials, and magnetic storage media.

[8] International patent application No. WO 2014/042142 relates to surface-modified ferromagnetic iron oxide particles that have heat generation properties dispersion stability in a medium for injection, and that are suitable for electromagnetic induction cancer cauterization.

[9] Nudelman H, et al., (Front Microbiol. 2014 Jan 29;5:9) have created three dimensional structural models of all known Magnetospirillum gryphiswaldense strain MSR-1 magnetosome associated proteins.

[10] C. Valverde-Tercedor, et al., (Appl Microbiol Biotechnol (2015) 99:5109-5121) reported that MamC plays an important role in the control of the size of magnetite crystals formed from magnetite nanoparticles synthesized inorganically in free-drift experiments.

[11] There is a need for methods enabling the reliable controlled production of monocrystalline nanoparticles having a defined size, and effective magnetic property suitable for a given application. SUMMARY OF THE INVENTION

[12] According to one aspect of the invention, there is provided peptide comprising not more than 50 amino acid residues, said peptide comprising the amino acid sequence having the formula:

X1 - A - X2- B -KE.

wherein:

Xi is selected from a negatively charged amino acid and an amino acid capable of binding a metal particle;

A is a tri-peptide selected from the group consisting of AAI, AVI, IVV, and VAL;

X2 is selected from a negatively charged amino acid and an amino acid capable of binding a metal particle; and

B is a bi-peptide selected from the group consisting of TG, SG, TA, and TV.

[13] In another embodiment, the peptide further comprising a C-terminus tri-peptide contiguous thereto, said tri-peptide being selected from the group consisting of: TVG, AAG, ALG, and ASG.

[14] In another embodiment, the peptide is selected from the group consisting of:

SEQ ID NO: 16 (LKEKRITNTX 1 A AIX2TGKET VG) ;

SEQ ID NO: 17 (LKDKQITGTX1AAIX2TGKEAAG);

SEQ ID NO: 18 (YNKGLVSPEX1AAIX2SGKEAAG);

SEQ ID NO: 19 ( KQRGEITTEX 1 A VIX2TGKE ALG) ;

SEQ ID NO: 20 (RETD GLSTEX 1 IV VX2TAKE A AG) ; and

SEQ ID NO: 21 (KSRGE ATNKX 1 V ALX2T VKE AS G) ; wherein Xi and X2 are, independently, selected from a negatively charged amino acid and an amino acid capable of binding a metal particle.

[15] In another embodiment, the peptide comprises an amino acid sequence selected from the group consisting of:

SEQ ID NO: 2 (LKEKRITNTEA AIDTGKETVG) ;

SEQ ID NO: 26 (LKEKRITNTEA AIETGKETVG);

SEQ ID NO: 3 (LKDKQITGTEAAIDTGKEA AG) ;

SEQ ID NO: 27 (LKDKQITGTEAAIETGKEAAG); SEQ ID NO: 4 (YNKGLVSPEEAAIDSGKEAAG);

SEQ ID NO: 28 ( YNKGLVSPEEAAIES GKEAAG) ;

SEQ ID NO: 5 (KQRGEITTEEAVIDTGKEALG);

SEQ ID NO: 29 (KQRGEITTEEA VIETGKEALG) ;

SEQ ID NO: 6 (RETDGLSTEAIVVDTAKEAAG);

SEQ ID NO: 30 (RETDGLSTEAIVVETAKEAAG); and

SEQ ID NO: 7 ( KSRGE ATNKD V ALHT VKE AS G) .

[16] In another embodiment, the peptide comprises the sequence as set forth in SEQ ID NO: 2.

[17] According to another aspect of the invention, there is provided peptide comprising not more than 50 amino acid residues, said peptide comprising the amino acid sequence as set forth in SEQ ID NO: 32 (GTPDLSDDALLAAAGEE) or SEQ ID NO: 11 (X1X2EVELRDALA), wherein Xi and X ₂ are selected from negatively charged amino acid or an amino acid capable of binding a metal particle. In another embodiment, the peptide is selected from SEQ ID NO: 15 (AQSDEEVELRDALA) or SEQ ID NO: 14 (MKSRDIESAQSDEEVELRDALA).

[18] In another embodiment, the peptide has a length of up to 30 amino acids. In another embodiment, the peptide has a length of at least 18 amino acids.

[19] In another embodiment, the peptide is linked to a cancer cell targeting moiety. In another embodiment, the cancer cell targeting moiety is a peptide. In another embodiment, the cancer cell targeting moiety is maltose binding protein (MBP).

[20] According to another aspect of the invention, there is provided composition comprising the peptide disclosed herein and an acceptable carrier. In another embodiment, the carrier is a pharmaceutically acceptable carrier. In another embodiment, the composition further comprising one or more magnetic nanoparticles.

[21] According to another aspect of the invention, there is provided composition comprising one or more magnetic nanoparticles and a magnetosome-derived peptide having a length of up to 50 amino acids, wherein at least 80% of said magnetic nanoparticles are characterized by a crystal size range of 30-60 nm. In another embodiment, the composition comprises a peptide selected from SEQ ID NO: 2-7, 11, 14-21, 26-30 and 32.

[22] In another embodiment, the composition comprises one or more magnetic nanoparticles selected from the group consisting of: magnetite, greigite, or combination thereof. [23] In another embodiment, the composition further comprises at least one agent selected from a cancer cell targeting agent or anti-cancer agent. In another embodiment, the cancer cell targeting agent is an antibody or an antigen binding fragment thereof.

[24] In another embodiment, the composition is for use in treatment or diagnosis of cancer in a subject in need thereof.

[25] According to another aspect of the invention, there is provided method of treating or diagnosing cancer in a subject in need thereof, the method comprising administering the composition of the invention to a target region of said subject, and applying a magnetic field to at least said target region, thereby treating or diagnosing cancer in said subject.

[26] In another embodiment, the composition and method of the invention is for diagnosing cancer, wherein said magnetic field is magnetic resonance imaging (MRI).

[27] In another embodiment, the composition and method of the invention is for treating cancer, wherein said applying magnetic field comprises applying an alternating magnetic field to generate heat by excitation of said magnetic nanoparticles.

[28] Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

[29] Figure 1A: Transmission electron microscopy (TEM) image of magnetic particles prepared by magnetite precipitation in-vitro with MamC.

[30] Figure IB: Transmission electron microscopy (TEM) image of magnetic particles prepared by magnetite precipitation in-vitro with MBP-Long.

[31] Figure 1C: Transmission electron microscopy (TEM) image of magnetic particles prepared by magnetite precipitation in-vitro with MBP-Short. [32] Figure ID: is a histogram presenting a quantitative analysis of the image presented in figure 1A.

[33] Figure IE: is a histogram presenting a quantitative analysis of the image presented in figure IB.

[34] Figure IF: is a histogram presenting a quantitative analysis of the image presented in figure 1C.

[35] Figure 1G: Transmission electron microscopy (TEM) image of magnetic particles prepared by magnetite precipitation in-vitro with MBP-Mms6.

[36] Figure 1H: Transmission electron microscopy (TEM) image of magnetic particles prepared by magnetite precipitation in-vitro with MBP.

[37] Figure II: Transmission electron microscopy (TEM) image of magnetic particles prepared by magnetite precipitation in-vitro with buffer alone.

[38] Figure 1J: is a histogram presenting a quantitative analysis of the image presented in figure 1G.

[39] Figure IK: is a histogram presenting a quantitative analysis of the image presented in figure 1H.

[40] Figure 1L: is a histogram presenting a quantitative analysis of the image presented in figure II.

[41] Figure 2A: is a graph showing ITC results of MBP-Long (0.2 mM) interaction with magnetite particles (0.019 mM), MBP-Short (0.2 mM) and MBP-Mms6 (0.2 mM) curves.

[42] Figure 2B: is a graph showing differences between MBP-Long (· symbol) to MBP (+ symbols). * The heat of dilution of MBP-Long, MBP-Mms6, MBP-Short into a clean buffer has been subtracted.

[43] Figure 3: Models of the structure of MamC-loop in electrostatic surface representation covering the structures represented as in figures 2 A and 2B.

[44] Figure 4A: TEM images of magnetic particles prepared by magnetite precipitation in-vitro with MBP-Mms6.

[45] Figure 4B: is a histogram showing quantification of particle size presented in figure 4A.

[46] Figure 4C: TEM images of magnetic particles prepared by magnetite precipitation in-vitro with MBP. [47] Figure 4D: is a histogram showing quantification of particle size presented in figure 4C.

[48] Figure 4E: TEM images of magnetic particles prepared by magnetite precipitation in-vitro with buffer alone.

[49] Figure 4F: is a histogram showing quantification of particle size presented in figure 4E.

[50] Figure 4G: TEM images of magnetic particles prepared by magnetite precipitation in-vitro with MBP-long.

[51] Figure 5: is the result of SDS-PAGE 15% analysis of MBP-Long, Short, MBP and MBP- Mms6.

[52] Figure 6: A table showing data collection and refinement statistics of MBP-Long. Last cell statistic is in brackets.

[53] Figures 7A: Isothermal Titration Calorimetry (ITC) results. Heat changes during ITC of MBP-Mms6 measurements with nano magnetic particles. * The heat of dilution of MBP-Mms6 and MBP-Short into a clean buffer has been subtracted.

[54] Figures 7B: Isothermal Titration Calorimetry (ITC) results. Heat changes during ITC of MBP-Short measurements with nano magnetic particles. * The heat of dilution of MBP-Mms6 and MBP-Short into a clean buffer has been subtracted.

[55] Figure 8: MamC multiple sequence alignment between MamC from M. magneticum AMB-1 to different strains: M. gryphiswaldense MSR-1, Magnetococcus marinus MC-1, Magnetovibrio blakemorei MV-1, Magnetofaba australis IT-1, Magnetospira sp. QH-2 and Magnetospirillium Sp. SO-1.

[56] Figure 9: Structural models for MamC-loop homologous proteins. Electrostatic potentials for all four strains: M. gryphiswaldense MSR-1, M. marinus MC-1, Magnetovibrio blakemorei MV-1, Magnetofaba australis IT-1, Magnetospira sp. QH-2 and Magnetospirillium Sp. SO-1 are modelled (dark grey represents negative charges and light grey represents positive charges).

[57] Figure 10A: is a histogram showing size distribution of magnetite particles co-precipitated with different protein mutants at 10 μg/ml protein concentration. Statistical significance of alterations from MBP-Long was tested using the ANOVA test.

[58] Figure 10B: is a ribbon-and-sticks representation of the D70A mutant.

[59] Figure IOC: are electrostatic surface representation images comparing D70A vs. MamC- MIC. [60] Figure 10D: is a graph showing ITC measurements of protein-magnetite nanoparticles interactions. Heat flow graph for each injection (μΐ/sec) as a function of time (s) of MBP-Long (light grey) and E66A/D70A (dark grey).

[61] Figure 10E: is a graph showing ITC measurements of protein-magnetite nanoparticles interactions. Heat flow graph for each injection (μΐ/sec) as a function of time (s) of MBP-Long (light grey) and E66A (dark grey).

[62] Figure 10F: is a graph showing heat rate normalization graph for each injection with the injected mole number (μΐ/ηιοΐ) for the interaction of MBP-Long (0.2 mM), and D70A (0.2 mM) with magnetite particles (0.019 mM). The heat of dilution of MBP-Long and all the three mutants into a clean buffer has been subtracted.

[63] Figure 11: is a graph showing circular dichroism (CD) spectrum analysis results of the different peptides at 25 °C in 20mM Tris 8 and 50mM NaCl.

[64] Figure 12. Magnetite particles size distribution. In vitro iron co-precipitation was performed using different peptides. Iron Solution of Fe ³⁺ and Fe ²⁺ , at a ratio of 2: 1 was titrated with 0.1 NaOH buffer. At pH 5, in each sample, peptide was added to a final concentration of lOuM and the titration stopped at pH 7. All samples were analyzed using TEM imaging and SEAD diffraction.

[65] Figure 13. Iron co-precipitation TEM images. Each peptide has different effect on the magnetite crystal shape rather than their size. The presense of mutant peptides in solution, both MamC and Mms6, revels more rounded particles than in the WT's and control samples.

[66] Figure 14. Magnetite binding of MBP-Mms6 peptide chimera. (Left) Western blot analysis of magnetite nanoparticles synthesized in the presence of MBP-Mms6 peptide chimera (C-term; SEQ ID NO: 14), MBP and without any protein (ø). Detection was done with anti-MBP-HRP conjugated antibody. (Right) Maltose detection of magnetite nanoparticles (MP) synthesized in the presence of the MBP-Mms6 peptide chimera (C) and without any protein (ø). Particles were washed several times after maltose binding (Wash 1-5) before color development. Maltose was detected (black arrows) via a sugar detecting protocol that involves the heating of thin layer chromatography film spotted with a mixture of H2SO4 and the solution in question. DETAILED DESCRIPTION OF THE INVENTION

[67] The present invention, in some embodiments thereof, relates to peptides, more particularly, but not exclusively, to peptides being capable of binding magnetic nanoparticles and using them for diagnostics and therapy.

[68] Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

[69] As discussed hereinabove, currently known surface-modified iron oxide nanoparticles used, for instance, for cancer cauterization are characterized in that a block copolymer, of polyethylene glycol or polystyrene is bound to the surface of ferromagnetic iron oxide nanoparticles, resulting in a reduced magnetic effect as well as high toxicity.

[70] In contrast, peptides and proteins possess many advantages over traditional polymers, making them highly desirable for use as biomaterials. In addition to controlling the number of repeat units, the sequence of the protein is designed such that the number of reactive side chains is manipulated. It is contemplated that these aforementioned properties allow the creation of defined protein polymers with customizable length, reactivity, architecture, and solubility. Additionally, it is contemplated that additional components (e.g., targeting moieties, cellular transporters) may be attachable to these customizable protein backbones. In some embodiments, the peptides described herein are biodegradable through normal protease activity into small, extractable components, thereby alleviating safety concerns.

[71] In some embodiments, the peptide has a length of up to 30 amino acids and comprises the amino acid sequence having the formula of: Xi, X ₂ , X ₃ , X ₄ , Xs, X ₆ , X7, Xs, X9, Xio, X11, X12, X13, Xi ₄ , Xi5, Xi6, K, E, X19, X20, G (SEQ ID NO: 1), wherein Xi = L, K, R or Y; X ₂ = K, Q, S, E or T; X ₃ = R, E, D, T or K; X ₄ = K, G or D; X ₅ = E, R, Q, G or L; X ₆ = I, A, L or V; Xy = T or S; X ₈ = T, N, G or P; X ₉ = E, T or K; X ₁₀ = E, D or A; X _n = A, V or I; X ₁₂ = A or V; X ₁₃ = I, V or L; X ₁₄ = D or H; X ₁₅ = T or S; X ₁₆ = G, V or A; X ₁₉ = A or T; and X20 = A, V, L or S. Table 1. amino acid sequences of the peptides of the invention

SEQ ID NO: Amino acid sequence

24 Xi, K, R, X ₂ , E, I, X ₃ , X ₄ , ¾, E, A, ¾, I, D, T, G, K, E, A, A, G

Xi = L or K; X ₂ = K or G; X ₃ = T or S; X ₄ = T or N; X ₅ = E or T and X ₆ = A or V

25 Xi, K, R, X ₂ , E, I, X ₃ , X ₄ , X ₅ , E, A, ¾, I, E, T, G, K, E, A, A, G,

Xi = L or K; X ₂ = K or G; X ₃ = T or S; X ₄ = T or N; X ₅ = E or T and X ₆ = A or V

16 LKEKRITNTXiAAIX ₂ TGKETVG

Xi and X ₂ = negatively charged amino acid or an amino acid capable of binding a metal particle.

2 LKEKRITNTEAAIDTGKETVG

17 LKDKQITGT XiAAI X ₂ TGKEAAG

Xi and X ₂ = negatively charged amino acid or an amino acid capable of binding ' a metal particle.

3 LKDKQITGTEAAIDTGKEAAG

11 XiX ₂ EVELRDALA

Xi and X ₂ = negatively charged amino acid or an amino acid capable of binding a metal particle

14 MKSRDIESAQSDEEVELRDALA

15 AQSDEEVELRDALA

26 LKEKRITNTEAAIETGKETVG

27 LKDKQITGTEAAIETGKEAAG

18 YNKGLVSPE XiAAI X ₂ SGKEAAG

Xi and X ₂ = negatively charged amino acid or an amino acid capable of binding a metal particle

4 YNKGLVSPEEAAIDSGKEAAG

28 YNKGLVSPEEAAIES GKEAAG

19 KQRGEITTE XiAVI X ₂ TGKEALG

Xi and X ₂ = negatively charged amino acid or an amino acid capable of binding a metal particle

5 KQRGEITTEEAVIDTGKEALG

29 KQRGEITTEEAVIETGKEALG

20 RETDGLSTE XiIVV X ₂ TAKEAAG

Xi and X ₂ = negatively charged amino acid or an amino acid capable of binding a metal particle

6 RETDGLSTEAIVVDTAKEAAG

30 RETDGLSTEAIVVETAKEAAG

21 KSRGEATNK XiVAL X ₂ TVKEASG

Xi and X ₂ = negatively charged amino acid or an amino acid capable of binding a metal particle

7 KSRGEATNKDVALHTVKEASG

32 GTPDLSDDALLAAAGEE [72] In some embodiments, the peptide has a length of up to 30 amino acids and comprises the amino acid sequence having the formula of Xi, K, R, X ₂ , E, I, X ₃ , X ₄ , Xs, E, A, X ₆ , 1, D, T, G, K, E, A, A, G (SEQ ID NO: 24), wherein: Xi = L or K; X ₂ = K or G; X ₃ = T or S; X ₄ = T or N; Xs = E or T and X ₆ = A or V.

[73] In some embodiments, the peptide has a length of up to 30 amino acids and comprises the amino acid sequence having the formula of Xi, K, R, X ₂ , E, I, X ₃ , X ₄ , Xs, E, A, X ₆ , I, E, T, G, K, E, A, A, G (SEQ ID NO: 25), wherein: Xi = L or K; X ₂ = K or G; X ₃ = T or S; X ₄ = T or N; Xs = E or T and X ₆ = A or V;

[74] In some embodiments, the peptide comprises or consists of the amino acid sequence having the formula of: Xi, X ₂ , X ₃ , X ₄ , Xs, X ₆ , X7, Xs, X9, Xio, X11, X12, X ₁₃ , X ₁₄ , Xis, Xi6, K, E, X19, X ₂ o, G (SEQ ID NO: 1), wherein Xi = L, K, R or Y; X ₂ = K, Q, S, E or T; X ₃ = R, E, D, T or K; X ₄ = K, G or D; X ₅ = E, R, Q, G or L; X ₆ = I, A, L or V; X ₇ = T or S; Xs = T, N, G or P; X ₉ = E, T or K; Xio = E, D or A; Xn = A, V or I; X ₁₂ = A or V; X ₁₃ = I, V or L; X ₁₄ = D or H; X ₁₅ = T or S; Xi6 = G, V or A; X ₁₉ = A or T; and X ₂₀ = A, V, L or S.

[75] In some embodiments, the peptide comprises or consists of the amino acid sequence having the formula of Xi, K, R, X ₂ , E, I, X ₃ , X ₄ , Xs, E, A, X ₆ , 1, D, T, G, K, E, A, A, G (SEQ ID NO: 24), wherein: Xi = L or K; X ₂ = K or G; X ₃ = T or S; X ₄ = T or N; Xs = E or T and X ₆ = A or V.

[76] In some embodiments, the peptide comprises or consists of the amino acid sequence having the formula of Xi, K, R, X ₂ , E, I, X ₃ , X ₄ , Xs, E, A, X ₆ , 1, E, T, G, K, E, A, A, G (SEQ ID NO: 25), wherein: Xi = L or K; X ₂ = K or G; X ₃ = T or S; X ₄ = T or N; Xs = E or T and X ₆ = A or V;

[77] In some embodiments, the peptide has a length of up to 50 amino acids and comprises the amino acid sequence having the formula of: V,G,G,A,A,A,L,A,K,N,V,R,L,Xi, X ₂ , X ₃ , X ₄ , Xs, X ₆ , X ₇ , Xs, X ₉ , Xio, X11, X12, Xi3, Xi4, Xis, Xie, K, E, X19, X ₂₀ , G,A,G,L,A,T,A,L,S (SEQ ID NO: 31), wherein Xi = L, K, R or Y; X ₂ = K, Q, S, E or T; X ₃ = R, E, D, T or K; X ₄ = K, G or D; X ₅ = E, R, Q, G or L; X ₆ = I, A, L or V; X ₇ = T or S; Xs = T, N, G or P; X ₉ = E, T or K; Xio = E, D or A; Xn = A, V or I; X ₁₂ = A or V; X ₁₃ = I, V or L; X ₁₄ = D or H; Xis = T or S; X ₁₆ = G, V or A; X ₁₉ = A or T; and X ₂₀ = A, V, L or S.

[78] In some embodiments, the peptide comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 2 (LKEKRITNTEAAIDTGKETVG). In some embodiments, the peptide comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 3 (LKDKQITGTEAAIDTGKEAAG). In some embodiments, the peptide comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 4 (YNKGLVSPEEAAIDSGKEAAG). In some embodiments, the peptide comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 5 (KQRGEITTEEAVIDTGKEALG). In some embodiments, the peptide comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 6 (RETDGLSTEAIVVDTAKEAAG). In some embodiments, the peptide comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 7 ( KSRGE ATNKD V ALHTVKE AS G) .

[79] In some embodiments, the peptide comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 26 (LKEKRITNTEAAIETGKETVG). In some embodiments, the peptide comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 27 (LKDKQITGTEAAIETGKEAAG). In some embodiments, the peptide comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 28 ( YNKGLVSPEE A AIES GKE A AG) . In some embodiments, the peptide comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 29 (KQRGEITTEEAVIETGKEALG). In some embodiments, the peptide comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 30 (RETDGLSTEAIVVETAKEAAG).

[80] In some embodiments, the peptide comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 15 ( AQSDEEVELRD ALA) . In some embodiments, the peptide comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 14 (MKSRDIESAQSDEEVELRDALA). In some embodiments, the peptide comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 32 (GTPDLSDDALLAAAGEE).

[81] In some embodiments, the peptide comprises or consists of a variant, an analog or a fragment of any one of SEQ ID NO: 2-7, 11, 14-21, 26-30 and 32. In one embodiment, said variant or analog or fragment refers to at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity to the amino acid sequence of any one of SEQ ID NO: 2 to SEQ ID NO: 7 and SEQ ID NO: 26 to SEQ ID NO: 30. In one embodiment, said variant or analog or fragment refers to at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity to the amino acid sequence of any one of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 32. [82] In some embodiments, said variant or analog has at least one amino acid substitution as compared to any one of SEQ ID NOs: 2-7, 11, 14-21, 26-30 and 32. In some embodiments, said variant or analog has at least 1, 2, 3, 4, or 5 amino acid substitution as compared to any one of SEQ ID NOs: 2-7, 11, 14-21, 26-30 and 32. In some embodiments, said variant or analog has at most 1, 2, 3, 4, or 5 amino acid substitution as compared to any one of SEQ ID NOs: 2-7, 11, 14- 21, 26-30 and 32. Each possibility represents a separate embodiment of the present invention. Different embodiments may be combined at will.

[83] In some embodiments, the peptide is a fragment of any one of SEQ ID NO: 2-7, 11, 14-21, 26-30 and 32 said fragment has at least 18 amino acid residues, at least 19 amino acid residues, or at least 20 amino acid residues, wherein said fragment is capable of binding a metal particle. Each possibility represents a separate embodiment of the present invention. Different embodiments may be combined at will.

[84] In some embodiments, the peptide of the invention comprises the amino acid sequence as set forth in SEQ ID NO: 16 (LKEKRITNTX i A AIX ₂ TGKET VG) , wherein Xi and X ₂ are, independently, selected from a negatively charged amino acid and an amino acid capable of binding a metal particle.

[85] In some embodiments, the peptide of the invention comprises the amino acid sequence as set forth in SEQ ID NO: 17 (LKDKQITGTX 1 A AIX2TGKEA AG) , wherein Xi and X ₂ are, independently, selected from a negatively charged amino acid and an amino acid capable of binding a metal particle.

[86] In some embodiments, the peptide of the invention comprises the amino acid sequence as set forth in SEQ ID NO: 18 ( YNKGLVSPEX 1 A AIX ₂ S GKE A AG) , wherein Xi and X ₂ are, independently, selected from a negatively charged amino acid and an amino acid capable of binding a metal particle.

[87] In some embodiments, the peptide of the invention comprises the amino acid sequence as set forth in SEQ ID NO: 19 (KQRGEITTEX 1 A VIX ₂ TGKE ALG) , wherein Xi and X ₂ are, independently, selected from a negatively charged amino acid and an amino acid capable of binding a metal particle.

[88] In some embodiments, the peptide of the invention comprises the amino acid sequence as set forth in SEQ ID NO: 20 (RETDGLSTEXiIVVX ₂ TAKEAAG), wherein Xi and X ₂ are, independently, selected from a negatively charged amino acid and an amino acid capable of binding a metal particle.

[89] In some embodiments, the peptide of the invention comprises the amino acid sequence as set forth in SEQ ID NO: 21 (KSRGE ATNKX i V ALX ₂ TVKE AS G) , wherein Xi and X ₂ are, independently, selected from a negatively charged amino acid and an amino acid capable of binding a metal particle.

[90] In some embodiments, the peptide of the invention comprises the amino acid sequence as set forth in SEQ ID NO: 11 (X iX ₂ EVELRD ALA) , wherein Xi and X ₂ are selected from negatively charged amino acid or an amino acid capable of binding a metal particle.

[91] In some embodiments, the negatively charged amino acids are, independently, selected form aspartic acid (D) or glutamic acid (E).

[92] An "amino acid capable of binding a metal particle", as used herein, is selected form the group consisting of: asparagine (N), glutamine acid (Q), tyrosine (Y), serine (S), threonine (T), histidine (H), cysteine (C) or methionine (M).

[93] In some embodiments, peptides of the present invention or fragments thereof are capable of forming an alpha-helix structure.

[94] In some embodiments, a peptide "capable of forming an alpha-helix structure" refers to a peptide that is predicted to form an alpha-helix structure by a structural model. In some embodiments, a peptide "capable of forming a a-helix structure" refers to a peptide that can be shown to form an alpha helix structure by experimental methods of the present invention e.g., circular dichroism or by other methods known in the art.

Peptides

[95] The terms, "peptide", "amino acid sequence" or "protein sequences", as used herein throughout interchangeably, refer to an amino acid sequence of a protein molecule.

[96] An "amino acid sequence" can be deduced from the nucleic acid sequence encoding the protein. However, terms such as "peptide", "polypeptide" or "protein" are not meant to limit the amino acid sequence to the deduced amino acid sequence, but include post-translational modifications of the deduced amino acid sequences, such as amino acid deletions, additions, and modifications such as glycolsylations and addition of lipid moieties. [97] The term "analog" includes any peptide having an amino acid sequence substantially identical to one of the sequences specifically shown herein in which one or more residues have been conservatively substituted with a functionally similar residue and which displays the abilities as described herein. Examples of conservative substitutions include the substitution of one non- polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another, the substitution of one aromatic residue for another such as between tyrosine, phenylalanine and histidine, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another. Each possibility represents a separate embodiment of the present invention.

[98] The phrase "conservative substitution" also includes the use of a chemically derivatized residue in place of a non-derivatized residue provided that such peptide displays the requisite function such as iron precipitation as specified herein.

[99] The term "derived from" or "corresponding to" refers to construction of an amino acid sequence based on the knowledge of a sequence using any one of the suitable means known to one skilled in the art, e.g. chemical synthesis in accordance with standard protocols in the art.

[100] Typically, the present invention encompasses derivatives of the peptides. The term "derivative" or "chemical derivative" includes any chemical derivative of the peptide having one or more residues chemically derivatized by reaction of side chains or functional groups. Such derivatized molecules include, for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t- butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine. Also included as chemical derivatives are those peptides, which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acid residues. For example: 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted or serine; and ornithine may be substituted for lysine.

[101] In addition, a peptide derivative can differ from the natural sequence of the peptides of the invention by chemical modifications including, but are not limited to, terminal-NH2 acylation, acetylation, or thioglycolic acid amidation, and by terminal-carboxlyamidation, e.g., with ammonia, methylamine, and the like. Peptides can be either linear, cyclic or branched and the like, which conformations can be achieved using methods well known in the art.

[102] The peptide derivatives and analogs according to the principles of the present invention can also include side chain bond modifications, including but not limited to -CH2-NH-, -CH2-S-, -CH2-S=0, OC-NH-, -CH2-0-, -CH2-CH2-, S=C-NH-, and -CH=CH-, and backbone modifications such as modified peptide bonds. Peptide bonds (-CO-NH-) within the peptide can be substituted, for example, by N-methylated bonds (-N(CH3)-CO-); ester bonds (-C(R)H-C-O-O- C(R)H-N); ketomethylene bonds (-CO-CH2-); a-aza bonds (-NH-N(R)-CO-), wherein R is any alkyl group, e.g., methyl; carba bonds (-CH2-NH-); hydroxyethylene bonds (-CH(OH)-CH2-); thioamide bonds (-CS-NH); olefmic double bonds (-CH=CH-); and peptide derivatives (-N(R)- CH2-CO-), wherein R is the "normal" side chain, naturally presented on the carbon atom. These modifications can occur at one or more of the bonds along the peptide chain and even at several (e.g., 2-3) at the same time.

[103] The present invention also encompasses peptide derivatives and analogs in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonylamino groups, carbobenzoxyamino groups, t-butyloxycarbonylamino groups, chloroacetylamino groups or formylamino groups. Free carboxyl groups may be derivatized to form, for example, salts, methyl and ethyl esters or other types of esters or hydrazides. The imidazole nitrogen of histidine can be derivatized to form N-im-benzylhistidine.

[104] The peptide analogs can also contain non-natural amino acids. Examples of non-natural amino acids include, but are not limited to, sarcosine (Sar), norleucine, ornithine, citrulline, diaminobutyric acid, homoserine, isopropyl Lys, 3-(2'-naphtyl)-Ala, nicotinyl Lys, amino isobutyric acid, and 3-(3'-pyridyl-Ala).

[105] Furthermore, the peptide analogs can contain other derivatized amino acid residues including, but not limited to, methylated amino acids, N-benzylated amino acids, O-benzylated amino acids, N-acetylated amino acids, O-acetylated amino acids, carbobenzoxy-substituted amino acids and the like. Specific examples include, but are not limited to, methyl- Ala (Me Ala), MeTyr, MeArg, MeGlu, MeVal, MeHis, N-acetyl-Lys, O-acetyl-Lys, carbobenzoxy-Lys, Tyr-O- Benzyl, Glu-O-Benzyl, Benzyl-His, Arg-Tosyl, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, and the like.

[106] The invention further includes peptide analogs, which can contain one or more D-isomer forms of the amino acids. Production of retro-inverso D-amino acid peptides where at least one amino acid and perhaps all amino acids are D-amino acids is well known in the art. When all of the amino acids in the peptide are D-amino acids, and the N- and C-terminals of the molecule are reversed, the result is a molecule having the same structural groups being at the same positions as in the L-amino acid form of the molecule. However, the molecule is more stable to proteolytic degradation and is therefore useful in many of the applications recited herein. Diastereomeric peptides may be highly advantageous over all L- or all D-amino acid peptides having the same amino acid sequence because of their higher water solubility, lower immunogenicity, and lower susceptibility to proteolytic degradation. The term "diastereomeric peptide" as used herein refers to a peptide comprising both L-amino acid residues and D-amino acid residues. The number and position of D-amino acid residues in a diastereomeric peptide of the preset invention may be variable so long as the peptide is capable of displaying the function of binding magnetite.

[107] As used herein the term "salts" refers to both salts of carboxyl groups and to acid addition salts of amino or guanido groups of the peptide molecule. Salts of carboxyl groups may be formed by means known in the art and include inorganic salts, for example sodium, calcium, ammonium, ferric or zinc salts, and the like, and salts with organic bases such as salts formed for example with amines such as triethanolamine, piperidine, procaine, and the like. Acid addition salts include, for example, salts with mineral acids such as, for example, acetic acid or oxalic acid. Salts describe here also ionic components added to the peptide solution to enhance hydrogel formation and /or mineralization of calcium minerals.

[108] According to one embodiment, the peptides of the present invention can be synthesized or prepared by any method and/or technique known in the art for peptide synthesis. According to another embodiment, the peptides can be synthesized by a solid phase peptide synthesis method of Merrifield (see J. Am. Chem. Soc, 85:2149, 1964). According to another embodiment, the peptides of the present invention can be synthesized using standard solution methods well known in the art (see, for example, Bodanszky, M., Principles of Peptide Synthesis, Springer- Verlag, 1984).

[109] In general, the synthesis methods comprise sequential addition of one or more amino acids or suitably protected amino acids to a growing peptide chain bound to a suitable resin. Normally, either the amino or carboxyl group of the first amino acid is protected by a suitable protecting group. The protected or derivatized amino acid can then be either attached to an inert solid support (resin) or utilized in solution by adding the next amino acid in the sequence having the complimentary (amino or carboxyl) group suitably protected, under conditions conductive for forming the amide linkage. The protecting group is then removed from this newly added amino acid residue and the next amino acid (suitably protected) is added, and so forth. After all the desired amino acids have been linked in the proper sequence, any remaining protecting groups are removed sequentially or concurrently, and the peptide chain, if synthesized by the solid phase method, is cleaved from the solid support to afford the final peptide.

[110] In the solid phase peptide synthesis method, the alpha-amino group of the amino acid is protected by an acid or base sensitive group. Such protecting groups should have the properties of being stable to the conditions of peptide linkage formation, while being readily removable without destruction of the growing peptide chain. Suitable protecting groups are t-butyloxycarbonyl (BOC), benzyloxycarbonyl (Cbz), biphenylisopropyloxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, (alpha,alpha)-dimethyl-3 ,5 dimethoxybenzyloxycarbonyl, o- nitrophenylsulfenyl, 2-cyano-t-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (FMOC) and the like. In the solid phase peptide synthesis method, the C-terminal amino acid is attached to a suitable solid support. Suitable solid supports useful for the above synthesis are those materials, which are inert to the reagents and reaction conditions of the stepwise condensation-deprotection reactions, as well as being insoluble in the solvent media used. Suitable solid supports are chloromethylpolystyrene-divinylbenzene polymer, hydroxymethyl-polystyrene-divinylbenzene polymer, and the like. The coupling reaction is accomplished in a solvent such as ethanol, acetonitrile, Ν,Ν-dimethylformamide (DMF), and the like. The coupling of successive protected amino acids can be carried out in an automatic polypeptide synthesizer as is well known in the art. [111] In another embodiment, peptides of the invention may be synthesized such that one or more of the bonds, which link the amino acid residues of the peptides are non-peptide bonds. In another embodiment, the non-peptide bonds include, but are not limited to, imino, ester, hydrazide, semicarbazide, and azo bonds, which can be formed by reactions well known to skilled in the art.

[112] In one embodiment, the peptides of the present invention, analogs or derivatives thereof produced by recombinant techniques can be purified so that the peptides will be substantially pure when administered to a subject.

[113] As used herein, the term "substantially pure" refers to a compound, e.g., a peptide, which has been separated from components, which naturally accompany it. Typically, a peptide is substantially pure when at least 50%, preferably at least 75%, more preferably at least 90%, and most preferably at least 99% of the total material (by volume, by wet or dry weight, or by mole percent or mole fraction) in a sample is the peptide of interest. Purity can be measured by any appropriate method, e.g., in the case of peptides by HPLC analysis.

[114] In one embodiment, the peptides of the invention are peptide conjugates, comprising the peptides of the present invention derivatives or analogs thereof joined at their amino or carboxy- terminus or at one of the side chains via a peptide bond to an amino acid sequence of a different protein. In another embodiment, conjugates comprising peptides of the invention and a different protein can be made by protein synthesis. In another embodiment, conjugates comprising peptides of the invention and a different protein can be made by use of a peptide synthesizer. In another embodiment, conjugates comprising peptides of the invention and a different protein can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the conjugate by methods commonly known in the art. In another embodiment, addition of amino acid residues may be performed at either terminus of the peptides of the invention for the purpose of providing a "linker" by which the peptides of this invention can be conveniently bound to a carrier. In another embodiment, the linkers are comprised of at least one amino acid residue. In another embodiment, the linkers can be of 40 or more residues. In another embodiment, the linkers are comprised of 1 to 10 residues. In another embodiment, amino acid residues used for linking are tyrosine, cysteine, lysine, glutamic and aspartic acid, or the like. [115] In some embodiments, a chimeric protein has a protein backbone having attached thereto a moiety at a binding site of the protein backbone. In some embodiments, the binding site of the protein backbone is the N-terminus of the protein backbone. In exemplary embodiments, the binding site of the protein backbone is the C-terminus of the protein backbone. The terms "protein construct", "construct", or "chimeric protein" as used herein throughout interchangeably, refer to an artificially made or recombinant molecule that comprises two or more protein sequences that are not naturally found within the same protein. The protein construct may be a fusion protein encoded by a single polynucleotide and may be made recombinantly.

[116] In some embodiments, the present invention provides chimeric peptides comprising a first peptide having an amino acid sequence as set forth in SEQ ID NO: 1, and a second peptide linked to said first peptide.

[117] In some embodiments, the present invention provides chimeric peptides comprising a first peptide having an amino acid sequence as set forth in any one of SEQ ID NO: 2-7, 11, 14-21, 26- 30 and 32, and a second peptide linked to said first peptide.

[118] In some embodiments, said second peptide is a cell targeting moiety. In some embodiments, said second peptide is a cancer cell targeting moiety.

[119] In some embodiments, the invention provides a composition comprising said chimeric peptide.

[120] In some embodiments, the present invention provides a composition comprising the peptide of the present invention (e.g., SEQ ID NO: 1, 2-7, 11, 14-21, 26-30 and 32), and a cell targeting agent.

[121] In some embodiments, the present invention provides a composition comprising the peptide of the present invention (e.g., SEQ ID NO: 1, 2-7, 11, 14-21, 26-30 and 32), and a second cell targeting moiety linked to said peptide.

[122] As used herein "cell targeting moiety" refers to a molecule i.e., a peptide that is capable of binding a target cell, or preferably has increased affinity and/or selectivity to a target cell. In come embodiments, said cell is a cancerous cell or a pre-cancerous cell.

[123] In some embodiments, the cell targeting moiety is a peptide or protein capable of specifically binding a cell. In an exemplary embodiment, the targeting moiety of the present invention is maltose binding protein (MBP). Non-limiting examples a peptide or protein which function as a cell targeting moiety are selected from the group consisting of: a peptide comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 22 (VPWMEPAYQRFL), a peptide comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 23 (ATWLPPR), Biotinylated-wGeyidvk (lowercase letters represent D-amino acids and VAR2CSA (a malarial protein that binds to distinct chondroitin sulfate (CS)). Other proteins and peptides capable of specifically binding a cancer cell are well known in the art. In some embodiments, any protein or peptide capable of specifically binding a cancer cell may be linked to peptides of the present invention and serve as a targeting moiety.

[124] In some embodiments, the cell targeting moiety or agent. In some embodiments, the antibody specifically binds a ligand presented on a target cancer cell. In some embodiments, the antibody has increased binding specificity to a ligand presented on a target cancer cell. In some embodiments, said ligand is a tumor associated antigen (TAA) or a tumor specific antigen (TSA). The terms TAA and TSA as used herein refer to any protein produced in a tumor cell that has an abnormal structure due to mutation or that is abnormally expressed by a cancer cell. Such abnormal proteins may be produced due to mutation of the concerned gene. Numerous TAAs and TSAs have been described in the art and may be used as a ligand for a cancer cell targeting moiety.

[125] In some embodiments, the present invention provides a composition comprising the peptide of the present invention (e.g., SEQ ID NO: 1, 2-7, 11, 14-21, 26-30 and 32), and an anticancer agent. In some embodiments, the anti-cancer agent is selected from the group consisting of chemotherapy, immunotherapy, and any combination thereof.

[126] Chemotherapeutic agents are well known to one skilled in the art. Non-limiting examples of chemotherapeutic agents includes doxorubicin, cyclophosphamide, mechlorethamine, chlorambucil, melphalan, dacarbazine, nitrosoureas, temozolomide, daunorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, paclitaxel, docetaxel, abraxane, taxotere, varinostat, romidepsin, irinotecan, topotecan, etoposide, teniposide, tafluposide, bortezomib, erlotinib, getitinib, imatinib, vermurafenib, vismodegib, azacytidine, azathioprine, capecitabine, cytarabine, doxifluridine, fluorouracil, gemcitabine, hydroxyurea, mercaptopurine, methotrexate, tioguanine, bleomycin, actinomycin, carboplatin, cisplatin, oxaliplatin, tretinoin, alitretinoin, bexarotene, vinblastine, vincristine, vindesine, and vinorelbine. [127] In exemplary embodiments, the peptide of the invention is characterized as having a loop shape. In some embodiments, the loop shape forms helical structure. In some embodiments, the loop shape forms helical structure on the helix C-terminal. In some embodiments, the helical structure is characterized as having a core of e.g., at least 12 amino acid. In some embodiments, one helix face is positively charged, while opposite side is mostly negatively charged. In another embodiment, the negatively charged face interacts with the magnetite particle having a positively charged surface. In some embodiments, there is provided a magnetic carrier comprising the peptide disclosed herein, optionally contiguous to a second peptide, e.g., MBP, further attached to one or more magnetic nanoparticles, in an effective amount.

[128] As exemplified in the example section that follows, the MamC loop adopts a helical structure which is necessary for its interaction with the magnetite surface.

Magnetotactic bacteria and magnetic nanoparticles

[129] The term "magnetotactic bacteria" as used herein refers to a class of bacteria that exhibit the ability to orient themselves along the magnetic field lines of Earth's magnetic field. Such bacteria include, but are not limited to the group consisting of: Magnetospirillium gryphiswaldenses, Magnetospirillim sp., Magnetovibrio blakemorei, Magnetofaba austalis, Magnetospira sp. and Magnetococus marinus.

[130] The term "magnetite" as used herein refers to a ferromagnetic mineral with chemical formula Fe ₃ 04, one of several iron oxides and a member of the spinel group. The chemical IUPAC name is iron (11,111) oxide and the common chemical name ferrous-ferric oxide. The formula for magnetite may also be written as FeO.Fe ₂ 0 ₃ , which is one part wustite (FeO) and one part hematite (Fe ₂ 0 ₃ ). This refers to the different oxidation states of the iron in one structure, not a solid solution.

[131] The term "magnetosome-derived peptide", as used herein, refers to peptides derived from a magnetotactic bacteria and having capability of binding one or more metal particles. A "peptide from a magnetotactic bacteria", as used herein refers to a peptide having at least 50%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity to a fragment of a MamC, Mms6, or Mms7 proteins.

[132] The term "magnetic nanoparticles", as used herein, denotes any particle having a size in the nanometer scale that exhibits magnetic properties (i.e. that orients in a magnetic field along the magnetic field lines). The particles may either be ferromagnetic or superparamagnetic or may show an intermediate characteristic.

[133] More particularly, the term "magnetic nanoparticles" refers to particles comprising one or more magnetic (nano)crystals. In some embodiments, a magnetic nanoparticle according to the invention comprises only a single nanocrystal. Such particles are also referred to as being "monocrystalline" or "monocrystals".

[134] In another embodiment, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of said magnetic nanoparticles are characterized by a crystal having a size range of 30-60 nm. In another embodiment, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of said magnetic nanoparticles are characterized by a crystal having a size range of 40-50 nm. In another embodiment, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of said magnetic nanoparticles are characterized by a crystal having a size range of 30-50 nm.

[135] Typically, but not exclusively, the magnetic nanoparticles used in the invention are of one or more metal oxides and/or metal sulfides, typically of the elements in the fourth row of the periodic table (i.e. chrome, manganese, iron, cobalt, and nickel). In some embodiments, the magnetic nanoparticles are made of a single metal oxide or a metal sulfide, or of an iron oxide which includes, without limitations, magnetite (Fe ₃ 0 ₄ ), or an iron sulfide, including, without limitations, greigite (Fe ₃ S ₄ ).

[136] In exemplary embodiments, the magnetic nanoparticles are magnetite.

[137] In some embodiments, there is provided use of the peptide of the invention for synthesis of magnetic nanoparticles. In some of the embodiments described herein, the magnetite are synthesized using the peptide of the invention as described herein, and can be synthesize, for example, and without limitation, by a titration of Fell/Felll, as exemplified in the "Example" section that follows.

[138] In some embodiments, the nanoparticles consist of iron oxides and particularly of magnetite (Fe ₃ 0 ₄ ), magnetite (gamma-Fe20 ₃ ) or mixtures of these two oxides and are preferably superparamagnetic. In general, the preferred nanoparticles can be represented by the formula FeOx, wherein X represents a number from 1 to 2. [ 139] In addition to the magnetic materials of the formula FeOx, wherein X is a number in the range of 1.0 to 2.0, materials of the general formula M(II)Fe204 with M=Co, Ni, Mn, Zn, Cu, Cd, Ba or other ferrites can also be used according to the invention. Preferably, metal atoms which differ from iron atoms are contained in a quantity of no more than 70 metal atom percent, particularly no more than 35 metal atom percent. Preferably, the nanoparticles consist no more than 98 percent per weight of iron oxide, containing both Fe(III) and Fe(II) in a ratio of preferably 1: 1 to 1:3. Additionally, silica or polymer particles, into which the magnetic materials such as those mentioned herein are incorporated and/or to which they are attached, are also suitable.

[140] In some embodiments, magnetic nanoparticles are nanoparticles, i.e., characterized as having a size in the nanometer scale. The term "nanometer scale", as used herein, refers to a particle size of e.g., less than 1000 nm (i.e. 1 μπι), less than 500 nm, less than 200 nm, less than 100 nm.

[141] The terms "size" and "dimension", "diameter", as used herein, are not solely to be interpreted literally (i.e. with regard to the length and width of the magnetic nanoparticles) but should also refer to the overall shape of the nanoparticles, including, without limitation, spherical, cubic, regular or deformed, and the like.

[142] In some embodiments, the size of e.g., at least 70%, at least 80%, at least 90% of the nanoparticles is about 40-50 nm within the range given by the mean diameter ± 10%.

[143] In some embodiments, the size of the complex comprising the first peptide, the targeting moiety and magnetite nanoparticles is between 10-400nm, between 50-300nm, between 100- 200nm or between 10-1 OOnm.

[144] In exemplary embodiments, the peptides of the invention are useful for forming magnetite crystals that are larger in size, compared to those achieved using native MamC protein, thereby indicating the peptide advantageous effect on the size of magnetite particle.

[145] MamC gene (or interchangeably mmsl3) is located on the magnetosome island (MAI) and encodes small (12.4 kDa) protein. Prediction of MamC secondary structure using XtalPred server shows two transmembrane helices with connecting loop which is directed to the magnetosome lumen. Without being bound by any particular theory or mechanism of action, 21 amino acids derived from MamC and homologs thereof were found to be highly rich in charged residues, forming an alpha helix loop structure capable of interacting with magnetite nanoparticles. [ 146] In some embodiments, the amino acids loop is derived from Magnetospirillum magneticum strain AMB-1. In some embodiments, the amino acids loop is from any MamC homolog derived from a magnetotactic bacteria. In some embodiments, the amino acid loop is derived from a MamC homolog magnetotactic bacteria.

Methods for treating cancer

[147] In some embodiments, the present invention provides a method for the treatment of cancer. In some embodiments, the treatment is a thermal therapy. The terms "thermal therapy" and "magnetic hyperthermia" as used herein interchangeably refer to a method for eliminating a target cell, i.e., a cancer cell. Thermal therapy relies on the property of magnetite nanoparticles to produce heat by oscillating under an alternating magnetic field (AMF). Using this property, thermotherapy is performed under an alternating magnetic field through direct injection into tumor tissues or administration of magnetic nanoparticles which are bound to a cancer targeting moiety.

[148] The term "AMF", as used herein, refers to a magnetic field that changes the direction of its field vector periodically, typically in a sinusoidal, triangular, rectangular or similar shape pattern. The AMF may also be added to a static magnetic field, such that only the AMF component of the resulting magnetic field vector changes direction. It will be appreciated that an alternating magnetic field is accompanied by an alternating electric field and is electromagnetic in nature.

[149] In some embodiments, the cancer cells are selectively damaged by subjecting the entire cell population to an alternating magnetic field or to a spinning magnetic field. One skilled in the art will appreciate that cancer cells have a far greater affinity for magnetic nanoparticles, relative to non-cancerous cells.

[150] In some embodiments, the term "treatment" as used herein refers to any response to, or anticipation of, a medical condition in a mammal, particularly a human, and includes but is not limited to: preventing the medical condition from occurring in a subject, which may or may not be predisposed to the condition, but has not yet been diagnosed with the condition and, accordingly, the treatment constitutes prophylactic treatment for the medical condition; inhibiting the medical condition, e.g., arresting, slowing or delaying the onset, development or progression of the medical condition; or relieving the medical condition, e.g., causing regression of the medical condition or reducing the symptoms of the medical condition. [151] The term "administering" as used herein, includes delivery of a composition or one or more pharmaceutically active ingredients to a subject, by any appropriate methods, which serve to deliver the composition or its active ingredients or other pharmaceutically active ingredients to the subject. In another embodiment, the method of administration may vary depending on various factors, such as for example, the components of the pharmaceutical composition or the nature of the pharmaceutically active or inert ingredients, the site of the potential or actual malady, age and physical condition of the subject.

[152] As used herein, the term "subject" refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

[153] Suitable routes of administration include, but are not limited to, parenteral injections, e.g., intradermal, intravenous, intramuscular, intralesional, subcutaneous, intrathecal, and any other mode of injection as known in the art. Although the bioavailability of peptides administered by other routes can be lower than when administered via parenteral injection, by using appropriate formulations it is envisaged that it will be possible to administer the compositions of the invention via transdermal, oral, rectal, vaginal, topical, nasal, inhalation and ocular modes of treatment. In addition, it may be desirable to introduce the pharmaceutical compositions of the invention by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer.

Pharmaceutical compositions

[154] In some embodiments, there is provided compositions (i.e., pharmaceutical compositions) comprising as an active ingredient a therapeutically effective amount of a peptide (i.e., polypeptides) of the present invention (e.g., SEQ ID NO: 1), and a pharmaceutically acceptable carrier.

[155] In some embodiments, the pharmaceutical composition further comprises magnetite. In some embodiments, the molar ratio of magnetic nanoparticle to peptide is between 1: 1 to 1:5, between 1: 1 to 1: 10, between 1: 10 to 1 : 100, between 1 : 100 to 1 : 500, between 1: 100 to 1: 1000, between 1 : 10 ³ to 1 : 10 ⁴ or between 10 ⁴ : 1 to 1: 10 ⁵ . [156] In some embodiments, the pharmaceutical composition is encapsulated within a liposome to enhance uptake by a target cell.

[157] In some embodiments, said liposome is one of a stealth liposome, a micellar system, an immunoliposome, a cationic liposome, or a fusogenic liposome.

[158] In some embodiments, the composition has a natural or anionic charge to reduce the rate of renal elimination.

[159] In some embodiments, the composition further comprising at least one agent selected from a cancer cell targeting agent or anti-cancer agent.

[160] In some embodiments, the nanoparticles of present invention are bound to therapeutically active substances wherein the separation of the therapeutically active substances from the nanoparticles is caused, initiated or substantially enhanced by an alternating magnetic field. In this context, the at least one therapeutically active substance is released by means of the direct influence of the alternating magnetic field or due to the local heating caused by the alternating magnetic field. Preferably, the release is caused by the fact that a thermally labile linker between the active ingredient, i.e. the therapeutically active substance and the nanoparticle is thermally cleaved and/or that a linker is used which is labile with respect to an alternating magnetic field. Therefore, the present invention consists of binding a therapeutically active substance, in particular a cytostatic, to a nanoparticle by means of a linker which can be cleaved thermally and/or by a magnetic field.

[161] In some embodiments, the present invention relates to nanoparticles, wherein at least one therapeutically active substance is covalently or ionically bound or bound via hydrogen bonds or via complexation (complex bond) or via intercalation or via lipophilic interactions by means of a linker and the linker can be cleaved due to thermal initiation or to initiation by an electromagnetic or respectively magnetic field.

[162] Thermally initiated cleavage means that a local heating under physiological conditions to a temperature of more than 45 degrees centigrade, preferably more than 50 degrees centigrade is sufficient to cleave the linker. Cleavage initiated by an electromagnetic or respectively magnetic field means that the application of an electromagnetic or respectively magnetic field under physiological conditions causes the linker to be cleaved, either only by the electromagnetic or respectively magnetic field and/or a local pH reduction induced by the electromagnetic or respectively magnetic field.

[163] The at least one therapeutically active substance, i.e. the molecules of at least one therapeutically active substance class or one particular active ingredient is preferably bound by means of a covalent or predominantly covalent bond and/or a sufficiently strong ionic bond, clathrate compounds or complexation (complex bonds) or respectively by means of an arrangement of a sufficient number of hydrogen bonds or hydrophobic interactions so that an uncontrolled release of therapeutically active substance can substantially be avoided. Uncontrolled release describes the separation of therapeutically active substance in healthy tissue, particularly separation without an alternating magnetic field being active.

[ 164] Such uncontrolled release results in therapeutically active substances being released at sites where they are more likely to cause detrimental side effects than therapeutic effects, that is outside of the carcinogenic tissue or respectively outside of the tumor cells.

[165] Thus, the therapeutically active substances remain fixedly bound to the nanoparticles and are transported to the cancer cell together with the nanoparticle. While the nanoparticles are transported to the cancer cells, only minor up to insignificant amounts of the therapeutically active substances are released. Once arrived in the cancer cells, the therapeutically active substances are released by means of an alternating magnetic field, particularly by means of an exterior alternating magnetic field or respectively an alternating magnetic field applied from the outside (impulse).

[166] In this context, "caused or initiated by an alternating magnetic field" means that the release or respectively separation is either directly caused by the alternating magnetic field or respectively the impulses or indirectly, for example by the activation or respectively induction of gene expression of enzymes or by the generation of heat.

[167] The nanoparticles consist of a magnetic material, preferably a ferromagnetic, antiferromagnetic, ferrimagnetic, antiferrimagnetic or superparamagnetic material, further preferred of iron oxides, particularly of superparamagnetic iron oxides or of pure iron provided with an oxide layer. Such nanoparticles can be heated by an alternating magnetic field. The tissue containing the nanoparticles can be heated to a temperature of more than 50 degrees centigrade. Such high temperatures can be achieved due to the fact that up to 800 pg and more of iron in form of the nanoparticles can be absorbed per tumor cell. [168] The pharmaceutical compositions of the invention can be formulated in the form of a pharmaceutically acceptable salt of the peptides of the invention or their analogs, or derivatives thereof. Pharmaceutically acceptable salts include those salts formed with free amino groups such as salts derived from non-toxic inorganic or organic acids such as hydrochloric, phosphoric, acetic, oxalic, tartaric acids, and the like, and those salts formed with free carboxyl groups such as salts derived from non-toxic inorganic or organic bases such as sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, and the like. In one embodiment, pharmaceutical compositions of the present invention are manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. In one embodiment, pharmaceutical compositions of the present invention undergo steam sterilization. In one embodiment, the compositions of the invention are processed under at least one step including high pressures and temperatures, such as using an autoclave.

[169] The term "pharmaceutically acceptable" means suitable for administration to a subject, e.g., a human. For example, the term "pharmaceutically acceptable" can mean approved by a regulatory agency of the Federal or a state government or listed in the U. S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates. Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; and agents for the adjustment of tonicity such as sodium chloride or dextrose are also envisioned.

[170] The compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, gels, creams, ointments, foams, pastes, sustained-release formulations and the like. The compositions can be formulated as a suppository, with traditional binders and carriers such as triglycerides, microcrystalline cellulose, gum tragacanth or gelatin. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in: Remington's Pharmaceutical Sciences" by E.W. Martin, the contents of which are hereby incorporated by reference herein. Such compositions will contain a therapeutically effective amount of the peptide of the invention, preferably in a substantially purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.

[171] An embodiment of the invention relates to a peptide presented in unit dosage form and are prepared by any of the methods well known in the art of pharmacy. In an embodiment of the invention, the unit dosage form is in the form of a tablet, capsule, lozenge, wafer, patch, ampoule, vial or pre-filled syringe. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the nature of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses can be extrapolated from dose-response curves derived from in-vitro or in-vivo animal model test bioassays or systems.

[172] Depending on the location of the tissue of interest, the peptides of the present invention can be supplied in any manner suitable for the provision of the peptide to cells within the tissue of interest. Thus, for example, a composition containing the peptides can be introduced, for example, into the systemic circulation, which will distribute said peptide to the tissue of interest. Alternatively, a composition can be applied topically to the tissue of interest (e.g., injected, or pumped as a continuous infusion, or as a bolus within a tissue, applied to all or a portion of the surface of the skin, etc.). [173] In an embodiment of the invention, peptides are administered via oral, rectal, vaginal, topical, nasal, ophthalmic, transdermal, subcutaneous, intramuscular, intraperitoneal or intravenous routes of administration. The route of administration of the pharmaceutical composition will depend on the disease or condition to be treated. Suitable routes of administration include, but are not limited to, parenteral injections, e.g., intradermal, intravenous, intramuscular, intralesional, subcutaneous, intrathecal, and any other mode of injection as known in the art. Although the bioavailability of peptides administered by other routes can be lower than when administered via parenteral injection, by using appropriate formulations it is envisaged that it will be possible to administer the compositions of the invention via transdermal, oral, rectal, vaginal, topical, nasal, inhalation and ocular modes of treatment. In addition, it may be desirable to introduce the pharmaceutical compositions of the invention by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer.

[174] For oral applications, the pharmaceutical composition may be in the form of tablets or capsules, which can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalhne cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate; or a glidant such as colloidal silicon dioxide. When the dosage unit form is a capsule, it can contain, in addition to materials of the above type, a liquid carrier such as fatty oil. In addition, dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or other enteric agents. The tablets of the invention can further be film coated.

[175] For purposes of parenteral administration, solutions in sesame or peanut oil or in aqueous propylene glycol can be employed, as well as sterile aqueous solutions of the corresponding water- soluble salts. Such aqueous solutions may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal injection purposes.

[176] The compositions of the present invention are generally administered in the form of a pharmaceutical composition comprising at least one of the active components of this invention together with a pharmaceutically acceptable carrier or diluent. Thus, the compositions of this invention can be administered either individually or together in any conventional oral, parenteral or transdermal dosage form.

[177] Pharmaceutical compositions according to embodiments of the invention may contain 0.1%-95% of the active components(s) of this invention, preferably l%-70%. In any event, the composition or formulation to be administered may contain a quantity of active components according to embodiments of the invention in an amount effective to treat the condition or disease of the subject being treated.

[178] The compositions also comprise preservatives, such as benzalkonium chloride and thimerosal and the like; chelating agents, such as EDTA sodium and others; buffers such as phosphate, citrate and acetate; tonicity agents such as sodium chloride, potassium chloride, glycerin, mannitol and others; antioxidants such as ascorbic acid, acetylcystine, sodium metabisulfote and others; aromatic agents; viscosity adjustors, such as polymers, including cellulose and derivatives thereof; and polyvinyl alcohol and acid and bases to adjust the pH of these aqueous compositions as needed. The compositions may also comprise local anesthetics or other actives.

[179] In addition, the compositions may further comprise binders (e.g. acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone), disintegrating agents (e.g. cornstarch, potato starch, alginic acid, silicon dioxide, croscarmelose sodium, crospovidone, guar gum, sodium starch glycolate), buffers (e.g., Tris-HCI., acetate, phosphate) of various pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), protease inhibitors, surfactants (e.g. sodium lauryl sulfate), permeation enhancers, solubilizing agents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g. hydroxypropyl cellulose, hyroxypropylmethyl cellulose), viscosity increasing agents(e.g. carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum), sweeteners (e.g. aspartame, citric acid), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), lubricants (e.g. stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), flow-aids (e.g. colloidal silicon dioxide), plasticizers (e.g. diethyl phthalate, triethyl citrate), emulsifiers (e.g. carbomer, hydroxypropyl cellulose, sodium lauryl sulfate), polymer coatings (e.g., poloxamers or poloxamines), coating and film forming agents (e.g. ethyl cellulose, acrylates, polymethacrylates) and/or adjuvants.

[180] The peptides of the present invention, derivatives, or analogs thereof can be delivered in a controlled release system. Thus, an infusion pump can be used to administer the peptide such as the one that is used, for example, for delivering insulin or chemotherapy to specific organs or tumors. In one embodiment, the peptide of the invention is administered in combination with a biodegradable, biocompatible polymeric implant, which releases the peptide over a controlled period of time at a selected site. Examples of preferred polymeric materials include, but are not limited to, polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, copolymers and blends thereof (See, Medical applications of controlled release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Fla., the contents of which are hereby incorporated by reference in their entirety). In yet another embodiment, a controlled release system can be placed in proximity to a therapeutic target, thus requiring only a fraction of the systemic dose.

[181] In one embodiment, compositions of the present invention are presented in a pack or dispenser device, such as an FDA approved kit, which contain one or more unit dosage forms containing the active ingredient. In one embodiment, the pack or dispenser device is accompanied by instructions for administration.

[ 182] In one embodiment, it will be appreciated that the peptides of the present invention can be provided to the individual with additional active agents to achieve an improved therapeutic effect as compared to treatment with each agent by itself. In another embodiment, measures (e.g., dosing and selection of the complementary agent) are taken to adverse side effects which are associated with combination therapies.

[183] A "therapeutically effective amount" of the peptide is that amount of peptide which is sufficient to provide a beneficial effect to the subject to which the peptide is administered. More specifically, a therapeutically effective amount means an amount of the peptide effective to prevent, alleviate or ameliorate tissue damage or symptoms of a disease of the subject being treated.

[184] In some embodiments, preparation of effective amount or dose can be estimated initially from in vitro assays. In one embodiment, a dose can be formulated in animal models and such information can be used to more accurately determine useful doses in humans. [185] In one embodiment, toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. In one embodiment, the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. In one embodiment, the dosages vary depending upon the dosage form employed and the route of administration utilized. In one embodiment, the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. [See e.g., Fingl, et al., (1975) "The Pharmacological Basis of Therapeutics", Ch. 1 p.l].

[ 186] In one embodiment, depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved. In one embodiment, the amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc. In one embodiment, compositions including the preparation of the present invention formulated in a compatible pharmaceutical carrier are also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

[187] In the discussion unless otherwise stated, adjectives such as "substantially" and "about" modifying a condition or relationship characteristic of a feature or features of an embodiment of the invention, are understood to mean that the condition or characteristic is defined to within tolerances that are acceptable for operation of the embodiment for an application for which it is intended. Unless otherwise indicated, the word "or" in the specification and claims is considered to be the inclusive "or" rather than the exclusive or, and indicates at least one of, or any combination of items it conjoins.

[188] In the description and claims of the present application, each of the verbs, "comprise," "include" and "have" and conjugates thereof, are used to indicate that the object or objects of the verb are not necessarily a complete listing of components, elements or parts of the subject or subjects of the verb.

Diagnostic use

[ 189] Due to the ability of the pharmaceutical composition described herein to target cancer cells, the pharmaceutical composition can be further utilized for monitoring the disease state within a body of a patient. The method, according to these embodiments of the invention, is effected by administering to the subject any of the pharmaceutical compositions described herein, and employing an imaging technique for monitoring a distribution of the magnetic nanoparticles within the body or a portion thereof. For example, the level of cancer progression in tumor sites can serve as a measure of the size of a tumor as well as the level of metabolic activity in the tumor cells.

[190] A "disease state" refers to the current status of a disease which may have been previously diagnosed, such prognosis, risk-stratification, assessment of ongoing drug therapy, prediction of outcomes, determining response to therapy, diagnosis of a disease or disease complication, following progression of a disease or providing any information relating to a patient's health status over time.

[191] In another aspect, the invention provides a method of imaging a neoplastic tissue, the method comprises administering to a subject having (or suspected of having) a neoplasia, a peptide e.g., a chimeric peptide, and detecting the compound following distribution thereof in-vivo. In some embodiments, said method of imaging includes the subsequent step (e.g., following the detection step) of generating an image of the detected distributed compound. The detection step may be performed using magnetic resonance imaging or any other method known for the detection of magnetic nanoparticles.

General:

[192] As used herein the term "about" refers to ± 10 %.

[193] The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to". The term "consisting of means "including and limited to". The term "consisting essentially of" means that the composition, method, or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[194] The word "exemplary" is used herein to mean "serving as an example, instance or illustration". Any embodiment described as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments and/or to exclude the incorporation of features from other embodiments. [195] The word "optionally" is used herein to mean "is provided in some embodiments and not provided in other embodiments". Any particular embodiment of the invention, may include a plurality of "optional" features unless such features conflict.

[196] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[197] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[198] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging/ranges between" a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between.

[199] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[200] As used herein, the term "treating" includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition. [201] 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 sub combination 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.

[202] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. EXAMPLES

[203] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.

MATERIALS AND METHODS

[204] Materials: 21 amino acid section of Magnetospirillum magneticum AMB- 1 strain mamC gene (Lue57-Gly77); LB medium; Kanamycin; EDTA pMBP-MamC; IPTG; SDS-PAGE;

[205] Cloning: 17 or 21 amino acid-long peptide derived from Ms. magneticum AMB-1 strain mamC gene (Lue57/Arg61-Gly77) and the MBP-Long mutants were cloned into pET28a containing MBP (maltose binding protein from Escherichia coli) at the MBP C-terminus (pMBP- MamC purchased from Biomatik Corporation, Ontario, Canada). Only the active C-terminal of Mms6 (MKSRDIESAQSDEEVELRDALA; SEQ ID NO: 14) from Ms. magneticum AMB-1 was cloned into plasmid pET28a on the maltose binding protein C-terminal (Novagen) with a 6XHis- tag on its N-terminal.

[206] Protein expression: Competent E. coli BL-21 or Rosetta cells were transformed with pMBP-MamC and grown in 12ml LB medium with 50 mg/ml kanamycin at 37°C. Protein production was induced with 0.5 mM IPTG at cell density corresponding to an absorbance of 0.6 at 600 nm, and the protein expressed at 20°C overnight. Cells were collected by centrifugation for 8 min at 6K rpm and frozen at -80°C. [207] Protein purification: Cells were thawed and resuspended in binding buffer (20 mM Tris pH 8, 200 mM NaCl, 1 mM EDTA). Protease inhibitors and DNase were added after cell resuspension. Cells were lysed by two rounds of high pressure of 172 MPausing the French Press apparatus (Thermo). The crude lysate was then centrifuged at 16,000 rpm for 40 min. The soluble lysate was loaded on amylose resin column followed by wash with five column volume (CV) of the binding buffer. Protein was eluted with elution buffer (20 mM Tris pH 8, 200 mM NaCl, 10 mM maltose). Elution fractions were analysed by 12.5% SDS-PAGE and were concentrated prior to the size exclusion step (superdex75 26/60 column). The protein eluted in size that correlated to a monomer protein (42 kDa). All fractions were collected and concentrated to final concentration of 28 mg/ml and have been frozen in liquid nitrogen and stored at -80°C.

[208] Crystal screening for MamC: Sitting-drop 96-well plate was set using the Index screen kit (Hampton Research, California, USA). Each well in the plate contained two protein drops, a high concentration (10 mg mL-1 for MBP-Long and Short and 18 mg mL-1 for MamC mutants) and a low concentration (5 mg/mL-1 for MBP-Long and Short and 9 mg/mL-1 for MamC mutants). The plates were stored at 20 °C in an automated imaging system (Rock Imager, Formulatrix, Bedford, Massachusetts, USA). After 12 days, positive hits were observed only for MBP-Long in one condition: 2 M ammonium sulphate with 0.1 M sodium acetate pH 3.8. Optimization plates were set around the obtained crystallization condition. For D70A, positive hits were observed after two days from two conditions: 0.1 M tri-sodium citrate pH 3.5 with 2 M ammonium sulfate and 0.1 M sodium citrate pH 4.5 with 2 M ammonium sulfate.

[209] Structure determination: Dataset was collected for MBP-Long and D70A using a single crystal on beamline ID14-4 and BM14, respectively, at the European Synchrotron Radiation Facility (ESRF), Grenoble, France. MBP-Long data set was collected with a wavelength of 0.939 A. 100 images were collected at an oscillation range of 1 .05 ^° , an exposure time of 0.5 s per image and a crystal-to-detector distance of 377.41 mm. D70A data set was collected with a wavelength of 0.953 A. 500 images were collected at an oscillation range of 0.5 "per image, an exposure time of 0.5 s per image and a crystal-to-detector distance of 275.73 mm. Data were reduced and scaled using the HKL2000 program suite. Phases were obtained via molecular replacement using PHASER followed by manual building cycles using Coot. Structures were refined in REFMAC5 (Vagin et al., 2004). Structural figures were prepared using PyMOL. [210] Magnetic Nanoparticles synthesis: Magnetite was synthesized with the modified co- precipitation method controlled by a titration system (Metrohm, 776 Dosimat and 719 S Titrino). Fe ⁿ /Fe ^m -chloride solution (1 M, Fe ⁿ :Fe ^m = 1:2) was added with 1 μΐ/min to a total volume of 10 ml. The pH and the temperature were kept constant during the process (pH = 11 ± 0.4 with 1 M NaOH; T = 25 ± 0.1 °C) and the synthesis lasted eight hours. All solutions were degassed before using and the system was kept under nitrogen atmosphere during the synthesis. The size was determined with Synchrotron X-ray diffraction at the μ-Spot beam line (Bessy II, Berlin) by Scherrer analysis of the 311 peak of magnetite.

[211] Isothermal Titration Calorimetry (ITC): Interaction of MamC constructs and magnetite is performed at a constant temperature by titrating the protein samples into a solution containing magnetite in the sample cell of the calorimeter as magnetite can clog the injection needle. The heat change is expressed as the electrical power (J s ^"1 ), KA and the stoichiometry, n, of the complex are also obtain by ITC. Controls are identical MamC buffer injection to the reference cell without protein.

[ 12] Expression and purification of full length recombinant MamC: MamC (as His-MamC) was expressed in Escherichia coli, purified in denaturing conditions and refolded by using the protocol described in Valverde-Tercedor et al. (2014a and b). Genomic DNA from Magnetococcus marinus was isolated following the method described by Martin-Platero et al. (2007). The gene that encodes for the MamC protein in Mc. marinus is designated as Mmcl_2265 in its genome. Primers for polymerase chain reaction (PCR) amplification of mamC were designed. PCR reaction mixtures (50 μί) contained 10-100 ng of genomic DNA, 10 pmol of each primer, 5 μL· of lx PCR buffer, 2.5 mM of each dNTP and 2 U of Taq polymerase (Dominion-MBL, Cordoba, Spain). The reaction mixture was preheated for 2 min at 95°C, followed by 25 cycles of amplification consisting of a denaturing step at 94°C for 1 min, an annealing step at 55°C for 1 min and an extension step at 72°C for 1 min.

[213] PCR products were purified using a MBL-PCR QuickClean Kit (Dominion-MBL) and sequenced using an ABI PRISM BigDye Terminator Cycle Sequencing Kit (Life Technologies: Applied Biosystems, Madrid, Spain), according to the manufacturer's instructions. They were cloned into pTrcHis-TOPO (Life Technologies: Invitrogen, Grand Island, NY) according to the manufacturer's instructions so the recombinant protein was produced as N-terminal hexahistidine tag fusion proteins. Recombinant clones were verified by dideoxynucleotide sequencing using ABI model 3100 sequencer (Life Technologies: Applied Biosystems).

[214] For the purification of the recombinant MamC protein, E. coli (TOP10) (TrcHisTOPO_mamC) was induced with 2 mM of IPTG for 5 h at 37°C. The obtained cell pellet was resuspended in Buffer A (20 mM phosphate buffer pH 8.0, 6 M urea and 0.5 M NaCl) and disrupted by sonication. After centrifugation of the cell lysate at 75,600 x g for 30 min at 4°C, supernatant was loaded on to a HiTrap chelating HP column (GE Healthcare) previously equilibrated with Buffer A using an AKTA Prime Plus FPLC System (GE Healthcare). The column was then washed with 15 bed volumes of Buffer A, followed by 10 bed volumes of Buffer A adjusted to pH 6.5 with HC1. Finally, the protein was eluted with 5 bed volumes of Buffer A adjusted to pH 4. The eluate was dialyzed overnight at 4°C against 1 L of Buffer B (Tris buffer pH 8.5, 4 M urea and 0.15 M NaCl). To reduce the concentration of urea, the dialysis buffer was diluted stepwise 1 :2 (four times) with fresh Buffer B without urea (named Buffer C) and dialyzed for another 2-4 h after each dilution step. The purity of the obtained protein preparations was estimated by Coomassie-stained SDS-PAGE.

[215] Circular dichroism: CD measurements were conducted with a J750 Spectropolarimeter (Jasco, Mary's Court, Easton, USA) equipped with a Pelletier device. All peptide samples were pre-diluted to different mg ml-1, in buffer containing 50 mM NaCl, 20 mM Tris-HCl, pH 8, and measured with a 0.1 cm optical path suprasil quartz cuvette (Hellma, Mullheim, Germany).

[216] Free drift biomineralization experiments. All reagents were purchased from Sigma- Aldrich. Deoxygenated solutions of NaHC03/Na2C03 (0.15 M/0.15 M), FeC13 (1 M), Fe(C104)2 (0.5 M) and NaOH (5 M) were prepared by using deoxygenated deionized water passed through a Millipore Milli-Q Plus water purification system. The deoxygenation of water was carried out by boiling the nanopurified water for 1 h and then cooling in an ice bath while continuously sparging with ultrapure N2. Once cool, the deoxygenated water was immediately placed inside a Coy anaerobic chamber (Coy Laboratory Products, Grass Lake, MI). Aliquots of all proteins used in the present study were also bubbled with purified N2 for 10 minutes, to remove residual 02, and placed inside the Coy chamber as well. Synthesis and materials' handling were carried out inside the anaerobic chamber (02 levels -40 ppb) to avoid potential oxidation. [217] Inorganic magnetite was precipitated from solutions in free drift experiments held at 25 °C and 1 atm total pressure, following the protocol described by Perez-Gonzalez et al. and Valverde- Tercedor et al., inside the Coy chamber filled with 4% H2 in N2 (Perez-Gonzalez et al., 2010; Valverde-Tercedor et al., 2015). Different volumes of the solutions listed above were mixed to prepare the precipitation solution to a final volume of NaHC03/Na2C03 (3.5 mM/3.5 mM), FeC13 (5.56 mM), Fe(C104)2 (2.78 mM). The final pH of all solutions was set at a value of 9 by the addition of the NaOH solution.

[218] The protein-bearing co-precipitation experiments were carried out in the presence of 10 μg mL-1 concentration of the relevant protein (MBP, MBP-Mms6, recombinant MamC, MBP-Long or MBP-Short). Each reaction was allowed to proceed inside the anaerobic chamber for 30 days, after which the precipitated materials were harvested. The solids were concentrated in the tubes with a magnet and the supernatants, transparent and containing no visible solids, were discarded. The precipitates were washed with deoxygenated water (10 mL). After removing the original supernatant liquid, each reaction flask was filled with the deoxygenated water, the magnet was removed and the water and precipitate were vigorously shaken for a few seconds, after which the precipitate was concentrated again with the magnet and the liquid removed. This rinsing process was repeated three times, after which the precipitate was collected and immediately freeze-dried (FLEXI-DRY-μΡ). Once freeze-dried, the precipitate was stored inside the anaerobic chamber until analyzed.

[219] X-Ray diffraction analysis: Powdered specimens were analyzed with an Xpert Pro X-ray diffractometer (PANalytical; The Netherlands) using the Cu Ka radiation, with the scan range set from 20 to 60 deg in 2 Θ (0.01 deg/step; 3 s per step). Identification of the precipitates was performed by using the XPowder software (Martin-Ramos 2004).

[220] Transmission electron microscopy. The morphology and size of the crystals collected in the experiments were studied by transmission electron microscopy (TEM) using a Philips Model CM20 electron microscope equipped with an energy dispersive X-ray spectrometer (ED AX). The synthesized magnetic powders were dehydrated with ethanol and embedded in Embed 812 resin. Ultrathin sections (50-70 nm) were prepared using a Reichert Ultracut S microtome (Leica Microsystems GmbH, Wetzlar, Germany), after which the sections were placed onto copper grids and carbon-coated using an EMITECK Model K975X Thermal Evaporator (Fall River, MA). Imaging and size distribution analysis was performed with more than 600 nanoparticles in each experiment. The size of the crystal was measured manually by using the ImageJ 1.47 software and the size distribution curves were determined from these measurements by using Origin Pro 9. The statistics calculations were performed using Statistical and MS Excel.

[221] Magnetic nanoparticles synthesis for protein interaction experiments. Magnetite was synthesized with the modified co-precipitation method controlled by a titration system (Metrohm, 776 Dosimat and 719 S Titrino). Fe+2/Fe+3 -chloride solution (1 M, FelLFelll = 1:2) was added at a rate of 1 \xL min-1 to a total volume of 10 mL. The pH and the temperature were kept constant during the process (pH = 11 ± 0.4 with 1 M NaOH; T = 25 ± 0.1 °C) and the synthesis lasted eight hours. All solutions were degased before use and the system was kept in an atmosphere of nitrogen during the synthesis. The size was determined with synchrotron X-ray diffraction at the μ-Spot beam line (Bessy II, Berlin) by Scherrer analysis of the 311 peak of magnetite (Baumgartner et al., 2013; Widdrat et al., 2014).

Example 1

The structure-function relationships of the M. magneticum AMB-1 MamC loop in the magnetite biomineralization process

[222] Two constructs that were fused to maltose binding protein (MBP) at its C-terminal, based on the loop sequence and its predicted secondary structure, were produced. The first construct is a short peptide that contains 17 amino acids (R61-G77, MBP-Short) whereas the second is a long peptide of 21 amino acid (L57-G77, MBP-Long).

[223] The effect of MamC, MBP-Short and MBP-Long during magnetite precipitation in-vitro was analysed. These precipitates were identified as magnetite using XRD (Fig. 1A, IB, 1C).

[224] Full-length MamC-derived magnetite nanoparticles display a size range of 20-50 nm, although most of the nanoparticles are 30-40 nm in size (Fig. ID). MamC solution concentrations of 10 μg/mL resulted in magnetite crystals better-faceted compared to these from the control experiment of buffer alone. Whilst rounded and isomorphic forms also showed, well -developed crystal faces displayed rhombic, rectangular and square 2D morphologies (Fig. 1A).

[225] To better define the MamC iron mineralization region, MBP-Long was compared to full MamC. Interestingly, MBP-Long-derived magnetites were well-faceted crystals varying in size depending on the protein concentration in solution (Fig. IB). At 10 μg/mL MBP-Long, magnetites exhibited a crystal size range of 20-50 nm, most of them being of 40-50 nm (Fig. IE). This size is significantly larger than that of the crystals from the control experiments of MBP-Mms6, MBP alone or buffer (Fig. 1J, IK, 1L) and is also larger than that of the full-length MamC derived magnetites. When the concentration of MBP-Long in the precipitating solution was increased to 30 μg/mL, the crystals size was comparable to these in the control experiment (Fig. 4G). Such size control implied a high degree of control over nucleation and crystal growth, probably promoted by the concentration of Fe cations in the negatively charged areas of the protein and may be by a templated growth mechanism. Since these effects were not produced by MBP (Fig. 1G, 1J), there is a specific effect observed due solely to the 21 amino acids linked to such a protein.

[226] On the contrary to MBP-Long, the presence of MBP-Short in the precipitating solution does not have a noticeable effect on the magnetite size. MBP-Short produces small nanoparticles with a size of 10-30 nm which most of them were in 10-20 nm range (Figs. 1C, IF). The effect of this protein is very similar to that of MBP, so it could be derived that either the effect of the 17 amino acids is minor or null, probably because such a fragment is not active or has a structure in which the charge amino acids are not exposed.

[227] In some of the 30 μg/ml protein samples an additional peak with low amplitude also appeared that most likely corresponded to siderite. However, the lack of other XRD peaks corresponding to other siderite reflections made it hard to unambiguously identify such a mineral phase. Therefore, the mineral identity of the samples observed by TEM was carefully analyzed by SAED before any further study, to ensure that the conclusions of this study were based on magnetite crystals. The precipitation of siderite is the result of a kinetic process in the precipitating solution in which both magnetite and siderite precipitation is thermodynamically favoured, the last due to the presence of the buffer carbonate in the precipitating solution. As reported in Valverde- Tercedor et al (2010), the absence of such a buffer yielded smaller (5-15 nm in size) magnetite crystals and showed no differences regardless the presence or absence of the different proteins in the precipitating solution. These carbonate free experiments are no further considered.

Example 2 MamC loop can interact with pre-formed magnetite nanoparticles in solution using the Isothermal Titration Calorimetry (ITC) system.

[228] Similar to the precipitation experiments MBP-Mms6 served as positive control and MBP served as negative control. Since magnetite nanoparticles agglomerate via magnetic interactions ITC, data can only indicate qualitatively for interactions. Analysis of the MamC-Long ITC curve indicates for exothermic binding reaction similar to that of MBP-Mms6 while MBP alone does not show any interactions (Fig. 2A and 2B). MamC-Short does not show any interaction with the nanoparticles which can support the previous results of its small effect on magnetite precipitation (Fig. 2A and 6B).

Example 3

MamC crystallization

[229] To understand better the structure-function relationship of MamC loop, MBP-Short and MBP-Long were subjected to crystallization trials. Only MBP-Long experiments yielded 2.8A protein diffracting crystals. The structure of the MBP-Long shows the native structure of MBP with an additional 20.3A alpha helix at the MBP C-terminal (Fig. 3A). In this structure, only 17 amino acids out of the 21 that were added onto the MBP C-terminal have a clear electron density (Fig. 3B). The lack of electron density map for the last four amino acids indicates on their flexibility. The MBP-Long structure can be described as a short helix-turn-helix which gives an overall curved structure. The surface electrostatic map of MamC loop shows two distinct electrostatic charged areas (Fig. 3C). The N-terminal side contains positive electrostatic charge and the C-terminal contains negative electrostatic charge. The electrostatic negatively charged helix face is predicted to interact with the magnetite particle which may have a positively charged surface due to the exposed iron ions while the positive charge may interact with negative lipid heads or other protein components.

[230] According to multiple sequence alignment, homologous MamC proteins (Ms. Magnetotacticum MS-1, Ms. Gryphiswaldense MSR -1, Ms. magnetotacticum MC-1 and Magnetite-containing magnetic vibrio) shows several medium-conserved residues located at the MamC loop, in contrast to the highly-conserved residues located at the integral membrane helices (Fig. 7). Structural loop models for these homologues based on our structure show similar charge distribution on their surfaces which can support the magnetite crystal-MamC loop interaction model (Fig. 8). Example 4

MamC mutant characterization

[231] Alanine point and double mutations at Glu66 and Asp70 were generated based on the previous results and according to a protein-magnetite interaction model. Three variants were generated: "E66A" having the amino acid sequence as set forth in SEQ ID NO: 8 (LKEKRITNTAAAIDTGKETVG ), "D70A" having the amino acid sequence as set forth in SEQ ID NO: 9 (LKEKRITNTEAAIATGKETVG) and "E66A/D70A" having the amino acid sequence as set forth in SEQ ID NO: 10 (LKEKRITNTAAAIATGKETVG). In-vitro iron precipitation with the three mutants; E66A, D70A and E66A/D70A, shows smaller magnetite crystals compared to MamC-Long. In ITC measurements, E66A and E66A/D70A did not show any interaction with the magnetite nanoparticles. In contrast, D70A presented a different ITC curve when compared to the MamC-Long curve (Figures 10A-F).

[232] Another reason for the ITC differences may be due to the helical structure that D70A creates despite the presence of the single mutation (Fig. 10B). Overlapping between E70A and the MamC-Long shows high similarity with a low RMS (0.44 A). Some minor changes are visible on the side chains at the N- and C-terminal sides of the MamC-MIL, which are not the same rotamers as in the MamC-Long. The main observable change was the surface charge alterations on the C- terminal side. The point mutation to alanine abolished the negative charge and did not affect the helical structure (Fig. IOC). Yet, an interaction of the positively charged surface with the negative ion layer formed on the magnetite surface cannot be fully excluded.

[233] Next, a number of peptides from a few sources and having variable lengths were tested in order to gain further insight into the 3D structure of the MamC loop. The following peptides were generated: "MamC, AMB-1" (21 a.a.) having the amino acid sequence as set forth in SEQ ID NO: 2 (LKEKRITNTEAAIDTGKETVG). "MamC, AMB-1 " (17 a.a. N-terminus) having the amino acid sequence as set forth in SEQ ID NO: 12 (RITNTE A AIDTGKET VG) . "MamC, AMB-1 " (17 a.a. C-terminus) having the amino acid sequence as set forth in SEQ ID NO: 13 (LKEKRITNTE A AIDTGK) . "MamC, MSR-1" (21 a.a.) having the amino acid sequence as set forth in SEQ ID NO: 3 (LKDKQITGTEAAIDTGKEAAG). "Mms6 (AMB-1)" (22 a.a) having the amino acid sequence as set forth in SEQ ID NO: 14 (MKSRDIESAQSDEEVELRDALA) and "Mms6 (AMB-1)" (14 a.a) having the amino acid sequence as set forth in SEQ ID NO: 15 ( AQSDEEVELRD ALA) .

[234] Circular dichroism (CD) was performed to determine whether these peptides have a helical structure in solution (Figure 11). Only two peptides show a signal of helical structure; MamC (AMB-1)- 21 a.a. and Mms6 (AMB-1)- 14 a.a. Those results support the previous results of the MamC-loop helical structure.

Example 5

Iron co-precipitation with different peptides derived from Mms6, MamC, and Mms7

[235] In vitro iron co-precipitation was performed using five different peptides: MamC from AMB_1, 21 amino acids; Mms6 from AMB-1, 22 amino acids; Mms7 from AMB-1, 17 amino acids and two mutants of Mms6 peptide (D12A and E13A). All the peptides were dissolved in Mq. Solution of Fe3 and Fe2, at a ratio of 2: 1, and continuously sparged with N2, stirred and slowly titrated with 0.1 M NaOH at room temperature. At pH 5, peptide was added into the iron solution to reach a final concentration of 1, 10 or 1.6 μg/mL, in order to detect the effect on crystal size and shape. All samples were analyzed by Transmission Electron Microscopy (TEM).

[236] Aspartic and glutamic acids are shown to be common amino acids in many biomineral related proteins which are the main players in biominerahzation process such as control nucleation and crystal growth. The negative charges of those amino acids can interact with positive ions (Ca, Mg, Fe, Na etc.), reducing the energetic barriers to crystal nucleation, and controlling the crystal direction growth into a specific structure.

[237] The present example illustrates site-specific mutation of the aspartic and glutamic amino acids into alanine in various MamC and Mms6 peptides (see, Figs. 12-13). It is contemplated that these amino acids are important for crystal growth and play a major role on the mineral structure. Both amino acids are negatively charge, and can interact with Fe ^+2/+3 ions and stabilize them which can lead to crystal nucleation. Furthermore, the proteins/peptides can direct the crystal growth in one direction by accelerating, switching and inhibiting their growth. In order to prevent mineral growth in one way, the protein/peptide needs to interact with the mineral surface and prevent sedimentation of more ions to the crystal layer. Without any interaction of protein/peptide, the mineral phases will grow spontaneously and will result in the most lower energy crystal form, sometime with non-defined mineral shape and size such we can see in the sample without any peptide.

Example 6

Binding characterization of MBP-Mms6 onto magnetite nanoparticles surfaces

[238] A Mms6-MBP chimera was tested for its magnetite coating. An MBP-Mms6 construct (thee Mms6 having the C-terminus active region of 22 amino acids) MKSRDIESAQSDEEVELRDALA; SEQ ID NO: 14). Synthetized magnetites under the presence of 0.8 mg/mL MBP, MBP-Mms6 or without any protein (control) were incubated in 96 well 250 uL plate with anti-MBP-HRP conjugated antibody 1:2000 dilution (abeam Ab49923 8.3 mg/ml) (Fig. 14 left) and after 5 washes with inert buffer (100 mM NaCl and 5 mM Tris pH 8.0) the antibody binding was detected via HRP chemiluminescent kit. Color was developed in MBP- Mms6 and magnetite free particles indicating that Mms6 is able to bind magnetites and that antibodies are bound to clean unprotected magnetite particles via unspecific binding.

[239] To further check the binding of MBP-Mms6 onto magnetites, the presence of maltose on synthesized magnetites was evaluated with MBP-Mms6 or without any protein. The synthesized magnetite particles were incubated with 5 mM maltose followed by 5 washing steps with buffer (as described above). All fractions and the magnetites were subjected to sugar detecting protocol (see, Chaplin, M. F., and Kennedy, J. F. (1994) Carbohydrate Analysis. A Practical Approach Oxford University Press) on a thin layer chromatography plate (Fig. 14 left). Only MBP-Mms6 showed a clear evidence for maltose presence indicating the binding of MBP to the magnetite via Mms6 while clean magnetites show no binding affinities to maltose. Example 7

Binding capabilities of magnetic nanoparticles to cell surfaces

[240] Binding capabilities of magnetic nanoparticles to cell surfaces is tested using fluorescence- activated cell sorting (FACS) and enzyme-linked immunosorbent assays (ELISA). For ELISA, Hek293 (non-cancer), HeLa (adenocarcinoma) and WiDr (adenocarcinoma colorectal) cell lines are fixed on a plate, followed by incubation with the relevant magnetic nanoparticles (coated chimeras or coated MamC as a reference) and washing steps. For detection of cell surface localization specific antibodies are used. For FACS, cells in suspension are incubated with magnetic nanoparticles, followed by washing steps and incubation with antibodies for FACS detection. The following procedure is then followed to validate the ability to differentiate between cells: Cells of several lines are mixed and magnetic nanoparticles are added; magnetic separation is then applied to differentiate between cells, and FACS for cell type sorting (using commercial antibodies) is used to detect cancer cells.

Example 8 Magnetic hyperthermia elimination of cancer cells by MamC-loop-targeting moiety chimeric protein

[241] As described before, MamC-loop is 21 amino acids long and can interact directly with the magnetite particle surface and adopts a helical structure. When MamC-loop is attached to MBP (maltose -binding protein) C-terminus, it can interact with magnetite nanoparticles and control magnetite size during in-vitro iron precipitation. Based on these results a recombinant chimeric protein that can interact with magnetite nanoparticles is generated. The chimeric protein can be used to target tissue or cells. This feature allows directing of the magnetite nanoparticles to any specific target, such as cancer cells.

[242] Next, In vitro iron co-precipitation with different targeting-protein MamC-loop constructs is performed with or without the chimeric constructs (target-protein-MamC loop) in order to control the magnetite particle size during nucleation and crystal growth. It was shown that the MamC loop can affect magnetite size during in-vitro iron precipitation and results in an average crystal size of 38 nm.

[243] Magnetic hyperthermia is a cancer treatment that uses the ability of magnetic nanoparticles to raise their heat due to external high-frequency of an electric magnetic energy field. The MamC- loop-MBP chimeric protein acts as a magnetite anchor. For hypothermia experiments, a DM- 100 magnetic hyperthermia device (Nanoscale Biomagnetics) will be used according to the manufacturer's instructions at varying magnetic field from 0 to 300/600 gauss, and variable frequencies. [244] MamC-loop-MBP or MBP or MamC-loop alone are introduced to various cancer cell lines. The cells are then subjected to magnetic hyperthermia. The rate of elimination of different populations of treated cells is quantified using a live-death assay (LIVE/BEAD kit for mammalian cells, Thermo Fisher scientific Inc.).

[245] 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 spirit and broad scope of the appended claims.