EpCAM targeted polypeptides, conjugates thereof, and methods of use thereof

1. A polypeptide comprising an amino acid sequence having at least about 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:61, and SEQ ID NO:62, wherein the polypeptide is capable of binding to epithelial cell adhesion molecule (EpCAM).

2. The polypeptide of claim 1 , comprising an amino acid sequence having at least about 97% sequence identity to a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:61, and SEQ ID NO:62.

3. The polypeptide of claim 1 , comprising an amino acid sequence having at least about 99% sequence identity to a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:61, and SEQ ID NO:62.

4. The polypeptide of claim 1 , comprising a non-paratopic cysteine variation.

5. The polypeptide of claim 1 , comprising an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:61, and SEQ ID NO:62.

6. A polypeptide comprising an amino acid sequence, wherein 1) the amino acid sequence has at least about 85% sequence identity to a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:61, and SEQ ID NO:62; and 2) the amino acid sequence comprises a combination of three loop regions selected from the group consisting of:

1) DYPNSASY (SEQ ID NO:16), GNTYN (SEQ ID NO:17), and TYRDNYSYSN (SEQ ID NO:18);

2) DYPNSASY (SEQ ID NO:16), GNTYN (SEQ ID NO:17), and TSRDNYSWSN (SEQ ID NO:32);

3) DYPNSASY (SEQ ID NO:16), GNTYN (SEQ ID NO:17), and TSRDNYSYLN (SEQ ID NO:33);

4) DDYTSASY (SEQ ID NO:34), GNTYN (SEQ ID NO:17), and TYRDNYSYSN (SEQ ID NO:18);

5) DYPNSASY (SEQ ID NO:16), GNTYN (SEQ ID NO:17), and TSRDNYSYSN (SEQ ID NO:35);

6) DNSNYASY (SEQ ID NO:36), GSTYN (SEQ ID NO:37), and TYRDNYSYSN (SEQ ID NO:18);

7) DDPDFASY (SEQ ID NO:39), GNTYS (SEQ ID NO:40), and TYRDNYSYSN (SEQ ID NO:18);

8) DYPNSASY (SEQ ID NO:16), GDTYN (SEQ ID NO:38), and TYRDNYSYSN (SEQ ID NO:18);

9) DYPNSASY (SEQ ID NO:16), GSKST (SEQ ID NO:3), and TGRGDSPASSK (SEQ ID NO:4);

10)DAPAVTVRY (SEQ ID NO:2), GNTYN (SEQ ID NO:17), and TGRGDSPASSK (SEQ ID NO:4); and

11) DAPAVTVRY (SEQ ID NO:2), GSKST (SEQ ID NO:3), and TYRDNYSYSN (SEQ ID NO:18); and

wherein the polypeptide is capable of binding to epithelial cell adhesion molecule (EpCAM).

7. The polypeptide of claim 5 , comprising an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:61, and SEQ ID NO:62.

8. A nucleic acid encoding a polypeptide of claim 1 .

9. A vector comprising an expression cassette, wherein the expression cassette comprises a nucleic acid sequence as described in claim 8 and a promoter operably linked to the nucleic acid.

10. A conjugate of formula (I):

P-(L-A) n   (I)

wherein:

P is a polypeptide as described in claim 1 that binds to epithelial cell adhesion molecule (EpCAM);

each L is independently a direct bond or a linking group;

each A is independently a detectable agent or a biologically active agent; and

n is 1 to 5.

11. The conjugate of claim 10 , wherein at least one A is a detectable agent.

12. The conjugate of claim 11 , wherein the detectable agent comprises a chelating group selected from the group consisting of p-SCN-Bn-TCMC, p-NO2-Bn-Cyclen, p-NO2-Bn-DOTA, p-NH2-Bn-DOTA, p-NH2-Bn-DOTA-tetra(t-Bu-ester), p-SCN-Bn-DOTA, DOTA-tris (t-Bu ester), DOTA-mono-NHS-tris(t-Bu ester), Maleimido-mono-amide-DOTA-tris (t-Bu ester), Maleimido-mono-amide-DOTA, Fmoc-L-Lys-mono-amide-DOTA-tris(t-Bu ester), 2-Aminoethyl-mono-amide-DOTA-tris(t-Bu ester), Azido-mono-amide-DOTA-tris(t-butyl ester), DOTA-NHS-ester, Azido-mono-amide-DOTA, p-NH2-Bn-DTPA, p-NH2-Bn-DTPA-penta (t-Bu ester), p-SCN-Bn-DTPA, p-NH2-CHX-A″-DTPA, CHX-A″-DTPA, DTPA-tetra (t-Bu ester), Maleimido-mono-amide-DTPA, p-NH2-Bn-PCTA, p-SCN-Bn-PCTA, p-NH2-Bn-oxo-DO3A, p-SCN-Bn-oxo-DO3A, p-NH2-Bn-NOTA, p-SCN-Bn-NOTA, NOTA-bis(t-Bu ester), Maleimido-mono-amide-NOTA, Deferoxamine-p-SCN, Deferoxamine-maleimide, DOTA-Biotin-Sarcosine, DO3A-Serotonin, Cyclen, Cyclam, DO2A, DO3A, DOTA, DOTA-NHS, DOTP, DOTMA, TETA, DOTAM, DiAmSar, CB-Cyclam, CB-TE2A, NOTA, TACN, Tm-DOTA, Gd-DOTA, Tm[DOTP]5-, Tm-DOTMA, Eu-DOTA-4AmC, Tm-p-SCN-Bn-DOTA, Gd-p-SCN-Bn-DOTA, Ho-p-SCN-Bn-DOTA, Tm-Maleimido-DOTA, Ho-Maleimido-DOTA, BisCBZ-Cyclen, TrisBOC-Cyclen, NO2A-(t-Bu ester), DO2A-t-Bu-ester, DO3A-t-Bu-ester and NODAGA.

13. The conjugate of claim 12 , wherein the chelating group is labeled with one or more radionuclides selected from the group consisting of Fluorine-18, Antimony-124, Antimony-125, Arsenic-74, Barium-103, Barium-140, Beryllium-7, Bismuth-206, Bismuth-207, Cadmium-109, Cadmium-115m, Calcium-45, Cerium-139, Cerium-141, Cerium-144, Cesium-137, Chromium-51, Cobalt-55, Cobalt-56, Cobalt-57, Cobalt-58, Cobalt-60, Cobalt-64, Copper-64, Copper-67, Erbium-169, Europium-152, Gallium-64, Gallium-68, Gadolinium-153, Gadolinium-157 Gold-195, Gold-199, Hafnium-175, Hafnium-175-181, Holmium-166, Indium-110, Indium-111, Iridium-192, Iron-55, Iron-59, Krypton-85, Lead-210, Manganese-54, Mercury-197, Mercury-203, Molybdenum-99, Neodymium-147, Neptunium-237, Nickel-63, Niobium-95, Osmium-185+191, Palladium-103, Platinum-195m, Praseodymium-143, Promethium-147, Protactinium-233, Radium-226, Rhenium-186, Rhenium-188, Rubidium-86, Ruthenium-103, Ruthenium-106, Scandium-44, Scandium-46, Selenium-75, Silver-110m, Silver-111, Sodium-22, Strontium-85, Strontium-89, Strontium-90, Sulfur-35, Tantalum-182, Technetium-99m, Tellurium-125, Tellurium-132, Thallium-204, Thorium-228, Thorium-232, Thallium-170, Tin-113, Tin-114, Tin-117m, Titanium-44, Tungsten-185, Vanadium-48, Vanadium-49, Ytterbium-169, Yttrium-86, Yttrium-88, Yttrium-90, Yttrium-91, Zinc-65, and Zirconium-95.

14. The conjugate of claim 11 , wherein the detectable agent comprises a fluorescent group.

15. The conjugate of claim 10 , wherein at least one A is a biologically active agent selected from the group consisting of biotin, a peptide, an antibody, chemotherapeutic agent, and an immunotherapeutic agent.

16. The conjugate of claim 10 , wherein L is a divalent, branched or unbranched, saturated or unsaturated, hydrocarbon chain, having from 1 to 25 carbon atoms, wherein one or more of the carbon atoms is optionally replaced by (—O—) or a divalent ring of formula:

and wherein the chain or ring is optionally substituted on carbon with one or more substituents selected from (C 1 -C 6 )alkoxy, (C 3 -C 6 )cycloalkyl, (C 1 -C 6 )alkanoyl, (C 1 -C 6 )alkanoyloxy, (C 1 -C 6 )alkoxycarbonyl, (C 1 -C 6 )alkylthio, azido, cyano, nitro, halo, hydroxy, oxo (═O), carboxy, aryl, aryloxy, heteroaryl, and heteroaryloxy.

17. The conjugate of claim 10 , wherein,

P is a polypeptide as described in claim 1 that binds to epithelial cell adhesion molecule (EpCAM);

L is a peptide linking group;

A is a polypeptide comprising a first dihydrofolate reductase (DHFR) peptide operably linked to a second DHFR peptide; and optionally, a tag peptide operably linked to either the first or second DHFR peptide; and

n is 1.

18. The conjugate of claim 17 , selected from the group consisting of SEQ ID NO: 53, SEQ ID NO: 55 and SEQ ID NO: 59.

19. A pharmaceutical composition comprising a conjugate of claim 10 and a pharmaceutically acceptable excipient.

20. A method for targeting a detectable agent or a biologically active agent to an EpCAM positive cell in an animal comprising administering a conjugate of claim 10 to the animal.

21. A method of detecting cancer in an animal, comprising administering a conjugate of claim 10 to the animal, wherein the conjugate binds to an EpCAM molecule; and detecting a signal from the detectable agent, wherein a signal greater than a signal from a control animal without cancer indicates the animal has cancer.

22. A chemically self-assembled nanoring (CSAN) comprising a plurality of conjugates as described in claim 17 and a plurality of bisMTX compounds.

23. The CSAN of claim 22 , operably linked to a biologically active agent or an immune cell.

24. A method for targeting a biologically active agent or an immune cell to an EpCAM positive cell in an animal comprising administering a CSAN as described in claim 23 to the animal.

25. The polypeptide of claim 5 , comprising SEQ ID NO:12.

26. The polypeptide of claim 5 , comprising SEQ ID NO:10.

27. A method for activating a T cell comprising contacting an EpCAM positive cell with a CSAN as described in claim 23 , wherein the CSAN is operably linked to an immune cell via CD3, and wherein the immune cell is a T cell.