阅读说明：本技术 Cd38特异性的双环肽配体 (CD 38-specific bicyclic peptide ligands ) 是由 L·巴尔达萨雷 R·拉尼 S·帕万 C·斯泰斯 D·托伊费尔 于 2020-01-15 设计创作，主要内容包括：本发明涉及与芳香族分子支架共价结合以使得两个或多个肽环在与支架的附着点之间相对的多肽。具体地,本发明描述的是CD38的高亲和力结合剂的肽。本发明还包括包含与一个或多个效应物和/或官能团缀合的所述肽的药物缀合物,包含所述肽配体和药物缀合物的药物组合物,以及所述肽配体和药物缀合物在预防、抑制或治疗CD38介导的疾病或病症中的用途。(The present invention relates to polypeptides covalently bound to an aromatic molecular scaffold such that two or more peptide loops are opposed between attachment points to the scaffold. In particular, described herein are peptides of high affinity binders to CD 38. The invention also includes drug conjugates comprising the peptides conjugated to one or more effectors and/or functional groups, pharmaceutical compositions comprising the peptide ligands and drug conjugates, and uses of the peptide ligands and drug conjugates in preventing, inhibiting, or treating CD 38-mediated diseases or disorders.)

1. A peptide ligand specific for CD38, comprising a polypeptide and an aromatic molecular scaffold, said polypeptide comprising at least three cysteine residues separated by at least two loop sequences, and said aromatic molecular scaffold forming covalent bonds with the cysteine residues of said polypeptide, thereby forming at least two polypeptide loops on said molecular scaffold.

2. A peptide ligand as defined in claim 1, wherein the loop sequence comprises 2,3,5,6 or 7 amino acids.

3. A peptide ligand as defined in claim 1 or claim 2, which comprises an amino acid sequence selected from:

C_i-F-X₁-L-D-X₂-X₃-C_ii-F-D-X₄-X₅-X₆-X₇-C_iii(SEQ ID NO：87)；

C_i-I-R/N-Y-G/A-D/N-I-C_ii-X₁-D/H-P/T-D/E-X₂-X₃-C_iii(SEQ ID NO：88)；

C_i-F-X₁-L-D-G-E-C_ii-F-X₂-X₃-G/P-X₄-X₅-C_iii(SEQ ID NO：89)；

C_iVNFGSVC_iiWDPDSRC_iii(SEQ ID NO：2)；

C_i-X₁-X₂-C_ii-A-D-F/M-P-I-X₃-X₄-C_iii(SEQ ID NO：90)；

C_iDYC_iiVRLGLTGC_iii(SEQ ID NO：74)；

C_iGWC_iiSDQIDGFC_iii(SEQ ID NO：75)；

C_iAWC_iiSDPIDGFC_iii(SEQ ID NO：76)；

C_iDWC_iiIDPGVSFC_iii(SEQ ID NO：77)；

C_iSWC_iiVDDGLPFC_iii(SEQ ID NO：78)；

C_iTWC_iiVDDGLSFC_iii(SEQ ID NO：79)；

C_iTWC_iiVDDETWNC_iii(SEQ ID NO：80)；

C_iDYC_iiIRLGLTGC_iii(SEQ ID NO：81)；

C_iDWC_iiTDNIPGIC_iii(SEQ ID NO：82)；

C_i-A/N-W/F-L-C_ii-P/D-N/D-L-C_iii(SEQ ID NO：91)；

C_i-D-F-T-M-P-C_ii-X₁-X₂-W-X₃-X₄-C_iii(SEQ ID NO: 92); and

C_iIFDYDC_iiDAWSAC_iii(SEQ ID NO：28)；

or a pharmaceutically acceptable salt thereof, wherein X₁-X₆Represents any amino acid residue, X₇Is absent or represents any amino acid, and C_i、C_iiAnd C_iiiRepresenting the first, second and third cysteine residues, respectively.

4. The peptide ligand as defined in any one of claims 1 to 3, wherein the loop sequence comprises three cysteine residues separated by two loop sequences, both loop sequences consisting of 6 amino acids, or one of the two loop sequences consisting of 5 amino acids and the other consisting of 6 amino acids, and the peptide ligand comprises an amino acid sequence selected from the group consisting of:

C_i-F-X₁-L-D-X₂-X₃-C_ii-F-D-X₄-X₅-X₆-X₇-C_iii(SEQ ID NO：87)；

C_i-I-R/N-Y-G/A-D/N-I-C_ii-X₁-D/H-P/T-D/E-X₂-X₃-C_iii(SEQ ID NO：88)；

C_i-F-X₁-L-D-G-E-C_ii-F-X₂-X₃-G/P-X₄-X₅-C_iii(SEQ ID NO: 89); and

C_iVNFGSVC_iiWDPDSRC_iii(SEQ ID NO：2)；

or a pharmaceutically acceptable salt thereof, wherein X₁-X₆Represents any amino acid residue, X₇Is absent or represents any amino acid, and C_i、C_iiAnd C_iiiRepresenting the first, second and third cysteine residues, respectively.

5. The peptide ligand as defined in any one of claims 1 to 3, wherein the loop sequence comprises three cysteine residues separated by two loop sequences, the first of the two loop sequences consisting of 2 amino acids and the second of the two loop sequences consisting of 7 amino acids, and the peptide ligand comprises an amino acid sequence selected from the group consisting of:

C_i-X₁-X₂-C_ii-A-D-F/M-P-I-X₃-X₄-C_iii(SEQ ID NO：90)；

C_iDYC_iiVRLGLTGC_iii(SEQ ID NO：74)；

C_iGWC_iiSDQIDGFC_iii(SEQ ID NO：75)；

C_iAWC_iiSDPIDGFC_iii(SEQ ID NO：76)；

C_iDWC_iiIDPGVSFC_iii(SEQ ID NO：77)；

C_iSWC_iiVDDGLPFC_iii(SEQ ID NO：78)；

C_iTWC_iiVDDGLSFC_iii(SEQ ID NO：79)；

C_iTWC_iiVDDETWNC_iii(SEQ ID NO：80)；

C_iDYC_iiIRLGLTGC_iii(SEQ ID NO: 81); and

C_iDWC_iiTDNIPGIC_iii(SEQ ID NO：82)；

or a pharmaceutically acceptable salt thereof, wherein X₁-X₄Represents any amino acid residue, and C_i、C_iiAnd C_iiiRepresenting the first, second and third cysteine residues, respectively.

6. A peptide ligand as defined in any one of claims 1 to 3, wherein the loop sequence comprises three cysteine residues separated by two loop sequences, both loop sequences consisting of 3 amino acids, and the peptide ligand comprises an amino acid sequence selected from:

C_i-A/N-W/F-L-C_ii-P/D-N/D-L-C_iii(SEQ ID NO：91)；

or a pharmaceutically acceptable salt thereof; wherein C is_i、C_iiAnd C_iiiRepresenting the first, second and third cysteine residues, respectively.

7. A peptide ligand as defined in any one of claims 1 to 3, wherein the loop sequence comprises three cysteine residues separated by two loop sequences, both loop sequences consisting of 5 amino acids, and the peptide ligand comprises an amino acid sequence selected from:

C_i-D-F-T-M-P-C_ii-X₁-X₂-W-X₃-X₄-C_iii(SEQ ID NO: 92); and

C_iIFDYDC_iiDAWSAC_iii(SEQ ID NO：28)；

or a pharmaceutically acceptable salt thereof, wherein X₁-X₄Represents any amino acid residue, and C_i、C_iiAnd C_iiiRepresenting the first, second and third cysteine residues, respectively.

8. A peptide ligand as defined in claim 3 or claim 4, wherein C_i-F-X₁-L-D-X₂-X₃-C_ii-F-D-X₄-X₅-X₆-X₇-C_iii(SEQ ID NO: 87)The peptide ligand comprises an amino acid sequence selected from the group consisting of:

C_iFELDGTC_iiFDWAQEC_iii(SEQ ID NO：1)；

C_iFWLDGEC_iiFDWNHEC_iii(SEQ ID NO：29)；

C_iFHLDGEC_iiFDLENTC_iii(SEQ ID NO：30)；

C_iFSLDGEC_iiFDLSGEC_iii(SEQ ID NO：32)；

C_iFTLDGEC_iiFDWTHEC_iii(SEQ ID NO：33)；

C_iFKLDGVC_iiFDLFHEC_iii(SEQ ID NO：34)；

C_iFMLDGEC_iiFDLNKEC_iii(SEQ ID NO：35)；

C_iFKLDGEC_iiFDWTHEC_iii(SEQ ID NO：36)；

C_iFTLDGEC_iiFDWDAEC_iii(SEQ ID NO：37)；

C_iFELDGSC_iiFDFDHEC_iii(SEQ ID NO：38)；

C_iFTLDGEC_iiFDVNREC_iii(SEQ ID NO：39)；

C_iFWLDHEC_iiFDWTHEC_iii(SEQ ID NO：40)；

C_iFQLDGEC_iiFDIYREC_iii(SEQ ID NO：41)；

C_iFELDGNC_iiFDWTHEC_iii(SEQ ID NO：42)；

C_iFHLDGEC_iiFDYEHEC_iii(SEQ ID NO：43)；

C_iFSLDGEC_iiFDIASEC_iii(SEQ ID NO：44)；

C_iFQLDGEC_iiFDTSHEC_iii(SEQ ID NO：45)；

C_iFSLDGAC_iiFDWTHEC_iii(SEQ ID NO：46)；

C_iFVLDGEC_iiFDYYEEC_iii(SEQ ID NO：47)；

C_iFRLDDEC_iiFDWTHEC_iii(SEQ ID NO：48)；

C_iFRLDGVC_iiFDLDDEC_iii(SEQ ID NO：49)；

C_iFRLDGEC_iiFDMGQEC_iii(SEQ ID NO：50)；

C_iFTLDGAC_iiFDLDGEC_iii(SEQ ID NO：51)；

C_iFTLDGQC_iiFDWTHEC_iii(SEQ ID NO：52)；

C_iFLLDGEC_iiFDWMQEC_iii(SEQ ID NO：53)；

C_iFELDGDC_iiFDWTHEC_iii(SEQ ID NO：54)；

C_iFTLDGTC_iiFDWTHEC_iii(SEQ ID NO：55)；

C_iFHLDGVC_iiFDWTHEC_iii(SEQ ID NO：56)；

C_iFYLDGTC_iiFDWTHEC_iii(SEQ ID NO：57)；

C_iFLLDGEC_iiFDWAQEC_iii(SEQ ID NO：58)；

C_iFHLDGEC_iiFDLAKTC_iii(SEQ ID NO：59)；

C_iFTLDGEC_iiFDLDGWC_iii(SEQ ID NO：62)；

C_iFLLDGEC_iiFDLIGEC_iii(SEQ ID NO：63)；

C_iFWLDGEC_iiFDLGGQC_iii(SEQ ID NO：67)；

C_iFELDGEC_iiFDLDNQC_iii(SEQ ID NO：68)；

C_iFWLDGEC_iiFDLYGGC_iii(SEQ ID NO：69)；

C_iFRLDGEC_iiFDISNEC_iii(SEQ ID NO：70)；

C_iFWLDGEC_iiFDFGGC_iii(SEq ID NO: 72) (ii) a And

C_iFTLDGAC_iiFDWTHEC_iii(SEQ ID NO：73)；

or a pharmaceutically acceptable salt thereof, wherein C_i、C_iiAnd C_iiiRepresents a first, second and third cysteine residue, respectively,

for example:

an amino acid sequence selected from:

a- (SEQ ID NO: 1) -A (referred to herein as 66-01-N002);

a- (SEQ ID NO: 29) -A (referred to herein as 66-08-01-N001);

ac- (SEQ ID NO: 29) (referred to herein as 66-08-01-N004);

Ac-(SEQ ID NO：29)-A-Sar₆-K (referred to herein as 66-08-01-N005);

Ac-(SEQ ID NO：29)-A-Sar₆-k (dota)) (referred to herein as 66-08-01-N016;

a- (SEQ ID NO: 30) -A (referred to herein as 66-08-44-N001);

Ac-A-(SEQ ID NO：30)-A-Sar₆-K (biotin) (referred to herein as 66-08-01-N003);

a- (SEQ ID NO: 32) -A (referred to herein as 66-08-02-N001);

a- (SEQ ID NO: 33) -A (referred to herein as 66-08-03-N001);

a- (SEQ ID NO: 34) -A (referred to herein as 66-08-04-N001);

Ac-A- (SEQ ID NO: 35) -A (referred to herein as 66-08-05-N001);

a- (SEQ ID NO: 36) -A (referred to herein as 66-08-06-N001);

a- (SEQ ID NO: 37) -A (referred to herein as 66-08-07-N001);

a- (SEQ ID NO: 38) -A (referred to herein as 66-08-09-N001);

a- (SEQ ID NO: 39) -A (referred to herein as 66-08-13-N001);

a- (SEQ ID NO: 40) -A (referred to herein as 66-08-15-N001);

a- (SEQ ID NO: 41) -A (referred to herein as 66-08-17-N001);

a- (SEQ ID NO: 42) -A (referred to herein as 66-08-18-N001);

a- (SEQ ID NO: 43) -A (referred to herein as 66-08-20-N001);

a- (SEQ ID NO: 44) -A (referred to herein as 66-08-22-N001);

a- (SEQ ID NO: 45) -A (referred to herein as 66-08-24-N001);

a- (SEQ ID NO: 46) -A (referred to herein as 66-08-26-N001);

a- (SEQ ID NO: 47) -A (referred to herein as 66-08-27-N001);

a- (SEQ ID NO: 48) -A (referred to herein as 66-08-28-N001);

a- (SEQ ID NO: 49) -A (referred to herein as 66-08-29-N001);

a- (SEQ ID NO: 50) -A (referred to herein as 66-08-30-N001);

a- (SEQ ID NO: 51) -A (referred to herein as 66-08-31-N001);

a- (SEQ ID NO: 52) -A (referred to herein as 66-08-32-N001);

a- (SEQ ID NO: 53) -A (referred to herein as 66-08-33-N001);

a- (SEQ ID NO: 54) -A (referred to herein as 66-08-34-N001);

a- (SEQ ID NO: 55) -A (referred to herein as 66-08-35-N001);

a- (SEQ ID NO: 56) -A (referred to herein as 66-08-36-N001);

a- (SEQ ID NO: 57) -A (referred to herein as 66-08-41-N001);

a- (SEQ ID NO: 58) -A (referred to herein as 66-08-43-N001);

a- (SEQ ID NO: 59) -A (referred to herein as 66-08-45-N001);

a- (SEQ ID NO: 62) -A (referred to herein as 66-08-48-N001);

a- (SEQ ID NO: 63) -A (referred to herein as 66-08-49-N001);

a- (SEQ ID NO: 67) -A (referred to herein as 66-08-53-N001);

a- (SEQ ID NO: 68) -A (referred to herein as 66-08-54-N001);

a- (SEQ ID NO: 69) -A (referred to herein as 66-08-55-N001);

a- (SEQ ID NO: 70) -A (referred to herein as 66-08-56-N001);

a- (SEQ ID NO: 72) -A (referred to herein as 66-08-58-N001); and

a- (SEQ ID NO: 73) -A (referred to herein as 66-08-N002).

9. A peptide ligand as defined in claim 3 or claim 4, wherein C_i-I-R/N-Y-G/A-D/N-I-C_ii-X₁-D/H-P/T-D/E-X₂-X₃-C_iii(SEQ ID NO: 88) comprises an amino acid sequence selected from the group consisting of:

C_iIRYGDIC_iiYDPDHSC_iii(SEQ ID NO：83)；

C_iIRYGDIC_iiFHPDYTC_iii(SEQ ID NO: 84); and

C_iINYANIC_iiLDTEKMC_iii(SEQ ID NO：85)；

or a pharmaceutically acceptable salt thereof, wherein C_i、C_iiAnd C_iiiRepresents a first, second and third cysteine residue, respectively,

for example:

an amino acid sequence selected from:

a- (SEQ ID NO: 83) -A (referred to herein as 66-17-01-N001);

a- (SEQ ID NO: 84) -A (referred to herein as 66-17-N001); and

a- (SEQ ID NO: 85) -A (referred to herein as 66-18-N001).

10. A peptide ligand as defined in claim 3 or claim 4, wherein C_i-F-X₁-L-D-G-E-C_ii-F-X₂-X₃-G/P-X₄-X₅-C_iii(SEQ ID NO: 89) comprises an amino acid sequence selected from the group consisting of:

C_iFELDGEC_iiFHFGEPC_iii(SEQ ID NO：60)；

C_iFVLDGEC_iiFEIGERC_iii(SEQ ID NO：61)；

C_iFELDGEC_iiFSFPGTC_iii(SEQ ID NO：64)；

C_iFELDGEC_iiFSWPYPC_iii(SEQ ID NO：65)；

C_iFTLDGEC_iiFLLGENC_iii(SEQ ID NO: 66); and

C_iFELDGEC_iiFNIGSKC_iii(SEQ ID NO：71)；

or a pharmaceutically acceptable salt thereof, wherein C_i、C_iiAnd C_iiiRepresents a first, second and third cysteine residue, respectively,

for example:

an amino acid sequence selected from:

a- (SEQ ID NO: 60) -A (referred to herein as 66-08-46-N001);

a- (SEQ ID NO: 61) -A (referred to herein as 66-08-47-N001);

a- (SEQ ID NO: 64) -A (referred to herein as 66-08-50-N001);

a- (SEQ ID NO: 65) -A (referred to herein as 66-08-51-N001);

a- (SEQ ID NO: 66) -A (referred to herein as 66-08-52-N001); and

a- (SEQ ID NO: 71) -A (referred to herein as 66-08-57-N001).

11. A peptide ligand as defined in claim 3 or claim 4, wherein C_iVNFGSVC_iiWDPDSRC_iii(SEQ ID NO: 2) comprises an amino acid sequence selected from the group consisting of:

a- (SEQ ID NO: 2) -A (referred to herein as 66-02-N002).

12. A peptide ligand as defined in claim 3 or claim 5, wherein C_i-X₁-X₂-A-D-F/M-C_ii-P-I-X₃-X₄-C_iii(SEQ ID NO: 90) comprises an amino acid sequence selected from the group consisting of:

C_iVPC_iiADFPIWYC_iii(SEQ ID NO：3)；

C_iVPC_iiADFPIWWC_iii(SEQ ID NO：4)；

C_iTPC_iiADFPIWSC_iii(SEQ ID NO：5)；

C_iTPC_iiADFPIHTC_iii(SEQ ID NO：6)；

C_iVHC_iiADFPIWGC_iii(SEQ ID NO：7)；

C_iVPC_iiADFPIWGC_iii(SEQ ID NO：8)；

C_iVMC_iiADFPIWGC_iii(SEQ ID NO：9)；

C_iTPC_iiADFPIWYC_iii(SEQ ID NO：10)；

C_iTPC_iiADFPILTC_iii(SEQ ID NO：11)；

C_iVAC_iiADFPIWGC_iii(SEQ ID NO：12)；

C_iTPC_iiADFPIYGC_iii(SEQ ID NO：13)；

C_iTPC_iiADFPILDC_iii(SEQ ID NO：14)；

C_iVKC_iiADFPIWGC_iii(SEQ ID NO：15)；

C_iTPC_iiADMPIWTC_iii(SEQ ID NO：16)；

C_iIPC_iiADFPIWGC_iii(SEQ ID NO：17)；

C_iIPC_iiADFPISVC_iii(SEQ ID NO：18)；

C_iVPC_iiADFPISFC_iii(SEQ ID NO：19)；

C_iIPC_iiADFPISFC_iii(SEQ ID NO：20)；

C_iVPC_iiADFPISVC_iii(SEQ ID NO：21)；

C_iVPC_iiADFPIFTC_iii(SEQ ID NO：22)；

C_iIPC_iiADFPIFTC_iii(SEQ ID NO: 23); and

C_iTPC_iiADFPIWGC_iii(SEQ ID NO：24)；

or a pharmaceutically acceptable salt thereof; wherein C is_i、C_iiAnd C_iiiRepresents a first, second and third cysteine residue, respectively,

for example:

an amino acid sequence selected from:

a- (SEQ ID NO: 3) -A (referred to herein as 66-03-00-N004);

(β-Ala)-Sar₁₀-A- (SEQ ID NO: 3) (referred to herein as 66-03-00-N006);

DOTA-(β-Ala)-Sar₁₀-A- (SEQ ID NO: 3) (referred to herein as 66-03-00-N007);

Ac-(SEQ ID NO：3)-A-Sar₆-K (referred to herein as 66-03-00-N008);

Ac-(SEQ ID NO：3)-A-Sar₆- (PG) (referred to herein as 66-03-00-N009);

a- (SEQ ID NO: 4) -A (referred to herein as 66-03-00-N005);

a- (SEQ ID NO: 5) -A (referred to herein as 66-03-01-N001);

a- (SEQ ID NO: 6) -A (referred to herein as 66-03-02-N001);

a- (SEQ ID NO: 7) -A (referred to herein as 66-03-03-N001);

a- (SEQ ID NO: 8) -A (referred to herein as 66-03-04-N001);

ac- (SEQ ID NO: 8) (referred to herein as 66-03-04-N002);

a- (SEQ ID NO: 9) -A (referred to herein as 66-03-05-N001);

a- (SEQ ID NO: 10) -A (referred to herein as 66-03-06-N001);

ac- (SEQ ID NO: 10) (referred to herein as 66-03-06-N002);

a- (SEQ ID NO: 11) -A (referred to herein as 66-03-07-N001);

a- (SEQ ID NO: 12) -A (referred to herein as 66-03-08-N001);

a- (SEQ ID NO: 13) -A (referred to herein as 66-03-09-N001);

a- (SEQ ID NO: 14) -A (referred to herein as 66-03-10-N001);

a- (SEQ ID NO: 15) -A (referred to herein as 66-03-11-N001);

ac- (SEQ ID NO: 15) (referred to herein as 66-03-11-N002);

a- (SEQ ID NO: 16) -A (referred to herein as 66-03-15-N001);

a- (SEQ ID NO: 17) -A (referred to herein as 66-03-16-N001);

a- (SEQ ID NO: 18) -A (referred to herein as 66-03-24-N003);

a- (SEQ ID NO: 19) -A (referred to herein as 66-03-25-N002);

a- (SEQ ID NO: 20) -A (referred to herein as 66-03-26-N003);

a- (SEQ ID NO: 21) -A (referred to herein as 66-03-27-N003);

a- (SEQ ID NO: 22) -A (referred to herein as 66-03-28-N002);

a- (SEQ ID NO: 23) -A (referred to herein as 66-03-29-N003);

a- (SEQ ID NO: 24) -A (referred to herein as 66-03-N002);

A-(SEQ ID NO：24)-A-Sar₆-K (biotin) (referred to herein as 66-03-N003);

a- (SEQ ID NO: 74) -A (referred to herein as 66-09-N001);

A-(SEQ ID NO：75)-A-Sar₆-K (biotin) (referred to herein as 66-10-01-N001);

A-(SEQ ID NO：76)-A-Sar₆-K (biotin) (referred to herein as 66-10-02-N001);

A-(SEQ ID NO：77)-A-Sar₆-K (biotin) (referred to herein as 66-10-03-N001);

A-(SEQ ID NO：78)-A-Sar₆-K (biotin) (referred to herein as 66-10-04-N001);

a- (SEQ ID NO: 79) -A (referred to herein as 66-10-N001);

a- (SEQ ID NO: 80) -A (referred to herein as 66-11-N001);

a- (SEQ ID NO: 81) -A (referred to herein as 66-12-N001); and

a- (SEQ ID NO: 82) -A (referred to herein as 66-13-N001).

13. A peptide ligand as defined in claim 3 or claim 6, wherein C_i-A/N-W/F-L-C_ii-P/D-N/D-L-C_iii(SEQ ID NO: 91) comprises an amino acid sequence selected from the group consisting of:

C_iAWLC_iiPNLC_iii(SEQ ID NO: 31); and

C_iNFLC_iiDDLC_iii(SEQ ID NO：86)；

or a pharmaceutically acceptable salt thereof; wherein C is_i、C_iiAnd C_iiiRepresents a first, second and third cysteine residue, respectively,

for example:

an amino acid sequence selected from:

SSQHG-(SEQ ID NO：31)-A-Sar₆-K (referred to herein as 66-08-01-N006); and

RHSNY-(SEQ ID NO：86)-A-Sar₆-K (biotin) (referred to herein as 66-20-00-T001-N001).

14. A peptide ligand as defined in claim 3 or claim 7, wherein C_i-D-F-T-M-P-C_ii-X₁-X₂-W-X₃-X₄-C_iii(SEQ ID NO: 92) comprises an amino acid sequence selected from the group consisting of:

C_iDFTMPC_iiENWKYC_iii(SEQ ID NO：25)；

C_iDFTMPC_iiPNWNAC_iii(SEQ ID NO: 26); and

C_iDFTMPC_iiQMWEQC_iii(SEQ ID NO：27)；

or a pharmaceutically acceptable salt thereof; wherein C is_i、C_iiAnd C_iiiRepresents a first, second and third cysteine residue, respectively,

for example:

an amino acid sequence selected from:

a- (SEQ ID NO: 25) -A (referred to herein as 66-05-07-N001);

a- (SEQ ID NO: 26) -A (referred to herein as 66-05-09-N001);

a- (SEQ ID NO: 27) -A (referred to herein as 66-05-N002); and

a- (SEQ ID NO: 28) -A (referred to herein as 66-06-N002).

15. The peptide ligand as defined in claim 1, wherein the molecular scaffold is selected from 1,3, 5-tris (bromomethyl) benzene (TBMB), and the peptide ligand comprises an amino acid sequence selected from:

a- (SEQ ID NO: 1) -A (referred to herein as 66-01-N002);

a- (SEQ ID NO: 2) -A (referred to herein as 66-02-N002);

a- (SEQ ID NO: 3) -A (referred to herein as 66-03-00-N004);

(β-Ala)-Sar₁₀-A- (SEQ ID NO: 3) (referred to herein as 66-03-00-N006);

DOTA-(β-Ala)-Sar₁₀-A- (SEQ ID NO: 3) (referred to herein as 66-03-00-N007);

Ac-(SEQ ID NO：3)-A-Sar₆-K (referred to herein as 66-03-00-N008);

Ac-(SEQ ID NO：3)-A-Sar₆- (PG) (referred to herein as 66-03-00-N009);

a- (SEQ ID NO: 4) -A (referred to herein as 66-03-00-N005);

a- (SEQ ID NO: 5) -A (referred to herein as 66-03-01-N001);

a- (SEQ ID NO: 6) -A (referred to herein as 66-03-02-N001);

a- (SEQ ID NO: 7) -A (referred to herein as 66-03-03-N001);

a- (SEQ ID NO: 8) -A (referred to herein as 66-03-04-N001);

ac- (SEQ ID NO: 8) (referred to herein as 66-03-04-N002);

a- (SEQ ID NO: 9) -A (referred to herein as 66-03-05-N001);

a- (SEQ ID NO: 10) -A (referred to herein as 66-03-06-N001);

ac- (SEQ ID NO: 10) (referred to herein as 66-03-06-N002);

a- (SEQ ID NO: 11) -A (referred to herein as 66-03-07-N001);

a- (SEQ ID NO: 12) -A (referred to herein as 66-03-08-N001);

a- (SEQ ID NO: 13) -A (referred to herein as 66-03-09-N001);

a- (SEQ ID NO: 14) -A (referred to herein as 66-03-10-N001);

a- (SEQ ID NO: 15) -A (referred to herein as 66-03-11-N001);

ac- (SEQ ID NO: 15) (referred to herein as 66-03-11-N002);

a- (SEQ ID NO: 16) -A (referred to herein as 66-03-15-N001);

a- (SEQ ID NO: 17) -A (referred to herein as 66-03-16-N001);

a- (SEQ ID NO: 18) -A (referred to herein as 66-03-24-N003);

a- (SEQ ID NO: 19) -A (referred to herein as 66-03-25-N002);

a- (SEQ ID NO: 20) -A (referred to herein as 66-03-26-N003);

a- (SEQ ID NO: 21) -A (referred to herein as 66-03-27-N003);

a- (SEQ ID NO: 22) -A (referred to herein as 66-03-28-N002);

a- (SEQ ID NO: 23) -A (referred to herein as 66-03-29-N003);

a- (SEQ ID NO: 24) -A (referred to herein as 66-03-N002);

A-(SEQ ID NO：24)-A-Sar₆-K (biotin) (referred to herein as 66-03-N003);

a- (SEQ ID NO: 25) -A (referred to herein as 66-05-07-N001);

a- (SEQ ID NO: 26) -A (referred to herein as 66-05-09-N001);

a- (SEQ ID NO: 27) -A (referred to herein as 66-05-N002);

a- (SEQ ID NO: 28) -A (referred to herein as 66-06-N002);

a- (SEQ ID NO: 29) -A (referred to herein as 66-08-01-N001);

ac- (SEQ ID NO: 29) (referred to herein as 66-08-01-N004);

Ac-(SEQ ID NO：29)-A-Sar₆-K (referred to herein as 66-08-01-N005);

Ac-(SEQ ID NO：29)-A-Sar₆-k (dota) (referred to herein as 66-08-01-N016);

Ac-A-(SEQ ID NO：30)-A-Sar₆-K (biotin) (referred to herein as 66-08-01-N003);

a- (SEQ ID NO: 30) -A (referred to herein as 66-08-44-N001);

SSQHG-(SEQ ID NO：31)-A-Sar₆-K (referred to herein as 66-08-01-N006);

a- (SEQ ID NO: 32) -A (referred to herein as 66-08-02-N001);

a- (SEQ ID NO: 33) -A (referred to herein as 66-08-03-N001);

a- (SEQ ID NO: 34) -A (referred to herein as 66-08-04-N001);

Ac-A- (SEQ ID NO: 35) -A (referred to herein as 66-08-05-N001);

a- (SEQ ID NO: 36) -A (referred to herein as 66-08-06-N001);

a- (SEQ ID NO: 37) -A (referred to herein as 66-08-07-N001);

a- (SEQ ID NO: 38) -A (referred to herein as 66-08-09-N001);

a- (SEQ ID NO: 39) -A (referred to herein as 66-08-13-N001);

a- (SEQ ID NO: 40) -A (referred to herein as 66-08-15-N001);

a- (SEQ ID NO: 41) -A (referred to herein as 66-08-17-N001);

a- (SEQ ID NO: 42) -A (referred to herein as 66-08-18-N001);

a- (SEQ ID NO: 43) -A (referred to herein as 66-08-20-N001);

a- (SEQ ID NO: 44) -A (referred to herein as 66-08-22-N001);

a- (SEQ ID NO: 45) -A (referred to herein as 66-08-24-N001);

a- (SEQ ID NO: 46) -A (referred to herein as 66-08-26-N001);

a- (SEQ ID NO: 47) -A (referred to herein as 66-08-27-N001);

a- (SEQ ID NO: 48) -A (referred to herein as 66-08-28-N001);

a- (SEQ ID NO: 49) -A (referred to herein as 66-08-29-N001);

a- (SEQ ID NO: 50) -A (referred to herein as 66-08-30-N001);

a- (SEQ ID NO: 51) -A (referred to herein as 66-08-31-N001);

a- (SEQ ID NO: 52) -A (referred to herein as 66-08-32-N001);

a- (SEQ ID NO: 53) -A (referred to herein as 66-08-33-N001);

a- (SEQ ID NO: 54) -A (referred to herein as 66-08-34-N001);

a- (SEQ ID NO: 55) -A (referred to herein as 66-08-35-N001);

a- (SEQ ID NO: 56) -A (referred to herein as 66-08-36-N001);

a- (SEQ ID NO: 57) -A (referred to herein as 66-08-41-N001);

a- (SEQ ID NO: 58) -A (referred to herein as 66-08-43-N001);

a- (SEQ ID NO: 59) -A (referred to herein as 66-08-45-N001);

a- (SEQ ID NO: 60) -A (referred to herein as 66-08-46-N001);

a- (SEQ ID NO: 61) -A (referred to herein as 66-08-47-N001);

a- (SEQ ID NO: 62) -A (referred to herein as 66-08-48-N001);

a- (SEQ ID NO: 63) -A (referred to herein as 66-08-49-N001);

a- (SEQ ID NO: 64) -A (referred to herein as 66-08-50-N001);

a- (SEQ ID NO: 65) -A (referred to herein as 66-08-51-N001);

a- (SEQ ID NO: 66) -A (referred to herein as 66-08-52-N001);

a- (SEQ ID NO: 67) -A (referred to herein as 66-08-53-N001);

a- (SEQ ID NO: 68) -A (referred to herein as 66-08-54-N001);

a- (SEQ ID NO: 69) -A (referred to herein as 66-08-55-N001);

a- (SEQ ID NO: 70) -A (referred to herein as 66-08-56-N001);

a- (SEQ ID NO: 71) -A (referred to herein as 66-08-57-N001);

a- (SEQ ID NO: 72) -A (referred to herein as 66-08-58-N001);

a- (SEQ ID NO: 73) -A (referred to herein as 66-08-N002);

a- (SEQ ID NO: 74) -A (referred to herein as 66-09-N001);

A-(SEQ ID NO：75)-A-Sar₆-K (biotin) (referred to herein as 66-10-01-N001);

A-(SEQ ID NO：76)-A-Sar₆-K (biotin) (referred to herein as 66-10-02-N001);

A-(SEQ ID NO：77)-A-Sar₆-K (biotin) (referred to herein as 66-10-03-N001);

A-(SEQ ID NO：78)-A-Sar₆-K (biotin) (referred to herein as 66-10-04-N001);

a- (SEQ ID NO: 79) -A (referred to herein as 66-10-N001);

a- (SEQ ID NO: 80) -A (referred to herein as 66-11-N001);

a- (SEQ ID NO: 81) -A (referred to herein as 66-12-N001);

a- (SEQ ID NO: 82) -A (referred to herein as 66-13-N001);

a- (SEQ ID NO: 83) -A (referred to herein as 66-17-01-N001);

a- (SEQ ID NO: 84) -A (referred to herein as 66-17-N001);

a- (SEQ ID NO: 85) -A (referred to herein as 66-18-N001); and

RHSNY-(SEQ ID NO：86)-A-Sar₆-K (biotin) (referred to herein as 66-20-00-T001-N001);

for example:

an amino acid sequence selected from:

a- (SEQ ID NO: 3) -A (referred to herein as 66-03-00-N004);

(β-Ala)-Sar₁₀-A- (SEQ ID NO: 3) (referred to herein as 66-03-00-N006);

DOTA-(β-Ala)-Sar₁₀-A- (SEQ ID NO: 3) (referred to herein as 66-03-00-N007);

Ac-(SEQ ID NO：3)-A-Sar₆-K (referred to herein as 66-03-00-N008);

Ac-(SEQ ID NO：3)-A-Sar₆- (PG) (referred to herein as 66-03-00-N009);

a- (SEQ ID NO: 7) -A (referred to herein as 66-03-03-N001);

a- (SEQ ID NO: 8) -A (referred to herein as 66-03-04-N001);

a- (SEQ ID NO: 10) -A (referred to herein as 66-03-06-N001);

a- (SEQ ID NO: 15) -A (referred to herein as 66-03-11-N001);

a- (SEQ ID NO: 22) -A (referred to herein as 66-03-28-N002);

a- (SEQ ID NO: 29) -A (referred to herein as 66-08-01-N001);

ac- (SEQ ID NO: 29) (referred to herein as 66-08-01-N004);

Ac-(SEQ ID NO：29)-A-Sar₆-k (dota) (referred to herein as 66-08-01-N016); and

Ac-A-(SEQ ID NO：30)-A-Sar₆-K (biotin) (referred to herein as 66-08-01-N003);

in particular:

an amino acid sequence selected from:

(β-Ala)-Sar₁₀-A- (SEQ ID NO: 3) (referred to herein as 66-03-00-N006).

16. A peptide ligand as defined in any one of claims 1 to 15, wherein the pharmaceutically acceptable salt is selected from the free acid or the sodium, potassium, calcium, ammonium salts.

17. The peptide ligand as defined in any one of claims 1 to 16, wherein the CD38 is human CD 38.

18. A drug conjugate comprising a peptide ligand as defined in any one of claims 1 to 17 conjugated to one or more effectors and/or functional groups.

19. A drug conjugate comprising a peptide ligand as defined in any one of claims 1 to 17 conjugated to one or more cytotoxic agents.

20. A drug conjugate as defined in claim 19, wherein the cytotoxic agent is selected from maytansine.

21. A drug conjugate as defined in claim 19 or claim 20, wherein the cytotoxic agent is selected from DM1 and the peptide ligand is selected from (β -Ala) -Sar₁₀-A- (SEQ ID NO: 5) (referred to herein as 66-03-00-N006):

22. a pharmaceutical composition comprising a peptide ligand as defined in any one of claims 1 to 17, or a drug conjugate as defined in any one of claims 18 to 21, in combination with one or more pharmaceutically acceptable excipients.

23. Use of a peptide ligand as defined in any one of claims 1 to 17, or a drug conjugate as defined in any one of claims 18 to 21, in the prevention, inhibition or treatment of a CD 38-mediated disease or condition.

Technical Field

The present invention relates to polypeptides covalently bound to an aromatic molecular scaffold such that two or more peptide loops are opposed between attachment points to the scaffold. In particular, described herein are peptides of high affinity binders to CD 38. The invention also includes drug conjugates comprising the peptides conjugated to one or more effectors and/or functional groups, pharmaceutical compositions comprising the peptide ligands and drug conjugates, and uses of the peptide ligands and drug conjugates in preventing, inhibiting, or treating CD 38-mediated diseases or disorders.

Background

Cyclic peptides are capable of binding to protein targets with high affinity and target specificity and are therefore an attractive class of molecules for the development of therapeutics. In fact, several cyclic peptides have been used successfully clinically, such as the antibacterial peptide vancomycin, the immunosuppressant Drug cyclosporin or the anticancer Drug octreotide (draggers et al (2008), Nat Rev Drug Discov 7(7), 608-24). Good binding properties result from the relatively large interaction surface formed between the peptide and the target and the reduced conformational flexibility of the cyclic structure. Typically, macrocycles are bound to surfaces of several hundred square angstroms, for example the cyclic peptide CXCR4 antagonist CVX15(Wu et al (2007), Science 330,1066-71), cyclic peptides with Arg-Gly-Asp motif that bind to integrin α Vb3(Xiong et al (2002), Science 296(5565), 151-5) Or the cyclic peptide inhibitor upain-1 (conjugated to urokinase-type plasminogen activator)Zhao et al (2007), J Structure Biol 160(1), 1-10).

Due to its cyclic structure, peptide macrocycles are less flexible than linear peptides, resulting in less entropy loss upon binding to the target and resulting in higher binding affinity. The reduced flexibility compared to linear peptides also results in locking of the target specific conformation, increasing the binding specificity. This effect has been exemplified by a potent and selective inhibitor of matrix metalloproteinase 8(MMP-8) which loses selectivity for other MMPs when its ring is opened (Cherney et al (1998), J Med Chem 41(11), 1749-51). The advantageous binding properties achieved by macrocyclization are more pronounced in polycyclic peptides with more than one peptide ring (e.g., in vancomycin, nisin and actinomycin).

Different research groups have previously attached polypeptides with cysteine residues to synthetic molecular structures (Kemp and McNamara (1985), J.Org.Chem; Timmerman et al (2005), ChemBioChem). Meloen and colleagues have used tris (bromomethyl) benzene and related molecules to rapidly and quantitatively cyclize multiple peptide loops onto synthetic scaffolds for structural simulation of protein surfaces (Timmerman et al (2005), ChemBiochem). Methods of producing drug candidate compounds are disclosed, for example, in WO 2004/077062 and WO 2006/078161, wherein the compounds are produced by attaching cysteine-containing polypeptides to a molecular scaffold (e.g., tris (bromomethyl) benzene).

Combinatorial methods based on phage display have been developed to generate and screen large libraries of bicyclic peptides against a target of interest (Heinis et al (2009), Nat Chem Biol 5(7), 502-7 and WO 2009/098450). Briefly, a linear peptide of six random amino acids (Cys- (Xaa) containing three cysteine residues and two regions was displayed on phage₆-Cys-(Xaa)₆-Cys) and cyclized by covalent attachment of the cysteine side chain to a small molecule (tris (bromomethyl) benzene).

Disclosure of Invention

According to a first aspect of the present invention there is provided a peptide ligand specific for CD38, comprising a polypeptide and an aromatic molecular scaffold, said polypeptide comprising at least three cysteine residues separated by at least two loop sequences, and said aromatic molecular scaffold forming covalent bonds with the cysteine residues of said polypeptide, thereby forming at least two polypeptide loops on said molecular scaffold.

According to another aspect of the present invention there is provided a drug conjugate comprising a peptide ligand as defined herein conjugated to one or more effectors and/or functional groups.

According to a further aspect of the invention there is provided a pharmaceutical composition comprising a peptide ligand or drug conjugate as defined herein in combination with one or more pharmaceutically acceptable excipients.

According to a further aspect of the present invention there is provided a peptide ligand or drug conjugate as defined herein for use in the prevention, inhibition or treatment of a disease or condition mediated by CD 38.

Drawings

FIG. 1: body weight change following administration of BT66BDC1 to female Balb/c nude mice bearing HT 1080. Data points represent group mean body weight. Error bars represent Standard Error (SEM) of the mean.

FIG. 2: tumor volume traces after administration of BT66BDC1 to female Balb/c nude mice bearing HT1080 xenografts. Data points represent group mean and error bars represent Standard Error (SEM) of mean.

FIG. 3: body weight change following administration of BT66BDC1 to female CB17-SCID mice carrying MOLP-8 xenografts. Data points represent group mean body weight. Error bars represent Standard Error (SEM) of the mean.

FIG. 4: tumor volume trajectories after administering BT66BDC1 to female CB17-SCID mice carrying MOLP-8 xenografts. Data points represent group mean and error bars represent Standard Error (SEM) of mean.

Detailed Description

In one embodiment, the loop sequence comprises 2,3,5,6 or 7 amino acids.

In another embodiment, the loop sequence comprises three cysteine residues separated by two loop sequences, one of which consists of 2 amino acids and the other of which consists of 7 amino acids.

In another embodiment, the loop sequence comprises three cysteine residues separated by two loop sequences, one of which consists of 5 amino acids and the other of which consists of 6 amino acids.

In another embodiment, the loop sequence comprises three cysteine residues separated by two loop sequences, both loop sequences consisting of 3 amino acids.

In another embodiment, the loop sequence comprises three cysteine residues separated by two loop sequences, both loop sequences consisting of 5 amino acids.

In another embodiment, the loop sequence comprises three cysteine residues separated by two loop sequences, both loop sequences consisting of 6 amino acids.

In one embodiment, the peptide ligand comprises an amino acid sequence selected from the group consisting of:

C_i-F-X₁-L-D-X₂-X₃-C_ii-F-D-X₄-X₅-X₆-X₇-C_iii(SEQ ID NO：87)；

C_i-I-R/N-Y-G/A-D/N-I-C_ii-X₁-D/H-P/T-D/E-X₂-X₃-C_iii(SEQ ID NO：88)；

C_i-F-X₁-L-D-G-E-C_ii-F-X₂-X₃-G/P-X₄-X₅-C_iii(SEQ ID NO：89)；

C_iVNFGSVC_iiWDPDSRC_iii(SEQ ID NO：2)；

C_i-X₁-X₂-C_ii-A-D-F/M-P-I-X₃-X₄-C_iii(SEQ ID NO：90)；

C_iDYC_iiVRLGLTGC_iii(SEQ ID NO：74)；

C_iGWC_iiSDQIDGFC_iii(SEQ ID NO：75)；

C_iAWC_iiSDPIDGFC_iii(SEQ ID NO：76)；

C_iDWC_iiIDPGVSFC_iii(SEQ ID NO：77)；

C_iSWC_iiVDDGLPFC_iii(SEQ ID NO：78)；

C_iTWC_iiVDDGLSFC_iii(SEQ ID NO：79)；

C_iTWC_iiVDDETWNC_iii(SEQ ID NO：80)；

C_iDYC_iiIRLGLTGC_iii(SEQ ID NO：81)；

C_iDWC_iiTDNIPGIC_iii(SEQ ID NO：82)；

C_i-A/N-W/F-L-C_ii-P/D-N/D-L-C_iii(SEQ ID NO：91)；

C_i-D-F-T-M-P-C_ii-X₁-X₂-W-X₃-X₄-C_iii(SEQ ID NO: 92); and

C_iIFDYDC_iiDAWSAC_iii(SEQ ID NO：28)；

or a pharmaceutically acceptable salt thereof, wherein X₁-X₆Represents any amino acid residue, X₇Is absent or represents any amino acid, and C_i、C_iiAnd C_iiiRepresenting the first, second and third cysteine residues, respectively.

In one embodiment, the loop sequence comprises three cysteine residues separated by two loop sequences, both of which consist of 6 amino acids, or one of which consists of 5 amino acids and the other of which consists of 6 amino acids, and the peptide ligand comprises an amino acid sequence selected from the group consisting of:

C_i-F-X₁-L-D-X₂-X₃-C_ii-F-D-X₄-X₅-X₆-X₇-C_iii(SEQ ID NO：87)；

C_i-I-R/N-Y-G/A-D/N-I-C_ii-X₁-D/H-P/T-D/E-X₂-X₃-C_iii(SEQ ID NO：88)；

C_i-F-X₁-L-D-G-E-C_ii-F-X₂-X₃-G/P-X₄-X₅-C_iii(SEQ ID NO: 89); and

C_iVNFGSVC_iiWDPDSRC_iii(SEQ ID NO：2)；

or a pharmaceutically acceptable salt thereof, wherein X₁-X₆Represents any amino acid residue, X₇Is absent or represents any amino acid, and C_i、C_iiAnd C_iiiRepresenting the first, second and third cysteine residues, respectively.

In one embodiment, the loop sequence comprises three cysteine residues separated by two loop sequences, the first of the two loop sequences consisting of 2 amino acids and the second of the two loop sequences consisting of 7 amino acids, and the peptide ligand comprises an amino acid sequence selected from the group consisting of:

C_i-X₁-X₂-C_ii-A-D-F/M-P-I-X₃-X₄-C_iii(SEQ ID NO：90)；

C_iDYC_iiVRLGLTGC_iii(SEQ ID NO：74)；

C_iGWC_iiSDQIDGFC_iii(SEQ ID NO：75)；

C_iAWC_iiSDPIDGFC_iii(SEQ ID NO：76)；

C_iDWC_iiIDPGVSFC_iii(SEQ ID NO：77)；

C_iSWC_iiVDDGLPFC_iii(SEQ ID NO：78)；

C_iTWC_iiVDDGLSFC_iii(SEQ ID NO：79)；

C_iTWC_iiVDDETWNC_iii(SEQ ID NO：80)；

C_iDYC_iiIRLGLTGC_iii(SEQ ID NO: 81); and

C_iDWC_iiTDNIPGIC_iii(SEQ ID NO：82)；

or a pharmaceutically acceptable salt thereof, wherein X₁-X₄Represents any amino acid residue, and C_i、C_iiAnd C_iiiRepresenting the first, second and third cysteine residues, respectively.

In one embodiment, the loop sequence comprises three cysteine residues separated by two loop sequences, both loop sequences consisting of 3 amino acids, and the peptide ligand comprises an amino acid sequence selected from the group consisting of:

C_i-A/N-W/F-L-C_ii-P/D-N/D-L-C_iii(SEQ ID NO：91)；

or a pharmaceutically acceptable salt thereof; wherein C is_i、C_iiAnd C_iiiRepresenting the first, second and third cysteine residues, respectively.

In one embodiment, the loop sequence comprises three cysteine residues separated by two loop sequences, both loop sequences consisting of 5 amino acids, and the peptide ligand comprises an amino acid sequence selected from the group consisting of:

C_i-D-F-T-M-P-C_ii-X₁-X₂-W-X₃-X₄-C_iii(SEQ ID NO: 92); and

C_iIFDYDC_iiDAWSAC_iii(SEQ ID NO：28)；

or a pharmaceutically acceptable salt thereof, wherein X₁-X₄Represents any amino acid residue, and C_i、C_iiAnd C_iiiRepresenting the first, second and third cysteine residues, respectively.

In another embodiment, C_i-F-X₁-L-D-X₂-X₃-C_ii-F-D-X₄-X₅-X₆-X₇-C_iiiPeptide ligands (SEQ ID NO: 87)Comprising an amino acid sequence selected from the group consisting of:

C_iFELDGTC_iiFDWAQEC_iii(SEQ ID NO：1)；

C_iFWLDGEC_iiFDWNHEC_iii(SEQ ID NO：29)；

C_iFHLDGEC_iiFDLENTC_iii(SEQ ID NO：30)；

C_iFSLDGEC_iiFDLSGEC_iii(SEQ ID NO：32)；

C_iFTLDGEC_iiFDWTHEC_iii(SEQ ID NO：33)；

C_iFKLDGVC_iiFDLFHEC_iii(SEQ ID NO：34)；

C_iFMLDGEC_iiFDLNKEC_iii(SEQ ID NO：35)；

C_iFKLDGEC_iiFDWTHEC_iii(SEQ ID NO：36)；

C_iFTLDGEC_iiFDWDAEC_iii(SEQ ID NO：37)；

C_iFELDGSC_iiFDFDHEC_iii(SEQ ID NO：38)；

C_iFTLDGEC_iiFDVNREC_iii(SEQ ID NO：39)；

C_iFWLDHEC_iiFDWTHEC_iii(SEQ ID NO：40)；

C_iFQLDGEC_iiFDIYREC_iii(SEQ ID NO：41)；

C_iFELDGNC_iiFDWTHEC_iii(SEQ ID NO：42)；

C_iFHLDGEC_iiFDYEHEC_iii(SEQ ID NO：43)；

C_iFSLDGEC_iiFDIASEC_iii(SEQ ID NO：44)；

C_iFQLDGEC_iiFDTSHEC_iii(SEQ ID NO：45)；

C_iFSLDGAC_iiFDWTHEC_iii(SEQ ID NO：46)；

C_iFVLDGEC_iiFDYYEEC_iii(SEQ ID NO：47)；

C_iFRLDDEC_iiFDWTHEC_iii(SEQ ID NO：48)；

C_iFRLDGVC_iiFDLDDEC_iii(SEQ ID NO：49)；

C_iFRLDGEC_iiFDMGQEC_iii(SEQ ID NO：50)；

C_iFTLDGAC_iiFDLDGEC_iii(SEQ ID NO：51)；

C_iFTLDGQC_iiFDWTHEC_iii(SEQ ID NO：52)；

C_iFLLDGEC_iiFDWMQEC_iii(SEQ ID NO：53)；

C_iFELDGDC_iiFDWTHEC_iii(SEQ ID NO：54)；

C_iFTLDGTC_iiFDWTHEC_iii(SEQ ID NO：55)；

C_iFHLDGVC_iiFDWTHEC_iii(SEQ ID NO：56)；

C_iFYLDGTC_iiFDWTHEC_iii(SEQ ID NO：57)；

C_iFLLDGEC_iiFDWAQEC_iii(SEQ ID NO：58)；

C_iFHLDGEC_iiFDLAKTC_iii(SEQ ID NO：59)；

C_iFTLDGEC_iiFDLDGWC_iii(SEQ ID NO：62)；

C_iFLLDGEC_iiFDLIGEC_iii(SEQ ID NO：63)；

C_iFWLDGEC_iiFDLGGQC_iii(SEQ ID NO：67)；

C_iFELDGEC_iiFDLDNQC_iii(SEQ ID NO：68)；

C_iFWLDGEC_iiFDLYGGC_iii(SEQ ID NO：69)；

C_iFRLDGEC_iiFDISNEC_iii(SEQ ID NO：70)；

C_iFWLDGEC_iiFDFGGC_iii(SEQ ID NO: 72); and

C_iFTLDGAC_iiFDWTHEC_iii(SEQ ID NO：73)；

or a pharmaceutically acceptable salt thereof; wherein C is_i、C_iiAnd C_iiiRepresenting the first, second and third cysteine residues, respectively.

In another embodiment, C_i-F-X₁-L-D-X₂-X₃-C_ii-F-D-X₄-X₅-X₆-X₇-C_iii(SEQ ID NO: 87) comprises an amino acid sequence selected from the group consisting of:

a- (SEQ ID NO: 1) -A (referred to herein as 66-01-N002);

a- (SEQ ID NO: 29) -A (referred to herein as 66-08-01-N001);

ac- (SEQ ID NO: 29) (referred to herein as 66-08-01-N004);

Ac-(SEQ ID NO：29)-A-Sar₆-K (referred to herein as 66-08-01-N005);

Ac-(SEQ ID NO：29)-A-Sar₆-k (dota) (referred to herein as 66-08-01-N016);

a- (SEQ ID NO: 30) -A (referred to herein as 66-08-44-N001);

Ac-A-(SEQ ID NO：30)-A-Sar₆-K (biotin) (referred to herein as 66-08-01-N003);

a- (SEQ ID NO: 32) -A (referred to herein as 66-08-02-N001);

a- (SEQ ID NO: 33) -A (referred to herein as 66-08-03-N001);

a- (SEQ ID NO: 34) -A (referred to herein as 66-08-04-N001);

Ac-A- (SEQ ID NO: 35) -A (referred to herein as 66-08-05-N001);

a- (SEQ ID NO: 36) -A (referred to herein as 66-08-06-N001);

a- (SEQ ID NO: 37) -A (referred to herein as 66-08-07-N001);

a- (SEQ ID NO: 38) -A (referred to herein as 66-08-09-N001);

a- (SEQ ID NO: 39) -A (referred to herein as 66-08-13-N001);

a- (SEQ ID NO: 40) -A (referred to herein as 66-08-15-N001);

a- (SEQ ID NO: 41) -A (referred to herein as 66-08-17-N001);

a- (SEQ ID NO: 42) -A (referred to herein as 66-08-18-N001);

a- (SEQ ID NO: 43) -A (referred to herein as 66-08-20-N001);

a- (SEQ ID NO: 44) -A (referred to herein as 66-08-22-N001);

a- (SEQ ID NO: 45) -A (referred to herein as 66-08-24-N001);

a- (SEQ ID NO: 46) -A (referred to herein as 66-08-26-N001);

a- (SEQ ID NO: 47) -A (referred to herein as 66-08-27-N001);

a- (SEQ ID NO: 48) -A (referred to herein as 66-08-28-N001);

a- (SEQ ID NO: 49) -A (referred to herein as 66-08-29-N001);

a- (SEQ ID NO: 50) -A (referred to herein as 66-08-30-N001);

a- (SEQ ID NO: 51) -A (referred to herein as 66-08-31-N001);

a- (SEQ ID NO: 52) -A (referred to herein as 66-08-32-N001);

a- (SEQ ID NO: 53) -A (referred to herein as 66-08-33-N001);

a- (SEQ ID NO: 54) -A (referred to herein as 66-08-34-N001);

a- (SEQ ID NO: 55) -A (referred to herein as 66-08-35-N001);

a- (SEQ ID NO: 56) -A (referred to herein as 66-08-36-N001);

a- (SEQ ID NO: 57) -A (referred to herein as 66-08-41-N001);

a- (SEQ ID NO: 58) -A (referred to herein as 66-08-43-N001);

a- (SEQ ID NO: 59) -A (referred to herein as 66-08-45-N001);

a- (SEQ ID NO: 62) -A (referred to herein as 66-08-48-N001);

a- (SEQ ID NO: 63) -A (referred to herein as 66-08-49-N001);

a- (SEQ ID NO: 67) -A (referred to herein as 66-08-53-N001);

a- (SEQ ID NO: 68) -A (referred to herein as 66-08-54-N001);

a- (SEQ ID NO: 69) -A (referred to herein as 66-08-55-N001);

a- (SEQ ID NO: 70) -A (referred to herein as 66-08-56-N001);

a- (SEQ ID NO: 72) -A (referred to herein as 66-08-58-N001); and

a- (SEQ ID NO: 73) -A (referred to herein as 66-08-N002).

In another embodiment, C_i-I-R/N-Y-G/A-D/N-I-C_ii-X₁-D/H-P/T-D/E-X₂-X₃-C_iii(SEQ ID NO: 88) comprises an amino acid sequence selected from the group consisting of:

C_iIRYGDIC_iiYDPDHSC_iii(SEQ ID NO：83)；

C_iIRYGDIC_iiFHPDYTC_iii(SEQ ID NO: 84); and

C_iINYANIC_iiLDTEKMC_iii(SEQ ID NO：85)；

or a pharmaceutically acceptable salt thereof; wherein C is_i、C_iiAnd C_iiiRepresenting the first, second and third cysteine residues, respectively.

In another embodiment, C_i-I-R/N-Y-G/A-D/N-I-C_ii-X₁-D/H-P/T-D/E-X₂-X₃-C_iii(SEQ ID NO: 88) comprises an amino acid sequence selected from the group consisting of:

a- (SEQ ID NO: 83) -A (referred to herein as 66-17-01-N001);

a- (SEQ ID NO: 84) -A (referred to herein as 66-17-N001); and

a- (SEQ ID NO: 85) -A (referred to herein as 66-18-N001).

In another embodiment, C_i-F-X₁-L-D-G-E-C_ii-F-X₂-X₃-G/P-X₄-X₅-C_iii(SEQ ID NO: 89) comprises an amino acid sequence selected from the group consisting of:

C_iFELDGEC_iiFHFGEPC_iii(SEQ ID NO：60)；

C_iFVLDGEC_iiFEIGERC_iii(SEQ ID NO：61)；

C_iFELDGEC_iiFSFPGTC_iii(SEQ ID NO：64)；

C_iFELDGEC_iiFSWPYPC_iii(SEQ ID NO：65)；

C_iFTLDGEC_iiFLLGENC_iii(SEQ ID NO: 66); and

C_iFELDGEC_iiFNIGSKC_iii(SEQ ID NO：71)；

or a pharmaceutically acceptable salt thereof; wherein C is_i、C_iiAnd C_iiiRepresenting the first, second and third cysteine residues, respectively.

In another embodiment, C_i-F-X₁-L-D-G-E-C_ii-F-X₂-X₃-G/P-X₄-X₅-C_iii(SEQ ID NO: 89) comprises an amino acid sequence selected from the group consisting of:

a- (SEQ ID NO: 60) -A (referred to herein as 66-08-46-N001);

a- (SEQ ID NO: 61) -A (referred to herein as 66-08-47-N001);

a- (SEQ ID NO: 64) -A (referred to herein as 66-08-50-N001);

a- (SEQ ID NO: 65) -A (referred to herein as 66-08-51-N001);

a- (SEQ ID NO: 66) -A (referred to herein as 66-08-52-N001); and

a- (SEQ ID NO: 71) -A (referred to herein as 66-08-57-N001).

In one embodiment, C_iVNFGSVC_iiWDPDSRC_iii(SEQ ID NO: 2) comprises an amino acid sequence selected from the group consisting of:

a- (SEQ ID NO: 2) -A (referred to herein as 66-02-N002).

In another embodiment, C_i-X₁-X₂-C_ii-A-D-F/M-P-I-X₃-X₄-C_iii(SEQ ID NO: 90) peptide ligands comprise C_i-X₁-X₂-C_ii-A-D-F-P-I-X₃-X₄-C_iiiPeptide ligands (SEQ ID NO: 91)。

In another embodiment, C_i-X₁-X₂-A-D-F/M-C_ii-P-I-X₃-X₄-C_iii(SEQ ID NO: 90) comprises an amino acid sequence selected from the group consisting of:

C_iVPC_iiADFPIWYC_iii(SEQ ID NO：3)；

C_iVPC_iiADFPIWWC_iii(SEQ ID NO：4)；

C_iTPC_iiADFPIWSC_iii(SEQ ID NO：5)；

C_iTPC_iiADFPIHTC_iii(SEQ ID NO：6)；

C_iVHC_iiADFPIWGC_iii(SEQ ID NO：7)；

C_iVPC_iiADFPIWGC_iii(SEQ ID NO：8)；

C_iVMC_iiADFPIWGC_iii(SEQ ID NO：9)；

C_iTPC_iiADFPIWYC_iii(SEQ ID NO：10)；

C_iTPC_iiADFPILTC_iii(SEQ ID NO：11)；

C_iVAC_iiADFPIWGC_iii(SEQ ID NO：12)；

C_iTPC_iiADFPIYGC_iii(SEQ ID NO：13)；

C_iTPC_iiADFPILDC_iii(SEQ ID NO：14)；

C_iVKC_iiADFPIWGC_iii(SEQ ID NO：15)；

C_iTPC_iiADMPIWTC_iii(SEQ ID NO：16)；

C_iIPC_iiADFPIWGC_iii(SEQ ID NO：17)；

C_iIPC_iiADFPISVC_iii(SEQ ID NO：18)；

C_iVPC_iiADFPISFC_iii(SEQ ID NO：19)；

C_iIPC_iiADFPISFC_iii(SEQ ID NO：20)；

C_iVPC_iiADFPISVC_iii(SEQ ID NO：21)；

C_iVPC_iiADFPIFTC_iii(SEQ ID NO：22)；

C_iIPC_iiADFPIFTC_iii(SEQ ID NO: 23); and

C_iTPC_iiADFPIWGC_iii(SEQ ID NO：24)；

or a pharmaceutically acceptable salt thereof, wherein C_i、C_iiAnd C_iiiRepresenting the first, second and third cysteine residues, respectively.

In one embodiment, C_i-X₁-X₂-A-D-F/M-C_ii-P-I-X₃-X₄-C_iii(SEQ ID NO: 90) or a peptide ligand of SEQ ID NO: 74-82 comprises an amino acid sequence selected from the group consisting of:

a- (SEQ ID NO: 3) -A (referred to herein as 66-03-00-N004);

(β-Ala)-Sar₁₀-A- (SEQ ID NO: 3) (referred to herein as 66-03-00-N006);

DOTA-(β-Ala)-Sar₁₀-A- (SEQ ID NO: 3) (referred to herein as 66-03-00-N007);

Ac-(SEQ ID NO：3)-A-Sar₆-K (referred to herein as 66-03-00-N008);

Ac-(SEQ ID NO：3)-A-Sar₆- (PG) (referred to herein as 66-03-00-N009);

a- (SEQ ID NO: 4) -A (referred to herein as 66-03-00-N005);

a- (SEQ ID NO: 5) -A (referred to herein as 66-03-01-N001);

a- (SEQ ID NO: 6) -A (referred to herein as 66-03-02-N001);

a- (SEQ ID NO: 7) -A (referred to herein as 66-03-03-N001);

a- (SEQ ID NO: 8) -A (referred to herein as 66-03-04-N001);

ac- (SEQ ID NO: 8) (referred to herein as 66-03-04-N002);

a- (SEQ ID NO: 9) -A (referred to herein as 66-03-05-N001);

a- (SEQ ID NO: 10) -A (referred to herein as 66-03-06-N001);

ac- (SEQ ID NO: 10) (referred to herein as 66-03-06-N002);

a- (SEQ ID NO: 11) -A (referred to herein as 66-03-07-N001);

a- (SEQ ID NO: 12) -A (referred to herein as 66-03-08-N001);

a- (SEQ ID NO: 13) -A (referred to herein as 66-03-09-N001);

a- (SEQ ID NO: 14) -A (referred to herein as 66-03-10-N001);

a- (SEQ ID NO: 15) -A (referred to herein as 66-03-11-N001);

ac- (SEQ ID NO: 15) (referred to herein as 66-03-11-N002);

a- (SEQ ID NO: 16) -A (referred to herein as 66-03-15-N001);

a- (SEQ ID NO: 17) -A (referred to herein as 66-03-16-N001);

a- (SEQ ID NO: 18) -A (referred to herein as 66-03-24-N003);

a- (SEQ ID NO: 19) -A (referred to herein as 66-03-25-N002);

a- (SEQ ID NO: 20) -A (referred to herein as 66-03-26-N003);

a- (SEQ ID NO: 21) -A (referred to herein as 66-03-27-N003);

a- (SEQ ID NO: 22) -A (referred to herein as 66-03-28-N002);

a- (SEQ ID NO: 23) -A (referred to herein as 66-03-29-N003);

a- (SEQ ID NO: 24) -A (referred to herein as 66-03-N002);

A-(SEQ ID NO：24)-A-Sar₆-K (biotin) (referred to herein as 66-03-N003);

a- (SEQ ID NO: 74) -A (referred to herein as 66-09-N001);

A-(SEQ ID NO：75)-A-Sar₆-K (biotin) (referred to herein as 66-10-01-N001);

A-(SEQ ID NO：76)-A-Sar₆-K (biotin) (referred to herein as 66-10-02-N001);

A-(SEQ ID NO：77)-A-Sar₆-K (biotin) (referred to herein as 66-10-03-N001);

A-(SEQ ID NO：78)-A-Sar₆-K (biotin) (referred to herein as 66-10-04-N001);

a- (SEQ ID NO: 79) -A (referred to herein as 66-10-N001);

a- (SEQ ID NO: 80) -A (referred to herein as 66-11-N001);

a- (SEQ ID NO: 81) -A (referred to herein as 66-12-N001); and

a- (SEQ ID NO: 82) -A (referred to herein as 66-13-N001).

In another embodiment, C_i-A/N-W/F-L-C_ii-P/D-N/D-L-C_iii(SEQ ID NO: 91) comprises an amino acid sequence selected from the group consisting of:

C_iAWLC_iiPNLC_iii(SEQ ID NO: 31); and

C_iNFLC_iiDDLC_iii(SEQ ID NO：86)；

or a pharmaceutically acceptable salt thereof; wherein C is_i、C_iiAnd C_iiiRepresenting the first, second and third cysteine residues, respectively.

In another embodiment, C_i-A/N-W/F-L-C_ii-P/D-N/D-L-C_iii(SEQ ID NO: 91) comprises an amino acid sequence selected from the group consisting of:

SSQHG-(SEQ ID NO：31)-A-Sar₆-K (referred to herein as 66-08-01-N006); and

RHSNY-(SEQ ID NO：86)-A-Sar₆-K (biotin) (referred to herein as 66-20-00-T001-N001).

In another embodiment, C_i-D-F-T-M-P-C_ii-X₁-X₂-W-X₃-X₄-C_iii(SEQ ID NO: 92) comprises an amino acid sequence selected from the group consisting of:

C_iDFTMPC_iiENWKYC_iii(SEQ ID NO：25)；

C_iDFTMPC_iiPNWNAC_iii(SEQ ID NO: 26); and

C_iDFTMPC_iiQMWEQC_iii(SEQ ID NO：27)；

or a pharmaceutically acceptable salt thereof; wherein C is_i、C_iiAnd C_iiiRepresenting the first, second and third cysteine residues, respectively.

In another embodiment, C_i-D-F-T-M-P-C_ii-X₁-X₂-W-X₃-X₄-C_iiiPeptide ligand of (SEQ ID NO: 92) or C_iIFDYDC_iiDAWSAC_iii(SEQ ID NO: 28) comprises an amino acid sequence selected from the group consisting of:

a- (SEQ ID NO: 25) -A (referred to herein as 66-05-07-N001);

a- (SEQ ID NO: 26) -A (referred to herein as 66-05-09-N001);

a- (SEQ ID NO: 27) -A (referred to herein as 66-05-N002); and

a- (SEQ ID NO: 28) -A (referred to herein as 66-06-N002).

In one embodiment, the molecular scaffold is selected from 1,3, 5-tris (bromomethyl) benzene (TBMB), and the peptide ligand comprises an amino acid sequence selected from:

a- (SEQ ID NO: 1) -A (referred to herein as 66-01-N002);

a- (SEQ ID NO: 2) -A (referred to herein as 66-02-N002);

a- (SEQ ID NO: 3) -A (referred to herein as 66-03-00-N004);

(β-Ala)-Sar₁₀-A- (SEQ ID NO: 3) (referred to herein as 66-03-00-N006);

DOTA-(β-Ala)-Sar₁₀-A- (SEQ ID NO: 3) (referred to herein as 66-03-00-N007);

Ac-(SEQ ID NO：3)-A-Sar₆-K (referred to herein as 66-03-00-N008);

Ac-(SEQ ID NO：3)-A-Sar₆- (PG) (referred to herein as 66-03-00-N009);

a- (SEQ ID NO: 4) -A (referred to herein as 66-03-00-N005);

a- (SEQ ID NO: 5) -A (referred to herein as 66-03-01-N001);

a- (SEQ ID NO: 6) -A (referred to herein as 66-03-02-N001);

a- (SEQ ID NO: 7) -A (referred to herein as 66-03-03-N001);

a- (SEQ ID NO: 8) -A (referred to herein as 66-03-04-N001);

ac- (SEQ ID NO: 8) (referred to herein as 66-03-04-N002);

a- (SEQ ID NO: 9) -A (referred to herein as 66-03-05-N001);

a- (SEQ ID NO: 10) -A (referred to herein as 66-03-06-N001);

ac- (SEQ ID NO: 10) (referred to herein as 66-03-06-N002);

a- (SEQ ID NO: 11) -A (referred to herein as 66-03-07-N001);

a- (SEQ ID NO: 12) -A (referred to herein as 66-03-08-N001);

a- (SEQ ID NO: 13) -A (referred to herein as 66-03-09-N001);

a- (SEQ ID NO: 14) -A (referred to herein as 66-03-10-N001);

a- (SEQ ID NO: 15) -A (referred to herein as 66-03-11-N001);

ac- (SEQ ID NO: 15) (referred to herein as 66-03-11-N002);

a- (SEQ ID NO: 16) -A (referred to herein as 66-03-15-N001);

a- (SEQ ID NO: 17) -A (referred to herein as 66-03-16-N001);

a- (SEQ ID NO: 18) -A (referred to herein as 66-03-24-N003);

a- (SEQ ID NO: 19) -A (referred to herein as 66-03-25-N002);

a- (SEQ ID NO: 20) -A (referred to herein as 66-03-26-N003);

a- (SEQ ID NO: 21) -A (referred to herein as 66-03-27-N003);

a- (SEQ ID NO: 22) -A (referred to herein as 66-03-28-N002);

a- (SEQ ID NO: 23) -A (referred to herein as 66-03-29-N003);

a- (SEQ ID NO: 24) -A (referred to herein as 66-03-N002);

A-(SEQ ID NO：24)-A-Sar₆-K (biotin) (referred to herein as 66-03-N003);

a- (SEQ ID NO: 25) -A (referred to herein as 66-05-07-N001);

a- (SEQ ID NO: 26) -A (referred to herein as 66-05-09-N001);

a- (SEQ ID NO: 27) -A (referred to herein as 66-05-N002);

a- (SEQ ID NO: 28) -A (referred to herein as 66-06-N002);

a- (SEQ ID NO: 29) -A (referred to herein as 66-08-01-N001);

ac- (SEQ ID NO: 29) (referred to herein as 66-08-01-N004);

Ac-(SEQ ID NO：29)-A-Sar₆-K (referred to herein as 66-08-01-N005);

Ac-(SEQ ID NO：29)-A-Sar₆-k (dota) (referred to herein as 66-08-01-N016);

Ac-A-(SEQ ID NO：30)-A-Sar₆-K (biotin) (referred to herein as 66-08-01-N003);

a- (SEQ ID NO: 30) -A (referred to herein as 66-08-44-N001);

SSQHG-(SEQ ID NO：31)-A-Sar₆-K (referred to herein as 66-08-01-N006);

a- (SEQ ID NO: 32) -A (referred to herein as 66-08-02-N001);

a- (SEQ ID NO: 33) -A (referred to herein as 66-08-03-N001);

a- (SEQ ID NO: 34) -A (referred to herein as 66-08-04-N001);

Ac-A- (SEQ ID NO: 35) -A (referred to herein as 66-08-05-N001);

a- (SEQ ID NO: 36) -A (referred to herein as 66-08-06-N001);

a- (SEQ ID NO: 37) -A (referred to herein as 66-08-07-N001);

a- (SEQ ID NO: 38) -A (referred to herein as 66-08-09-N001);

a- (SEQ ID NO: 39) -A (referred to herein as 66-08-13-N001);

a- (SEQ ID NO: 40) -A (referred to herein as 66-08-15-N001);

a- (SEQ ID NO: 41) -A (referred to herein as 66-08-17-N001);

a- (SEQ ID NO: 42) -A (referred to herein as 66-08-18-N001);

a- (SEQ ID NO: 43) -A (referred to herein as 66-08-20-N001);

a- (SEQ ID NO: 44) -A (referred to herein as 66-08-22-N001);

a- (SEQ ID NO: 45) -A (referred to herein as 66-08-24-N001);

a- (SEQ ID NO: 46) -A (referred to herein as 66-08-26-N001);

a- (SEQ ID NO: 47) -A (referred to herein as 66-08-27-N001);

a- (SEQ ID NO: 48) -A (referred to herein as 66-08-28-N001);

a- (SEQ ID NO: 49) -A (referred to herein as 66-08-29-N001);

a- (SEQ ID NO: 50) -A (referred to herein as 66-08-30-N001);

a- (SEQ ID NO: 51) -A (referred to herein as 66-08-31-N001);

a- (SEQ ID NO: 52) -A (referred to herein as 66-08-32-N001);

a- (SEQ ID NO: 53) -A (referred to herein as 66-08-33-N001);

a- (SEQ ID NO: 54) -A (referred to herein as 66-08-34-N001);

a- (SEQ ID NO: 55) -A (referred to herein as 66-08-35-N001);

a- (SEQ ID NO: 56) -A (referred to herein as 66-08-36-N001);

a- (SEQ ID NO: 57) -A (referred to herein as 66-08-41-N001);

a- (SEQ ID NO: 58) -A (referred to herein as 66-08-43-N001);

a- (SEQ ID NO: 59) -A (referred to herein as 66-08-45-N001);

a- (SEQ ID NO: 60) -A (referred to herein as 66-08-46-N001);

a- (SEQ ID NO: 61) -A (referred to herein as 66-08-47-N001);

a- (SEQ ID NO: 62) -A (referred to herein as 66-08-48-N001);

a- (SEQ ID NO: 63) -A (referred to herein as 66-08-49-N001);

a- (SEQ ID NO: 64) -A (referred to herein as 66-08-50-N001);

a- (SEQ ID NO: 65) -A (referred to herein as 66-08-51-N001);

a- (SEQ ID NO: 66) -A (referred to herein as 66-08-52-N001);

a- (SEQ ID NO: 67) -A (referred to herein as 66-08-53-N001);

a- (SEQ ID NO: 68) -A (referred to herein as 66-08-54-N001);

a- (SEQ ID NO: 69) -A (referred to herein as 66-08-55-N001);

a- (SEQ ID NO: 70) -A (referred to herein as 66-08-56-N001);

a- (SEQ ID NO: 71) -A (referred to herein as 66-08-57-N001);

a- (SEQ ID NO: 72) -A (referred to herein as 66-08-58-N001);

a- (SEQ ID NO: 73) -A (referred to herein as 66-08-N002);

a- (SEQ ID NO: 74) -A (referred to herein as 66-09-N001);

A-(SEQ ID NO：75)-A-Sar₆-K (biotin) (referred to herein as 66-10-01-N001);

A-(SEQ ID NO：76)-A-Sar₆-K (biotin) (referred to herein as 66-10-02-N001);

A-(SEQ ID NO：77)-A-Sar₆-K (biotin) (referred to herein as 66-10-03-N001);

A-(SEQ ID NO：78)-A-Sar₆-K (biotin) (referred to herein as 66-10-04-N001);

a- (SEQ ID NO: 79) -A (referred to herein as 66-10-N001);

a- (SEQ ID NO: 80) -A (referred to herein as 66-11-N001);

a- (SEQ ID NO: 81) -A (referred to herein as 66-12-N001);

a- (SEQ ID NO: 82) -A (referred to herein as 66-13-N001);

a- (SEQ ID NO: 83) -A (referred to herein as 66-17-01-N001);

a- (SEQ ID NO: 84) -A (referred to herein as 66-17-N001);

a- (SEQ ID NO: 85) -A (referred to herein as 66-18-N001); and

RHSNY-(SEQ ID NO：86)-A-Sar₆-K (biotin) (referred to herein as 66-20-00-T001-N001).

In another embodiment, the molecular scaffold is selected from 1,3, 5-tris (bromomethyl) benzene (TBMB), and the peptide ligand comprises an amino acid sequence selected from the group consisting of:

a- (SEQ ID NO: 3) -A (referred to herein as 66-03-00-N004);

(β-Ala)-Sar₁₀-A- (SEQ ID NO: 3) (referred to herein as 66-03-00-N006);

DOTA-(β-Ala)-Sar₁₀-A- (SEQ ID NO: 3) (referred to herein as 66-03-00-N007);

Ac-(SEQ ID NO：3)-A-Sar₆-K (referred to herein as 66-03-00-N008);

Ac-(SEQ ID NO：3)-A-Sar₆- (PG) (referred to herein as 66-03-00-N009);

a- (SEQ ID NO: 7) -A (referred to herein as 66-03-03-N001);

a- (SEQ ID NO: 8) -A (referred to herein as 66-03-04-N001);

a- (SEQ ID NO: 10) -A (referred to herein as 66-03-06-N001);

a- (SEQ ID NO: 15) -A (referred to herein as 66-03-11-N001);

a- (SEQ ID NO: 22) -A (referred to herein as 66-03-28-N002);

a- (SEQ ID NO: 29) -A (referred to herein as 66-08-01-N001);

ac- (SEQ ID NO: 29) (referred to herein as 66-08-01-N004);

Ac-(SEQ ID NO：29)-A-Sar₆-k (dota) (referred to herein as 66-08-01-N016); and

Ac-A-(SEQ ID NO：30)-A-Sar₆-K (biotin) (referred to herein as 66-08-01-N003).

As shown in table 1 herein, the scaffold/peptide ligands of this embodiment showed excellent competitive binding of CD 38.

In yet another embodiment, the molecular scaffold is selected from 1,3, 5-tris (bromomethyl) benzene (TBMB), and the peptide ligand comprises an amino acid sequence selected from the group consisting of:

(β-Ala)-Sar₁₀-A- (SEQ ID NO: 3) (referred to herein as 66-03-00-N006);

the scaffold/peptide ligands of this embodiment, alone, and when conjugated to toxin DM-1 (as shown in table 2 herein), showed excellent competitive binding of integrin CD38 (as shown in table 1 herein).

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in the fields of peptide chemistry, cell culture and phage display, nucleic acid chemistry and biochemistry). Standard techniques are used for Molecular Biology, genetic and biochemical approaches (see Sambrook et al, Molecular Cloning: A Laboratory Manual, 3 rd edition, 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al, Short Protocols in Molecular Biology (1999) 4 th edition, John Wiley & Sons, Inc.), which are incorporated herein by reference.

Nomenclature

Numbering

When referring to amino acid residue positions within the peptides of the invention, due to cysteine residues (C)_i、C_iiAnd C_iii) Are invariant and thus the cysteine residues are omitted from the numbering, so the numbering of the amino acid residues within the peptides of the invention is as follows:

-C_i-F₁-E₂-L₃-D₄-G₅-T₆-C_ii-F₇-D₈-W₉-A₁₀-Q₁₁-E₁₂-C_iii-(SEQ ID NO：1)。

for the purposes of this specification, it is assumed that all bicyclic peptides are cyclized by TBMB (1,3, 5-tris (bromomethyl) benzene) and generate a trisubstituted structure. Cyclization with TBMB takes place at C_i、C_iiAnd C_iiiThe above.

Molecular formula

N-or C-terminal extensions of the bicyclic core sequence are added to the left or right side of the sequence, separated by hyphens. For example, the N-terminal β Ala-Sar10-Ala tail will be expressed as:

βAla-Sar10-A-(SEQ ID NO：X)。

reverse peptide sequence

It is envisaged that the peptide sequences disclosed herein will also be used in their reverse form, as disclosed in Nair et al (2003) J Immunol 170(3), 1362-1373. For example, the sequence is inverted (i.e., N-terminal to C-terminal and vice versa), their stereochemistry is also inverted (i.e., D-amino acid to L-amino acid and vice versa).

Peptide ligands

As referred to herein, a peptide ligand refers to a peptide covalently bound to a molecular scaffold.Typically, such peptides comprise two or more reactive groups (i.e., cysteine residues) capable of forming covalent bonds with the scaffold, and a sequence that is opposite (subtensed) between the reactive groups, since the sequence forms a loop when the peptide is bound to the scaffold, it is referred to as a loop sequence. In the present case, the peptide comprises at least three cysteine residues (referred to herein as C)_i、C_iiAnd C_iii) And forming at least two rings on the stent.

Advantages of peptide ligands

Certain bicyclic peptides of the present invention have a number of advantageous properties that enable them to be considered suitable drug-like molecules for injection, inhalation, nasal, ocular, oral or topical administration. These advantageous properties include:

species cross-reactivity. This is a typical requirement for preclinical pharmacodynamic and pharmacokinetic assessments;

-protease stability. Bicyclic peptide ligands ideally should exhibit stability to plasma proteases, epithelial ("membrane-anchored") proteases, gastric and intestinal proteases, lung surface proteases, intracellular proteases, and the like. Protease stability should be maintained between different species so that bicyclic lead candidates can be developed in animal models and administered with confidence to humans;

-ideal solubility curve. This is a function of the ratio of charged hydrophilic residues to hydrophobic residues and intramolecular/intermolecular H bonds, which are important for formulation and absorption purposes;

optimal plasma half-life in circulation. Depending on the clinical indication and treatment regimen, it may be desirable to develop bicyclic peptides for short-term exposure in an acute disease management setting, or to develop bicyclic peptides with enhanced retention in circulation, so as to be optimal for management of more chronic disease states. Other factors driving the desired plasma half-life are the requirement for sustained exposure for maximum therapeutic efficiency versus the concomitant toxicology caused by sustained exposure of the agent; and

-selectivity. Certain peptide ligands of the invention show good selectivity compared to other CDs.

Pharmaceutically acceptable salts

It is understood that salt forms are within the scope of the invention and reference to peptide ligands includes salt forms of the ligands.

Salts of the invention may be synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods, for example, as described in Pharmaceutical Salts: properties, Selection, and Use, p.heinrich Stahl (eds.), camile g.wermuth (eds.) ISBN: 3-90639-026-8, hardcover 388, 8 months 2002. In general, these salts can be prepared by reacting the free acid or base forms of these compounds with the appropriate base or acid in water, or in an organic solvent, or in a mixture of the two.

Acid addition salts (mono-or di-salts) can be formed with a variety of acids (inorganic and organic). Examples of acid addition salts include mono-or di-salts with acids selected from: acetic acid, 2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid (e.g., L-ascorbic acid), L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, butyric acid, (+) camphoric acid, camphorsulfonic acid, (+) - (1S) -camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1, 2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, hemi-lactic acid, gentisic acid, glucoheptonic acid, D-gluconic acid, glucuronic acid (e.g., D-glucuronic acid), glutamic acid (e.g., L-glutamic acid), alpha-oxoglutaric acid, glycolic acid, hippuric acid, hydrohalic acid (e.g., hydrobromic acid, hydrochloric acid, citric acid, malic acid, citric acid, malic acid, citric acid, malic acid, citric acid, and citric acid, malic acid, citric acid, malic acid, citric acid, malic acid, citric acid, and citric acid, malic acid, citric acid, hydroiodic acid), isethionic acid, lactic acid (e.g., (+) -L-lactic acid, (+ -) -DL-lactic acid), lactobionic acid, maleic acid, malic acid, (-) -L-malic acid, malonic acid, (+ -) -DL-mandelic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1, 5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, pyruvic acid, L-pyroglutamic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, (+) -L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, undecylenic acid and valeric acid, as well as acylated amino acids and cation exchange resins.

One particular group of salts consists of the salts formed as follows: acetic acid, hydrochloric acid, hydroiodic acid, phosphoric acid, nitric acid, sulfuric acid, citric acid, lactic acid, succinic acid, maleic acid, malic acid, isethionic acid, fumaric acid, benzenesulfonic acid, toluenesulfonic acid, sulfuric acid, methanesulfonic acid (methanesulfonate), ethanesulfonic acid, naphthalenesulfonic acid, valeric acid, propionic acid, butyric acid, malonic acid, glucuronic acid and lactobionic acid. One specific salt is the hydrochloride salt. Another specific salt is acetate.

If the compound is an anionic compound, or has a functional group which may be anionic (for example, -COOH may be-COO)^-) Salts may be formed with organic or inorganic bases to produce suitable cations. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions, such as Li⁺、Na⁺And K⁺(ii) a Alkaline earth metal cations, e.g. Ca²⁺And Mg²⁺(ii) a And other cations, e.g. Al³⁺Or Zn⁺. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH)₄ ⁺) And substituted ammonium ions (e.g., NH)₃R⁺、NH₂R₂ ⁺、NHR₃ ⁺、NR₄ ⁺). Some examples of suitable substituted ammonium ions are those derived from: methylamine, ethylamine, diethylamine, propylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine and tromethamine, and amino acids such as lysine and arginine. An example of a common quaternary ammonium ion is N (CH)₃)₄ ⁺。

When the peptides of the invention contain amine functional groups, these may form quaternary ammonium salts, for example by reaction with alkylating agents according to methods well known to those skilled in the art. Such quaternary ammonium compounds are within the scope of the peptides of the invention.

Modified derivatives

It will be appreciated that modified derivatives of the peptide ligands defined herein are within the scope of the invention. Examples of such suitable modified derivatives include one or more modifications selected from: n-terminal and/or C-terminal modifications; substitution of one or more amino acid residues with one or more unnatural amino acid residue (e.g., substitution of one or more polar amino acid residues with one or more allelic or isoelectronic amino acids; substitution of one or more nonpolar amino acid residues with other unnatural allelic or isoelectronic amino acids); adding a spacer group; replacing one or more oxidation-sensitive amino acid residues with one or more oxidation-resistant amino acid residues; (ii) one or more amino acid residues are replaced with alanine, one or more L-amino acid residues are replaced with one or more D-amino acid residues; n-alkylation of one or more amide bonds within bicyclic peptide ligands; replacing one or more peptide bonds with an alternative bond; peptide backbone length modification; substitution of one or more amino acid residues for a hydrogen on the alpha-carbon with another chemical group, modification of amino acids (e.g., cysteine, lysine, glutamic/aspartic acids, and tyrosine) with suitable amine, thiol, carboxylic acid, and phenol reactive reagents to functionalize the amino acids, and introduction or substitution of amino acids that bring orthogonal reactivity suitable for functionalization, such as amino acids with an azide or alkyne group, which respectively allow functionalization with alkyne or azide-bearing moieties.

In one embodiment, the modified derivative comprises an N-terminal and/or C-terminal modification. In another embodiment, wherein the modified derivative comprises an N-terminal modification using a suitable amino reaction chemistry, and/or a C-terminal modification using a suitable carboxy reaction chemistry. In another embodiment, the N-terminal or C-terminal modification comprises the addition of an effector group including, but not limited to, a cytotoxic agent, a radio-chelator, or a chromophore.

In another embodiment, the modified derivative comprises an N-terminal modification. In another embodiment, the N-terminal modification comprises an N-terminal acetyl group. In this embodiment, the N-terminal cysteine group (referred to herein as C) is present during peptide synthesis_iWith acetic anhydride or other suitable reagent, thereby producing an N-terminally acetylated molecule. This embodiment offers the advantage of removing potential recognition points for aminopeptidases, avoiding the possibility of degradation of bicyclic peptides.

In an alternative embodiment, the N-terminal modification comprises the addition of a molecular spacer group that facilitates conjugation of the effector group and retains the potency of the bicyclic peptide on its target.

In another embodiment, the modified derivative comprises a C-terminal modification. In another embodiment, the C-terminal modification comprises an amide group. In this embodiment, the C-terminal cysteine group (referred to herein as C)_iiiGroup (C) is synthesized during peptide synthesis as an amide, thereby producing a molecule whose C-terminus is amidated. This embodiment provides the advantage of removing the potential recognition site for carboxypeptidases, reducing the possibility of proteolytic degradation of bicyclic peptides.

In one embodiment, the modified derivative comprises the replacement of one or more amino acid residues with one or more non-natural amino acid residues. In this embodiment, unnatural amino acids with allelic/iso-electronic side chains can be selected that are neither recognized by degrading proteases nor have any adverse effect on target potency.

Alternatively, unnatural amino acids with constrained amino acid side chains can be used such that proteolytic hydrolysis of nearby peptide bonds is conformationally and sterically hindered. In particular, this relates to proline analogues, bulky side chains, C α -disubstituted derivatives (e.g. aminoisobutyric acid, Aib) and cyclic amino acids, the simple derivatives being amino-cyclopropyl carboxylic acids.

In one embodiment, the modified derivative comprises the addition of a spacer group. In another embodiment, the modified derivative comprises a cysteine (C) towards the N-terminus_i) And/or a C-terminal cysteine (C)_iii) A spacer group is added.

In one embodiment, the modified derivative comprises the replacement of one or more oxidation-sensitive amino acid residues with one or more oxidation-resistant amino acid residues. In another embodiment, the modified derivative comprises replacing a tryptophan residue with a naphthylalanine or alanine residue. This embodiment provides the advantage of improving the drug stability profile of the resulting bicyclic peptide ligands.

In one embodiment, the modified derivative comprises the replacement of one or more charged amino acid residues with one or more hydrophobic amino acid residues. In alternative embodiments, the modified derivative comprises the replacement of one or more hydrophobic amino acid residues with one or more charged amino acid residues. The correct balance of charged amino acid residues and hydrophobic amino acid residues is an important feature of bicyclic peptide ligands. For example, hydrophobic amino acid residues affect the degree of plasma protein binding and thus the concentration of the available free fraction in plasma, whereas charged amino acid residues (in particular arginine) may affect the interaction of peptides with cell surface phospholipid membranes. The combination of the two may affect the half-life, volume of distribution and exposure of the peptide drug and may be adjusted according to the clinical endpoint. In addition, the correct combination and number of charged amino acid residues and hydrophobic amino acid residues (if the peptide drug has been administered subcutaneously) can reduce irritation at the injection site.

In one embodiment, the modified derivative comprises the replacement of one or more L-amino acid residues with one or more D-amino acid residues. This embodiment is believed to increase proteolytic stability by steric hindrance and the propensity to stabilize the beta turn conformation by D-amino acids (Tugyi et al (2005) PNAS, 102(2), 413-418).

In one embodiment, the modified derivative comprises removing any amino acid residues and substituting with alanine. This embodiment provides the advantage of removing potential proteolytic attack sites.

It should be noted that each of the above-mentioned modifications is used to intentionally improve the efficacy or stability of the peptide. Further efficacy improvement based on modification can be achieved by the following mechanism:

-introducing hydrophobic moieties that exploit the hydrophobic effect and reduce the dissociation rate, thereby achieving higher affinity;

introduction of charged groups, which utilize long range ionic interactions, leading to faster rates and higher affinities (see e.g. Schreiber et al Rapid, electronically associated association of proteins (1996), Nature struct. biol.3, 427-31); and

introducing other constraints into the peptide, for example by correctly constraining the side chains of the amino acids so that the loss of entropy upon target binding is minimal, limiting the twist angle of the backbone so that the loss of entropy upon target binding is minimal, and introducing other circularizations in the molecule for the same reason.

(for review see Gentilucci et al, Current pharmaceutical Design, (2010), 16, 3185-.

Isotopic variation

The present invention includes all pharmaceutically acceptable (radio) isotopically-labelled peptide ligands of the present invention in which one or more atoms are replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature; the peptide ligands of the invention, wherein the attached metal chelating group (termed "effector") is capable of retaining the relevant (radioactive) isotope; and peptide ligands of the invention in which certain functional groups are covalently replaced by related (radio) isotopes or isotopically labeled functional groups.

Examples of isotopes suitable for inclusion in a peptide ligand of the invention include isotopes of hydrogen, such as²H, (D) and³h (T); carbon, e.g.¹¹C、¹³C and¹⁴c; chlorine, e.g.³⁶Cl; fluorine, e.g.¹⁸F; iodine, e.g.¹²³I、¹²⁵I and¹³¹i; nitrogen, e.g.¹³N and¹⁵n; oxygen, e.g.¹⁵O、¹⁷O and¹⁸o; phosphorus, e.g.³²P; sulfur, e.g. of³⁵S; copper, e.g. of⁶⁴Cu; gallium, e.g.⁶⁷Ga or⁶⁸Ga; yttrium, e.g.⁹⁰Y and lutetium, e.g.¹⁷⁷Lu; and bismuth, e.g.²¹³Bi。

Certain isotopically-labeled peptide ligands of the present invention, for example those into which a radioactive isotope has been introduced, are useful in studies of drug and/or substrate tissue distribution, and in clinical assessments of the presence and/or absence of the CD38 target in diseased tissue. The peptide ligands of the invention may also have valuable diagnostic propertiesIn that they can be used to detect or identify the formation of complexes between the marker compounds and other molecules, peptides, proteins, enzymes or receptors. The detection or identification method may use a compound labeled with a labeling agent such as a radioisotope, an enzyme, a fluorescent substance, a luminescent substance (e.g., luminol, a luminol derivative, luciferin, aequorin, and luciferase), or the like. In view of the radioactive isotope tritium (i.e. tritium³H (T)) and carbon-14 (i.e.¹⁴C) Easy to introduce and ready-to-use detection means, which are particularly suitable for this purpose.

With heavier isotopes such as deuterium (i.e.²H (d)) substitution may provide certain therapeutic advantages due to higher metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and may therefore be preferred in certain circumstances.

Using positron emitting isotopes (e.g. of the type¹¹C、¹⁸F、¹⁵O and¹³n) substitution can be used in Positron Emission Tomography (PET) studies to examine target occupancy.

Isotopically-labelled compounds of the peptide ligands of the present invention can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying examples using an appropriate isotopically-labelled reagent in place of the unlabelled reagent previously used.

Aromatic molecular scaffold

The term "aromatic molecular scaffold" as referred to herein refers to any molecular scaffold defined herein, comprising an aromatic carbocyclic or heterocyclic ring system.

It will be understood that the aromatic molecular scaffold will comprise aromatic moieties. Examples of suitable aromatic moieties within the aromatic scaffold include biphenylene, triphenylene, naphthalene, or anthracene.

It will be understood that the aromatic molecular scaffold will comprise a heteroaromatic moiety. Examples of suitable heteroaromatic moieties within the aromatic scaffold include pyridine, pyrimidine, pyrrole, furan and thiophene.

It is also understood that the aromatic molecular scaffold may comprise halomethyl arene moieties such as bis (bromomethyl) benzene, tris (bromomethyl) benzene, tetrakis (bromomethyl) benzene, or derivatives thereof.

Non-limiting examples of aromatic molecular scaffolds include: bis-, tri-, or tetra (halomethyl) benzene; bis-, tris-, or tetrakis (halomethyl) pyridines; bis-, tri-, or tetra (halomethyl) pyridazine; bis-, tri-, or tetra (halomethyl) pyrimidines; bis-, tris-, or tetrakis (halomethyl) pyrazines; bis-, tris-, or tetrakis (halomethyl) -1,2, 3-triazine; bis-, tris-, or tetrakis (halomethyl) -1,2, 4-triazine; bis-, tri-, or tetra (halomethyl) pyrrole, -furan, -thiophene; bis-, tri-, or tetra (halomethyl) imidazole, -oxazole, -thiazole; bis-, tris-, or tetrakis (halomethyl) -3H-pyrazole, -isoxazole, -isothiazole; bis-, tri-, or tetra (halomethyl) biphenylene; bis-, tri-or tetra (halomethyl) triphenylene; 1, 8-bis (halomethyl) naphthalene; bis-, tris-, or tetrakis (halomethyl) anthracene; and bis-, tris-or tetrakis (2-halomethylphenyl) methane.

More specific examples of aromatic molecular scaffolds include: 1, 2-bis (halomethyl) benzene; 3, 4-bis (halomethyl) pyridine; 3, 4-bis (halomethyl) pyridazine; 4, 5-bis (halomethyl) pyrimidine; 4, 5-bis (halomethyl) pyrazines; 4, 5-bis (halomethyl) -1,2, 3-triazine; 5, 6-bis (halomethyl) -1,2, 4-triazine; 3, 4-bis (halomethyl) pyrrole, -furan, -thiophene, and other regioisomers; 4, 5-bis (halomethyl) imidazole, -oxazole, -thiazole; 4, 5-bis (halomethyl) -3H-pyrazole, -isoxazole, -isothiazole; 2,2' -bis (halomethyl) biphenylene; 2,2 "-bis (halomethyl) triphenylene; 1, 8-bis (halomethyl) naphthalene; 1, 10-bis (halomethyl) anthracene; bis (2-halomethylphenyl) methane; 1,2, 3-tris (halomethyl) benzene; 2,3, 4-tris (halomethyl) pyridine; 2,3, 4-tris (halomethyl) pyridazine; 3,4, 5-tris (halomethyl) pyrimidine; 4,5, 6-tris (halomethyl) -1,2, 3-triazine; 2,3, 4-tris (halomethyl) pyrrole, -furan, -thiophene; 2,4, 5-bis (halomethyl) imidazole, -oxazole, -thiazole; 3,4, 5-bis (halomethyl) -1H-pyrazole, -isoxazole, -isothiazole; 2,4, 2' -tris (halomethyl) biphenylene; 2,3',2 "-tris (halomethyl) triphenylene; 1,3, 8-tris (halomethyl) naphthalene; 1,3, 10-tris (halomethyl) anthracene; bis (2-halomethylphenyl) methane; 1,2,4, 5-tetrakis (halomethyl) benzene; 1,2,4, 5-tetrakis (halomethyl) pyridine; 2,4,5, 6-tetrakis (halomethyl) pyrimidine; 2,3,4, 5-tetrakis (halomethyl) pyrrole, -furan, -thiophene; 2,2',6,6' -tetrakis (halomethyl) biphenylene; 2, 2", 6, 6" -tetrakis (halomethyl) triphenylene; 2,3,5, 6-tetrakis (halomethyl) naphthalene and 2,3,7, 8-tetrakis (halomethyl) anthracene; and bis (2, 4-bis (halomethyl) phenyl) methane.

As described in the aforementioned documents, the molecular scaffold may be a small molecule, such as an organic small molecule.

In one embodiment, the molecular scaffold may be a macromolecule. In one embodiment, the molecular scaffold is a macromolecule consisting of amino acids, nucleotides, or carbohydrates.

In one embodiment, the molecular scaffold comprises a reactive group capable of reacting with a functional group of the polypeptide to form a covalent bond.

The molecular scaffold may comprise chemical groups that form links to peptides, such as amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, azides, anhydrides, succinimides, maleimides, alkyl halides, and acyl halides.

In one embodiment, the molecular scaffold may comprise or may consist of tris (bromomethyl) benzene, particularly 1,3, 5-tris (bromomethyl) benzene ('TBMB') or a derivative thereof.

In one embodiment, the molecular scaffold is 2,4, 6-tris (bromomethyl) mesitylene. The molecule is similar to 1,3, 5-tris (bromomethyl) benzene, but contains three additional methyl groups attached to the benzene ring. This has the advantage that additional methyl groups can form further contacts with the polypeptide, thus adding additional structural constraints.

The molecular scaffold of the invention comprises chemical groups that allow the functional groups of the polypeptides of the encoded library of the invention to form covalent linkages to the molecular scaffold. The chemical group is selected from a variety of functional groups including amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, anhydrides, succinimides, maleimides, azides, alkyl halides, and acyl halides.

Scaffold reactive groups that can be used to react with the thiol groups of cysteine on a molecular scaffold are alkyl halides (or also known as halohydrocarbons or haloalkanes).

Examples include bromomethylbenzene (a scaffold reactive group such as TBMB) or iodoacetamide. Other scaffold reactive groups for selectively coupling compounds to cysteines in proteins are maleimides, compounds containing α β unsaturated carbonyl groups and compounds containing α halomethylcarbonyl groups. Examples of maleimides that may be used as molecular scaffolds in the present invention include: tris- (2-maleimidoethyl) amine, tris- (2-maleimidoethyl) benzene, tris- (maleimido) benzene. An example of a compound containing an α -halomethylcarbonyl group is N, N', N "- (benzene-1, 3, 5-triyl) tris (2-bromoacetamide). Selenocysteine is also a natural amino acid, which has similar reactivity with cysteine and can be used in the same reaction. Thus, wherever cysteine is mentioned, selenocysteine can generally be substituted unless the context indicates otherwise.

Effectors and functional groups

According to another aspect of the present invention there is provided a drug conjugate comprising a peptide ligand as defined herein conjugated to one or more effectors and/or functional groups.

The effector and/or functional group may be attached, for example, to the N and/or C terminus of the polypeptide, or to an amino acid within the polypeptide, or to a molecular scaffold.

Suitable effector groups include antibodies and portions thereof or fragments thereof. For example, the effector group may include an antibody light chain constant region (CL), an antibody CH1 heavy chain domain, an antibody CH2 heavy chain domain, an antibody CH3 heavy chain domain, or any combination thereof, in addition to one or more constant region domains. The effector group may also comprise the hinge region of an antibody (such a region is typically found between the CH1 and CH2 domains of an IgG molecule).

In other embodiments of this aspect of the invention, the effector group according to the invention is the Fc region of an IgG molecule. Advantageously, the peptide ligand-effector group according to the invention comprises or consists of a peptide ligand Fc fusion having a t β -half-life of 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, or 7 days or more. Most advantageously, the peptide ligand according to the invention comprises or consists of a peptide ligand Fc-fusion having a t β -half-life of 1 day or more.

Functional groups typically include binding groups, drugs, reactive groups for attachment of other entities, functional groups that facilitate uptake of the macrocyclic peptide into a cell, and the like.

The ability of the peptide to penetrate into the cell will render the peptide effective against the target within the cell. Targets that can be accessed by peptides with the ability to penetrate into cells include transcription factors, intracellular signal transduction molecules (such as tyrosine kinases), and molecules involved in apoptotic pathways. Functional groups capable of penetrating cells include peptides or chemical groups that have been added to peptide or molecular scaffolds. Such as peptides derived from VP22, HIV-Tat, Drosophila's homeobox protein (antennapedia), etc., e.g.such as Chen and Harrison, Biochemical Society Transactions (2007) Vol.35, part 4, page 821; gupta et al, Advanced Drug Discovery Reviews (2004), volume 57 9637. Examples of short peptides that have been shown to be effective for translocation across the plasma membrane include the 16 amino acid penetrating peptide from drosophila antennapedia protein (desrossi et al (1994) J biol. chem. 269, p. 10444), the 18 amino acid 'model amphipathic peptide' (Oehlke et al (1998) Biochim biophysis Acts, p. 1414, p. 127) and the arginine-rich region of the HIV TAT protein. Non-peptide Methods include the use of small molecule mimetics or SMOCs that can be easily attached to biomolecules (Okuyama et al (2007) Nature Methods, vol 4, page 153). Other chemical strategies to add guanidine groups to the molecule also enhance cell penetration (Elson-Scwab et al (2007) J Biol Chem, Vol.282, page 13585). Small molecular weight molecules (e.g., steroids) can be added to the molecular scaffold to enhance uptake by the cells.

One class of functional groups that can be attached to a peptide ligand includes antibodies and binding fragments thereof, such as Fab, Fv or single domain fragments. In particular, antibodies can be used which bind to proteins capable of increasing the half-life of the peptide ligand in vivo.

In one embodiment, the peptide ligand-effector group according to the invention has a t β -half-life selected from: 12 hours or more, 24 hours or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more, 14 days or more, 15 days or more, or 20 days or more. Advantageously, the peptide ligand-effector group or composition according to the invention will have a t β -half-life of 12 to 60 hours. In other embodiments, it will have a t β -half life of one day or more. In still other embodiments, it will be in the range of 12 to 26 hours.

In a particular embodiment of the invention, the functional group is selected from metal chelators, which are suitable for complexing pharmaceutically relevant metal radioisotopes.

Possible effector groups also include enzymes, such as carboxypeptidase G2 for enzyme/prodrug therapy, in which a peptide ligand replaces an antibody in ADEPT.

In a particular embodiment of the invention, the functional group is selected from drugs, e.g. cytotoxic agents for cancer therapy. Suitable examples include: alkylating agents, such as cisplatin and carboplatin, and oxaliplatin, dichloromethyldiethylamine, cyclophosphamide, chlorambucil, ifosfamide; antimetabolites include the purine analogs azathioprine and mercaptopurine or pyrimidine analogs; plant alkaloids and terpenoids include vinca alkaloids, such as vincristine, vinblastine, vinorelbine, and vindesine; etoposide and teniposide, which are derivatives of podophyllotoxin; taxanes, including paclitaxel, originally called Taxol; topoisomerase inhibitors include camptothecin: irinotecan and topotecan, and type II inhibitors, including amsacrine, etoposide phosphate, and teniposide. Other agents may include antitumor antibiotics including the immunosuppressive agents actinomycin D (for kidney transplantation), doxorubicin, epirubicin, bleomycin, calicheamicin and the like.

In another specific embodiment according to the present invention, the cytotoxic agent is selected from maytansinoids (e.g., DM1) or monomethyl auristatin (e.g., MMAE).

DM1 is a cytotoxic agent, which is a thiol-containing derivative of maytansine, having the following structure:

monomethyl auristatin e (mmae) is a synthetic antitumor agent with the following structure:

in another specific embodiment according to the present invention, the cytotoxic agent is selected from maytansinoids (e.g., DM 1). The data herein are shown in table 2, which demonstrates the effect of peptide ligands conjugated to DM 1-containing toxins.

In one embodiment, the cytotoxic agent is linked to the bicyclic peptide through a cleavable bond (e.g., a disulfide bond or a protease-sensitive bond). In other embodiments, groups adjacent to the disulfide bonds are modified to control steric hindrance of the disulfide bonds and thereby control the rate of cleavage and concomitant release of the cytotoxic agent.

The published literature establishes the possibility of modifying the susceptibility of disulfide bonds to reduction by introducing steric hindrance on either side of the disulfide bond (Kellogg et al (2011) Bioconjugate Chemistry, 22, 717). A greater degree of steric hindrance reduces the rate of reduction of intracellular glutathione and also of extracellular (systemic) reducing agents, thereby reducing the ease with which toxins are released intracellularly and extracellularly. Thus, by carefully selecting the degree of steric hindrance on either side of the disulfide bond, optimal selection of disulfide stability in circulation (minimizing undesirable side effects of the toxin) and efficient release in the intracellular environment (maximizing therapeutic effect) can be achieved.

Steric hindrance on either side of the disulfide bond is modulated by the introduction of one or more methyl groups on the targeting entity (here, a bicyclic peptide) or toxin side of the molecular construct.

In one embodiment, the cytotoxic agent and linker are selected from any combination of those described in WO2016/067035 (wherein the cytotoxic agent and linker are incorporated herein by reference).

In one embodiment, the cytotoxic agent is DM1 and the bicyclic peptide is (β -Ala) -Sar₁₀-A- (SEQ ID NO: 3) (referred to herein as 66-03-00-N006) and conjugates comprise compounds of formula (I):

the BDC of formula (I) is known herein as BT66 BDC-1. Data are shown herein that demonstrate excellent competitive binding for BT66BDC-1, as measured by the CD38 competitive binding shown in table 2. Also shown herein are in vivo data demonstrating that BT66BDC-1 at 1mg/kg and 3mg/kg produces dose-dependent anti-tumor activity in MOLP-8 xenograft models, as shown in tables 9, 10, and figure 4. Also shown herein are in vivo data demonstrating that BT66BDC-1 at 3mg/kg completely eradicates tumors at 14 days in HT1080 xenograft model, as shown in tables 5,6, and fig. 2.

Synthesis of

The peptides of the invention can be prepared synthetically by standard techniques and then reacted with the molecular scaffold in vitro. In doing so, standard chemical methods may be used. This allows rapid large-scale preparation of soluble materials for further downstream experiments or validation. Such methods are accomplished using conventional chemistry such as that disclosed in Timmerman et al, supra.

Thus, the invention also relates to the preparation of a polypeptide or conjugate selected as described herein, wherein said preparation optionally comprises further steps as described below. In one embodiment, these steps are performed on the final product polypeptide/conjugate prepared by chemical synthesis.

When preparing the conjugate or complex, amino acid residues in the polypeptide of interest may be optionally substituted.

The peptide may also be extended to incorporate, for example, another loop and thus introduce multiple specificities.

For extension of the peptide, it can be chemically extended using standard solid or solution phase chemistry, using orthogonally protected lysines (and the like), simply at its N-or C-terminus or within a loop. The activated or activatable N-or C-terminus can be introduced using standard (bio) conjugation techniques. Alternatively, it may be added by fragment condensation or Native Chemical ligation, for example, as described (Dawson et al, 1994.Synthesis of Proteins by Nature Chemical ligation. science 266: 776-.

Alternatively, the peptide may be extended or modified by further conjugation of disulfide bonds. This has the further advantage of allowing the first and second peptides to be separated from one another once in the cell reducing environment. In this case, a molecular scaffold (e.g., TBMB) may be added during the chemical synthesis of the first peptide to react with the three cysteine groups; an additional cysteine or thiol may then be added to the N-or C-terminus of the first peptide such that the cysteine or thiol reacts only with the free cysteine or thiol of the second peptide to form a disulfide-linked bicyclic peptide-peptide conjugate.

Similar techniques are equally applicable to the synthesis/coupling of two bicyclic and bispecific macrocycles, possibly resulting in a tetraspecific molecule.

Furthermore, the addition of further functional or effector groups can be done in the same way, using appropriate chemical methods, at the N-or C-terminus or by coupling at the side chain. In one embodiment, the coupling is performed in a manner that does not block the activity of either entity.

Pharmaceutical composition

According to another aspect of the present invention there is provided a pharmaceutical composition comprising a peptide ligand as defined herein and a drug conjugate in combination with one or more pharmaceutically acceptable excipients.

Generally, the peptide ligands of the invention will be used in purified form together with a pharmacologically appropriate excipient or carrier. Typically, such excipients or carriers include aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and/or buffered media. Parenteral carriers (vehicles) include sodium chloride solution, ringer's dextrose, dextrose and sodium chloride, and lactated ringer's solution. Suitable physiologically acceptable adjuvants (if necessary to maintain the polypeptide complex in suspension) may be selected from thickening agents such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates.

Intravenous carriers include liquid and nutritional supplements and electrolyte supplements such as those based on ringer's dextrose. Preservatives and other additives may also be present, such as antimicrobials, antioxidants, chelating agents and inert gases (Mack (1982) Remington's Pharmaceutical Sciences, 16 th edition).

The peptide ligands of the invention may be used as compositions administered alone or in combination with other agents. These may include antibodies, antibody fragments and various immunotherapeutic drugs such as cyclosporine, methotrexate, doxorubicin or cisplatin as well as immunotoxins. The pharmaceutical composition may comprise a "mixture" of: various cytotoxic or other agents are associated with the protein ligands of the invention, or even combinations of selected polypeptides according to the invention with different specificities (e.g., polypeptides selected using different target ligands), whether or not they are combined prior to administration.

The route of administration of the pharmaceutical composition according to the present invention may be any one known to those of ordinary skill in the art. For treatment, the peptide ligands of the invention may be administered to any patient according to standard techniques. Administration may be by any suitable means, including parenterally, intravenously, intramuscularly, intraperitoneally, transdermally, by pulmonary route, or also by direct infusion with a catheter as appropriate. Preferably, the pharmaceutical composition according to the invention is administered by inhalation. The dose and frequency of administration will depend on the age, sex and condition of the patient, concurrent administration of other drugs, contraindications (counter-indication) and other parameters that should be considered by the clinician.

The peptide ligands of the invention may be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has proven effective and lyophilization and reconstitution techniques known in the art can be employed. Those skilled in the art will appreciate that lyophilization and reconstitution can result in varying degrees of loss of activity, and that levels may have to be adjusted upward to compensate.

Compositions containing the peptide ligands of the invention or mixtures thereof may be administered for prophylactic and/or therapeutic treatment. In certain therapeutic applications, a sufficient amount to achieve at least partial inhibition, suppression, modulation, killing, or some other measurable parameter of a selected cell population is defined as a "therapeutically effective dose". The amount required to achieve this dose will depend on the severity of the disease and the general state of the patient's own immune system, but will generally range from 0.005 to 5.0mg of the selected peptide ligand per kilogram of body weight, with doses of from 0.05 to 2.0 mg/kg/dose being more commonly used. For prophylactic applications, compositions containing the peptide ligands of the invention or mixtures thereof may also be administered at similar or slightly lower doses.

Compositions containing peptide ligands according to the invention can be used in prophylactic and therapeutic settings to help alter, inactivate, kill or ablate a selected target cell population in a mammal. In addition, the peptide ligands described herein can be selectively used ex vivo or in vitro for killing, depleting, or otherwise effectively removing a target cell population from a heterogeneous collection of cells. Blood from the mammal can be combined ex vivo with selected peptide ligands, thereby killing or otherwise removing unwanted cells from the blood, and returning the blood to the mammal according to standard techniques.

Therapeutic uses

The bicyclic peptides of the invention have a specific role as binding agents for CD 38.

CD38 is a 45kD type II transmembrane glycoprotein with a long C-terminal extracellular domain and a short N-terminal cytoplasmic domain. The CD38 protein is a bifunctional ectoenzyme that catalyzes the conversion of NAD + to cyclic ADP-ribose (cADPR) and also hydrolyzes cADPR to ADP-ribose. During ontogeny, CD38 appears on CD34+ committed stem cells and lineage-committed progenitors of lymphoid, erythroid and myeloid cells. CD38 expression persists primarily in the lymphoid lineage, with different expression levels at different stages of T and B cell development.

CD38 is upregulated in a number of hematopoietic malignancies, including non-hodgkin's lymphoma (NHL), Burkitt's Lymphoma (BL), Multiple Myeloma (MM), B chronic lymphocytic leukemia (B-CLL), B and T Acute Lymphocytic Leukemia (ALL), T Cell Lymphoma (TCL), Acute Myeloid Leukemia (AML), Hairy Cell Leukemia (HCL), Hodgkin's Lymphoma (HL), and Chronic Myeloid Leukemia (CML), and cell lines derived from a variety of hematopoietic malignancies. On the other hand, most primitive pluripotent stem cells of the hematopoietic system are CD 38-. CD38 expression in hematopoietic malignancies and its association with disease progression make CD38 an attractive target for antibody therapy.

CD38 has been reported to be involved in Ca²⁺Mobilization (Morra et al (1998) FASEB J.12; 581-592; Zilber et al (2000) Proc Natl Acad Sci USA 97, 2840-2845) and signal transduction in lymphoid and myeloid cells or cell lines by tyrosine phosphorylation of a variety of signal transduction molecules, including phospholipase C-gamma, ZAP-70, syk and C-cbl (Funaro et al (1993) Eur J Immunol 23, 2407-2411; Morra et al (1998), supra; Funaro et al (1990) J Immunol 145, 2390-2396; Zubiur et al (1997) J Immunol 159, 193-205; Deaglio et al (2003) Blood 102, 2146-2155; Todisco et al (2000) Blood 95, 535-542; Konova et al (1998) J Immunol 161, 1997-4708; Kilbo et al 28161, 4708; Natl et al J-47159; Jun J-4708; Jun et al (1990) J-47159), 184- > 192; kitanaka et al (1999) J Immunol162, 1952-1958; mallone et al (2001) Int Immunol 13, 397-. Based on these observations, CD38 is considered to be an important signaling molecule for the maturation and activation of lymphoid and myeloid cells during normal development.

The exact role of CD38 in signal transduction and hematopoiesis remains unclear, particularly since most of these signaling studies use cell lines that ectopically overexpress CD38 and the anti-CD 38 monoclonal antibody, CD38 and anti-CD 38 monoclonal antibodies being non-physiological ligands. Since the CD38 protein has the enzymatic activity of producing cADPR, it is inducible Ca²⁺Division of mobilizationSon (Lee et al (1989) J Biol Chem 264, 1608-1615; Lee and Arhus (1991) Cell Regul 2, 203-209), it was proposed that CD38 ligation of monoclonal antibodies trigger Ca in lymphocytes by increasing cADPR production²⁺Mobilization and signaling (Lee et al (1997) Adv Exp Med Biol 419, 411-419). In contrast to this hypothesis, truncation and point mutation analysis of the CD38 protein revealed that neither its cytoplasmic tail nor its enzymatic activity were essential for signal transduction mediated by anti-CD 38 antibodies (Kitanaka et al, (1999) J Immunol162, 1952-1958; Lund et al (1999) J Immunol162, 2693-2702; Hoshino et al (1997) J Immunol 158, 741-747).

The best evidence for CD38 function came from CD 38-/-knockout mice that were deficient in their innate Immunity due to dendritic cell migration defects and have reduced T cell-dependent humoral responses (Partida-Sanchez et al, (2004) Immunity 20, 279-291; Partida-Sanchez et al, (2001) Nat Med 7, 1209-1216). Nevertheless, it is not clear whether the mouse functions as CD38 in humans, since the pattern of CD38 expression in hematopoiesis varies greatly between humans and mice: a) unlike human immature progenitor stem cells, similar progenitor stem cells express high levels of CD38 in mice (Randall et al (1996) Blood 87, 4057-4067; dagher et al (1998) Biol Blood Marrow Transplant 4, 69-74), B) whereas high levels of CD38 expression are found in germinal center B cells and plasma cells during human B cell development (Uckun (1990) Blood 76, 1908-1923; kumagai et al, (1995) J Exp Med 181, 1101-containing 1110), in mice, the expression level of CD38 was low in the corresponding cells (Oliver et al, (1997) J Immunol 158, 1108-containing 1115; ridderstad and Tarlinton (1998) J Immunol 160, 4688-.

Several anti-human CD38 antibodies with different proliferative properties on various tumor cells and cell lines have been described in the literature. For example, chimeric OKT10 antibodies with mouse Fab and human IgG1 Fc were very effective in mediating antibody-dependent cell-mediated cytotoxicity (ADCC) against lymphocytes in the presence of peripheral Blood mononuclear effector cells from MM patients or normal individuals (Stevenson et al, (1991) Blood77, 1071-. CDR-grafted humanized versions of the anti-CD 38 antibody AT13/5 have demonstrated potent ADCC activity against a CD38 positive cell line (U.S. patent application No. 09/797,941). The human monoclonal anti-CD 38 antibody has been shown to mediate killing of the CD38 positive cell line in vitro by ADCC and/or Complement Dependent Cytotoxicity (CDC) and to delay tumor growth in SCID mice bearing the MM cell line RPMI-8226 (WO 2005/103083). On the other hand, several anti-CD 38 antibodies (IB4, SUN-4B7 and OKT10, but not IB6, AT1 or AT2) induced proliferation of Peripheral Blood Mononuclear Cells (PBMC) from normal individuals (Ausiello et al, (2000) Tissue antibodies 56, 539-547).

Some antibodies of the prior art have been shown to trigger apoptosis in CD38+ B cells. However, they can only do so in the presence of stromal cells or stromal derived cytokines. An agonistic anti-CD 38 antibody (IB4) was reported to prevent B cell apoptosis in human development center (GC) (Zupo et al, (1994) Eur J Immunol 24, 1218-. Another anti-CD 38 antibody, T16, induced apoptosis of immature lymphocytes and leukemia lymphoblasts from ALL patients (Kumagai et al, (1995) J Exp Med 181, 1101-542), and leukemia myeloblasts from AML patients (Todisco et al, (2000) Blood 95, 535-542), but T16 induced apoptosis only in the presence of stromal cells or stroma-derived cytokines (IL-7, IL-3, stem cell factor).

Polypeptide ligands selected according to the methods of the invention are useful for in vivo therapeutic and prophylactic applications, in vitro and in vivo diagnostic applications, in vitro assays and reagent applications, and the like. Ligands with selected levels of specificity may be used in applications involving testing in non-human animals where cross-reactivity is desired, or in diagnostic applications where careful control of cross-reactivity with homologues or paralogs is desired. In some applications, such as vaccine applications, the ability to elicit an immune response to a predetermined range of antigens can be exploited to tailor vaccines against specific diseases and pathogens.

Substantially pure peptide ligands having at least 90 to 95% homogeneity are preferred for mammalian administration, and 98 to 99% or more homogeneity are most preferred for pharmaceutical administration (especially when the mammal is a human). Once purified, partially purified, or to homogeneity as desired, the selected polypeptide may be used for diagnosis or therapy (including ex vivo), or for development and performance of assay procedures, immunofluorescent staining, and the like (Lefkovite and Pernis, (1979 and 1981), Immunological Methods, Vol.I and II, Academic Press, NY).

According to another aspect of the present invention there is provided a peptide ligand or drug conjugate as defined herein for use in the prevention, inhibition or treatment of a disease or condition mediated by CD 38.

According to another aspect of the present invention there is provided a method of preventing, inhibiting or treating a disease or condition mediated by CD38, the method comprising administering to a patient in need thereof an effector group of a peptide ligand as defined herein and a drug conjugate.

In one embodiment, CD38 is mammalian CD 38. In another embodiment, the mammalian CD38 is human CD38(hCD 38).

In one embodiment, the disease or disorder mediated by CD38 is selected from cancer.

Examples of cancers (and their benign counterparts) that can be treated (or inhibited) include, but are not limited to, tumors of epithelial origin (various types of adenomas and carcinomas, including adenocarcinomas, squamous carcinomas, transitional cell carcinomas, and other carcinomas), such as bladder and urinary tract cancers, breast cancers, gastrointestinal cancers (including esophagus, stomach, small intestine, colon, rectum, and anus), liver cancers (hepatocellular carcinoma), gallbladder and biliary tract cancers, exocrine pancreatic cancers, kidney cancers, lung cancers (e.g., adenocarcinoma, small cell lung cancer, non-small cell lung cancer, bronchioloalveolar cancer, and mesothelioma), head and neck cancers (e.g., tongue cancer, oral cancer, larynx cancer, pharynx cancer, nasopharynx cancer, tonsillar cancer, salivary gland cancer, nasal cavity cancer, and paranasal sinus cancer), ovarian cancer, fallopian tube cancer, peritoneal cancer, vaginal cancer, vulval cancer, penile cancer, cervical cancer, myometrial cancer, endometrial cancer, and other cancers, Thyroid cancer (e.g., thyroid follicular cancer), adrenal cancer, prostate cancer, skin and adnexal cancers (e.g., melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, dysplastic nevi); hematologic malignancies (i.e., leukemias, lymphomas) and diseases of premalignant hematologic and marginal malignancies include hematologic malignancies and disorders associated with the lymphatic system (e.g., acute lymphocytic leukemia [ ALL ], chronic lymphocytic leukemia [ CLL ], B-cell lymphomas such as diffuse large B-cell lymphoma [ DLBCL ], follicular lymphoma, Burkitt's lymphoma, mantle cell lymphoma, T-cell lymphoma and leukemia, Natural killer [ NK ] cell lymphoma, Hodgkin's lymphoma, hairy cell leukemia, nonsignificant monoclonal gammoproteinemia, plasmacytoma, multiple myeloma, and post-transplant lymphoproliferative disorders), and hematologic malignancies and disorders associated with the bone marrow system (e.g., acute myelogenous leukemia [ AML ], chronic myelogenous leukemia [ CML ], chronic myelogenous leukemia [ ML ], "CML Eosinophilic syndrome, myeloproliferative disorders such as polycythemia vera, primary thrombocythemia and primary myelofibrosis, myeloproliferative syndrome, myelodysplastic syndrome and promyelocytic leukemia); tumors of mesenchymal origin, for example sarcomas of soft tissue, bone or cartilage such as osteosarcoma, fibrosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, angiosarcoma, kaposi's sarcoma, ewing's sarcoma, synovial sarcoma, epithelioid sarcoma, gastrointestinal stromal tumors, benign and malignant tissue cell tumors, and dermatofibrosarcoma protruberans; tumors of the central or peripheral nervous system (e.g., astrocytomas, gliomas and glioblastomas, meningiomas, ependymomas, pinealomas, and schwannomas); endocrine tumors (e.g., pituitary tumors, adrenal tumors, islet cell tumors, parathyroid tumors, carcinoid tumors, and medullary thyroid cancers); ocular and accessory tumors (e.g., retinoblastoma); germ cell and trophoblastic tumors (e.g., teratoma, seminoma, dysgerminoma, hydatidiform mole, and choriocarcinoma); and pediatric and embryonic tumors (e.g., medulloblastoma, neuroblastoma, wilms' tumor, and primitive neuroectodermal tumors); or congenital or other syndromes that predispose a patient to a malignancy (e.g., xeroderma pigmentosum).

In another embodiment, the cancer is selected from hematopoietic malignancies, for example selected from: non-hodgkin's lymphoma (NHL), Burkitt's Lymphoma (BL), Multiple Myeloma (MM), B chronic lymphocytic leukemia (B-CLL), B and T Acute Lymphocytic Leukemia (ALL), T Cell Lymphoma (TCL), Acute Myeloid Leukemia (AML), Hairy Cell Leukemia (HCL), Hodgkin's Lymphoma (HL), and Chronic Myeloid Leukemia (CML).

Reference herein to the term "prevention" relates to the administration of a protective composition prior to induction of the disease. By "inhibit" is meant administration of the composition after the induction event but prior to clinical manifestation of the disease. "treatment" refers to the administration of a protective composition after symptoms of the disease become apparent.

Animal model systems are available that can be used to screen peptide ligands for effectiveness in protecting against or treating disease. The present invention facilitates the use of animal model systems that allow the development of polypeptide ligands that are cross-reactive with human and animal targets to allow the use of animal models.

The invention is further described with reference to the following examples.

Examples

Materials and methods

Peptide synthesis

Peptide synthesis was performed based on the Fmoc chemistry method using a Symphony Peptide synthesizer manufactured by Peptide Instruments and a Syro II synthesizer manufactured by MultiSynTech. Standard Fmoc-amino acids (Sigma, Merck) were used, with appropriate side chain protecting groups: in each case using the standard coupling conditions, and then using standard methods for deprotection. The peptide was purified using HPLC and modified after isolation using 1,3, 5-tris (bromomethyl) benzene (TBMB, Sigma). For this purpose, the linear peptide is treated with H₂O to about 35mL, add about 500. mu.L of 100mM TBMB in acetonitrile and add 5mL of 1M NH₄HCO₃H of (A) to (B)₂The reaction is initiated by the O solution. The reaction was allowed to proceed at room temperature for about 30-60 minutes and was immediately lyophilized (as judged by MALDI) upon completion of the reaction. After the freeze-drying, the mixture is dried,the modified peptide was purified as above while replacing Luna C8 with a Gemini C18 column (Phenomenex) and changing the acid to 0.1% trifluoroacetic acid. Pure fractions containing the correct TMB modifying substance were pooled, lyophilized and stored at-20 ℃.

Unless otherwise indicated, all amino acids are used in the L-configuration.

In some cases, the peptide is first converted to an activated disulfide and then coupled to the free thiol group of the toxin using the following method; a solution of 4-methyl (succinimidyl 4- (2-pyridylthio) valerate) (100mM) in dry DMSO (1.25mol eq) was added to a solution of peptide (20mM) in dry DMSO (1mol eq). The reaction was mixed well and DIPEA (20mol eq) was added. The reaction was monitored by LC/MS until completion.

Preparation of bicyclic peptide drug conjugate BT66BDC-1

The reaction scheme is as follows:

66-03-00-N041(56mg) was dissolved in DMF (0.8 ml). DM1(19mg) in DMF (0.78ml) was added followed by diisopropylethylamine (26. mu.l) and the mixture was stirred at room temperature for 2 h. Water (17.5ml) was added, the mixture was filtered and then applied to a Luna C18(3) preparative HPLC column (250 mm. times.20 mm). The product was obtained by elution with a gradient of 16% to 55% acetonitrile (containing 1% trifluoroacetic acid) and water (containing 1% trifluoroacetic acid) over 50 minutes. The pure fractions were lyophilized to give 37.5mg of product.

LC/MS(ES⁺) Calculated as MH + 3234.4; 3234.4 are displayed.

Biological data

Competitive binding assay for CD38

The affinity (Ki) of the peptides of the invention for human CD38 was determined using fluorescence polarimetry using the method reported in Lea et al (Expert Opin Drug Discov.20116 (1): 17-3), and the following fluorescently labeled peptide ACTPCADFPIWGCA-Sar was used when Fl is a fluorescein molecule₆-K(Fl)((SEQ ID NO：93)-Sar₆-K (Fl)) for TBMB derivatives.

The peptide ligands of the invention were tested in the CD38 competitive binding assay described above, with the results shown in table 1:

table 1: bioassay data for peptide ligands of the invention

The bi-cyclic drug conjugates of BT66BDC-1 were tested in the CD38 competitive binding assay described above, with the results shown in table 2:

table 2: bioassay data for the bicyclic drug conjugates of the invention

*n＝1

In vivo efficacy test of BT66BDC1 in treating HT1080 xenografts in BALB/c nude mice

2.1 purpose of study

The purpose of this study was to evaluate the in vivo antitumor efficacy of BT66BDC1 in the treatment of HT1080 xenografts in BALB/c nude mice.

2.2 design of the experiment

TABLE 3

2.3 materials

2.3.1 animals and feeding conditions

2.3.1.1 animal

Species: little mouse (Mus Musculus)

And (2) breeding: balb/c nude mice

The week age is as follows: 6-8 weeks

Sex: female

Weight: 18-22 g

Animal number: adding 12 mice for later use

Animal suppliers: shanghai LC Laboratory Animal Co., LTD.

2.3.1.2 feeding conditions

Mice were kept in separate ventilated cages at constant temperature and humidity, 3 animals per cage.

Temperature: 20 ℃ to 26 ℃.

Humidity 40% to 70%.

Cage: is made of polycarbonate. Size: 300mm X180 mm X150 mm. The bedding material was corncobs, changed twice a week.

Diet: the animals were free to eat the radiation sterilized dry particulate food throughout the study.

Drinking water: animals can freely drink sterile drinking water.

Cage identification: the identification tag of each cage contains the following information: animal number, sex, species line, date of receipt, treatment, study number, group number, and date of treatment initiation.

Animal identification: the animals were marked with ear codes.

2.3.2 test and Positive controls

Product identification: BT66BDC1

Physical description: freeze-dried powder

Molecular weight: 3234.4

Packaging and storage conditions: stored at-80 deg.C

2.4 Experimental methods and procedures

2.4.1 cell culture

HT1080 tumor cells were maintained in monolayer culture in vitro at 37 ℃ in air with 0% CO₂In an EMEM medium supplemented with 10% heat-inactivated fetal bovine serum. Tumor cells were routinely passaged twice weekly by trypsin-EDTA treatment. Cells grown in the exponential growth phase were harvested and counted for tumor inoculation.

2.4.2 tumor inoculation

Each mouse was inoculated subcutaneously in the right flank with 0.2ml of HT1080 tumor cells (5X 10) in PBS⁶) For use in tumor formation. For efficacy studies, when the mean tumor volume reached about 180mm³At that time, animals were randomized and treatment was initiated. Test article administration and animal numbers for each group are shown in table 4.

Table 4: test article preparation

2.4.3 observations

Procedures related to Animal handling, Care and treatment in all studies were performed according to guidelines approved by the Wuxi ApTec Institutional Animal Care and Use Committee (IACUC) and following the guidelines of the Association for the Assessment and characterization of Laboratory Animal Care (AAALAC). In routine monitoring, animals are examined daily for tumor growth and any effect of treatment on normal behavior, such as activity, food and water consumption (by observation only), weight gain/loss (body weight measured daily), eye/hair shine, and any other abnormal effects stated in the protocol. Mortality and observed clinical signs were recorded based on the number of animals in each subset.

2.4.4 tumor measurements and endpoints

The primary endpoint was to see if tumor growth could be delayed or if mice could be cured. Tumor volume was measured in two dimensions using a caliper three times a week and in mm using the following formula³Represents the volume: v is 0.5a × b²Wherein a and b are the major and minor diameters of the tumor, respectively. Tumor size was then used for T/C value calculation. The T/C value (percentage) is an index of the antitumor efficacy; t and C are the average volumes of the treatment and control groups, respectively, on the indicated days.

TGI was calculated for each group using the following formula: TGI (%) [1- (Ti-T0)/(Vi-V0) ] × 100; ti is the mean tumor volume of the treatment group on the day specified, T0 is the mean tumor volume of the treatment group on the day of initiation of treatment, Vi is the mean tumor volume of the vehicle control group on the same day as Ti, and V0 is the mean tumor volume of the vehicle group on the day of initiation of treatment.

2.4.5 plasma Collection

At PG-D14, mice of groups 2,3 were re-dosed and plasma was collected for PK analysis at 5 min, 15 min, 30 min, 60 min and 120 min post-dose.

2.4.6 statistical analysis

A summary of statistical data including mean and Standard Error of Mean (SEM) for each group of tumor volumes at each time point is provided.

Statistical analysis of tumor volume differences between groups was performed based on data obtained at the optimal treatment time point after the final dose.

One-way ANOVA was performed to compare tumor volumes between groups, and when significant F-statistics (ratio of treatment variance to error variance) were obtained, comparisons between groups were performed using the Games-Howell test. All data were analyzed using Prism. P <0.05 was considered statistically significant.

2.5 results

2.5.1 mortality, morbidity and weight gain or loss

Body weight was monitored periodically as an indirect measure of toxicity. The body weight change of BT66BDC1 administered to female Balb/c nude mice carrying HT1080 is shown in FIG. 1. Mice treated with 10mg/kg of BT66BDC1 showed severe weight loss and death of 3/3 animals.

2.5.2 tumor volume trajectories

The mean tumor volume as a function of time in female Balb/c nude mice bearing HT1080 xenografts is shown in Table 5 below.

Table 5: trajectory of tumor volume over time

2.5.3 tumor growth curves

The tumor growth curve is shown in figure 2.

2.5.4 tumor growth inhibition assay

Tumor growth inhibition rate of BT66BDC1 in HT1080 xenograft model was calculated based on tumor volume measurements at day 14 after the start of treatment.

Table 6: tumor growth inhibition assay (T/C and TGI)

a. Mean. + -. SEM.

b. Tumor growth inhibition was calculated by dividing the group mean tumor volume of the treated group by the group mean tumor volume of the control group (T/C).

2.6 results summary and discussion

In this study, BT66BDC1 was evaluated for therapeutic efficacy in an HT1080 xenograft model. The measured body weight and body weight changes are shown in figure 1. Tumor volumes for all treatment groups at various time points are shown in tables 5,6 and fig. 2.

On day 14, mean tumor size of vehicle-treated mice reached 2232mm³. The dose-dependent antitumor activity produced at 1mg/kg and 3mg/kg of BT66BDC1, respectively, had a measurement of 967mm³(TGI＝61.8％，p>0.05) and 0mm³(TGI＝108.8％,p<0.01). In this, tumors were completely eradicated at 3mg/kg of BT66BDC1 on day 14. Is treated with 10mg/kg of BT66BDC1Tumor regression was also followed rapidly, but resulted in severe weight loss and 3/3 animal death.

In vivo efficacy test of BT66BDC1 in treating MOLP-8 xenografts in CB17-SCID mice

3.1 purpose of study

The objective of this study was to evaluate the in vivo antitumor efficacy of BT66BDC1 in the treatment of subcutaneous MOLP-8 xenograft models in CB17-SCID mice.

3.2 design of the experiment

TABLE 7

Note that: n: the number of animals; administration volume: the adjusted dose volume was based on body weight 10. mu.l/g.

3.3 materials

3.3.1 animals and feeding conditions

3.3.1.1 animals

Species: little mouse

And (2) breeding: CB17-SCID

The week age is as follows: 6-8 weeks

Sex: female

Weight: 18-22 g

Animal number: adding 18 mice for later use

Animal suppliers: shanghai SLAC Laboratory Animal Co., LTD.

3.3.1.2 Breeding conditions

Mice were kept in separate ventilated cages at constant temperature and humidity, 3 animals per cage.

Temperature: 20 to 26 ℃.

Humidity 40-70%.

Cage: is made of polycarbonate. Size: 300mm X180 mm X150 mm. The bedding material was corncobs, changed twice a week.

Diet: the animals were free to eat the radiation sterilized dry particulate food throughout the study.

Drinking water: animals can freely drink sterile drinking water.

Cage identification: the identification tag of each cage contains the following information: animal number, sex, species line, date of receipt, treatment, study number, group number, and date of treatment initiation.

Animal identification: the animals were marked with ear codes.

3.3.2 test and Positive controls

Product identification: BT66BDC1

Physical description: stock DMSO solution

MW:3234.4, purity > 95%

Concentration: 20mg/ml

Packaging and storage conditions: the storage was at-80 ℃.

3.4 Experimental methods and procedures

3.4.1 cell culture

In RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 5% CO in air at 37 ℃₂As a monolayer culture in vitro, MOLP-8 tumor cells were maintained. Tumor cells were routinely passaged twice weekly by trypsin-EDTA treatment. Cells grown in the exponential growth phase were harvested and counted for tumor inoculation.

3.4.2 tumor inoculation

Each mouse was inoculated subcutaneously in the right flank with 0.2ml of MOLP-8 tumor cells (10X 10) in PBS⁶) And 50% matrigel to form tumors. For efficacy studies, when the mean tumor volume reached about 150-³At that time, animals were randomized and treatment was initiated. Test substance administration and animal numbers per group are shown in table 8:

table 8: test article preparation

3.4.3 observations

Procedures related to animal handling, care and treatment in all studies were performed according to the guidelines approved by the Wuxi ApTec Institutes of Animal Care and Use (IACUC) and following the guidelines of the Association for assessment and characterization of laboratory animal Care (AAALAC). In routine monitoring, animals are examined daily for tumor growth and any effect of treatment on normal behavior, such as activity, food and water consumption (by observation only), weight gain/loss (body weight measured twice weekly), eye/hair shine, and any other abnormal effects stated in the protocol. Mortality and observed clinical signs were recorded based on the number of animals in each subset.

3.4.4 tumor measurements and endpoints

The primary endpoint was to see if tumor growth could be delayed or if mice could be cured. Tumor size was measured in two dimensions using calipers three times a week and in mm using the following formula³Represents the volume: v is 0.5a × b²Wherein a and b are the major and minor diameters of the tumor, respectively. Tumor size was then used for the calculation of T/C values. The T/C value (percentage) is an index of the antitumor efficacy; t and C are the average volumes of the treatment and control groups, respectively, on the indicated days.

TGI was calculated for each group using the following formula: TGI (%) [1- (Ti-T0)/(Vi-V0) ] × 100; ti is the mean tumor volume of the treatment group on the day specified, T0 is the mean tumor volume of the treatment group on the day of initiation of treatment, Vi is the mean tumor volume of the vehicle control group on the same day as Ti, and V0 is the mean tumor volume of the vehicle group on the day of initiation of treatment.

3.4.5 sample Collection

At PG-D14, mice of groups 2,3 were re-dosed and plasma was collected for PK analysis at 5 min, 15 min, 30 min, 60 min and 120 min post-dose.

3.4.6 statistical analysis

A summary of statistical data including mean and Standard Error of Mean (SEM) for each group of tumor volumes at each time point is provided.

Statistical analysis of tumor volume differences between groups was performed based on data obtained at the optimal treatment time point after final dosing.

One-way ANOVA was performed to compare tumor volumes between groups, and when significant F-statistics (ratio of treatment variance to error variance) were obtained, multiple comparison methods were applied after ANOVA. Potential synergistic effects between treatments will be analyzed by two-way ANOVA. All data will be analyzed using SPSS 17.0. P <0.05 was considered statistically significant.

3.5 results

3.5.1 mortality, morbidity and weight gain or loss

Animals were monitored regularly for body weight as an indirect measure of toxicity. The change in body weight of BT66BDC1 administered to female CB17-SCID mice bearing MOLP-8 tumors is shown in FIG. 3. Treatment of mice with 10mg/kg of BT66BDC1 showed weight loss and animal death.

3.5.2 tumor volume trajectories

The mean tumor volume as a function of time in female CB17-SCID mice carrying MOLP-8 xenografts is shown in table 9.

Table 9: trajectory of tumor volume over time

3.5.3 tumor growth curve

The tumor growth curve is shown in fig. 4.

3.5.4 tumor growth inhibition assay

The inhibition rate of BT66BDC1 on tumor growth in the MOLP-8 xenograft model was calculated based on tumor volume measurements at day 14 after the start of treatment.

Table 10: tumor growth inhibition assay (T/C and TGI)

a. Mean. + -. SEM.

b. Tumor growth inhibition was calculated by dividing the group mean tumor volume of the treated group by the group mean tumor volume of the control group (T/C).

3.6 results summary and discussion

In this study, BT66BDC1 was evaluated for therapeutic efficacy in the MOLP-8 xenograft model. The measured body weight and body weight change are shown in fig. 3. Tumor volumes for all treatment groups at various time points are shown in tables 9, 10 and fig. 4.

On day 14, mean tumor size of vehicle-treated mice reached 1771mm³. The dose-dependent antitumor activity produced at BT66BDC1 of 1mg/kg and 3mg/kg had a measurement of 1114mm, respectively³(TGI＝41％，p<0.05) and 77mm³(TGI＝106％，p<0.001) of the tumor. BT66BDC1 at 10mg/kg rapidly regressed tumors after treatment, but resulted in severe weight loss and 3/3 animal death.

Sequence listing

<110> Betheskoaddi Ltd

<120> bicyclic peptide ligands specific for CD38

<130> BIC-C-P2516PCT

<150> GB1900530.5

<151> 2019-01-15

<160> 93

<170> PatentIn version 3.5

<210> 1

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 1

Cys Phe Glu Leu Asp Gly Thr Cys Phe Asp Trp Ala Gln Glu Cys

1 5 10 15

<210> 2

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 2

Cys Val Asn Phe Gly Ser Val Cys Trp Asp Pro Asp Ser Arg Cys

1 5 10 15

<210> 3

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 3

Cys Val Pro Cys Ala Asp Phe Pro Ile Trp Tyr Cys

1 5 10

<210> 4

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 4

Cys Val Pro Cys Ala Asp Phe Pro Ile Trp Trp Cys

1 5 10

<210> 5

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 5

Cys Thr Pro Cys Ala Asp Phe Pro Ile Trp Ser Cys

1 5 10

<210> 6

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 6

Cys Thr Pro Cys Ala Asp Phe Pro Ile His Thr Cys

1 5 10

<210> 7

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 7

Cys Val His Cys Ala Asp Phe Pro Ile Trp Gly Cys

1 5 10

<210> 8

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 8

Cys Val Pro Cys Ala Asp Phe Pro Ile Trp Gly Cys

1 5 10

<210> 9

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 9

Cys Val Met Cys Ala Asp Phe Pro Ile Trp Gly Cys

1 5 10

<210> 10

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 10

Cys Thr Pro Cys Ala Asp Phe Pro Ile Trp Tyr Cys

1 5 10

<210> 11

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 11

Cys Thr Pro Cys Ala Asp Phe Pro Ile Leu Thr Cys

1 5 10

<210> 12

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 12

Cys Val Ala Cys Ala Asp Phe Pro Ile Trp Gly Cys

1 5 10

<210> 13

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 13

Cys Thr Pro Cys Ala Asp Phe Pro Ile Tyr Gly Cys

1 5 10

<210> 14

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 14

Cys Thr Pro Cys Ala Asp Phe Pro Ile Leu Asp Cys

1 5 10

<210> 15

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 15

Cys Val Lys Cys Ala Asp Phe Pro Ile Trp Gly Cys

1 5 10

<210> 16

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 16

Cys Thr Pro Cys Ala Asp Met Pro Ile Trp Thr Cys

1 5 10

<210> 17

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 17

Cys Ile Pro Cys Ala Asp Phe Pro Ile Trp Gly Cys

1 5 10

<210> 18

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 18

Cys Ile Pro Cys Ala Asp Phe Pro Ile Ser Val Cys

1 5 10

<210> 19

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 19

Cys Val Pro Cys Ala Asp Phe Pro Ile Ser Phe Cys

1 5 10

<210> 20

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 20

Cys Ile Pro Cys Ala Asp Phe Pro Ile Ser Phe Cys

1 5 10

<210> 21

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 21

Cys Val Pro Cys Ala Asp Phe Pro Ile Ser Val Cys

1 5 10

<210> 22

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 22

Cys Val Pro Cys Ala Asp Phe Pro Ile Phe Thr Cys

1 5 10

<210> 23

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 23

Cys Ile Pro Cys Ala Asp Phe Pro Ile Phe Thr Cys

1 5 10

<210> 24

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 24

Cys Thr Pro Cys Ala Asp Phe Pro Ile Trp Gly Cys

1 5 10

<210> 25

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 25

Cys Asp Phe Thr Met Pro Cys Glu Asn Trp Lys Tyr Cys

1 5 10

<210> 26

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 26

Cys Asp Phe Thr Met Pro Cys Pro Asn Trp Asn Ala Cys

1 5 10

<210> 27

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 27

Cys Asp Phe Thr Met Pro Cys Gln Met Trp Glu Gln Cys

1 5 10

<210> 28

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 28

Cys Ile Phe Asp Tyr Asp Cys Asp Ala Trp Ser Ala Cys

1 5 10

<210> 29

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 29

Cys Phe Trp Leu Asp Gly Glu Cys Phe Asp Trp Asn His Glu Cys

1 5 10 15

<210> 30

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 30

Cys Phe His Leu Asp Gly Glu Cys Phe Asp Leu Glu Asn Thr Cys

1 5 10 15

<210> 31

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 31

Cys Ala Trp Leu Cys Pro Asn Leu Cys

1 5

<210> 32

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 32

Cys Phe Ser Leu Asp Gly Glu Cys Phe Asp Leu Ser Gly Glu Cys

1 5 10 15

<210> 33

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 33

Cys Phe Thr Leu Asp Gly Glu Cys Phe Asp Trp Thr His Glu Cys

1 5 10 15

<210> 34

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 34

Cys Phe Lys Leu Asp Gly Val Cys Phe Asp Leu Phe His Glu Cys

1 5 10 15

<210> 35

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 35

Cys Phe Met Leu Asp Gly Glu Cys Phe Asp Leu Asn Lys Glu Cys

1 5 10 15

<210> 36

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 36

Cys Phe Lys Leu Asp Gly Glu Cys Phe Asp Trp Thr His Glu Cys

1 5 10 15

<210> 37

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 37

Cys Phe Thr Leu Asp Gly Glu Cys Phe Asp Trp Asp Ala Glu Cys

1 5 10 15

<210> 38

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 38

Cys Phe Glu Leu Asp Gly Ser Cys Phe Asp Phe Asp His Glu Cys

1 5 10 15

<210> 39

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 39

Cys Phe Thr Leu Asp Gly Glu Cys Phe Asp Val Asn Arg Glu Cys

1 5 10 15

<210> 40

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 40

Cys Phe Trp Leu Asp His Glu Cys Phe Asp Trp Thr His Glu Cys

1 5 10 15

<210> 41

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 41

Cys Phe Gln Leu Asp Gly Glu Cys Phe Asp Ile Tyr Arg Glu Cys

1 5 10 15

<210> 42

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 42

Cys Phe Glu Leu Asp Gly Asn Cys Phe Asp Trp Thr His Glu Cys

1 5 10 15

<210> 43

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 43

Cys Phe His Leu Asp Gly Glu Cys Phe Asp Tyr Glu His Glu Cys

1 5 10 15

<210> 44

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 44

Cys Phe Ser Leu Asp Gly Glu Cys Phe Asp Ile Ala Ser Glu Cys

1 5 10 15

<210> 45

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 45

Cys Phe Gln Leu Asp Gly Glu Cys Phe Asp Thr Ser His Glu Cys

1 5 10 15

<210> 46

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 46

Cys Phe Ser Leu Asp Gly Ala Cys Phe Asp Trp Thr His Glu Cys

1 5 10 15

<210> 47

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 47

Cys Phe Val Leu Asp Gly Glu Cys Phe Asp Tyr Tyr Glu Glu Cys

1 5 10 15

<210> 48

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 48

Cys Phe Arg Leu Asp Asp Glu Cys Phe Asp Trp Thr His Glu Cys

1 5 10 15

<210> 49

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 49

Cys Phe Arg Leu Asp Gly Val Cys Phe Asp Leu Asp Asp Glu Cys

1 5 10 15

<210> 50

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 50

Cys Phe Arg Leu Asp Gly Glu Cys Phe Asp Met Gly Gln Glu Cys

1 5 10 15

<210> 51

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 51

Cys Phe Thr Leu Asp Gly Ala Cys Phe Asp Leu Asp Gly Glu Cys

1 5 10 15

<210> 52

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 52

Cys Phe Thr Leu Asp Gly Gln Cys Phe Asp Trp Thr His Glu Cys

1 5 10 15

<210> 53

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 53

Cys Phe Leu Leu Asp Gly Glu Cys Phe Asp Trp Met Gln Glu Cys

1 5 10 15

<210> 54

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 54

Cys Phe Glu Leu Asp Gly Asp Cys Phe Asp Trp Thr His Glu Cys

1 5 10 15

<210> 55

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 55

Cys Phe Thr Leu Asp Gly Thr Cys Phe Asp Trp Thr His Glu Cys

1 5 10 15

<210> 56

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 56

Cys Phe His Leu Asp Gly Val Cys Phe Asp Trp Thr His Glu Cys

1 5 10 15

<210> 57

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 57

Cys Phe Tyr Leu Asp Gly Thr Cys Phe Asp Trp Thr His Glu Cys

1 5 10 15

<210> 58

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 58

Cys Phe Leu Leu Asp Gly Glu Cys Phe Asp Trp Ala Gln Glu Cys

1 5 10 15

<210> 59

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 59

Cys Phe His Leu Asp Gly Glu Cys Phe Asp Leu Ala Lys Thr Cys

1 5 10 15

<210> 60

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 60

Cys Phe Glu Leu Asp Gly Glu Cys Phe His Phe Gly Glu Pro Cys

1 5 10 15

<210> 61

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 61

Cys Phe Val Leu Asp Gly Glu Cys Phe Glu Ile Gly Glu Arg Cys

1 5 10 15

<210> 62

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 62

Cys Phe Thr Leu Asp Gly Glu Cys Phe Asp Leu Asp Gly Trp Cys

1 5 10 15

<210> 63

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 63

Cys Phe Leu Leu Asp Gly Glu Cys Phe Asp Leu Ile Gly Glu Cys

1 5 10 15

<210> 64

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 64

Cys Phe Glu Leu Asp Gly Glu Cys Phe Ser Phe Pro Gly Thr Cys

1 5 10 15

<210> 65

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 65

Cys Phe Glu Leu Asp Gly Glu Cys Phe Ser Trp Pro Tyr Pro Cys

1 5 10 15

<210> 66

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 66

Cys Phe Thr Leu Asp Gly Glu Cys Phe Leu Leu Gly Glu Asn Cys

1 5 10 15

<210> 67

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 67

Cys Phe Trp Leu Asp Gly Glu Cys Phe Asp Leu Gly Gly Gln Cys

1 5 10 15

<210> 68

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 68

Cys Phe Glu Leu Asp Gly Glu Cys Phe Asp Leu Asp Asn Gln Cys

1 5 10 15

<210> 69

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 69

Cys Phe Trp Leu Asp Gly Glu Cys Phe Asp Leu Tyr Gly Gly Cys

1 5 10 15

<210> 70

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 70

Cys Phe Arg Leu Asp Gly Glu Cys Phe Asp Ile Ser Asn Glu Cys

1 5 10 15

<210> 71

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 71

Cys Phe Glu Leu Asp Gly Glu Cys Phe Asn Ile Gly Ser Lys Cys

1 5 10 15

<210> 72

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 72

Cys Phe Trp Leu Asp Gly Glu Cys Phe Asp Phe Gly Gly Cys

1 5 10

<210> 73

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 73

Cys Phe Thr Leu Asp Gly Ala Cys Phe Asp Trp Thr His Glu Cys

1 5 10 15

<210> 74

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 74

Cys Asp Tyr Cys Val Arg Leu Gly Leu Thr Gly Cys

1 5 10

<210> 75

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 75

Cys Gly Trp Cys Ser Asp Gln Ile Asp Gly Phe Cys

1 5 10

<210> 76

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 76

Cys Ala Trp Cys Ser Asp Pro Ile Asp Gly Phe Cys

1 5 10

<210> 77

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 77

Cys Asp Trp Cys Ile Asp Pro Gly Val Ser Phe Cys

1 5 10

<210> 78

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 78

Cys Ser Trp Cys Val Asp Asp Gly Leu Pro Phe Cys

1 5 10

<210> 79

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 79

Cys Thr Trp Cys Val Asp Asp Gly Leu Ser Phe Cys

1 5 10

<210> 80

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 80

Cys Thr Trp Cys Val Asp Asp Glu Thr Trp Asn Cys

1 5 10

<210> 81

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 81

Cys Asp Tyr Cys Ile Arg Leu Gly Leu Thr Gly Cys

1 5 10

<210> 82

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 82

Cys Asp Trp Cys Thr Asp Asn Ile Pro Gly Ile Cys

1 5 10

<210> 83

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 83

Cys Ile Arg Tyr Gly Asp Ile Cys Tyr Asp Pro Asp His Ser Cys

1 5 10 15

<210> 84

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 84

Cys Ile Arg Tyr Gly Asp Ile Cys Phe His Pro Asp Tyr Thr Cys

1 5 10 15

<210> 85

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 85

Cys Ile Asn Tyr Ala Asn Ile Cys Leu Asp Thr Glu Lys Met Cys

1 5 10 15

<210> 86

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 86

Cys Asn Phe Leu Cys Asp Asp Leu Cys

1 5

<210> 87

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<221> Xaa

<222> (3)..(3)

<223> Xaa represents any amino acid residue

<220>

<221> Xaa

<222> (6)..(7)

<223> Xaa represents any amino acid residue

<220>

<221> Xaa

<222> (11)..(13)

<223> Xaa represents any amino acid residue

<220>

<221> Xaa

<222> (14)..(14)

<223> Xaa is absent or represents any amino acid residue

<400> 87

Cys Phe Xaa Leu Asp Xaa Xaa Cys Phe Asp Xaa Xaa Xaa Xaa Cys

1 5 10 15

<210> 88

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<221> Xaa

<222> (3)..(3)

<223> Xaa is R or N

<220>

<221> Xaa

<222> (5)..(5)

<223> Xaa is G or A

<220>

<221> Xaa

<222> (6)..(6)

<223> Xaa is D or N

<220>

<221> Xaa

<222> (9)..(9)

<223> Xaa represents any amino acid residue

<220>

<221> Xaa

<222> (10)..(10)

<223> Xaa is D or H

<220>

<221> Xaa

<222> (11)..(11)

<223> Xaa is P or T

<220>

<221> Xaa

<222> (12)..(12)

<223> Xaa is D or E

<220>

<221> Xaa

<222> (13)..(14)

<223> Xaa represents any amino acid residue

<400> 88

Cys Ile Xaa Tyr Xaa Xaa Ile Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys

1 5 10 15

<210> 89

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<221> Xaa

<222> (3)..(3)

<223> Xaa represents any amino acid residue

<220>

<221> Xaa

<222> (10)..(11)

<223> Xaa represents any amino acid residue

<220>

<221> Xaa

<222> (12)..(12)

<223> Xaa is G or P

<220>

<221> Xaa

<222> (13)..(14)

<223> Xaa represents any amino acid residue

<400> 89

Cys Phe Xaa Leu Asp Gly Glu Cys Phe Xaa Xaa Xaa Xaa Xaa Cys

1 5 10 15

<210> 90

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<221> Xaa

<222> (2)..(3)

<223> Xaa represents any amino acid residue

<220>

<221> Xaa

<222> (7)..(7)

<223> Xaa is F or M

<220>

<221> Xaa

<222> (10)..(11)

<223> Xaa represents any amino acid residue

<400> 90

Cys Xaa Xaa Cys Ala Asp Xaa Pro Ile Xaa Xaa Cys

1 5 10

<210> 91

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<221> Xaa

<222> (2)..(2)

<223> Xaa is A or N

<220>

<221> Xaa

<222> (3)..(3)

<223> Xaa is W or F

<220>

<221> Xaa

<222> (6)..(6)

<223> Xaa is P or D

<220>

<221> Xaa

<222> (7)..(7)

<223> Xaa is N or D

<400> 91

Cys Xaa Xaa Leu Cys Xaa Xaa Leu Cys

1 5

<210> 92

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<220>

<221> Xaa

<222> (8)..(9)

<223> Xaa represents any amino acid residue

<220>

<221> Xaa

<222> (11)..(12)

<223> Xaa represents any amino acid residue

<400> 92

Cys Asp Phe Thr Met Pro Cys Xaa Xaa Trp Xaa Xaa Cys

1 5 10

<210> 93

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 93

Ala Cys Thr Pro Cys Ala Asp Phe Pro Ile Trp Gly Cys Ala

1 5 10