Methods and compositions for heptameric targeting ligands

Patent metadata

Field	Value
Publication number	US-9340616-B2
Application number	US-201314088243-A
Country	US
Kind code	B2
Filing date	Nov 22, 2013
Priority date	May 23, 2011
Publication date	May 17, 2016
Grant date	May 17, 2016

Abstract

The present invention provides a self assembly molecule having an affinity for one or more target molecules, for use in formation of a heptameric complex, comprising: a) a monomer comprising a multimerization domain of Archaeal Sm1 (AF-Sm1) protein or SM-like ribonucleoprotein from other organisms, able to interact with other molecules of the same monomer comprising a multimerization domain of AF-Sm1 protein or SM-like ribonucleoprotein to self-assemble into a heptamer; and b) a target binding domain or peptide attached directly or via a linker to the monomer of (a). Also provided are heptamers comprising these self assembly molecules and methods for their use in therapy, imaging and diagnostics.

First claim

What is claimed is:

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 detect

Assignees

• Univ North Carolina

Inventors

• Liu Rihe

Classifications

• G01N33/5759 Primary

  involving compounds localised on the membrane of tumour or cancer cells · CPC title

• A61K49/0056

  Peptides, proteins, polyamino acids · CPC title

• C07K2319/735

  containing a domain for self-assembly, e.g. a viral coat protein (includes phage display) · CPC title

• G01N33/57492

  Physics · mapped topic

• C07K14/47

  from mammals · CPC title