Dll3 targeting peptides and constructs thereof

Abstract

The present disclosure relates to targeting moieties such as peptides and antibodies that can bind to DLL3. The disclosure also provides targeting constructs, which may include a targeting moiety attached, via an optional linker, to a chelating agent for association of a cargo. Methods of making the constructs and formulations thereof are also provided. Methods of using the constructs and / or formulations thereof to treat subjects, for example, to treat or prevent cancer, are also described.

Description

Abstract This disclosure relates to targeting groups, such as peptides and antibodies, that can bind to DLL3. This disclosure also provides targeting constructs that may include a targeting group linked to a chelating agent via an optional linker for binding a load. This disclosure also provides methods for preparing the constructs and formulations thereof. Furthermore, this disclosure describes methods for treating subjects using the constructs and / or formulations thereof, for example, for treating or preventing cancer.

Claims

CLAIMS1. A cyclic peptide of Formula B:or a pharmaceutically acceptable salt thereof, wherein:P1is selected from: -L1-Chelator,D1is -NR”-Chelator;L1is absent or selected fromwherein the amino group of L1connects to the carbonyl group of P1or Chelator to form an amide bond;P2is selected from C(O)NH2, C(O)OH,D2is OH or NH2;L2is absent or selected from:wherein the amino group of L2connects to the carbonyl group of P2to form an amide bond;P3is selected from H,L3is absent or independently selected fromwherein the carbonyl group of L3connects to an amine group of P2to form an amide bond;D3is independently selected from: CH3, C(O)OH, andX is halogen;R0is an amino acid side chain of a natural amino acid or an amino acid side chain of an unnatural amino acid;R1is an amino acid side chain of a natural amino acid or an amino acid side chain of an unnatural amino acid;R2is an amino acid side chain of a natural amino acid or an amino acid side chain of an unnatural amino acid;R3is an amino acid side chain of a natural amino acid or an amino acid side chain of an unnatural amino acid;B1is C1-6alkylene;C1is C1-6alkylene;A1is selected from:wherein w is selected from 1 , 2, or 3;R4is an amino acid side chain of a natural amino acid or an amino acid side chain of an unnatural amino acid, both of which are optionally substituted with CH2C(O)OH or C(O)(CH2CH2O)p(CH2)2N(CH3)3+;R5is an amino acid side chain of a natural amino acid or an amino acid side chain of an unnatural amino acid;R6is an amino acid side chain of a natural amino acid or an amino acid side chain of an unnatural amino acid;R7is selected from:(i) an amino acid side chain of a natural amino acid,(ii) an amino acid side chain of an unnatural amino acid, or(iii) selected from the group consisting of:R8is an amino acid side chain of a natural amino acid or an amino acid side chain of an unnatural amino acid;R9is an amino acid side chain of a natural amino acid or an amino acid side chain of an unnatural amino acid;R10is an amino acid side chain of a natural amino acid or an amino acid side chain of an unnatural amino acid; m is 0 or 1 ; each n, q, and u are independently an integer from 0 to 16; each p is independently an integer from 0 to 24; each s is independently an integer from 0 to 16; each t is independently 1 , 2, 3, 4, 5, or 6; each R’ is independently selected from H, C(O)OH, (CH2)OH, and NHAc; and each R” is independently selected from H and CH3; wherein when a variable group R0, R1, R2, R3, R4, R5, R6, R7, R8, R9, or R10is defined as the amino acid side chain of a cyclic amino acid, the corresponding amino acid nitrogen of the peptide backbone of the generic formula forms part of the cyclic group; andand wherein the cyclic peptide optionally comprises a radionuclide.

2. The cyclic peptide of claim 1 , wherein the cyclic peptide of Formula B is a cyclic peptide of Formula la:or a pharmaceutically acceptable salt thereof.

3. The cyclic peptide of claim 1 , wherein the cyclic peptide of Formula B is a cyclic peptide of Formula lb:or a pharmaceutically acceptable salt thereof.

4. The cyclic peptide of any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein:P1is selected fromwherein:D1is NR”-Chelator;L1is absent orn and s are independently an integer from 2 to 15; and p is 8, 9, 10, 11 , or 12.

5. The cyclic peptide of any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein P1is -L1-Chelator.

6. The cyclic peptide of any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein P1is selected from DOTA,7. The cyclic peptide of any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein P2is C(O)NH2 or C(O)OH.

8. The cyclic peptide of any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein P2is selected from C(O)NH2, C(O)OH, and9. The cyclic peptide of claim 8, or a pharmaceutically acceptable salt thereof, wherein P2is selected from C(O)NH2, C(O)OH,P3is Ac or; andX is halo.

10. The cyclic peptide of claim 1 , or a pharmaceutically acceptable salt thereof, wherein the cyclic peptide of Formula B is a cyclic peptide of Formula II:or a pharmaceutically acceptable salt thereof, wherein P2is C(O)NH2or C(O)OH.

11. The cyclic peptide of claim 1 , or a pharmaceutically acceptable salt thereof, wherein m is 0;P1is selected from DOTA,P2is selected from C(O)NH2, C(O)OH,P3is Ac orX is halo;R1is selected from the group consisting of an amino acid side chain of Trp, 2Nal, 1 Nal, 4CF3-Phe, 7Aza-Trp, 1 Me-Trp, 5OH-Trp, BIP, 5OMe-Trp, 4F-Phe, 3Pya, 4Pya, PAF, MAF, OAF, 5Qui, 7MeO-Trp, 7Me-Trp, 5F-Trp, 7CI-Trp, D-Ala, Ala, alpha-Me-Trp, and NMe-Trp;R2is selected from the group consisting of an amino acid side chain of Thr, D-Ala, Ala, alpha-Me-Thr, Lys, and NMe-Thr;R3is selected from the group consisting of an amino acid side chain of lie, Env, CHA, CBA, Nle, Tbg, THPG, Chg, 2Nal, 1Nal, 2CF3-Phe, 2PhEt-Ala, D-Ala, Ala, Leu, t-Bu-Ala, NMe- Nle, α-tert-amylGly, Allo-lle, Lys(C12), Lys(C14), Lys(C16), alpha-Me-lle, and NMe-tBuAla;A1is selected from the group consisting of:R4is selected from the group consisting of an amino acid side chain of Asn, D-Ala, Ala, DAB-4-NHCOC5H11, DAB-4-NHCOC7H15, Asp, Ser, Lys, 3-(4-piperidinyl)-Ala, 3-(1-morpholinyl)- Ala, 3Pya, 4Pya, Glu, NMe-Asn, PipA(acetic), and Pip(PegNMe3)Ala;R5is selected from the group consisting of an amino acid side chain of Asn, Ala, D-Ala, Trp, Asp, Lys, 3Pya, 4Pya, 3-(4-piperidinyl)-Ala, 3-(1-morpholinyl)-Ala, Glu, NMe-Asn, and Ser;R6is selected from the group consisting of an amino acid side chain of Trp, 4CF3-Phe, 1 Me-Trp, 7Aza-Trp, BIP, 2Nal, 1 Nal, alpha-Me-Trp, D-Ala, Ala, 4F-Phe, , 5F-Trp, 5Ome-Trp, Asn, 5OH-Trp, 7Me-Trp, 7MeO-Trp, 7CI-Trp, and NMe-Trp;R7is:(i) selected from the group consisting of an amino acid side chain of 3Pya, 4Pya, Lys(Me)3, His, Ala, D-Ala, Gin, Lys, Glu, Arg, Orn, NMe-His, and Ser; or(ii) selected from the group consisting of:each s is independently 3, 5,10, 12, or 14;R8is selected from the group consisting of an amino acid side chain of Asp, D-Ala, Ala, Asn, Thr, NMe-Asp, and alpha-Me-Asp;R9is selected from the group consisting of an amino acid side chain of Trp, 7Aza-Trp, 1 Me-Trp, D-Ala, Ala, 4F-Phe, 1 Nal, 2Nal, 5F-Trp, 5Ome-Trp, alpha-Me-Trp, 7CI-Trp, 5OH-Trp, 7Me-Trp, 7MeO-Trp, and NMe-Trp; andR10is selected from the group consisting of an amino acid side chain of Pro, D-Ala, Ala, alpha-Me-Pro, trans4Fluoro-Pro, cis4Fluoro-Pro, trans4OH-Pro, cis4OH-Pro, Pip, 5,5-diMe-Pro, NMe-Ser, trans4NH2-Pro, cis4NH2-Pro, Sar, Aze, NMe-Ala, NMe-Leu, R-3Me-Aze, alpha-Me- Aze, ACI, and 3Me2-Aze.

12. The cyclic peptide of claim 1 or 3, or a pharmaceutically acceptable salt thereof, wherein m is 1 ;P1is selected from DOTA,P2is selected from C(O)NH2, C(O)OH,P3is selected from Ac andX is halo;R0is selected from the group consisting of an amino acid side chain of Gly, Met, D-Ala, Ala, Nle, and Nva;R1is selected from the group consisting of an amino acid side chain of T rp, 2Nal, 1 Nal, 4CF3-Phe, 7Aza-Trp, 1 Me-Trp, 5OH-Trp, BIP, 5Ome-Trp, 4F-Phe, 3Pya, 4Pya, PAF, MAF, OAF, 5Qui, 7MeO-Trp, 7Me-Trp, 5F-Trp, 7CI-Trp, D-Ala, Ala, alpha-Me-Trp, and NMe-Trp;R2is selected from the group consisting of an amino acid side chain of Thr, D-Ala, Ala, alpha-Me-Thr, Lys, and NMe-Thr;R3is selected from the group consisting of an amino acid side chain of lie, Env, CHA, CBA, Nle, Tbg, THPG, Chg, 2Nal, 1 Nal, 2CF3-Phe, 2PhEt-Ala, D-Ala, Ala, Leu, t-Bu-Ala, NMe- Nle, α-tert-amylGly, Allo-lle, Lys(C12), Lys(C14), Lys(C16), alpha-Me-lle, and NMe-tBuAla;A1is selected from the group consisting ofR4is selected from the group consisting of an amino acid side chain of Asn, D-Ala, Ala, DAB-4-NHCOC5H11, DAB-4-NHCOC7H15, Asp, Ser, Lys, 3-(4-piperidinyl)-Ala, 3-(1-morpholinyl)- Ala, 3Pya, 4Pya, Glu, and NMe-Asn;R5is selected from the group consisting of an amino acid side chain of Asn, Ala, D-Ala, Trp, Asp, Lys, 3Pya, 4Pya, 3-(4-piperidinyl)-Ala, 3-(1-morpholinyl)-Ala, Glu, NMe-Asn, and Ser;R6is selected from the group consisting of an amino acid side chain of Trp, 4CF3-Phe, 1 Me-Trp, 7Aza-Trp, BIP, 2Nal, 1 Nal, alpha-Me-Trp, D-Ala, Ala, 4F-Phe, , 5F-Trp, 5Ome-Trp, Asn, 5OH-Trp, 7Me-Trp, 7MeO-Trp, 7CI-Trp, and NMe-Trp;R7is selected from the group consisting of an amino acid side chain of 3Pya, 4Pya, Lys(Me)3, His, Ala, D-Ala, Gin, Lys, Glu, Arg, Orn, NMe-His, and Ser; orR7is selected from the group consisting ofeach s is independently 3, 5,10, 12, or 14;R8is selected from the group consisting of an amino acid side chain of Asp, D-Ala, Ala, Asn, Thr, NMe-Asp, and alpha-Me-Asp;R9is selected from the group consisting of an amino acid side chain of Trp, 7Aza-Trp, 1 Me-Trp, D-Ala, Ala, 4F-Phe, 1 Nal, 2Nal, 5F-Trp, 5Ome-Trp, alpha-Me-Trp, 7CI-Trp, 5OH-Trp, 7Me-Trp, 7MeO-Trp, and NMe-Trp; andR10is selected from the group consisting of an amino acid side chain of Pro, D-Ala, Ala, alpha-Me-Pro, trans4Fluoro-Pro, cis4Fluoro-Pro, trans4OH-Pro, cis4OH-Pro, Pip, 5,5-diMe-Pro, NMe-Ser, trans4NH2-Pro, cis4NH2-Pro, Sar, Aze, NMe-Ala, NMe-Leu, R-3Me-Aze, alpha-Me- Aze, ACI, and 3Me2-Aze.

13. The cyclic peptide of any one of claims 1 to 9, 11 , and 12, or a pharmaceutically acceptable salt thereof, whereinB1is CH2or C(CH3)2; andC1is CH2or C(CH3)2.

14. The cyclic peptide of any one of claims 1 to 9, 11 , and 12, or a pharmaceutically acceptable salt thereof, whereinB1is CH2; andC1is CH2.

15. The cyclic peptide of any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof, wherein Chelator is independently selected from a group consisting of ethylenediamine tetraacetic acid (EDTA), diethylenetriamine pentaacetic acid (DTPA), 1,4,7,10-tetra- azacylcododecane-N,N',N",N"'-tetraacetic acid (DOTA), 6-((16-((6-Carboxypyridin-2-yl)methyl)- 1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)-4-isothiocyanatopicolinic acid (Macropa), Macrodipa, 2,2’,2”,2”’-(1,10-dioxa-4,7,13,16-tetraazacyclooctadecane-4,7,13,16- tetrayl)tetraacetic acid) (Crown), 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid, α- (2-carboxyethyl) (DOTAGA), 1,4,7-Triazacyclononane-N,N',N"-triacetic acid (NOTA), 1,4,7,10- tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (TETA), 1,4,7,10,13- pentaazacyclopentadecane-N,N',N",N"',N""-pentaacetic acid (PEPA), and 1,4,7,10,13,16- hexaazacyclohexadecane-N,N',N",N"',N"",N""'-hexaacetic acid (HEHA).

16. The cyclic peptide of any one of claims 1 to 15, or a pharmaceutically acceptable salt thereof, wherein Formula B is substituted by at least one chelator.

17. The cyclic peptide of any one of claims 1 to 16, or a pharmaceutically acceptable salt thereof, wherein the cyclic peptide of Formula B is selected from a cyclic peptide in Table A.

18. The cyclic peptide of any one of claims 1 to 16, or a pharmaceutically acceptable salt thereof, wherein the cyclic peptide of Formula B is selected from a cyclic peptide in Table B.

19. The cyclic peptide of any one of claims 1 to 18, or a pharmaceutically acceptable salt thereof, wherein the cyclic peptide further comprises a radioisotope.

20. The cyclic peptide of claim 19, or a pharmaceutically acceptable salt thereof, wherein the radioisotope is selected from a radioisotope in Table 3.

21. A pharmaceutical composition comprising the cyclic peptide of any one of claims 1 to 20, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

22. A method of treating cancer in a subject in need thereof comprising administering to the subject the cyclic peptide of any one of claims 1 to 20, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 21 to the subject.

23. The method of claim 22, wherein the cancer is a DLL3-mediated cancer.

24. The method of claim 22 or 23, wherein the cancer is a neuroendocrine neoplasm, melanoma, or primary brain cancer.

25. The method of claim 24, wherein the neuroendocrine neoplasm is selected from small cell lung cancer (SCLC), medullary thyroid carcinoma (MTC), large cell neuroendocrine cancer (LCNEC), gastroenteropancreatic neuroendocrine carcinoma (GEP NEC), neuroendocrine prostate cancer (NEPC), small cell prostate cancer (SCPC), Merkel cell carcinoma (MCC), neuroendocrine cervical carcinoma, and Grade 3 neuroendocrine tumors (NETs).

26. A peptide having binding specificity for DLL3, wherein the peptide binds to one or more amino acids of A81 , L83, G106, A85, and R61 of a DLL3 amino acid sequence of SEQ ID NO: 1 , wherein the peptide comprises the amino acid sequence of Formula A:or a pharmaceutically acceptable salt thereof, wherein: X0is any natural or unnatural amino acid or X0is absent; X1is selected from Trp, 7Aza-Trp, 1 Me-Trp, 5OH-Trp, 5OMe-Trp, 7OMe-Trp, 7Me-Trp, 5F-Trp, 7CI-Trp, alpha-Me-Trp, and NMe-Trp;X2 and X3 are each independently any natural or unnatural amino acids;Y1 is Cys;X4, X5, X6, X7and X8 are each independently any natural or unnatural amino acids; Y2is Cys;X9 is selected from Trp, 7Aza-Trp, 1 Me-Trp, 5OH-Trp, 5OMe-Trp, 7OMe-Trp, 7Me-Trp, 5F-Trp, 7CI-Trp, alpha-Me-Trp, and NMe-Trp;X10 is selected from Pro, alpha-Me-Pro, trans4Fluoro-Pro, cis4Fluoro-Pro, trans4OH-Pro, cis4OH-Pro, 5,5-diMe-Pro, trans4NH2-Pro, and cis4NH2-Pro;P1is selected from: -L1-Chelator,D1is -NR”-Chelator;L1is absent or selected fromwherein the amino group of L1connects to the carbonyl group of P1or Chelator to form an amide bond; each n, q, and u are independently an integer from 0 to 16; each p is independently an integer from 0 to 24; each s is independently an integer from 0 to 16; each t is independently 1 , 2, 3, 4, 5, or 6; each R’ is independently selected from H, C(O)OH, (CH2)OH, and NHAc; and each R” is independently selected from H and CH3; wherein the cyclic peptide is cyclized via a linker between Y1and Y2; and wherein the cyclic peptide binds to DLL3.

27. The peptide of claim 26, binding to amino acids A81 , L83, G106, A85, and R61 of a DLL3 amino acid sequence of SEQ ID NO: 1 .

28. The peptide of claim 26 or 27, binding to main chain atoms of amino acids A81 , L83, G106, and A85 of a DLL3 amino acid sequence of SEQ ID NO: 1.

29. The peptide of any one of claims 26 to 28, binding to side chain atoms of amino acid R61 of a DLL3 amino acid sequence of SEQ ID NO: 1 .

30. The peptide of any one of claims 26 to 29, binding to main chain atoms of amino acids A81 , L83, G106, and A85 of a DLL3 amino acid sequence of SEQ ID NO: 1 , and binding to side chain atoms of amino acid R61 of a DLL3 amino acid sequence of SEQ ID NO: 1 .

31. The peptide of any one of claims 26 to 30, capable of binding DLL3 with an SPR KD value of about 1 x 10-8M to about 1 x 10-10M.

32. The peptide of any one of claims 26 to 31 , comprising an amino acid sequence of WTACANAKDCWP, or a derivative thereof comprising one or more unnatural amino acids.

33. The peptide of 32, wherein amino acids W1 , A3, A7, and W11 bind to DLL3.

34. A pharmaceutical composition comprising the peptide of any one of claims 26 to 33 and a pharmaceutically acceptable carrier.

35. A method of treating cancer in a subject in need thereof comprising administering to the subject the peptide of any one of claims 26 to 33 or the pharmaceutical composition of claim 34 to the subject.

36. The method of claim 35, wherein the cancer is a DLL3-mediated cancer.

37. The method of claim 35 or 36, wherein the cancer is a neuroendocrine neoplasm, melanoma, or primary brain cancer.

38. The method of claim 37, wherein the neuroendocrine neoplasm is selected from small cell lung cancer (SOLO), medullary thyroid carcinoma (MTC), large cell neuroendocrine cancer (LCNEC), gastroenteropancreatic neuroendocrine carcinoma (GEP NEC), neuroendocrine prostate cancer (NEPC), small cell prostate cancer (SCPC), Merkel cell carcinoma (MCC), neuroendocrine cervical carcinoma, and Grade 3 neuroendocrine tumors (NETs).

39. The method of claim 24 or 37, wherein the neuroendocrine neoplasm is extrapulmonary neuroendocrine carcinoma (NEC) of the cervix.

40. The cyclic peptide of claim 17, wherein the cyclic peptide is radiolabeled with F-18, Ga- 68, In-111 , Lu-177, or Ac-225, and pharmaceutically acceptable salts and solvates thereof.