Methods and compositions for heptameric targeting ligands

1. A self-assembling molecule in heptameric form, comprising:

a) a monomer comprising a multimerization domain of Archaeal Sm1 (AF-Sm1) protein or SM-like ribonucleoprotein, comprising an amino acid sequence selected from the group consisting of:

A) at least about 50 residues of the amino acid sequence of SEQ ID NO:1 (MPPRPLDVLN RSLKSPVIVR LKGGREFRGT LDGYDIHMNL VLLDAEEIQN GEVVRKVGSV VIRGDTVVFV SPAPGGE);

B) at least about 50 residues of the amino acid sequence of SEQ ID NO:2 (MARPLDVLNK ALKTPVLVRL KGGREFRGTL DGYDIHMNLV LVDAEEIQNG EVVRKLGSVV IRGDTVVFVS PSQ);

C) at least about 50 residues of the amino acid sequence of SEQ ID NO:3 (MAKRPLDVLN KALQTPVLVR LKGGREFRGI LNGYDIHMNI VLENAEEIQN GEVVRKLGSV VIRGDTVVFV SPSE);

D) at least about 50 residues of the amino acid sequence of SEQ ID NO:4 (MANRPLDVLN KALQTPVLVR LKGGREFRGI LNGYDIHMNL VLQNAEEIQG GEVIRKLGSV VIRGDTVVFV SPSP);

E) at least about 50 residues of the amino acid sequence of SEQ ID NO:5 (MGNRPLDILN NALNTAVIVR LKGAREFRGT LQGYDVHMNL VLDEAEEIKE GEIIRKIGSV VVRGDNVVYV SP);

F) at least about 50 residues of the amino acid sequence of SEQ ID NO:6 (MANRPLDILN NALNTPVIVR LKGAREFRGE LQGYDVHMNL VLDNAEELKD GEIVRKLGSV VIRGDNVVYL SP);

G) at least about 50 residues of the amino acid sequence of SEQ ID NO:7 (RPLDAL GNSLNSPVII KLKGDREFRG VLKSFDLHMN LVLNDAEELE DGEVTRRLGT VLIRGDNIVY ISP); and

H) at least about 50 residues of the amino acid sequence of SEQ ID NO:8 (RPLDALGN SLNSPVIIKL KGDREFRGVL KSFDLHMNLV LNDAEELEDG EVTRRLGTVL IRGDNIVYIS), and

b) a target binding domain or peptide attached to the monomer of (a), wherein the target binding domain or peptide is selected from the group consisting of:

A) an epidermal growth factor receptor (EGFR)-binding Z domain comprising the amino acid sequence: VDNKFNKEMWAAWEEIRNLPNLNGWQMTAFIASLVDDPSQSANLLAEAKKLNDAQAPK (SEQ ID NO:20);

B) a human epidermal growth factor receptor 2 (HER2) binding Z domain comprising the amino acid sequence: VDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSLYDDPSQSANLLAEAKKLNDAQAPK (SEQ ID NO:21);

C) a prostate specific membrane antigen (PSMA)-binding peptide comprising the amino acid sequence: QKHHNYL (amino acid residues 79-85 of SEQ ID NO: 22);

D) a PSMA-binding fibronectin type III (FN3) domain comprising the amino acid sequence: MGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTA TISGLKPGVDYTITVYAVTQKHHNYLPISINYRTEIDKPSQ (SEQ ID NO:22);

E) a gastrin-releasing (GRP78) binding peptide comprising the amino acid sequence: WIFPWIQLGGS (SEQ ID NO:23);

F) an EGFR-binding peptide comprising the amino acid sequence: YHWYGYTPQNVI (SEQ ID NO:24);

G) a plectin-1-binding peptide comprising the amino acid sequence: KTLLPTPGGS (SEQ ID NO:25);

H) a human epidermal growth factor receptor 3 (HER3) binding Z domain comprising the amino acid sequence: VDNKFNKERYSAYYEIWQLPNLNVRQKAAFIGSLQDDPSQSANLLAEAKKLNDAQAPK (SEQ ID NO:26);

I) an α v β 3 -binding FN3 domain comprising the amino acid sequence: MGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTA TISGLKPGVDYTITVYAVTPRGDWNEGSKPISINYRT (SEQ ID NO:27);

J) a TNFα-binding FN3 domain comprising the amino acid sequence: MGVSDVPRDLEVVAATPTSLLISWRPTSNPPRYYRITYGETGGNSPVQEFTVPPWASTAT ISGLKPGVDYTITVYAVTAQTGHHLHDKPISINYRT (SEQ ID NO:28);

K) a vascular endothelial growth factor receptor (VEGFR)-binding FN3 domain comprising the amino acid sequence: MGVSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTAT ISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRT (SEQ ID NO:29);

L) a gastrin-releasing peptide comprising the amino acid sequence: GNHWAVGHLM (SEQ ID NO:30);

M) an EGFR-binding Z domain (Z EGFR ) comprising the amino acid sequence: MVDNKFNKEM WAAWEEIRNL PNLNGWQMTA FIASLVDDPS QSANLLAEAK KLNDAQAPK (SEQ ID NO:31)

N) a HER2-binding Z domain (Z EGFR ) comprising the amino acid sequence: MVDNKFNKEM RNAYWEIALL PNLNNQQKRA FIRSLYGDPS QSANLLAEAK KLNDAQAPK (SEQ ID NO:32); and

O) a PSMA-binding peptide (SP PSMA ) comprising the amino acid sequence: MWQPDTAHHWATL (SEQ ID N0:33).

2. The molecule of claim 1 , wherein the target binding domain or peptide of (b) is attached via a linker peptide to the monomer of (a).

3. The molecule of claim 2 , wherein the linker peptide is selected from the group consisting of:

a) a linker peptide comprising the amino acid sequence of SEQ ID NO:9 (GPQPQPKPQPK);

b) a linker peptide comprising the amino acid sequence of SEQ ID NO:10 (GGGGS) n , wherein n is any number;

c) a linker peptide comprising the amino acid sequence of SEQ ID NO:11 (TPPTPSPSTPPTPSP);

d) a linker peptide comprising the amino acid sequence of SEQ ID NO:12 (EFPKPSTPPGSSGGAP);

e) a linker peptide comprising the amino acid sequence of SEQ ID NO:13 (PQPQPQPKPQPKPEPE);

f) a linker peptide comprising the amino acid sequence of SEQ ID NO:14 (GGGS) n , wherein n is any number;

g) a linker peptide comprising the amino acid sequence of SEQ ID NO: 15 (GSGSGS) n , wherein n is any number;

h) a linker peptide comprising of the amino acid sequence of SEQ ID NO:011116 (TPPTPSP) n , wherein n is any number;

i) a linker peptide comprising the amino acid sequence of SEQ ID NO:17 ((PQPQPK) n , wherein n is any number;

j) a linker peptide comprising the amino acid sequence of SEQ ID NO:18 (PQPQPE) n , wherein n is any number;

k) a linker peptide comprising the amino acid sequence of SEQ ID NO:19 (PEPEPQPQGG);

l) a linker peptide comprising the amino acid sequence of SEQ ID NO:34 (GPQPQPKPQPKPEPEPQPQGG); and

m) any combination of (a)-(l) above.

4. The molecule of claim 1 , wherein the target binding domain or peptide of (b) is attached to the monomer of (a) at the amino terminus and/or at the carboxy terminus.

5. The molecule of claim 1 , further comprising a histidine tag.

6. The molecule of claim 1 , further comprising a diagnostic molecule, a therapeutic molecule, an imaging molecule or any combination thereof.

7. The molecule of claim 1 , further comprising an amino-terminal and/or a carboxy-terminal cysteine for site-specific conjugation with a diagnostic molecule, a therapeutic molecule, an imaging molecule, a nanoparticle or any combination thereof.

8. The self-assembling molecule of claim 1 , in heptameric form in the absence of any cysteine residues that maintain the oligomeric state.

9. A heptamer comprising seven self assembly molecules of claim 1 .

10. A heptamer comprising seven self assembly molecules of claim 1 , lacking any cysteine residues that maintain the oligomeric state.

11. The heptamer of claim 9 , having a binding strength for a target molecule that is increased from about 100 fold to about 10,000 fold, as compared with a monomer control.

12. A method of producing a heptamer having a binding strength for a target molecule that is increased from about 100 fold to about 10,000 fold as compared with a monomer control, comprising:

a) combining a plurality of the self assembly molecules of claim 1 under conditions whereby the molecules self assemble into heptamers; and

b) optionally isolating the heptamers, thereby producing the heptamer.

13. A heptamer produced by the method of claim 12 .

14. A method of detecting and/or localizing cancer cells in a subject, comprising administering to the subject an effective amount of the heptamer of claim 9 , wherein the targeting domain or peptide is specific for a target molecule on the surface of cancer cells in the subject and the heptamer further comprises an imaging molecule and/or detectable molecule, whereby the heptamer binds the target molecule on the surface of cancer cells in the subject and the imaging molecule is visualized and/or the detectable molecule is detected at its binding location in the subject, thereby detecting and/or localizing cancer cells in the subject.

15. A method of diagnosing cancer in a subject, comprising administering to the subject an effective amount of the heptamer of claim 9 , wherein the targeting domain or peptide is specific for a target molecule on the surface of cancer cells in the subject and the heptamer further comprises an imaging molecule and/or detectable molecule, whereby the heptamer binds the target molecule on the surface of cancer cells in the subject and the imaging molecule is visualized and/or the detectable molecule is detected on cancer cells in the subject, thereby diagnosing cancer in the subject.

16. The method of claim 14 , wherein the imaging molecule comprises an MRI contrast agent, a radioisotope, 64 Cu-ATSM, 18 F-FDG, fluoride, FLT, FMISO, gallium, technetium-99m, a near-infrared (IR) fluorescence molecule, a nanoparticle-containing imaging agent or any combination thereof.

17. The method of claim 14 , wherein the target molecule comprises an extracellular domain of a cell surface receptor selected from the group consisting of an epidermal growth factor receptor family member, EGFR, HER2, HER3, c-MET, VEGFR, insulin receptor, insulin-like growth factor receptor, prostate specific membrane antigen, mesothelin, hepsin, an integrin, mucin, MUC16, a cell surface cluster of differentiation (CD) molecule, CD20, CD22, CD30, CD33, CD44, CD56, and any combination thereof.

18. A kit comprising the heptamer of claim 9 .

19. A composition comprising the heptamer of claim 9 in a pharmaceutically acceptable carrier.

20. The self-assembling molecule of claim 1 , wherein the monomer of (a) comprises the amino acid sequence MPPRPLDVLN RSLKSPVIVR LKGGREFRGT LDGYDIHMNL VLLDAEEIQN GEVVRKVGSV VIRGDTVVFV SPAPGGE (SEQ ID NO:1) and the target binding domain or peptide of (b) comprises the amino acid sequence MVDNKFNKEM WAAWEEIRNL PNLNGWQMTA FIASLVDDPS QSANLLAEAK KLNDAQAPK (SEQ ID NO:31) and the monomer of (a) is attached to the target binding domain of (b) via a linker comprising the amino acid sequence GPQPQPKPQPK PEPEPQPQGG (SEQ ID NO:34).

21. The self-assembling molecule of claim 1 , wherein the monomer of (a) comprises the amino acid sequence MPPRPLDVLN RSLKSPVIVR LKGGREFRGT LDGYDIHMNL VLLDAEEIQN GEVVRKVGSV VIRGDTVVFV SPAPGGE (SEQ ID NO:1) and the target binding domain or peptide of (b) comprises the amino acid sequence MVDNKFNKEM RNAYWEIALL PNLNNQQKRA FIRSLYGDPS QSANLLAEAK KLNDAQAPK (SEQ ID NO:32) and the monomer of (a) is attached to the target binding domain of (b) via a linker comprising the amino acid sequence GPQPQPKPQPK PEPEPQPQGG (SEQ ID NO:34).

22. The self-assembling molecule of claim 1 , wherein the monomer of (a) comprises the amino acid sequence MPPRPLDVLN RSLKSPVIVR LKGGREFRGT LDGYDIHMNL VLLDAEEIQN GEVVRKVGSV VIRGDTVVFV SPAPGGE (SEQ ID NO:1) and the target binding domain or peptide of (b) comprises the amino acid sequence MWQPDTAHHWATL (SEQ ID NO:33) and the monomer of (a) is attached to the target binding domain of (b) via a linker comprising the amino acid sequence GPQPQPKPQPK PEPEPQPQGG (SEQ ID NO:34).