EA046487B1 - BICYCLIC PEPTIDE LIGANDS SPECIFIC TO MT1-MMP - Google Patents

Description

Field of technology to which the invention relates

The present invention relates to polypeptides covalently linked to molecular scaffolds such that two or more peptide loops are located between the points of attachment to the scaffold. In particular, the invention describes peptides capable of binding with high affinity to membrane metalloprotease type 1 (MT1-MMP). The invention also describes drug conjugates containing these peptides conjugated to one or more effector and/or functional groups that can be used for imaging and targeted cancer therapy.

BACKGROUND OF THE INVENTION

Cyclic peptides are capable of binding to target proteins with high affinity and specificity and therefore represent a promising class of molecules for the development of therapeutics. In fact, several cyclic peptides are already successfully used in the clinic, such as the antibacterial peptide vancomycin, the immunosuppressant cyclosporine, or the anticancer drug octreotide (Driggers et al. (2008), Nat Rev Drug Discov 7 (7), 608-24). Good binding properties are determined by the relatively large interaction surface between the peptide and the target, as well as the reduced conformational flexibility of cyclic structures. Macrocycles typically bind to surfaces of several hundred square angstroms, such as the cyclic peptide antagonist CVX15 CXCR4 (400 A ² ; Wu et al. (2007), Science 330, 1066-71), a cyclic peptide with an Arg-Gly- motif. Asp, which binds to integrin aVb3 (355 A ² ) (Xiong et al. (2002), Science 296 (5565), 151-5), or the cyclic peptide inhibitor upain-1, which binds to urokinase-type plasminogen activator (603 A ² ; Zhao et al (2007), J Struct Biol 160 (1), 1-10).

Due to the cyclic configuration, peptide macrocycles are less flexible than linear peptides, resulting in reduced entropy loss upon binding to targets and higher binding affinities. Reduced flexibility also leads to fixation of target-specific conformations, increasing binding specificity compared to linear peptides. This effect can be illustrated by the potent and selective inhibitor of matrix metalloproteinase 8 (MMP-8), which loses selectivity over other MMPs after ring opening (Cherney et al. (1998), J Med Chem 41 (11), 1749-51 ). The beneficial binding properties achieved through macrocyclization are even more pronounced for polycyclic peptides containing more than one peptide ring, such as, for example, vancomycin, nisin and actinomycin.

Various research groups have previously attached polypeptides containing cysteine residues to a synthetic molecular structure (Kemp and McNamara (1985), J. Org. Chem; Timmerman et al. (2005), ChemBioChem). Meloen et al. used tris(bromomethyl)benzene and related molecules to rapidly and quantitatively cyclize multiple peptide loops on synthetic scaffolds to structurally mimic protein surfaces (Timmerman et al. (2005), ChemBioChem). Methods for the preparation of candidate drug compounds, in which the compounds are prepared by coupling cysteine-containing polypeptides to a molecular scaffold such as, for example, tris(bromomethyl)benzene, are described in WO 2004/077062 and WO 2006/078161. Other suitable examples of molecular scaffolds include non-aromatic scaffolds described in Heinis et al (2014) Angewandte Chemie, International Edition 53(6) 1602-1606.

Combinatorial approaches using phage display have been developed to generate and screen large libraries of bicyclic peptides to identify targets of interest (Heinis et al. (2009), Nat Chem Biol 5 (7), 502-7 and WO 2009/098450). Briefly, combinatorial libraries of linear peptides containing three cysteine residues and two stretches of six random amino acids (Cys-(Xaa) ₆ -Cys-(Xaa) ₆ -Cys) are presented on phage and subjected to cyclization by covalently linking cysteine side chains to low molecular weight frame.

The essence of the invention

According to a first aspect, the invention provides a MT1MMP-specific peptide ligand comprising a polypeptide containing at least three cysteine residues separated by at least two loop sequences, and a molecular scaffold that forms covalent bonds with the cysteine residues of the polypeptide to produce at least two polypeptide loops on a molecular scaffold, wherein said molecular scaffold is 1,1',1(1,3,5-triazinan-1,3,5-triyl)triprop-2-en-1-one (T AT A).

In accordance with another aspect, the invention relates to a drug conjugate containing a peptide ligand described herein conjugated to one or more effector and/or functional groups.

In accordance with a further aspect, the invention relates to a pharmaceutical composition containing a peptide ligand or drug conjugate described herein, in combination with one or more pharmaceutically acceptable excipients.

In accordance with another aspect, the invention relates to a peptide ligand or drug conjugate described herein for the prevention, suppression or treatment of a disease or disorder mediated by MT1-MMP.

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Brief description of drawings

The drawing shows the change in body weight and registration of tumor volume after administration of BT17BDC58 to female BALB/c nude mice carrying the HT1080 xenograft. Experimental points correspond to the average body weight in the group. Error bars indicate standard error of the mean (SEM).

Detailed Description of the Invention

In one embodiment, said loop sequences comprise 2, 3, 5, 6, 7 or 9 amino acids. In another embodiment, said loop sequences comprise 3 or 7 amino acids.

In another embodiment, said loop sequences comprise three cysteine residues separated by two loop sequences, the first loop consisting of 7 amino acids and the second loop consisting of 2 amino acids, for example:

CEESFYPECDHC (SEQ ID NO: 1);

in particular

A-(SEQ ID NO: 1)-A (referred to here as 17-108-02).

In another embodiment, said loop sequences comprise three cysteine residues separated by two loop sequences, the first loop consisting of 3 amino acids and the second loop consisting of 6 amino acids, for example:

CPDLCLDLFPNC (SEQ ID NO: 2); And

CPELCVDLYPHC (SEQ ID NO: 3);

in particular:

A-(SEQ ID NO: 2)-A (referred to here as 17-111-01).

A-(SEQ ID NO: 3)-A (referred to here as 17-111-02).

In another embodiment, said loop sequences comprise three cysteine residues separated by two loop sequences, the first loop consisting of 6 amino acids and the second loop consisting of 3 amino acids, for example:

CHPEWVSCEFHC (SEQ ID NO: 4);

in particular

A-(SEQ ID NO: 4)-A (referred to here as 17-116-01).

In another embodiment, said loop sequences comprise three cysteine residues separated by two loop sequences, the first loop consisting of 3 amino acids and the second loop consisting of 7 amino acids, for example

CSHECALLFPKTC(SEQ ID NO: 5);

CFDECQLLFPKTC (SEQ ID NO: 6);

CLDECKLLFPKTC(SEQ ID NO: 7);

CREECMLLFPKTC(SEQ ID NO: 8);

CETECALLFPRSC (SEQ ID NO: 9);

CADECRLLFPKTC(SEQ ID NO: 10);

CDVECRLLFPRSC (SEQ ID NO: 11);

CIDECRLLFPRSC (SEQ ID NO: 12);

CVRECALLFPKTC(SEQ ID NO: 13);

CV[HArg]ECALLFPKTC (SEQ ID NO: 14);

CVRECALLFPRTC(SEQ ID NO: 15);

CVRECALLFP[HArg]TC (SEQ ID NO: 16);

CV[HArg]ECALLFP[HArg]TC (SEQ ID NO: 17);

CV[HArg]ECALLFPATC (SEQ ID NO: 18);

CVAECALLFP[HArg]TC (SEQ ID NO: 19);

CVTECQLLFPKTC (SEQ ID NO: 20);

CRHECELLFPKTC (SEQ ID NO: 21);

CQRECALLFPKTC(SEQ ID NO: 22);

CVRECTLLFPKTC (SEQ ID NO: 23);

CTIECALLFPKTC(SEQ ID NO: 24);

CARECALLFPKTC(SEQ ID NO: 25);

CINECRLLFPKTC (SEQ ID NO: 26);

CYTECSLLFPKTC(SEQ ID NO: 27);

CHEECRLLFPKTC(SEQ ID NO: 28);

CLEECKLLFPKTC (SEQ ID NO: 29);

CIDECALLFPRTC(SEQ ID NO: 30);

CYEECRLLFPRTC(SEQ ID NO: 31);

CVRECRLLFPKTC(SEQ ID NO: 32);

CHIECALLFPKTC (SEQ ID NO: 33);

CKRECMLLFPKTC(SEQ ID NO: 34);

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CYRECALLFPKTC(SEQ ID NO: 35);

CLTECALLFPKTC(SEQ ID NO: 36);

CEVECRLLFPKTC (SEQ ID NO: 37);

CEAECRLLFPKTC (SEQ ID NO: 38);

CVQECALLFPKTC(SEQ ID NO: 39);

CIRECSLLFPKTC (SEQ ID NO: 40);

CVTECALLFPKTC (SEQ ID NO: 41);

CVAECKLLFPKTC (SEQ ID NO: 42);

CVGECALLFPKTC(SEQ ID NO: 43);

CVVECALLFPKTC(SEQ ID NO: 44);

CVFECALLFPKTC(SEQ ID NO: 45);

CA[HArg]ECALLFP[HArg]TC (SEQ ID NO: 46);

CV[HArg]ECALLFA[HArg]TC (SEQ ID NO: 47);

CV[HArg]ECALLFP[HArg]AC (SEQ ID NO: 48);

CV[HArg]ECALL[1Nal]P[HArg]TC (SEQ ID NO: 49);

CV[HArg]ECALL[Cha]P[HArg]TC (SEQ ID NO: 50);

CV[HArg]ECALLF[Pip][HArg]TC (SEQ ID NO: 51);

CV[HArg]ECALLFP[HArg]SC (SEQ ID NO: 52);

CV[HArg]ECALLFP[HArg][HSer]C (SEQ ID NO: 53);

CV[HArg]ECALLF[HyP][HArg]TC (SEQ ID NO: 54);

CV[HArg]EC[Aib]LLFP[HArg]TC (SEQ ID NO: 55);

CV[HArg]ECAL[Nle]FP[HArg]TC (SEQ ID NO: 56);

CV[HArg]ECA[tBuAla]LFP[HArg]TC (SEQ ID NO: 57);

CV[HArg]ECA[Nle]LFP[HArg]TC (SEQ ID NO: 58);

CV[Aad2]ECALLFP[HArg]TC (SEQ ID NO: 59);

CP[HArg]ECALLFP[HArg]TC (SEQ ID NO: 60);

CV[HArg]ECALL[4FlPhe]P[HArg]TC (SEQ NO: 61);

CV[HArg]ECAL[tBuGly]FP[HArg]TC (SEQ ID NO: 62);

CV[HArg]ECAL[Cha]FP[HArg]TC (SEQ ID NO: 63);

CV[HArg]ECALL[2Nal]P[HArg]TC (SEQ ID NO: 64);

CV[HArg]ECALLFP[HArg][HyV]C (SEQ ID NO: 65);

C[tBuGly][HArg]ECALLFP[HArg]TC (SEQ ID NO: 66);

CVEECALLFP[HArg]TC (SEQ ID NO: 67);

CV[HArg]ECA[Cpa]LFP[HArg]TC (SEQ ID NO: 68);

CV[HArg]ECA[Cba]LFP[HArg]TC (SEQ ID NO: 69);

CV[HArg]ECA[C5A]LFP[HArg]TC (SEQ ID NO: 70);

CV[HArg]ECA[Cha]LFP[HArg]TC (SEQ ID NO: 71);

CV[HArg]ECA[tBuGly]LFP[HArg]TC (SEQ ID NO: 72);

CV[HArg]ECALLF[cis-HyP][HArg]TC (SEQ ID NO: 73);

CV[HArg]ECAL[Cpa]FP[HArg]TC (SEQ ID NO: 74);

CV[HArg]ECAL[C5A]FP[HArg]TC (SEQ ID NO: 75);

CV[HArg]ECA[tBuAla]LF[HyP][HArg]TC (SEQ ID NO: 76);

CV[HArg]ECA[tBuAla][tBuGly]F[HyP][HArg]TC (SEQ ID NO: 77) and

C[tBuGly][HArg]ECA[tBuAla]LFP[HArg]TC (SEQ ID NO: 78);

where Aad is alpha-E-aminoadipic acid, Aib is aminoisobutyric acid, C5a is beta-cyclopentyl-E-alanine, Cba is β-cyclobutylalanine, Cha is 3-cyclohexyl-E-alanine, Cpa is betacyclopropyl-E -alanine, 4FlPhe is 4-fluoro-E-phenylalanine, HArg is homoarginine, HyP is hydroxyproline, HyV is 3-hydroxy-E-valine, HSer is homoserine, 1Nal is 1-naphthylalanine, 2Nal is 2-naphthylalanine, Nle is norleucine, P is pipecolic acid, tBuAla is tert-butylalanine, tBuGly is tert-butylglycine;

in particular

A-(SEQ ID NO: 5)-A (referred to here as 17-120-00);

A-(SEQ ID NO: 6)-A (herein designated 17-120-01);

A-(SEQ ID NO: 7)-A (herein designated 17-120-02);

A-(SEQ ID NO: 8)-A (herein designated 17-120-03);

A-(SEQ ID NO: 9)-A (herein designated 17-120-04);

A-(SEQ ID NO: 10)-A (herein designated 17-120-05);

A-(SEQ ID NO: 11)-A (herein designated 17-120-07);

A-(SEQ ID NO: 12)-A (herein designated 17-120-08);

APPP-(SEQ ID NO: 13)-A (referred to here as 17-120-09-T01);

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QISP-(SEQ ID NO: 13)-A (referred to here as 17-120-09-T02);

ALPP-(SEQ ID NO: 13)-A (referred to here as 17-120-09-T03 and BCY1124);

Ac-ALPP-(SEQ ID NO: 13) (herein referred to as Ac-(17-120-09-T03) and BCY1125);

Sar3-ALPP-(SEQ ID NO: 13) (herein referred to as Sar3-A-(17-120-09-T03));

GPPP-(SEQ ID NO: 13)-A (herein designated 17-120-09-T04);

SPPP-(SEQ ID NO: 13)-A (herein designated 17-120-09-T05);

NPPP-(SEQ ID NO: 13)-A (referred to here as 17-120-09-T06);

EPPP-(SEQ ID NO: 13)-A (referred to here as 17-120-09-T07);

HPPP-(SEQ ID NO: 13)-A (herein designated 17-120-09-T08);

APNP-(SEQ ID NO: 13)-A (referred to here as 17-120-09-T09);

APDP-(SEQ ID NO: 13)-A (herein designated 17-120-09-T10);

APLP-(SEQ ID NO: 13)-A (herein designated 17-120-09-T11);

APAP-(SEQ ID NO: 13)-A (referred to here as 17-120-09-T12);

APHP-(SEQ ID NO: 13)-A (referred to here as 17-120-09-T13);

Sar3-ALPP-(SEQ ID NO: 14) (herein referred to as Sar3-A-(17-120-09-T03)HArg2);

Sar3-ALPP-(SEQ ID NO: 15) (herein referred to as Sar3-A-(17-120-09-T03) Arg9);

Sar3-ALPP-(SEQ ID NO: 16) (herein referred to as Sar3-A-(17-120-09-T03)HArg9);

(B-Ala)-Sar10-ALPP-(SEQ ID NO: 17) (herein referred to as (B-Ala)-Sar10-A-(17-120-09-T03)HArg2 HArg9);

Ac-(B-Ala)-Sar10-ALPP-(SEQ ID NO: 17) (herein referred to as Ac-(B-Ala)-Sar10-A-(17-120-09-T03)HArg2 HArg9);

ALPP-(SEQ ID NO: 17) (herein referred to as BCY3959);

[Ac]LPP-(SEQ ID NO: 17) (herein referred to as BCY9933);

[Ac]APP-(SEQ ID NO: 17) (herein referred to as BCY9934);

[Ac]LAP-(SEQ ID NO: 17) (herein referred to as BCY9935);

[Ac]LPA-(SEQ ID NO: 17) (herein referred to as BCY9936);

[Ac]-(SEQ ID NO: 17) (herein referred to as BCY9968);

[Ac]LYP-(SEQ ID NO: 17) (herein referred to as BCY11147);

[Ac]LPY-(SEQ ID NO: 17) (herein referred to as BCY11148);

[Ac][dA]PP-(SEQ ID NO: 17) (herein referred to as BCY11165);

[Ac]L[dA]P-(SEQ ID NO: 17) (herein referred to as BCY11166);

[Ac]LP[dA]-(SEQ ID NO: 17) (herein referred to as BCY11167);

ALPP-(SEQ ID NO: 17)-A (herein designated BCY10288).

(B-Ala)-Sar10-ALPP-(SEQ ID NO: 18) (herein referred to as (B-Ala)-Sar10-A-(17-120-09-T03)HArg2 Ala9);

(B-Ala)-Sar10-ALPP-(SEQ ID NO: 19) (referred to here as (B-Ala)-Sar10-A-(17-120-09-T03) Ala2 HArg9);

[Ac]LPP-(SEQ ID NO: 19) (herein referred to as BCY9938);

APMP-(SEQ ID NO: 20)-A (referred to here as 17-120-10-T01);

APSP-(SEQ ID NO: 21)-A (referred to here as 17-120-11-T01);

AALP-(SEQ ID NO: 22)-A (herein designated 17-120-12-T01);

ALDP-(SEQ ID NO: 23)-A (referred to here as 17-120-13-T01);

ADRP-(SEQ ID NO: 24)-A (herein designated 17-120-14-T01);

ATQP-(SEQ ID NO: 25)-A (referred to here as 17-120-15-T01);

SPPP-(SEQ ID NO: 25)-A (referred to here as 17-120-15-T02);

ARHP-(SEQ ID NO: 26)-A (referred to here as 17-120-16-T01);

ALPP-(SEQ ID NO: 27)-A (herein designated 17-120-17-T01);

A-(SEQ ID NO: 28)-A (herein designated 17-120-18);

A-(SEQ ID NO: 29)-A (herein designated 17-120-19);

A-(SEQ ID NO: 30)-A (herein designated 17-120-20);

A-(SEQ ID NO: 31)-A (herein designated 17-120-21);

APPP-(SEQ ID NO: 31)-A (referred to here as 17-120-21-T01);

APSP-(SEQ ID NO: 32)-A (referred to here as 17-120-22-T01);

PLPP-(SEQ ID NO: 32)-A (referred to here as 17-120-22-T02);

APAP-(SEQ ID NO: 33)-A (herein designated 17-120-23-T01);

AVEP-(SEQ ID NO: 34)-A (referred to here as 17-120-24-T01);

AEPA-(SEQ ID NO: 35)-A (referred to here as 17-120-25-T01);

ASPP-(SEQ ID NO: 36)-A (herein designated 17-120-26-T01);

AAPP-(SEQ ID NO: 37)-A (referred to herein as 17-120-27-T01);

APPP-(SEQ ID NO: 38)-A (referred to here as 17-120-28-T01);

AVPP-(SEQ ID NO: 39)-A (herein designated 17-120-29-T01);

SPPP-(SEQ ID NO: 40)-A (referred to here as 17-120-30-T01);

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HLPP-(SEQ ID NO: 41)-A (herein designated 17-120-31-T01);

RLPP-(SEQ ID NO: 41)-A (referred to here as 17-120-31-T02);

APPP-(SEQ ID NO: 41)-A (herein designated 17-120-31-T03);

MPPP-(SEQ ID NO: 42)-A (referred to here as 17-120-32-T01);

SPPP-(SEQ ID NO: 43)-A (referred to here as 17-120-33-T01);

APPP-(SEQ ID NO: 44)-A (referred to here as 17-120-34-T01);

APPP-(SEQ ID NO: 45)-A (referred to here as 17-120-35-T01);

[Ac]LPP-(SEQ ID NO: 46) (herein referred to as BCY9937);

[Ac]LPP-(SEQ ID NO: 47) (herein referred to as BCY9943);

[Ac]LPP-(SEQ ID NO: 48) (herein referred to as BCY9945);

[Ac]LPP-(SEQ ID NO: 49) (herein referred to as BCY9946);

[Ac]LPP-(SEQ ID NO: 50) (herein referred to as BCY9949);

[Ac]LPP-(SEQ ID NO: 51) (herein referred to as BCY9951);

[Ac]LPP-(SEQ ID NO: 52) (herein referred to as BCY9952);

[Ac]LPP-(SEQ ID NO: 53) (herein referred to as BCY9953);

[Ac]LPP-(SEQ ID NO: 54) (herein referred to as BCY9954);

[Ac]LPP-(SEQ ID NO: 55) (herein referred to as BCY9955);

[Ac]LPP-(SEQ ID NO: 56) (herein referred to as BCY9957);

[Ac]LPP-(SEQ ID NO: 57) (herein referred to as BCY9959);

[Ac]LYP-(SEQ ID NO: 57) (herein referred to as BCY12401);

[Ac]EYP-(SEQ ID NO: 57) (herein referred to as BCY12405);

[Ac]LPP-(SEQ ID NO: 58) (herein referred to as BCY9960);

[Ac]LPP-(SEQ ID NO: 59) (herein referred to as BCY9961);

[Ac]LPP-(SEQ ID NO: 60) (herein referred to as BCY9963);

[Ac]LPP-(SEQ ID NO: 61) (herein referred to as BCY9964);

[Ac]LPP-(SEQ ID NO: 62) (herein referred to as BCY9965);

[Ac]LPP-(SEQ ID NO: 63) (herein referred to as BCY9966);

[Ac]LPP-(SEQ ID NO: 64) (herein referred to as BCY10223);

[Ac]LPP-(SEQ ID NO: 65) (herein referred to as BCY10224);

[Ac]LPP-(SEQ ID NO: 66) (herein referred to as BCY11149);

[Ac]LPP-(SEQ ID NO: 67) (herein referred to as BCY11150);

[Ac]LPP-(SEQ ID NO: 68) (herein referred to as BCY11151);

[Ac]LPP-(SEQ ID NO: 69) (herein referred to as BCY11152);

[Ac]LPP-(SEQ ID NO: 70) (herein referred to as BCY11153);

[Ac]LPP-(SEQ ID NO: 71) (herein referred to as BCY11154);

[Ac]LPP-(SEQ ID NO: 72) (herein referred to as BCY11155);

[Ac]LPP-(SEQ ID NO: 73) (herein referred to as BCY11163);

[Ac]LPP-(SEQ ID NO: 74) (herein referred to as BCY11158);

[Ac]LPP-(SEQ ID NO: 75) (herein referred to as BCY11160);

[Ac]LYP-(SEQ ID NO: 76) (herein referred to as BCY12402);

[Ac]LYP-(SEQ ID NO: 77) (herein referred to as BCY12403); And

[Ac]LYP-(SEQ ID NO: 78) (herein referred to as BCY12404).

In another embodiment, the peptide ligand contains an amino acid sequence that is (B-Ala)-Sar10-ALPP-(SEQ ID NO: 17) (herein referred to as (B-Ala)-Sar10-A-(17-120 -09-T03)HArg2HArg9).

The data shown in Fig. 1 and in table. 4 and 5 indicate that a bicyclic peptide drug conjugate containing the indicated peptide ligand (BT17BDC58) exhibits dose-dependent antitumor activity.

In another embodiment, said loop sequences comprise three cysteine residues separated by two loop sequences, the first loop consisting of 7 amino acids and the second loop consisting of 3 amino acids, for example:

CSSWDKLMCHPYC(SEQ ID NO: 79);

in particular

A-(SEQ ID NO: 79)-A (referred to here as 17-121-00).

In another embodiment, said loop sequences comprise three cysteine residues separated by two loop sequences, the first loop consisting of 3 amino acids and the second loop consisting of 9 amino acids, for example

CPEECFYLPPHPMSC(SEQ ID NO: 80);

CPQECFYLPGHSLYC (SEQ ID NO: 81);

CPGECFYPPGHPLAC (SEQ ID NO: 82);

CPGECFYPTNHPLYC (SEQ ID NO: 83);

CPQECFYPIGHPLAC (SEQ ID NO: 84);

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CPEECFYPPGHKLHC (SEQ ID NO: 85);

CPQECFYPPGHRLRC (SEQ ID NO: 86);

CPQECFYPPGHPYHC (SEQ ID NO: 87);

CPQECFYPSTHPLYC(SEQ ID NO: 88);

CPGECFYPSNHRLYC(SEQ ID NO: 89);

CPDECFYPPEHPLAC (SEQ ID NO: 90);

CPGECFYPPGHHLSC (SEQ ID NO: 91);

CPGECFYPPGHHLGC (SEQ ID NO: 92);

CPEECFYPPNHPLYC (SEQ ID NO: 93);

CPGECFYPPDHPLYC(SEQ ID NO: 94);

CPGECFYPPGHPLYC(SEQ ID NO: 95);

CPGECFYPPNHPFYC (SEQ ID NO: 96);

CPGECFYPPNHPLYC (SEQ ID NO: 97);

CPEECFYPPGHPLAC (SEQ ID NO: 98);

CWMECFYPPGHPLAC (SEQ ID NO: 99);

CFEECFYPPGHPLAC(SEQ ID NO: 100);

CPGECFYPPGHPLRC (SEQ ID NO: 101);

CPGECFYPPGHPREC (SEQ ID NO: 102);

CPGECFYPPGHRFHC (SEQ ID NO: 103) and

CPGECFYPPGHRLYC (SEQ ID NO: 104);

in particular

A-(SEQ ID NO: 80)-A (herein designated 17-127-01);

A-(SEQ ID NO: 81)-A (referred to here as 17-129-00);

SQT-(SEQ ID NO: 82)-A (referred to here as 17-129-01-T01);

SMT-(SEQ ID NO: 82)-A (referred to here as 17-129-01-T02);

SLV-(SEQ ID NO: 82)-A (referred to here as 17-129-01-T03);

ISSYG-(SEQ ID NO: 82)-A (referred to here as 17-129-01-T04);

ENITT-(SEQ ID NO: 82)-A (referred to here as 17-129-01-T05);

A-(SEQ ID NO: 83)-A (herein designated 17-129-02);

A-(SEQ ID NO: 84)-A (herein designated 17-129-03);

A-(SEQ ID NO: 85)-A (herein designated 17-129-04);

A-(SEQ ID NO: 86)-A (herein designated 17-129-05);

A-(SEQ ID NO: 87)-A (herein designated 17-129-06);

A-(SEQ ID NO: 88)-A (herein designated 17-129-07);

A-(SEQ ID NO: 89)-A (herein designated 17-129-08);

A-(SEQ ID NO: 90)-A (herein designated 17-129-09);

A-(SEQ ID NO: 91)-A (herein designated 17-129-10);

A-(SEQ ID NO: 92)-A (herein designated 17-129-11);

L-(SEQ ID NO: 93)-HA (herein designated 17-129-12-T01);

T-(SEQ ID NO: 94)-NA (referred to here as 17-129-13-T01);

Q-(SEQ ID NO: 95)-NA (referred to here as 17-129-14-T01);

A-(SEQ ID NO: 95)-NVI (referred to here as 17-129-14-T02);

N-(SEQ ID NO: 96)-NA (herein designated 17-129-15-T01);

D-(SEQ ID NO: 97)-RA (herein designated 17-129-16-T01);

SRM-(SEQ ID NO: 98)-A (referred to here as 17-129-17-T01);

SRS-(SEQ ID NO: 98)-A (herein designated 17-129-17-T02);

RYMTR-(SEQ ID NO: 98)-A (referred to herein as 17-129-17-T03);

REE-(SEQ ID NO: 99)-A (referred to herein as 17-129-18-T01);

DNM-(SEQ ID NO: 99)-A (herein designated 17-129-18-T02);

QES-(SEQ ID NO: 99)-A (herein designated 17-129-18-T03);

ADY-(SEQ ID NO: 99)-A (referred to herein as 17-129-18-T04);

MAN-(SEQ ID NO: 100)-A (referred to here as 17-129-19-T01);

SQN-(SEQ ID NO: 100)-A (referred to here as 17-129-19-T02);

A-(SEQ ID NO: 101)-TVL (referred to here as 17-129-20-T01);

A-(SEQ ID NO: 102)-SWL (referred to here as 17-129-21-T01);

A-(SEQ ID NO: 103)-LTE (referred to here as 17-129-22-T01);

A-(SEQ ID NO: 104)-YSE (referred to here as 17-129-23-T01) and

Ac-(SEQ ID NO: 104)-YSE (herein referred to as Ac(17-129-23-T01)).

In another embodiment, said loop sequences comprise three cysteine residues separated by two loop sequences, the first loop consisting of 6 amino acids and the second loop consisting of 6 amino acids, for example

CEEEFYPCGHPLYVC(SEQ ID NO: 105);

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CEEQFYPCTHALYTC (SEQ ID NO: 106);

CVEEFYPCDHPLYSC (SEQ ID NO: 107);

CEEEFYPCGHPMHPC (SEQ ID NO: 108);

CDEQFYPCHHRLYSC (SEQ ID NO: 109);

CEEEFYPCGHPFHPC (SEQ ID NO: 110);

CLEQFYPCEHPLFSC (SEQ ID NO: 111);

CVEQFYPCGHRHYIC (SEQ ID NO: 112);

CEEQFYPCSHPLYTC (SEQ ID NO: 113);

CEEQFYPCNHPLNVC (SEQ ID NO: 114);

CEEEFYPCSHPLNPC (SEQ ID NO: 115);

CEEQFYPCGHKLSPC (SEQ ID NO: 116);

CPEQFYPCDHRLYIC (SEQ ID NO: 117);

CQEQFYPCNHPLSPC (SEQ ID NO: 118);

CDEQFYPCNHRLNTC (SEQ ID NO: 119);

CEEAFYPCHHPLYRC (SEQ ID NO: 120);

CDEDFYPCGHYLNQC (SEQ ID NO: 121);

CEEQFYPCTHPLYVC (SEQ ID NO: 122);

CPEQFYPCTHRLYQC (SEQ ID NO: 123);

CEEQFYPCSHPLYRC (SEQ ID NO: 124);

CAEQFYPCDHPLYRC (SEQ ID NO: 125);

CAEEFYPCDHPLYRC (SEQ ID NO: 126);

CEEAFYPCNHPLYTC (SEQ ID NO: 127);

CAEAFYPCDHPLYVC (SEQ ID NO: 128);

CEEAFYPCSHPLFIC(SEQ ID NO: 129);

CEEAFYPCSHPLHPC (SEQ ID NO: 130);

CEEAFYPCSHPLFVC(SEQ ID NO: 131);

CEEQFYPCSHPLYSC (SEQ ID NO: 132);

CEEAFYPCEHPLYMC (SEQ ID NO: 133) and CEEQFYPCNHPLYMC (SEQ ID NO: 134); in particular

A-(SEQ ID NO: 105)-A (herein designated 17-126-01);

A-(SEQ ID NO: 106)-A (herein designated 17-126-02);

A-(SEQ ID NO: 107)-A (herein designated 17-126-03);

A-(SEQ ID NO: 108)-A (herein designated 17-126-06);

A-(SEQ ID NO: 109)-A (herein designated 17-126-07);

A-(SEQ ID NO: 110)-A (herein designated 17-126-08);

A-(SEQ ID NO: 111)-A (herein designated 17-126-09);

A-(SEQ ID NO: 112)-A (herein designated 17-126-10);

A-(SEQ ID NO: 113)-A (herein designated 17-126-18);

A-(SEQ ID NO: 114)-A (herein designated 17-126-19);

A-(SEQ ID NO: 115)-A (herein designated 17-126-20);

A-(SEQ ID NO: 116)-A (herein designated 17-126-21);

A-(SEQ ID NO: 117)-A (herein designated 17-126-22);

A-(SEQ ID NO: 118)-A (herein designated 17-126-23);

A-(SEQ ID NO: 119)-A (herein designated 17-126-24);

A-(SEQ ID NO: 120)-A (herein designated 17-126-25);

Ac-A-(SEQ ID NO: 120)-A (herein referred to as Ac-(17-126-25));

A-(SEQ ID NO: 121)-A (herein designated 17-126-26);

A-(SEQ ID NO: 122)-A (herein designated 17-126-27);

A-(SEQ ID NO: 123)-A (herein designated 17-126-28);

HSP-(SEQ ID NO: 124)-A (herein designated 17-126-30-T01);

GPH-(SEQ ID NO: 125)-A (referred to herein as 17-126-31-T01);

IHS-(SEQ ID NO: 126)-A (referred to herein as 17-126-32-T01);

WSP-(SEQ ID NO: 127)-A (referred to here as 17-126-33-T01);

SHS-(SEQ ID NO: 127)-A (herein designated 17-126-33-T02);

DLH-(SEQ ID NO: 128)-A (referred to here as 17-126-35-T01);

ANE-(SEQ ID NO: 129)-A (referred to here as 17-126-36-T01);

AVW-(SEQ ID NO: 130)-A (referred to here as 17-126-37-T01);

KVQ-(SEQ ID NO: 131)-A (referred to here as 17-126-38-T01);

A-(SEQ ID NO: 132)-PDVA (herein designated 17-126-39-T01);

A-(SEQ ID NO: 133)-HQAA (referred to here as 17-126-40-T01) and A-(SEQ ID NO: 134)-RENA (referred to here as 17-126-41-T01).

- 7 046487

In another embodiment, said loop sequences comprise three cysteine residues separated by two loop sequences, the first loop consisting of 6 amino acids and the second loop consisting of 5 amino acids, for example

CLEQFYPCGDPRLC (SEQ ID NO: 135) and CEEQFYPCGHHLLC (SEQ ID NO: 136);

in particular

A-(SEQ ID NO: 135)-A (herein designated 17-126-11) and

A-(SEQ ID NO: 136)-A (herein designated 17-126-12).

In another embodiment, said loop sequences comprise three cysteine residues separated by two loop sequences, the first loop consisting of 5 amino acids and the second loop consisting of 5 amino acids, for example

CLEPDECFYPMEC (SEQ ID NO: 137);

CKEPQECFYPLKC (SEQ ID NO: 138) and

CDSPEECFYPLEC(SEQ ID NO: 139);

in particular

A-(SEQ ID NO: 137)-A (herein designated 17-122-02);

A-(SEQ ID NO: 138)-A (herein designated 17-122-03); And

A-(SEQ ID NO: 139)-A (referred to here as 17-122-04).

In one embodiment, the peptide ligand is selected from the peptide ligands listed in table. 2 or 3.

Unless otherwise noted, all technical and scientific terms used herein have their traditional meanings known to those skilled in the art, such as peptide chemistry, cell culture and phage display, nucleic acid chemistry, and biochemistry. Use standard techniques of molecular biology, genetics, and biochemistry (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., Short Protocols in Molecular Biology (1999) ^4th ed., John Wiley & Sons, Inc.), which are incorporated herein by reference.

Nomenclature

Numbering

When mentioning the positions of amino acid residues in the peptide ligands of the present invention, cysteine residues (Ci, Cii and Ciii) are not included in the numbering because they are invariant, therefore the numbering of amino acid residues in the peptide ligands of the present invention is carried out as follows:

C ₁ -Ei-E2-S3-F4-Y5-P6-E7-C ₁₁ -D8-H9-C ₁₁₁ (SEQ ID NO: 1).

For the purposes of the present invention, all bicyclic peptides are assumed to be cyclized with 1,1',1''(1,3,5-triazinan-1,3,5-triyl)tripropan-1-one (TATA) to give a trisubstituted structure. Cyclization involving TATA is carried out at Ci, Cii and Ciii. TATA is an example of an αβ-unsaturated carbonyl-containing molecular framework (Angewandte Chemie, International Edition (2014), 53 (6), 16021606).

Molecular format

N- or C-terminal extensions of the bicyclic core sequence are added to the left or right side of the sequence and separated by a hyphen. For example, the N-terminal tail of eAla-Sar10-Ala is designated as follows:

eAla-Sar10-A-(SEQ ID NO: X).

Inverted peptide sequences

Information given in Nair et al (2003) J Immunol 170 (3), 1362-1373 suggests that the peptide sequences described here will also find use in their retroinverted form. For example, a reverse sequence is used (ie, the N-terminus becomes the C-terminus and vice versa), the stereochemistry of which is also reversed (ie, D-amino acids become L-amino acids and vice versa).

Peptide ligands

A peptide ligand as used herein refers to a peptide covalently linked to a molecular framework. Typically, such peptides contain two or more reactive groups (such as cysteine residues) that are capable of forming covalent bonds with the scaffold, and a sequence between these reactive groups, which is called a loop sequence because it forms a loop when the peptide binds to the scaffold. In this case, the peptides contain at least three cysteine residues (denoted here as Ci, Cii and Ciii) and form at least two loops on the framework.

Benefits of Peptide Ligands

Some of the bicyclic peptides of the present invention have a number of useful properties that allow them to be considered drug molecules suitable for injectable,

- 8 046487 inhalation, nasal, ocular, oral or local administration. These beneficial properties include:

Interspecies cross-reactivity

Some ligands show cross-reactivity against PBPs of different bacterial species and can therefore be used to treat infections caused by multiple bacterial species. Other ligands may be highly specific for PBPs of certain bacterial species, which may be useful in treating the infection without collateral damage to the patient's beneficial flora;

Protease stability

Bicyclic peptide ligands should ideally exhibit resistance to plasma proteases, epithelial (membrane-anchored) proteases, gastric and intestinal proteases, lung surface proteases, intracellular proteases, and the like. Protease stability must be maintained across species so that a leading bicyclic candidate can be developed in animal models and also confidently administered to humans;

Desired solubility profile

Solubility depends on the ratio of charged and hydrophilic residues to hydrophobic residues and the number of intra/intermolecular hydrogen bonds, it is a parameter important for preparing compositions and achieving absorption;

Optimal half-life in circulating plasma

Depending on the clinical indication and treatment regimen, it may be necessary to develop a bicyclic peptide for short-term action in the treatment of acute disease, or to develop a bicyclic peptide with an increased retention time in the bloodstream that is optimal for the treatment of chronic disease conditions. Other factors that determine the desired plasma half-life are the requirements of prolonged exposure to ensure maximum therapeutic efficacy compared to associated toxicology due to prolonged exposure to the drug.

Selectivity: Certain peptide ligands of the present invention exhibit selectivity for MT1-MMP but are unable to cross-react with MMP isoforms such as MMP-1, MMP-2, MMP-15 and MMP-16.

Pharmaceutically acceptable salts

It should be understood that salt forms are within the scope of this invention, and references to peptide ligands include salt forms of said ligands.

The salts of the present invention can be synthesized from a starting compound containing a basic or acid moiety using conventional chemical methods, such as those described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8, hardcover, 388 pages, August 2002. Typically, such salts are prepared by reacting the free acid or base forms of the compounds with a suitable base or suitable acid in water or an organic solvent, or in a mixture thereof.

Acid addition salts (mono- or di-salts) can be prepared using a wide range of acids, both inorganic and organic. Examples of acid addition salts include mono or di-salts prepared using an acid selected from the group consisting of acetic, 2,2-dichloroacetic, adipic, alginic, ascorbic (eg, L-ascorbic), L-aspartic, benzenesulfonic, benzoic, 4 -acetamidobenzoic, butane, (+)camphor, camphorosulfonic, (+)-(U)-camphor-10-sulfonic, capric, capronic, caprylic, cinnamic, citric, cyclamine, dodecylsulfuric, ethane-1,2-disulfonic, ethanesulfonic, 2-hydroxyethanesulfonic, formic, fumaric, galactic, gentisic, glucoheptonic, D-gluconic, glucuronic (e.g. D-glucuronic acid), glutamic (e.g. L-glutamic), α-oxoglutaric, glycolic, hippuric, hydrohalic acid (e.g. hydrobromic, hydrochloric, hydroiodic), isethionic, lactic (for example, (+)-b-lactic, A-DL-lactic), lactobionic, maleic, malic, (-)-E-malic. malonic, A-DL-mandelic, methanesulfonic, naphthalene-2-sulfonic, naphthalene-1,5-disulfonic, 1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, pyruvic, L-pyroglutamic, salicylic, 4-aminosalicylic sebacic, stearic, succinic, sulfuric, tannic, (+)-b-tartaric, thiocyanic, p-toluenesulfonic, undecylenic and valeric acids, as well as acylated amino acids and cation exchange resins.

One particular group of salts consists of salts derived from acetic, hydrochloric, hydroiodic, phosphoric, nitric, sulfuric, citric, lactic, succinic, maleic, malic, isethionic, fumaric, benzenesulfonic, toluenesulfonic, sulfuric, methanesulfonic (mesylate), ethanesulfonic, naphthalene sulfonic , valeric, propane, butanoic, malonic, glucuronic and lactobionic acids. The specific salt is a hydrochloride. Another specific salt is acetate.

If the compound is anionic or contains a functional group that can be

- 9 046487 anionic (for example, -COOH can be converted to ^-COO- ), then a salt formed by an organic or inorganic base generating a suitable cation can be obtained. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Li ⁺ , Na ⁺ and K ⁺ , alkaline earth metal cations such as Ca ²⁺ and Mg ²⁺ , and other cations such as Al ³⁺ or Zn ⁺ . Examples of suitable organic cations include, but are not limited to, ammonium ion (ie, NH4 ⁺ ) and substituted ammonium ions (eg, NH3R ⁺ , NH2R2 ⁺ , NHR3 ⁺ , NR ₄ ⁺ ). Examples of some 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, as well as amino acids such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3) ⁴⁺ .

If the peptides of the present invention contain an amino group, they can form quaternary ammonium salts, for example, by reaction with an alkylating agent according to methods well known to one skilled in the art. Such quaternary ammonium compounds are included within the scope of the peptides of the present invention.

Modified derivatives

It should be understood that the modified peptide ligand derivatives defined herein are within the scope of the present invention. Examples of such suitable modified derivatives include one or more modifications selected from: N-terminal and/or C-terminal modifications; replacing one or more amino acid residues with one or more unnatural amino acid residues (for example, replacing one or more polar amino acid residues with one or more isosteric or isoelectronic amino acids; replacing one or more non-polar amino acid residues with other unnatural isosteric 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; replacing one or more amino acid residues with alanine; replacing one or more L-amino acid residues with one or more D-amino acid residues; N-alkylation of one or more amide bonds within the bicyclic peptide ligand; replacing one or more peptide bonds with a surrogate bond; changes in the length of the peptide backbone; substituting a hydrogen on the alpha carbon of one or more amino acid residues with another chemical group, modifying amino acids such as cysteine, lysine, glutamate/aspartate and tyrosine with suitable amines, thiols, carboxylic acid and phenol-reactive reagents to functionalize said amino acids, as well as the introduction or replacement of amino acids to achieve orthogonal reaction properties that provide functionalization, for example, amino acids bearing azide or alkyne groups, which allow functionalization with alkyne or azide-containing moieties, respectively.

In one embodiment, the modified derivative contains a modification at the N-terminus and/or the C-terminus. In another embodiment, the modified derivative is prepared by N-terminal modification using suitable amino reactants and/or C-terminal modification using suitable carboxy reactants. In another embodiment, said N-terminal or C-terminal modification includes the addition of an effector group, non-limiting examples of which include a cytotoxic agent, a radiochelator, or a chromophore.

In another embodiment, the modified derivative contains an N-terminal modification. In another embodiment, the N-terminal modification includes the introduction of an N-terminal acetyl group. In this embodiment, the N-terminal cysteine group (referred to here as Ci) is capped with acetic anhydride or other suitable reagents during peptide synthesis to produce an N-terminally acetylated molecule. This embodiment has the advantage of removing a potential aminopeptidase recognition site and avoiding possible degradation of the bicyclic peptide.

In an alternative embodiment, the N-terminal modification involves the addition of a molecular spacer group that facilitates the conjugation of effector groups and maintains the activity of the bicyclic peptide towards its target.

In another embodiment, the modified derivative contains a C-terminal modification. In another embodiment, the C-terminal modification includes the introduction of an amide group. In this embodiment, a C-terminal cysteine group (herein referred to as Ciii) is added as an amide during peptide synthesis to produce a C-terminally amidated molecule. This embodiment has the advantage of removing a potential carboxypeptidase recognition site and reducing the potential for proteolytic degradation of the bicyclic peptide.

In one embodiment, the modified derivative comprises replacing one or more amino acid residues with one or more unnatural amino acid residues. In this embodiment, unnatural amino acids containing isosteric

- 10 046487 sic/isoelectronic side chains that are not recognized by degradative proteases and do not have any adverse effect on the activity towards the target.

Alternatively, unnatural amino acids may be used that contain spatially restricted side chains that provide conformational and steric hindrance to proteolytic hydrolysis of the adjacent peptide bond. In particular, such amino acids can be proline analogs, amino acids with bulky side chains, C-disubstituted derivatives (such as aminoisobutyric acid, Aib) and cycloamino acids, a simple derivative being aminocyclopropylcarboxylic acid.

In one embodiment, the modified derivative is prepared by adding a spacer group. In another embodiment, the modified derivative is prepared by adding a spacer group to the N-terminal cysteine (Ci) and/or the C-terminal cysteine ( _Ciii ).

In one embodiment, the modified derivative comprises replacing one or more oxidation-sensitive amino acid residues with one or more oxidation-stable amino acid residues.

In one embodiment, the modified derivative comprises replacing one or more charged amino acid residues with one or more hydrophobic amino acid residues. In an alternative embodiment, the modified derivative comprises replacing one or more hydrophobic amino acid residues with one or more charged amino acid residues. The correct balance of charged and hydrophobic amino acid residues is an important characteristic of bicyclic peptide ligands. For example, hydrophobic amino acid residues influence the degree of binding of plasma proteins and, consequently, the concentration of the free accessible fraction in plasma, while charged amino acid residues (in particular, arginine) can influence the interaction of the peptide with phospholipid membranes on the cell surface. The combination of these two types of amino acids can influence the half-life, volume of distribution and exposure of the peptide drug and can be optimized according to the clinical outcome. In addition, the correct combination of charged amino acid residues and hydrophobic amino acid residues and their number can reduce irritation at the injection site (if the peptide drug is administered subcutaneously).

In one embodiment, the modified derivative comprises replacing one or more L-amino acid residues with one or more D-amino acid residues. This embodiment is believed to increase proteolytic stability through steric hindrance and through the ability of D-amino acids to stabilize β-bend conformations (Tugyi et al. (2005) PNAS, 102 (2), 413-418).

In one embodiment, the modified derivative is prepared by removing any amino acid residues and replacing them with alanine residues. This embodiment has the advantage of removing potential proteolytic attack site(s).

It should be noted that each of the above modifications are intentionally carried out to increase the activity or stability of the peptide. Further modification-induced increases in activity can be achieved through the following mechanisms:

Introduction of hydrophobic fragments, the hydrophobicity of which leads to a decrease in dissociation rates and, as a consequence, to an increase in affinity;

Introduction of charged groups that are characterized by long-range ionic interactions leading to increased association rates and hence affinities (see, e.g., Schreiber et al., Rapid, electrostatically assisted association of proteins (1996), Nature Struct. Biol. 3, 427-31); And

Introducing additional steric constraint into the peptide, for example by appropriately sterically constraining the amino acid side chains so that entropy loss is minimal upon binding to the target, sterically constraining the torsion angles of the main chain so that entropy loss is minimal upon binding to the target, and introducing additional cyclizations into the peptide. molecule for identical reasons.

(reviews can be found in Gentilucci et al., Curr. Pharmaceutical Design, (2010), 16, 3185-203 and Nestor et al., Curr. Medicinal Chem (2009), 16, 4399-418).

Isotopic options

The present invention includes all pharmaceutically acceptable (radio)isotopically labeled peptide ligands of the present invention in which one or more atoms are replaced by atoms having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number of the isotope typically naturally occurring, as well as peptide ligands of the present invention to which are attached metal chelating groups (called effectors) that are capable of retaining relevant (radio)isotopes, and peptide ligands of the present invention in which certain functional groups are covalently substituted by the corresponding (radio)isotopes or functional groups labeled with isotopes.

Examples of isotopes suitable for inclusion in the peptide ligands of the present invention include isotopes of hydrogen such as ² H (D) and ³ H (T), carbon such as ¹¹ C, ¹³ C and ¹⁴ C, chlorine such

- 11 046487 as ³⁶ Cl, fluorine such as ¹⁸ F, iodine such as ¹²³ I, ¹²⁵ I and ¹³¹ I, nitrogen such as ¹³ N and ¹⁵ N, oxygen such as ¹⁵ 0, ¹⁷ O and ¹⁸ O, phosphorus such as ³² P, sulfur such as ³⁵ S, copper such as ⁶⁴ Cu, gallium such as ⁶⁷ Ga or ⁶⁸ Ga, yttrium such as ⁹⁰ Y, lutetium such as ¹⁷⁷ Lu, and bismuth such as ²¹³ Bi.

Certain isotopically labeled peptide ligands of the present invention, for example those containing a radioactive isotope, can be used to study the distribution of drugs and/or substrates in tissues. The peptide ligands of the present invention may additionally have valuable diagnostic properties such that they can be used to detect or identify complex formation between a labeled compound and other molecules such as peptides, proteins, enzymes or receptors. Detection or identification methods can use compounds labeled with such means as radioisotopes, enzymes, fluorescent substances, luminescent substances (for example, luminol, luminol derivatives, luciferin, aequorin and luciferase), etc. Radioactive isotopes such as tritium, those. ³ H (T), and carbon-14, i.e. ¹⁴ C, due to the ease of their administration and detection.

Substitution with heavier isotopes such as deuterium, such as ^2H (D), may provide certain therapeutic benefits due to greater metabolic stability, such as increased half-life in vivo, or the ability to use lower doses, and may therefore be preferred in some cases. circumstances.

Substitution with positron-emitting isotopes such as ¹¹ C, ¹⁸ F, ¹⁵ O and ¹³ N can be used to study target occupancy using positron emission tomography (PET).

The isotope-labeled peptide ligand compounds of the present invention can generally be prepared using conventional methods known to those skilled in the art, or methods similar to those described in the accompanying examples using an appropriately isotope-labeled reagent in place of a previously used unlabeled reagent.

Effector and functional groups

In accordance with another aspect, the invention relates to a drug conjugate containing a peptide ligand as defined herein conjugated to one or more effector and/or functional groups.

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

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

In a further embodiment of this aspect of the invention, the effector group of the present invention is the Fc region of an IgG molecule. Preferably, the peptide ligand-effector group of the present invention contains or includes a peptide ligand-Fc hybrid having a half-life of a day or more, two 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 preferably, the peptide ligand of the present invention includes or contains a peptide ligand-Fc hybrid having a half-life te of one day or more.

Functional groups typically include linking groups, drugs, reactive groups for attaching other moieties, functional groups that promote the uptake of macrocyclic peptides into cells, and the like.

The ability of peptides to penetrate cells allows peptides to act against intracellular targets. Targets that can be targeted by cell-penetrating peptides include transcription factors, intracellular signaling molecules such as tyrosine kinases, and molecules involved in the apoptotic pathway. Functional groups that provide cell penetration include peptides or chemical groups attached to either a peptide or a molecular scaffold. Peptides may be used such as derivatives of, for example, VP22, HIV-Tat, Drosophila homeobox protein (Antennapedia), for example, as described in Chen and Harrison, Biochemical Society Transactions (2007) Volume 35, part 4, p821; Gupta et al. in Advanced Drug Discovery Reviews (2004) Volume 57 9637. Examples of short peptides that have been shown to be efficiently transported across plasma membranes include the peptide penetratin from the Drosophila Antennapedia protein (Derossi et al (1994) J Biol. Chem. Volume 269 p10444 ), containing 16 amino acids, a model amphipathic peptide (Oehlke et al (1998) Biochim Biophys Acts Volume 1414 p127) containing 18 amino acids, and arginine-rich regions of the HIV TAT protein. Non-peptide approaches include the use of small molecule mimics or SMOCs that can be easily attached to biomolecules (Okuyama et al (2007) Nature Methods Volume 4 p153). Other chemical strategies of adding guanidinium groups to molecules also increase cell penetration (Elson-Schwab et al (2007) J Biol

- 12 046487

Chem Volume 282 p13585). Low molecular weight molecules, such as steroids, can be attached to a molecular scaffold to increase cellular uptake.

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

In one embodiment, the peptide ligand effector moiety of the invention has a half-life te selected from the group consisting of: 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. Preferably, the peptide ligand effector group or composition of the invention has a half-life te ranging from 12 to 60 hours. In another embodiment, its half-life te is one day or more. In a further embodiment, the half-life is in the range of 12 to 26 hours.

In one particular embodiment of the invention, the functional group is selected from a metal chelator capable of complexing metal radioisotopes used in medicine.

Possible effector groups also include enzymes, such as carboxypeptidase G2, for use in enzyme/prodrug therapy where a peptide ligand replaces the antibodies in ADEPT.

In one particular embodiment of the invention, the functional group is selected from a drug, such as a cytotoxic agent for the treatment of cancer. Suitable examples include alkylating agents such as cisplatin and carboplatin, as well as oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide; antimetabolites such as purine analogs, azathioprine and mercaptopurine, or pyrimidine analogs; plant alkaloids and terpenoids including vinca alkaloids such as vincristine, vinblastine, vinorelbine and vindesine; podophyllotoxin and its derivatives etoposide and teniposide; taxanes such as paclitaxel, originally known as taxol; topoisomerase inhibitors such as the camptothecins: irinotecan and topotecan, and type II inhibitors such as amsacrine, etoposide, etoposide phosphate and teniposide. Other agents may include antitumor antibiotics such as the immunosuppressant dactinomycin (which is used in kidney transplants), doxorubicin, epirubicin, bleomycin, calichemycins, and others.

In another specific embodiment of the invention, the cytotoxic agent is selected from maytansinoids (such as DM1) or monomethyl auristatins (such as MMAE).

DM1 is a cytotoxic agent that is a thiol-containing derivative of maytansine and has the following structure:

Monomethyl auristatin E (MMAE) is a synthetic anticancer agent and has the following structure:

- 13 046487

In the following specific embodiment of the invention, the cytotoxic agent is selected from monomethyl auristatin E (MMAE). The data presented in Fig. 1 and in table. 4 and 5 demonstrate the effects of peptide ligands conjugated to a toxin containing MMAE.

In one embodiment, the cytotoxic agent is linked to the bicyclic peptide by a cleavable bond, such as a disulfide bond or a protease-sensitive bond. In a further embodiment, the groups adjacent to the disulfide bond are modified to control blocking of the disulfide bond and thus the rate of cleavage and concomitant release of the cytotoxic agent.

Published work identifies the possibility of changing the sensitivity of a disulfide bond to reduction by introducing steric hindrance on both sides of the disulfide bond (Kellogg et al. (2011) Bioconjugate Chemistry, 22, 717). A higher degree of steric hindrance reduces the rate of reduction by intracellular glutathione as well as by extracellular (systemic) reducing agents, resulting in reduced ease of release of the toxin both inside and outside the cell. Thus, the optimal degree of disulfide stability in the bloodstream (which minimizes unwanted side effects of the toxin) versus efficient release into the intracellular environment (which maximizes therapeutic effect) can be achieved by carefully selecting the degree of steric hindrance on both sides of the disulfide bond.

The hindrance on both sides of the disulfide bond is modulated by introducing one or more methyl groups either on the targeting moiety (here a bicyclic peptide) or on the toxin side of the molecular construct.

In one embodiment, the cytotoxic agent and linker are selected from any combinations described in WO 2016/067035 (cytotoxic agents and linkers are incorporated herein by reference).

In one embodiment, the linker between said cytotoxic agent and said bicyclic peptide contains one or more amino acid residues. Examples of amino acid residues suitable as linkers include Ala, Cit, Lys, Trp and Val.

In one embodiment, the cytotoxic agent is selected from MMAE, and said drug conjugate further comprises a linker selected from -PABC-Cit-Val-glutaryl- or -PABC-cyclobutyl-Ala-Cit-βAla-, wherein PABC is p-aminobenzylcarbamate. Complete information on the cyclobutyl-containing linker can be found in Wei et al. (2018) J. Med. Chem. 61, 9891000. In another embodiment, the cytotoxic agent is selected from MMAE and the linker is -PABC-Cit-Val-glutaryl-.

In one embodiment, the cytotoxic agent is MMAE, the bicyclic peptide is selected from (B-Ala)-Sar10-ALPP- (SEQ ID NO: 17), and the linker is selected from -PABC-Cit-Valglutaryl-. The said BDC is known here as BT17BDC58, which can be schematically represented as follows:

- 14 046487

where BICYCLE-N007 is (B-Ala)-SarlO-ALPP-(SEQ ID NO: 17), also known as (B-Ala)Sar10-A-(17-120-09-T03)HArg2HArg9).

The data shown in Fig. 1 and in table. 4 and 5 indicate the presence of antitumor activity.

Synthesis

The peptides of the present invention can be obtained synthetically using standard methods followed by interaction with a molecular scaffold in vitro. standard chemical methods can then be used. This allows for rapid large-scale production of soluble material for subsequent experiments or testing. Such methods can be carried out using conventional chemical methods such as those described in Timmerman et al. (higher).

Thus, the invention also relates to the production of polypeptides selected in accordance with the present invention, where the production includes optional additional steps described below. In one embodiment, these steps are carried out on the final polypeptide product obtained by chemical synthesis.

Peptides can also be extended, for example to add another loop and therefore introduce multiple specificities.

The peptide can be extended using conventional chemical methods at the N-terminus or C-terminus, or within loops using orthogonally protected lysines (and their analogues) using standard solid-phase chemistry or liquid-phase chemistry methods. Standard (bio)conjugation techniques can be used to introduce an activated or activated N- or C-terminus. Alternatively, additions can be introduced by condensation of fragments or by native chemical ligation, for example as described in (Dawson et al. 1994. Synthesis of Proteins by Native Chemical Ligation. Science 266:776-779), or using enzymes such as subtiligases such as described in (Chang et al. Proc Natl Acad Sci USA. 1994 Dec 20; 91(26):12544-8, or Hikari et al Bioorganic & Medicinal Chemistry Letters Volume 18, Issue 22, 15 November 2008, Pages 6000-6003) .

Alternatively, the peptides can be extended or modified by further conjugation via disulfide bonds. An additional advantage of such conjugation is that it allows the first and second peptides to be separated from each other in the reducing environment of the cell. In this case, a molecular scaffold (eg TATA) can be added during chemical synthesis of the first peptide so that it reacts with the three cysteine groups; an additional cysteine or thiol residue can 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 methods are equally applicable to the synthesis/coupling of two bicyclic and bispecific macrocycles, possibly resulting in a tetraspecific molecule.

In addition, the addition of other functional or effector groups can be accomplished in a similar manner by attaching them to the N- or C-terminus or to the side chains using appropriate chemical methods. In one embodiment, the coupling is carried out in such a way that it does not block the activity of either moiety.

Pharmaceutical compositions

In accordance with another aspect, the invention relates to a pharmaceutical composition containing a peptide ligand described herein in combination with one or more pharmaceutically acceptable excipients.

Typically, the peptide ligands of the present invention are used in purified form together with pharmacologically acceptable excipients or carriers. Typically, such excipients or carriers include aqueous or alcohol/aqueous solutions, emulsions or suspensions such as saline and/or buffered media. Parenteral media include sodium chloride solution, Ringer's solution containing dextrose, Ringer's solution containing dextrose and sodium chloride, and lactate. If it is necessary to maintain the polypeptide complex in suspension, suitable physiologically acceptable excipients selected from thickening agents such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates are added.

Intravenous media include liquid and nutrient replenishment media, as well as replenishment media containing electrolytes, such as dextrose-containing Ringer's solution. Media may also contain preservatives and other additives such as antimicrobials, antioxidants, chelating agents and inert gases (Mack (1982)

- 15 046487

Remington's Pharmaceutical Sciences, 16th edition).

The compounds of the invention may be used alone or in combination with another agent or agents. Another agent for use in combination may be, for example, another antibiotic or an antibiotic adjuvant, such as a Gram-negative bacterial penetration enhancer, an inhibitor of resistance determinants, or an inhibitor of virulence mechanisms.

Antibiotics suitable for use in combination with the compounds of the present invention include, but are not limited to:

Beta-lactams such as penicillins, cephalosporins, carbapenems or monobactams. Suitable penicillins include oxacillin, methicillin, ampicillin, cloxacillin, carbenicillin, piperacillin, tricarcillin, flucloxacillin and nafcillin; suitable cephalosporins include cefazolin, cephalexin, cephalothin, ceftazidime, cefepime, ceftobiprole, ceftaroline, ceftolozane and cefiderocol; suitable carbapenems include meropenem, doripenem, imipenem, ertapenem, biapenem and tebipenem; Suitable monobactams include aztreonam;

Lincosamides such as clindamycin and lincomycin;

Macrolides such as azithromycin, clarithromycin, erythromycin, telithromycin and solithromycin;

Tetracyclines such as tigecycline, omadacycline, eravacycline, doxycycline and minocycline;

Quinolones such as ciprofloxacin, levofloxacin, moxifloxacin and delafloxacin;

Rifamycins such as rifampicin, rifabutin, rifalazil, rifapentine and rifaximin;

Aminoglycosides such as gentamicin, streptomycin, tobramycin, amikacin and plazomycin;

Glycopeptides such as vancomycin, teichoplanin, telavancin, dalbavancin and oritavancin, Pleuromutilins such as lefamulin,

Oxazolidinones, such as linezolid or tedizolid,

Polymyxins such as polymyxin B or colistin;

trimethoprim, iclaprim, sulfamethoxazole;

metronidazole;

fidaxomicin;

mupirocin;

fusidic acid;

daptomycin;

murepavidin;

fosfomycin and nitrofurantoin.

Suitable antibiotic adjuvants include, but are not limited to, agents known to enhance bacterial penetration, such as outer membrane permeabilizers or efflux pump inhibitors; outer membrane permeabilizers may include polymyxin B nonapeptide or other polymyxin analogues, or sodium edetate;

inhibitors of resistance mechanisms such as beta-lactamase inhibitors; Suitable beta-lactamase inhibitors include clavulanic acid, tazobactam, sulbactam, avibactam, relebactam and nacubactam and inhibitors of virulence mechanisms such as toxins and secretion systems including antibodies.

The compounds of the present invention can also be used in combination with biological therapies, such as nucleic acid, antibody, bacteriophage or phage lysine therapies.

The method of administering the pharmaceutical compositions of the invention may be any of the usual methods known to those skilled in the art. For the purpose of treatment, the peptide ligands of the invention can be administered to any patient in accordance with standard methods. Routes of administration include, but are not limited to, oral (eg, by oral administration); buccal; sublingual; transdermal (for example, using a patch, plaster, etc.); transmucosal (for example, using a patch, plaster, etc.); intranasal (for example, using a nasal spray); ophthalmic (for example, using eye drops); pulmonary (for example, by inhalation or insufflation therapy using, for example, an aerosol, for example, through the mouth or nose); rectal (for example, using a suppository or enema); vaginal (for example, using a pessary); parenteral, for example, by injection, such as subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardial, intrathecal, intraspinal, intracapsular, subcapsular, intraocular, intraperitoneal, intratracheal, subcutaneous, intraarticular, subarachnoid and intrasternal; by implanting a depot or reservoir, for example, subcutaneously or intramuscularly. Preferably, the pharmaceutical compositions according to the invention are administered parenterally. The dosage and frequency of administration depend on the age, gender and condition of the patient, concomitant use of other medications, contraindications

- 16 046487 and other parameters that should be taken into account by the clinician.

The peptide ligands of the present invention can be lyophilized for storage and redissolved in a suitable carrier before use. This method has been shown to be effective and can be carried out using lyophilization and redissolution methods known in the art. Those skilled in the art will recognize that lyophilization and redissolution can result in varying degrees of loss of activity, which may require upward dosage adjustments to compensate.

Compositions containing the peptide ligands of the present invention, or a mixture thereof, can be administered for the purpose of therapeutic treatment. For certain therapeutic applications, an amount sufficient to achieve at least partial inhibition, suppression, modulation, killing, or other measurable parameter of the population of selected cells is defined as a therapeutically effective dose. The amount required to achieve this dose depends on the severity of the disease and the general condition of the patient's own immune system, but usually ranges from 10 μg to 250 mg of the selected peptide ligand per kilogram of body weight, with a dose of 100 μg to 25 mg/kg being commonly used. dose.

A composition containing a peptide ligand of the present invention can be used therapeutically for the treatment of a microbial infection, or for prophylaxis in an individual at risk of infection, for example, undergoing surgery, chemotherapy, mechanical ventilation or other condition or planned intervention. In addition, the peptide ligands described herein can be used in vitro or in vitro to selectively kill, deplete, or otherwise effectively reduce a population of target cells in a heterogeneous mixture of cells. The mammal's blood can be combined extracorporeally with selected peptide ligands to kill or otherwise remove unwanted cells from the blood, followed by the blood being returned to the mammal according to standard techniques.

Therapeutic Use

The bicyclic peptides of the present invention have a specific use as high-affinity membrane metalloproteinase type 1 (MT1-MMP, also known as MMP14) binding agents. MT1-MMP is a transmembrane metalloproteinase that plays an important role in the remodeling of the extracellular matrix, directly by degrading some of its components and indirectly by activating pro-MMP2. MT1-MMP also plays a key role in tumor angiogenesis (Sounni et al. (2002) FASEB J. 16 (6), 555-564) and is expressed at elevated levels in a number of solid tumors, so drug conjugates containing MT1-MMP-binding bicyclic peptides of the present invention may be specifically used for the targeted treatment of cancer, in particular solid tumors such as non-small cell lung carcinomas. In one embodiment, the bicyclic peptide of the present invention is specific for human MT1-MMP. In a further embodiment, the bicyclic peptide of the present invention is specific for murine MT1-MMP. In another embodiment, the bicyclic peptide of the present invention is specific for human and murine MT1-MMP. In a further embodiment, the bicyclic peptide of the present invention is specific for human, murine and canine MT1-MMP.

The polypeptide ligands of the present invention can be used for in vivo therapeutic and prophylactic purposes, in vitro and in vivo diagnostic purposes, in vitro assays, as reagents, and the like. Ligands having a certain level of specificity can be used in applications that involve testing non-human animals where cross-reactivity is desired, or in diagnostic applications where cross-reactivity with homologs or paralogs must be carefully monitored. In some applications, such as vaccines, the ability to elicit an immune response to predetermined ranges of antigens can be used to tailor the vaccine to specific diseases and pathogens.

Substantially pure peptide ligands with at least 90 to 95% homogeneity are preferred for administration to a mammal, and homogeneity of 98 to 99% or greater is most preferred for pharmaceutical use, particularly when the mammal is a human. Once purified, partially or to the desired degree of homogeneity, the selected polypeptides can be used for diagnostic or therapeutic purposes (including in vitro), or for the development and implementation of analytical procedures, immunofluorescence staining, etc. (Lefkovite and Pernis, (1979 and 1981) Immunological Methods, Volumes I and II, Academic Press, NY).

The peptide ligand conjugates of the present invention generally find use in methods of preventing, suppressing or treating cancer, particularly solid tumors such as non-small cell lung carcinomas.

Thus, in another aspect, the invention provides drug conjugates containing a peptide ligand described herein for use in methods of preventing, suppressing or treating cancer, particularly solid tumors such as non-small cell lung carcinomas.

- 17 046487

In accordance with another aspect, the invention relates to a method of preventing, suppressing or treating cancer, in particular solid tumors such as non-small cell lung carcinomas, which includes administering to a patient in need thereof a drug conjugate containing a peptide ligand described herein.

Examples of cancers (and their benign counterparts) that can be treated (or suppressed) include, but are not limited to, tumors of epithelial origin (adenomas and carcinomas of various types, including adenocarcinomas, squamous cell, transitional cell and other carcinomas), such as bladder carcinomas and urinary tract, breast, gastrointestinal tract (including the esophagus, stomach, small intestine, colon, rectum and anus), liver (hepatocellular carcinoma), gallbladder and biliary system, exocrine pancreas, kidney, lung (eg, adenocarcinomas, small cell lung carcinomas, non-small cell lung carcinomas, bronchoalveolar carcinomas, and mesotheliomas), head and neck (eg, cancers of the tongue, mouth, larynx, pharynx, nasopharynx, tonsils, salivary glands, nasal cavity, and paranasal sinuses) , ovary, fallopian tubes, peritoneum, vagina, vulva, penis, cervix, myometrium, endometrium, thyroid (eg, follicular thyroid carcinoma), adrenal gland, prostate, skin and adnexa (eg, melanoma, basal cell carcinoma, squamous cell carcinoma , keratoacanthoma, dysplastic nevus); hematologic malignancies (such as leukemias, lymphomas) and precancerous hematologic disorders and borderline malignancies such as hematologic malignancies and related conditions of the lymphoid lineage (eg, acute lymphocytic leukemia [ALL], chronic lymphocytic leukemia [CLL], B-cell lymphomas, such such as diffuse large cell lymphoma [DLBCL], follicular lymphoma, Burkitt lymphoma, mantle cell lymphoma, T-cell lymphomas and leukemias, natural killer cell [NK] lymphomas, Hodgkin lymphomas, hairy cell leukemia, monoclonal gammopathy of unknown origin, plasmacytoma, multiple myeloma and posttrans plantation lymphoproliferative disorders), hematologic malignancies, and related conditions of myeloid origin (eg, acute myelogenous leukemia [AML], chronic myelogenous leukemia [CML], chronic myelomonocytic leukemia [CMML], hypereosinophilic syndrome, myeloproliferative disorders such as polycythemia vera, essential 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 osteosarcomas, fibrosarcomas, chondrosarcomas, rhabdomyosarcomas, leiomyosarcoma, liposarcoma, angiosarcoma, Kaposi's sarcoma, Ewing's sarcoma, synovial sarcomas, epithelioid sarcomas, gastrointestinal stromal tumors, benign and malignant histiocytomas and elevated dermatofibrosarcomas; tumors of the central or peripheral nervous system (eg, astrocytomas, gliomas and glioblastomas, meningiomas, ependymomas, pineal tumors and schwannomas); endocrine tumors (eg, pituitary tumors, adrenal tumors, islet cell tumors, parathyroid tumors, carcinoid tumors and medullary thyroid carcinoma); tumors of the eye and appendages (for example, retinoblastoma); germ cell and trophoblast tumors (eg, teratomas, seminomas, dysgerminomas, benign gestational trophoblastic disease and choriocarcinomas); as well as pediatric and embryonal tumors (eg, medulloblastoma, neuroblastoma, Wilms tumor, and primitive neuroectodermal tumors); or syndromes, congenital or otherwise, that make the patient susceptible to malignancy (eg, xeroderma pigmentosum).

References herein to the term prophylaxis include administration of the protective composition prior to induction of disease. The term suppression refers to the administration of the composition after an induction event, but before the clinical manifestation of the disease. The term treatment includes administration of the protective composition after the onset of symptoms of the disease.

Animal model systems exist that can be used to screen for the effectiveness of drug conjugates in preventing or treating disease. The present invention facilitates the use of animal model systems because the polypeptide ligands of the invention can cross-react with human and animal targets.

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

Examples

Materials and methods Peptide synthesis

Peptide synthesis was carried out using the Fmoc strategy on a Symphony peptide synthesizer manufactured by Peptide Instruments and a Syro II synthesizer manufactured by MultiSynTech. Standard Fmoc amino acids (Sigma, Merck) with appropriate side chain protecting groups are used: in each case, standard condensation conditions are used whenever possible, followed by deprotection using standard methods.

- 18 046487

Alternatively, the peptides are purified by HPLC and, after isolation, modified with 1,3,5 triacryloylhexahydro-1,3,5-triazine (TATA, Sigma). To do this, the linear peptide is diluted 50:50 MeCN:H2O to -35 ml, add -500 µl of 100 mM TATA in acetonitrile and initiate the reaction with 5 ml of 1 M NH4HCO3 in H2O. The reaction is allowed to proceed for -30-60 minutes at room temperature and after completion of the reaction (determined by MALDI) is lyophilized. After completion of the reaction, 1 mL of a 1 M solution of L-cysteine hydrochloride monohydrate (Sigma) in H2O was added to the reaction mixture and kept for -60 min at room temperature to quench any excess TATA.

After lyophilization, the modified peptide is purified as described above, replacing the Luna C8 column with a Gemini C18 column (Phenomenex) and the acid with 0.1% trifluoroacetic acid. Pure fractions containing the target TATA-modified substance are pooled, lyophilized and stored at -20 °C.

Unless otherwise noted, all amino acids used are of the L configuration.

In some cases, before binding to the free thiol group of the toxin, the peptides are converted to activated disulfides using the following method: a solution of 4-methyl(succinimidyl-4(2-pyridylthio)pentanoate) (100 mM) in dry DMSO (1.25 mol-eq. ) is added to a solution of the peptide (20 mM) in dry DMSO (1 mol equiv.). The reaction mixture is thoroughly stirred and DIPEA (20 mol-eq.) is added. The reaction is monitored by LC/MS until completion.

Preparation of bicyclic drug conjugate BT17BDC58

A 50 mL round bottom flask containing BICYCLE-NH2 ((B-Ala)-Sar10-ALPP-(SEQ ID NO: 17), also known as (B-Ala)-Sar10-A-(17-120-09-T03 )HArg2HArg9) (66 mg, 22.4 µmol, 1.00 eq.) in DMA (5 ml) was purged with nitrogen from a balloon. DIEA (2.91 mg, 112.4 µmol, 19.6 µl, 5 eq.) was then added at 25°C with stirring. Compound 8 (which can be prepared by the method described for the preparation of compound 8 in WO 2018/127699) (30.00 mg, 22.48 µmol, 1.00 eq.) is then added and the reaction mixture is stirred under a nitrogen-rich atmosphere at 25°C for 16 h. LC-MS results demonstrate that compound 8 is completely consumed and one major peak is present with the desired MS. The resulting reaction mixture was purified by preparative HPLC (in the presence of TFA). Compound BT17BDC58 (20.2 mg, 4.85 µmol, 21.56% yield) was obtained as a white solid.

Biological test results

MT1-MMP Competitive Binding Assay with Fluorescence Polarization

Due to their high affinity for the hemopexin domain of MT1-MMP (PEX), fluorescein-labeled derivatives 17-69-07 and 17-69-12 (referred to as 17-69-07-N040, 17-69-07-N041 and 17-69-12 -N004) can be used for competition assays (FP detection). Here, a preformed PEX complex with a fluorescent PEX-binding tag is titrated with a free fluorescein-free bicyclic peptide. Since all peptides based on 17-69 are expected to bind at the same site, the titrant will displace the fluorescent tag from the PEX. The dissociation of the complex can be measured quantitatively and the Kd of the competitor (titrant) of the target protein can be determined. The advantage of the competition method is that the affinity of fluorescein-free bicyclic peptides can be determined accurately and quickly.

The concentration of the label is typically at or below the Kd (here 1 nM) and the binding protein (here hemopexin MT1-MMP) is in 15-fold excess so that >90% of the label is bound. A non-fluorescent competing bicyclic peptide (usually just the bicyclic core sequence) is then titrated so that it displaces the fluorescent tag from the target protein. Label displacement is measured and correlated with a decrease in fluorescence polarization. The decrease in fluorescence polarization is proportional to the fraction of the target protein bound to the non-fluorescent titrant and is therefore a measure of the affinity of the titrant for the target protein.

In some experiments, collagen binding compounds (i.e. 17-88-N006 and 17-88-226

- 19 046487

N002) are used similarly to hemopexin binding compounds.

The raw data is fitted to an analytical solution to a cubic equation that describes the equilibrium between the fluorescent label, the titrant, and the binding protein. Fitting requires the affinity of the fluorescent tag for the target protein, which can be determined separately using direct FP binding experiments (see previous section). Curve fitting is performed using Sigmaplot 12.0 and is used as an adaptation of the equation described by Zhi-Xin Wang (FEBS Letters 360 (1995), 111-114).

Table 1. Characteristic parameters of labels used in competition analysis with fluorescence polarization

Name Subsequence Frame K _d (hM) (direct e binding) Binding area 17-69-07- ACYNEFGCEDFYDICA[S TBMB 0.52 Hemopexin N040 ar] ₆ [KFl] ((SEQ ID NO: 140)[Sar] ₆ [KFl]) 17-69-12- N004 [Fl]G[Sar] ₅ ACMNQFGCED FYDICA ([Fl]G[Sar] ₅ -(SEQ ID NO: 141)) TBMB 1.0 Hemopexin 17-69-07- N041 [Fl]G[Sar] ₅ ACYNEFACED FYDICA ([Fl]G[Sar] ₅ -(SEQ ID NO: 142)) TBMB 3.4 Hemopexin 17-88- N006 ACPYSWETCLFGDYRCA [Sar] ₆ [KFl] ((SEQ ID NO: 143)-[Sar] ₆ [KFl]) TBMB 14 Collagen 17-88- 226-N002 ACPYDWATCLFGDYRCA [Sar] ₆ [KFl] ((SEQ ID NO: 144)- [Sar] ₆ [KFl]) TBMB 50 Collagen

The results of testing some of the peptide ligands of the invention using the above binding assay are shown in table. 2.

Table 2. Results of competitive binding assay for selected __________peptide ligands of the present invention__________

Name of the bicyclic fragment Label Kd (nM±95% C1) 17-108-02 17-88-N006 4488.4±545.85 17-111-01 17-69-07-N041 1800 p=1 17-111-02 17-69-07-N041 2300 p=1 17-116-01 17-69-07-N040 352 p=1 17-120-00 17-88-N006 923±287.43 17-120-01 17-88-N006 310.33±30.89 17-120-02 17-88-N006 190.2±37.57 17-120-03 17-88-N006 603.56±35 17-120-04 17-88-N006 224.5 p=1 17-120-05 17-69-07-N040 >279.5±69.3 17-120-07 17-88-N006 273±119.56 17-120-08 17-88-N006 258±101.92 17-120-09-T01 17-88-N006 53.83±13.33

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17-120-09-ΊΌ2 17-88-N006 55.2 n=l 17-120-09-T03 (BCY1124) 17-88-N006 35.6 n=l 17-120-09-ΊΌ3 (BCY1124) 17-88-226-N002 32+20.24 Ac-(17-120-09-T03) (BCY1125) 17-88-226-N002 18.65+6.96 Sar3-A-(17-120-09-T03) 17-88-226-N002 16.33+5.73 Sar3-A-(17-120-09-T03)HArg2 17-88-226-N002 26.63+9.31 Sar3-A-(17-120-09-T03) Arg9 17-88-226-N002 8.28+3.53 Sar3-A-(17-120-09-T03)HArg9 17-88-226-N002 30.9+10 (B-Ala)-Sarl0-A-(17-120-09-T03) HArg2 HArg9 17-88-226-N002 39.5+16.43 (B-Ala)-Sarl0-A-(17-120-09-T03) HArg2 Ala9 17-88-226-N002 189.55+32.24 (B-Ala)-Sarl0-A-(17-120-09-T03) Ala2HArg9 17-88-226-N002 89.55+10.09 Ac-(B-Ala)-Sarl0-A-(17-120-09- T03) HArg2 HArg9 17-88-226-N002 43.9+15.48 17-120-09-T04 17-88-N006 34.15+10.2 17-120-09-T05 17-88-N006 32.3+6.81 17-120-09-ΊΌ6 17-88-N006 49.8 n=l 17-120-09-ΊΌ7 17-88-N006 48.1 n=l 17-120-09-T08 17-88-N006 37.5 n=l 17-120-09-T09 17-88-N006 77.2 n=l 17-120-09-T10 17-88-N006 38.5 n=l 17-120-09-T11 17-88-N006 44.2 n=l 17-120-09-T12 17-88-N006 62.2 n=l 17-120-09-T13 17-88-N006 69.3 n=l 17-120-10-T01 17-88-N006 132.3+103.29 17-120-11-T01 17-88-N006 612+760.47 17-120-12-T01 17-88-N006 183 n=l 17-120-13-T01 17-88-N006 189+123.48 17-120-14-T01 17-88-N006 148 n=l 17-120-15-T01 17-88-N006 178 n=l 17-120-15-T02 17-88-N006 76.67+72.87 17-120-16-T01 17-88-N006 74.4+17.64

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17-120-17-T01 17-88-N006 157 n=l 17-120-18 17-88-N006 252±92.12 17-120-19 17-88-N006 303±258.72 17-120-20 17-88-N006 248.5±14.7 17-120-21 17-88-N006 >82.75±4.61 17-120-21-TOI 17-88-N006 113 p=1 17-120-22-TO 1 17-88-N006 62.35±30.48 17-120-22-T02 17-88-N006 46.1±26.5 17-120-23-TO 1 17-88-N006 127±7.84 17-120-24-TO 1 17-88-N006 126±35.28 17-120-25-T01 17-88-N006 194.5±161.7 17-120-26-TO 1 17-88-N006 598 p=1 17-120-27-TO 1 17-88-N006 394 p=1 17-120-28-TO 1 17-88-N006 191.5±4.9 17-120-29-TO 1 17-88-N006 162±68.6 17-120-30-T01 17-88-N006 78.7 p=1 17-120-31-T01 17-88-N006 50.2 p=1 17-120-31-T02 17-88-N006 68.3 p=1 17-120-31-TOZ 17-88-N006 41.47±5.32 17-120-32-T01 17-88-N006 63.8±26.49 17-120-33-T01 17-88-N006 77.6 p=1 17-120-34-T01 17-88-N006 59.87±8.58 17-120-35-T01 17-88-N006 23.33±9.48 17-121-00 17-69-07-N040 678 p=1 17-122-02 17-69-07-N040 >929 p=3 17-122-03 17-69-07-N040 >378 p=3 17-122-04 17-69-07-N040 >2100 17-126-01 17-69-07-N041 >316±9.8 17-126-02 17-69-07-N041 >282± 172.48 17-126-03 17-69-07-N041 >43 0± 11.76 17-126-06 17-69-07-N040 675±192.08 17-126-07 17-69-07-N040 197.5±85.26 17-126-08 17-69-07-N040 711 p=1 17-126-09 17-69-07-N040 165 p=1

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17-126-10 17-69-07-N040 737 n=l 17-126-11 17-69-07-N040 971 n=l 17-126-12 17-69-07-N040 2900 n=l 17-126-18 17-69-07-N040 147 n=l 17-126-19 17-69-07-N040 199 n=l 17-126-20 17-69-07-N040 246 n=1 17-126-21 17-69-07-N040 131 n=l 17-126-22 17-69-07-N040 295 n=l 17-126-23 17-69-07-N040 409 n=l 17-126-24 17-69-07-N040 200 n=l 17-126-25 17-69-07-N040 >138.76 Ac-(17-126-25) 17-69-12-N004 60.5 n=l 17-126-26 17-69-07-N040 1200 n=l 17-126-27 17-69-07-N040 143 n=l 17-126-28 17-69-07-N040 250 n=l 17-126-30-T01 17-69-07-N040 239 n=l 17-126-31-T01 17-69-07-N040 295 n=l 17-126-32-T01 17-69-07-N040 390 n=l 17-126-33-T01 17-69-07-N040 244 n=l 17-126-33-T02 17-69-07-N040 296 n=l 17-126-35-T01 17-69-07-N040 263 n=l 17-126-36-T01 17-69-07-N040 149 n=l 17-126-37-T01 17-69-07-N040 155 n=l 17-126-38-T01 17-69-07-N040 162 n=l 17-126-39-T01 17-69-07-N040 187 n=l 17-126-40-T01 17-69-07-N040 310 n=l 17-126-41-TOI 17-69-07-N040 202 n=1 17-127-01 17-69-07-N040 2200 n=l 17-129-00 17-69-07-N040 446.0 17-129-01-T01 17-69-07-N040 499 n=l 17-129-01-T02 17-69-07-N040 525 n=l 17-129-01-T03 17-69-07-N040 598 n=l 17-129-01-T04 17-69-07-N040 705 n=l 17-129-01-T05 17-69-07-N040 324 n=l

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17-129-02 17-69-07-N040 877±650.71 17-129-03 17-69-07-N040 536.5±126.42 17-129-04 17-69-07-N040 595±372.39 17-129-05 17-69-07-N040 136.17±22.27 17-129-06 17-69-07-N040 566 p=1 17-129-07 17-69-07-N040 582 p=1 17-129-08 17-69-07-N040 516 p=1 17-129-09 17-69-07-N040 1092 p=1 17-129-10 17-69-07-N040 781 p=1 17-129-11 17-69-07-N040 912 p=1 17-129-12-T01 17-69-07-N040 187±86.24 17-129-13-T01 17-69-07-N040 248 p=1 17-129-14-T01 17-69-07-N040 245.5±46.06 17-129-14-T02 17-69-07-N040 318±27.44 17-129-15-T01 17-69-07-N040 278 p=1 17-129-16-T01 17-69-07-N040 263 p=1 17-129-17-T01 17-69-07-N040 418 p=1 17-129-17-T02 17-69-07-N040 369 p=1 17-129-17-TOZ 17-69-07-N040 312 p=1 17-129-18-T01 17-69-07-N040 138.33±43.96 17-129-18-T02 17-69-07-N040 334 p=1 17-129-18-TOZ 17-69-07-N040 202±92.96 17-129-18-T04 17-69-07-N040 171.5±53.9 17-129-19-T01 17-69-07-N040 754 p=1 17-129-19-T02 17-69-07-N040 458 p=1 17-129-20-TO 1 17-69-07-N040 213 p=1 17-129-21-T01 17-69-07-N040 110.8±33.71 17-129-22-TO 1 17-69-07-N040 53.7 17-129-23-T01 17-69-07-N040 54.8 Ac-(17-129-23-T01) 17-69-12-N004 8.9 p=1

SPR Binding Analysis

Biacore experiments were performed to determine KD values (nM) for monomeric peptides binding to the hemopexin domain of the human MT1 protein MMP14 (obtained from Merck Millipore).

The protein was randomly biotinylated in PBS using EZ-Link™ Sulfo-NHS-LC-LCbiotin reagent (Thermo Fisher) according to the protocol suggested by the manufacturer. The protein is thoroughly desalted to remove unbound biotin using spin columns in PBS.

For the peptide binding assay, a Biacore 3000 instrument with a CM5 chip (GE Healthcare) was used. Streptavidin was immobilized on the chip using standard amine coupling chemistry at 25°C using HBS-N (10 mM HEPES, 0.15 M NaCl, pH 7.4) as a running buffer. Briefly, the carboxymethyldextran surface is activated by a 7-minute injection of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)/0.1 M N-hydroxysuccinimide (NHS) in a 1:1 ratio at a flow rate of 10 µl/min. To capture streptavidin, the protein is diluted to 0.2 mg/ml in 10 mM sodium acetate (pH 4.5), capture is carried out by injecting 120 μl of streptavidin onto the activated surface of the chip. The remaining activated groups were blocked by a 7-min injection of 1 M ethanolamine (pH 8.5) and biotinylated MT1 MMP14 was captured to 12001800 RU. The buffer is replaced with PBS/0.05% Tween 20 and a series of dilutions of the peptides are carried out in this buffer with a final DMSO concentration of 0.5%. The maximum peptide concentration is 100 nM with 6 additional 2-fold dilutions. The SPR assay is performed at 25°C with a flow rate of 50 μL/min, an association time of 60 s, and a dissociation time of 400 to 1200 s depending on the individual peptide. Data are adjusted for the effect of DMSO. All data are obtained using dual control blank injections and control surfaces using standard methods, data processing and fitting of kinetic results is carried out using Scrubber software, version 2.0c (BioLogic software). The data is adjusted from the

- 24 046487 using a simple 1:1 coupling model, taking into account mass transfer effects if necessary.

The results of testing some of the peptide ligands of the present invention using the above SPR and competitive binding assays are shown in table. 3.

Table 3. Results of analysis of selected peptide ligands of the present invention _____________by SPR and competitive binding methods_____________

Tablet 1 Tablet 2 Name of the bicyclic fragment KD (SPR)/hM Ki (PP-connection)/nM Ki (PP-connection)/nM BCY1124 14.8 78 107 BCY1125 15.1 BCY3959 27.7 BCY9933 30.7 BCY9934 36.9 BCY9935 39.2 BCY9936 38 is* BCY9937 77.8 BCY9938 80.9 BCY9943 299 BCY9945 1360 BCY9946 372 BCY9949 190 BCY9951 364 BCY9952 870 BCY9953 296 BCY9954 28.3 BCY9955 73.5 BCY9957 304.2 BCY9959 5.87 BCY9960 72.7 BCY9961 13000 BCY9963 7100 BCY9964 35 BCY9965 77.6 BCY9966 240 BCY9968 163 BCY10223 400 BCY10224 97.8 BCY9965 76 BCY11147 53 BCY11148 45 BCY11149 80 BCY11150 396 AND*. BCY11151 35 BCY11152 68 BCY11153 43 BCY11154 129

- 25 046487

BCY11155 459 BCY11163 54 BCY11158 124 BCY11160 — — 289 BCY11165 — — 72 BCY11166 92 BCY11167 135 BCY10288 52.5 BCY12401 10,834 BCY12402 8.9004 BCY12403 56.125 BCY12404 27.44 BCY12405 14.7

In vivo testing of BT17BDC58 efficacy against HT1080 xenograft treatment in BALB/c nude mice

1. Purpose of the study

The purpose of this study is to analyze the in vivo antitumor efficacy of BT17BDC58 against the HT1080 xenograft model in BALB/c nude mice.

2. Experimental design

Gr. Treatment Dose (mg/kg) η Dose volume (µl/g) Way introduction Scheme 1 Wednesday - 3 10 i.v. biw*2 weeks 2 BT17BDC58 1 3 10 i.v. biw*2 weeks 3 BT17BDC58 3 3 10 i.v. biw*2 weeks 4 BT17BDC58 10 3 10 i.v. biw*2 weeks

Note: n - number of animals;

Dose volume - dose volume is adjusted according to body weight 10 µl/g;

biw - twice a week.

3. Materials

3.1 Animals and conditions for their keeping

3.1.1. Animals

Species: Mus Musculus

Strain: Balb/c naked

Age: 6-8 weeks

Female gender

Body weight: 18-22 g

Number of animals: 21 mice plus stock

Animal supplier: Shanghai LC Laboratory Animal Co., LTD.

3.1.2. Conditions of detention

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

Temperature: 20-26°C.

Humidity: 40-70%.

Cages: Made of polycarbonate. Size 300 mm x 180 mm x 150 mm. Corn cobs are used as bedding, which are changed twice a week.

Diet: Animals have free access to irradiation-sterilized dry pelleted food throughout the study period.

Water: Animals have free access to sterile drinking water.

Cage Identification: Identification labels for each cage contain the following information: number of animals, sex, strain, date received, treatment, study number, group number and treatment start date.

Animal identification: Animals are marked by marking their ears.

3.2 Experimental samples and positive controls

Product Identification: BT17BDC58

Manufacturer: Bicycle Therapeutics

Lot number: 1

- 26 046487

Physical characteristics: lyophilized powder

Molecular weight: 7.6 mg

Purity: 98.36%

Packaging and storage conditions: store at -80°C.

4. Experimental methods and procedures

4.1 Cell culture

HT1080 tumor cells are maintained in vitro as a monolayer culture in medium containing 10% heat-inactivated fetal bovine serum at 37°C in air containing 5% CO2. Tumor cells are traditionally subcultured twice weekly by trypsin-EDTA treatment. Cells growing in the exponential growth phase are collected and counted, after which the tumor is inoculated.

4.2 Tumor inoculation

Each mouse is inoculated subcutaneously in the right flank with HT1080 tumor cells (5x 10 ⁶ ) in 0.2 ml PBS for tumor development. 21 animals are randomly assigned to groups when the average tumor volume reaches 174 mm ³ . The administration of the test substance and the number of animals in each group are shown in the Experimental Design table.

4.3 Preparation of test substance composition

Treatment Dose (mg/ml) Composition Wednesday - 25 mM histidine pH 7.0, 10% sucrose (in the absence of DMSO) BT17BDC58 1 Dissolve 7.6 mg of BT17BDC58 in 7.475 ml of composition dilution buffer BT17BDC58 0.3 Dilute 240 µl of BT17BDC58 solution, 1 mg/ml, in 560 µl of composition dilution buffer BT17BDC58 OD Dilute 80 µl of BT17BDC58 solution, 1 mg/ml, in 720 µl of composition dilution buffer

4.4 Observations

All research procedures related to animal handling, animal care and treatment of animals are conducted in accordance with guidelines approved by the WuXi AppTec Institutional Animal Care and Use Committee (IACUC), following the guidelines of the International Association for the Assessment and Accreditation of Laboratory Animal Research ( AAALAC). During routine monitoring, animals are monitored daily for the effects of tumor growth and treatment on normal behavior such as mobility, food and water intake (visual only), weight gain/loss, dull eyes/matted hair, and any other abnormal effects noted in protocol. Death and observed clinical signs are recorded based on the number of animals in each subgroup.

4.5 Tumor measurements and results

The main outcome is the observed delay in tumor growth or cure of mice. The tumor volume is measured three times a week in two dimensions using a caliper, the volume is expressed in mm ³ using the formula:

V=0.5axb ² , where a and b denote the long and short diameters of the tumor, respectively. Tumor size is then used to calculate the T/C value. The T/C value (percentage) indicates antitumor efficacy; T and C are the average tumor volumes in the experimental and control groups, respectively, on a specific day.

TGI was calculated for each group using the formula:

TGI (%) = [1-(Ti-T0)/(Vi-V0)]x100;

Ti denotes the mean tumor volume in the experimental group on a particular day, T0 denotes the mean tumor volume in the experimental group on the day of treatment, Vi denotes the mean tumor volume in the control group receiving the medium on the day Ti is determined, and V0 denotes the mean tumor volume in the group receiving Wednesday, on the day of treatment.

4.6 Sample collection

At the end of the study, plasma is collected at 5, 15, 30, 60 and 120 minutes after dosing.

4.7 Statistical analysis

Summary statistics including mean and standard error of the mean (SEM) are provided to estimate tumor volume in each group at each time point.

Statistical analysis of differences in tumor volume among different groups is performed based on data obtained at the best therapeutic time point after the last dose.

-

Claims (3)

To compare tumor volume among different groups, one-way ANOVA is performed, after obtaining a significant F-statistic (the ratio of the variance of the experimental results to the error variance), the comparison of different groups is carried out using the Games-Howell test. All data is analyzed using Prism. Values corresponding to P<0.05 are considered statistically significant. 5. Results 5.1 Change in body weight and tumor growth curve Body weight and tumor growth are shown in Fig. 1. 5.2 Registration of tumor volume The mean tumor volume over time in female Balb/c nude mice bearing the HT1080 xenograft is shown in Table 1. 4. Table 4. Registration of tumor volume over time Treatment Number of days after start of treatment 0 2 4 7 9 AND 14 Wednesday, biw 174±1 8 318±30 473±31 688±87 859±148 975±167 1075±164 BT17BDC58, 1 mg/kg, biw 174±2 2 265±46 351±69 488±75 577±62 676±79 785±58 BT17BDC58, 3 mg/kg, biw 174±2 5 151=1=16 73±19 42±11 50±4 67±9 128=1=16 BT17BDC58, 10 mg/kg, biw 173±2 5 153±29 58±20 27±1 18±4 10±1 2±2 5.3 Tumor growth inhibition assay The rate of tumor growth inhibition by BT17BDC58 in the HT1080 xenograft model was calculated based on tumor volume measurements at day 14 after initiation of treatment. Table 5. Tumor growth inhibition assay Gr- Treatment Tumor volume (mm ³ ) ^a T/C ^b (%) TGI (%) P-value 1 Wednesday, biw 1075±164 - - - 2 BT17BDC58, 1 mg/kg, biw 785±58 73 32 p>0.05 3 BT17BDC58, 3 mg/kg, biw 128±16 12 105 p<0.001 4 BT17BDC58, 10 mg/kg, biw 2±2 0.2 119 p<0.001 a) Mean±SEM. b) Tumor growth inhibition is calculated by dividing the mean tumor volume in the experimental group by the mean tumor volume in the control group (T/C). 6. Results and discussion This study evaluates the therapeutic efficacy of BT17BDC58 in the HT1080 xenograft model. The measured values of body weight and tumor volume in all experimental groups at different time points are shown in Fig. 1 and in table. 4 and 5. The average tumor size in mice receiving the medium reaches 1075 mm ³ on day 14. BT17BDC58 in doses of 1 mg/kg (TV=785 ^mm3 , TGI=32.2%, p>0.05), 3 mg/kg (TV=128 ^mm3 , TGI=105.1%, p<0.001) and 10 mg/kg (TV=2 mm ³ , TGI=84.3%, p<0.001) exhibits dose-dependent antitumor activity. Among them, BT17BDC58 at a dose of 10 mg/kg causes complete remission of 2/3 tumors and regression of 1/3 tumors to 7 mm ³ on day 14. CLAIM 1. An MT1-MMP-specific peptide ligand comprising a polypeptide containing at least three cysteine residues separated by at least two loop sequences, and a molecular scaffold that forms covalent bonds with the cysteine residues of the polypeptide to produce at least two polypeptide loops on a molecular framework, wherein said molecular framework is 1,1',1''-(1,3,5-triazinan-1,3,5-triyl)triprop-2-en-1-one (TATA), and said peptide ligand contains an amino acid sequence selected from - 28 046487 CEESFYPECDHC (SEQ ID NO:1); CPDLCLDLFPNC (SEQ ID NO:2); CPELCVDLYPHC (SEQ ID NO:3); CHPEWVSCEFHC (SEQ ID NO:4); CSHECALLFPKTC(SEQ ID NO: 5); CFDECQLLFPKTC (SEQ ID NO: 6); CLDECKLLFPKTC(SEQ ID NO: 7); CREECMLLFPKTC(SEQ ID NO: 8); CETECALLFPRSC(SEQ ID NO: 9); CADECRLLFPKTC (SEQ ID NO:10); CDVECRLLFPRSC (SEQ ID NO:11); CIDECRLLFPRSC (SEQ ID NO:12); CVRECALLFPKTC (SEQ ID NO:13); CV[HArg]ECALLFPKTC (SEQ ID NO: 14); CVRECALLFPRTC(SEQ ID NO: 15); CVRECALLFP[HArg]TO (SEQ ID NO: 16); CV[HArg]ECALLFP[HArg]TO (SEQ ID NO: 17); CV[HArg]ECALLFPATC (SEQ ID NO: 18); CVAECALLFP[HArg]TO (SEQ ID NO: 19); CVTECQLLFPKTC (SEQ ID NO:20); CRHECELLFPKTC (SEQ ID NO:21); CQRECALLFPKTC(SEQ ID NO:22); CVRECTLLFPKTC (SEQ ID NO:23); CTIECALLFPKTC (SEQ ID NO:24); - 29 046487 CARECALLFPKTC (SEQ ID NO: 25) ; CINECRLLFPKTC (SEQ ID NO: 26) ; CYTECSLLFPKTC (SEQ ID NO: 27) ; CHEECRLLFPKTC (SEQ ID NO: 28) ; CLEECKLLFPKTC (SEQ ID NO: 29) ; CIDECALLFPRTC (SEQ ID NO: thirty) ; CYEECRLLFPRTC (SEQ ID NO: 31) ; CVRECRLLFPKTC (SEQ ID NO: 32) ; CHIECALLFPKTC (SEQ ID NO: 33); CKRECMLLFPKTC (SEQ ID NO: 34); CYRECALLFPKTC (SEQ ID NO: 35) ; CLTECALLFPKTC (SEQ ID NO: 36) ; CEVECRLLFPKTC (SEQ ID NO: 37) ; CEAECRLLPKTC (SEQ ID NO: 38) ; CVQECALLFPKTC (SEQ ID NO: 39) ; CIRECSLLFPKTC (SEQ ID NO: 40) ; CVTECALLFPKTC (SEQ ID NO: 41) ; CVAECKLLFPKTC (SEQ ID NO: 42) ; CVGECALLFPKTC (SEQ ID NO: 43); CWECALLFPKTC (SEQ ID NO: 44); CVFECALLFPKTC (SEQ ID NO: 45) ; CA[HArg]ECALLFP[HArg]TC (SEQ ID NO: 46); CVfHArg]ECALLFA[HArg]TC (SEQ ID NO: 47); CV[HArg]ECALLFP[HArg]AC (SEQ ID NO: 48); CV[HArg]ECALL[INal]P[HArg]TC (SEQ ID NO: 49); CV[HArg]ECALL[Cha]P[HArg]TC (SEQ ID NO: 50); CV[HArg]ECALLF[Pip][HArg]TC (SEQ ID NO: 51); CV[HArg]ECALLFP[HArg]SC (SEQ ID NO: 52); CV[HArg]ECALLFP[HArg][HSer]C (SEQ ID NO: 53); CV[HArg]ECALLF[HyP][HArg]TC (SEQ ID NO: 54); CV[HArg]EC[Aib]LLFP[HArg]TC (SEQ ID NO: 55); CV[HArg]ECAL[Nie]FP[HArg]TC (SEQ ID NO: 56); CV[HArg]ECA[tBuAla]LFP[HArg]TC (SEQ ID NO: 57) CV[HArg]ECA[Nle]LFP[HArg]TC (SEQ ID NO: 58); CV[Aad2]ECALLFP[HArg]TC (SEQ ID NO: 59); - 30 046487 CP[HArg]ECALLFP[HArg]TC (SEQ ID NO: 60); CV[HArg]ECALL[4FlPhe]P[HArg]TC (SEQ NO: 61); CV[HArg]ECAL[tBuGly]FP[HArg]TC (SEQ ID NO: 62); CV[HArg]ECAL[Cha]FP[HArg]TC (SEQ ID NO: 63); CV[HArg]ECALL[2Nal]P[HArg]TC (SEQ ID NO: 64); CV[HArg]ECALLFP[HArg][HyV]C (SEQ ID NO: 65); C[tBuGly][HArg]ECALLFP[HArg]TC (SEQ ID NO: 66); CVEECALLFP[HArg]TC (SEQ ID NO: 67); CV[HArg]EGA[Cpa]LFP[HArg]TC (SEQ ID NO: 68); CV[HArg]EGA[Cba]LFP[HArg]TC (SEQ ID NO: 69); CV[HArg]ECA[C5A]LFP[HArg]TC (SEQ ID NO: 70); CV[HArg]EGA[Cha]LFP[HArg]TC (SEQ ID NO: 71); CV[HArg]ECA[tBuGly]LFP[HArg]TC (SEQ ID NO: 72); CV[HArg]ECALLF[Cis-HyP][HArg]TC (SEQ ID NO: 73); CV[HArg]ECAL[Cpa]FP[HArg]TC (SEQ ID NO: 74); CV[HArg]ECAL[C5A]FP[HArg]TC (SEQ ID NO: 75); CV[HArg]ECA[tBuAla]LF[HyP][HArg]TC (SEQ ID NO: 76); CV[HArg]EGA[tBuAla][tBuGly]F[HyP][HArg]TC (SEQ ID NO: 77); C[tBuGly][HArg]ECA[tBuAla]LFP[HArg]TC (SEQ ID NO: 78); CSSWDKLMCHPYC (SEQ ID NO: 79) ; CPEECFYLPPHPMSC (SEQ ID NO: 80) ; CPQECFYLPGHSLYC (SEQ ID NO: 81) ; CPGECFYPPGHPLAC (SEQ ID NO:82); CPGECFYPTNHPLYC (SEQ ID NO:83); CPQECFYPIGHPLAC (SEQ ID NO:84); CPEECFYPPGHKLHC (SEQ ID NO:85) ; CPQECFYPPGHRLRC (SEQ ID NO: 8 6) ; CPQECFYPPGHPYHC (SEQ ID NO:87); CPQECFYPSTHPLYC(SEQ ID NO:88); CPGECFYPSNHRLYC (SEQ ID NO:89) ; CPDECFYPPEHPLAC (SEQ ID NO:90); CPGECFYPPGHHLSC (SEQ ID NO:91); CPGECFYPPGHHLGC (SEQ ID NO:92); CPEECFYPPNHPLYC (SEQ ID NO:93); CPGECFYPPDHPLYC (SEQ ID NO:94); - 31 046487 CPGECFYPPGHPLYC (SEQ ID NO: 95) ; CPGECFYPPNHPFYC (SEQ ID NO: 96) ; CPGECFYPPNHPLYC (SEQ ID NO: 97) ; CPEECFYPPGHPLAC (SEQ ID NO: 98) ; CWMECFYPPGHPLAC (SEQ ID NO: 99) ; CFEECFYPPGHPLAC (SEQ ID NO: 100) ; CPGECFYPPGHPLRC (SEQ ID NO: 101); CPGECFYPPGHPREC (SEQ ID NO: 102) ; CPGECFYPPGHRFHC (SEQ ID NO: 103) ; CPGECFYPPGHRLYC (SEQ ID NO: 104); CEEEFYPCGHPLYVC (SEQ ID NO: 105) ; CEEQFYPCTHALYTC (SEQ ID NO: 10 6); CVEEFYPCDHPLYSC (SEQ ID NO: 107) ; CEEEFYPCGHPMHPC (SEQ ID NO: 10 8); CDEQFYPCHHRLYSC (SEQ ID NO: 10 9); CEEEFYPCGHPFHPC (SEQ ID NO: 110); CLEQFYPCEHPLFSC (SEQ ID NO: ill); CVEQFYPCGHRHYIC (SEQ ID NO: 112) ; CEEQFYPCSHPLYTC (SEQ ID NO: 113); CEEQFYPCNHPLNVC (SEQ ID NO: 114); CEEEFYPCSHPLNPC (SEQ ID NO: 115); CEEQFYPCGHKLSPC (SEQ ID NO: 11 6) ; CPEQFYPCDHRLYIC (SEQ ID NO: 117); CQEQFYPCNHPLSPC (SEQ ID NO: 118); CDEQFYPCNHRLNTC (SEQ ID NO: 119) ; CEEAFYPCHHPLYRC (SEQ ID NO: 120); CDEDFYPCGHYLNQC (SEQ ID NO: 121) ; CEEQFYPCTHPLYVC (SEQ ID NO: 122); CPEQFYPCTHRLYQC (SEQ ID NO: 12 3) ; CEEQFYPCSHPLYRC (SEQ ID NO: 12 4); CAEQFYPCDHPLYRC (SEQ ID NO: 12 5) ; CAEEFYPCDHPLYRC (SEQ ID NO: 12 6); CEEAFYPCNHPLYTC (SEQ ID NO: 127); CAEAFYPCDHPLYVC (SEQ ID NO: 128); CEEAFYPCSHPLFIC (SEQ ID NO: 129) ; CEEAFYPCSHPLHPC (SEQ ID NO: 130); CEEAFYPCSHPLFVC (SEQ ID NO: 131) ; CEEQFYPCSHPLYSC (SEQ ID NO: 132) ; CEEAFYPCEHPLYMC (SEQ ID NO: 133); CEEQFYPCNHPLYMC (SEQ ID NO: 134); CLEQFYPCGDPRLC (SEQ ID NO: : 135); CEEQFYPCGHHLLC (SEQ ID NO: : 136) ; CLEPDECFYPMEC (SEQ ID NO: 137); CKEPQECFYPLKC (SEQ ID NO: 138); And CDSPEECFYPLEC (SEQ ID NO: 139) ; where Aad denotes alpha-A-aminoadipic acid, Aib denotes aminoisobutyric acid, C5a denotes beta-cyclopentyl-c-alanine, Cba denotes β-cyclobutylalanine, Cha denotes 3-cyclohexyl-c-alanine, Cpa denotes beta-cyclopentyl-c-alanine , 4FlPhe stands for 4-fluoro-cphenylalanine, HArg stands for homoarginine, HuP stands for hydroxyproline, HyV stands for 3hydroxy-k-valine, HSer stands for homoserine, 1Nal stands for 1-naphthylalanine, 2Nal stands for 2-naphthylalanine, Nle stands for norleucine, Pip stands for pipecolic acid, tBuAla stands for tbutylalanine, tBuGly stands for t-butylglycine. 2. The peptide ligand of claim 1, wherein the peptide ligand comprises an amino acid sequence that is CV[HArg]ECALLFP[HArg]TC (SEQ ID NO: 17). 3. The peptide ligand according to claim 1, where the peptide ligand contains an amino acid sequence selected from A-(SEQ ID NO: 1)-A (referred to herein as 17-108-02); A-(SEQ ID NO: 2)-A (referred to herein as 17-111-01); A-(SEQ ID NO: 3)-A (referred to herein as 17-111-02); A-(SEQ ID NO: 4)-A (referred to herein as 17-116-01); A-(SEQ ID NO: 5)-A (referred to herein as 17-120-00); A-(SEQ ID NO: 6)-A (referred to herein as 17-120-01); A-(SEQ ID NO: 7)-A (referred to herein as 17-120-02); A-(SEQ ID NO: 8)-A (referred to herein as 17-120-03); A-(SEQ ID NO: 9)-A (referred to herein as 17-120-04); A-(SEQ ID NO: 10)-A (referred to herein as 17-120-05); A-(SEQ ID NO: 11)-A (referred to herein as 17-120-07); A-(SEQ ID NO: 12)-A (referred to herein as 17-120-08); APPP-(SEQ ID NO: 13)-A (referred to here as 17-120-09-T01); QISP-(SEQ ID NO: 13)-A (referred to herein as 17-120-09-T02); ALPP-(SEQ ID NO: 13)-A (referred to herein as 17-120-09-T03 and BCY1124); Ac-ALPP-(SEQ ID NO: 13) (herein referred to as Ac-(17-120-09-T03) and BCY1125); Sar3-ALPP-(SEQ ID NO: 13) (referred to here as Sar3-A-(17-120-09-T03)); GPPP-(SEQ ID NO: 13)-A (referred to here as 17-120-09-T04); SPPP-(SEQ ID NO: 13)-A (referred to here as 17-120-09-T05); NPPP-(SEQ ID NO: 13)-A (referred to here as 17-120-09-T06); EPPP-(SEQ ID NO: 13)-A (referred to here as 17-120-09-T07); HPPP-(SEQ ID NO: 13)-A (referred to here as 17-120-09-T08); APNP-(SEQ ID NO: 13)-A (herein referred to as 17-120-09-T09); APDP-(SEQ ID NO: 13)-A (referred to here as 17-120-09-T10); APLP-(SEQ ID NO: 13)-A (referred to herein as 17-120-09-T11); APAP-(SEQ ID NO: 13)-A (referred to here as 17-120-09-T12); APHP-(SEQ ID NO: 13)-A (referred to here as 17-120-09-T13); Sar3-ALPP-(SEQ ID NO: 14) (referred to here as Sar3-A-(17-120-09-T03)HArg2); Sar3-ALPP-(SEQ ID NO: 15) (referred to here as Sar3-A-(17-120-09-T03) Arg9); Sar3-ALPP-(SEQ ID NO: 16) (referred to here as Sar3-A-(17-120-09-T03)HArg9); (B-Ala)-Sar10-ALPP-(SEQ ID NO: 17) (referred to here as (B-Ala)-Sar10-A-(17-120-09-T03) HArg2 HArg9); Ac-(B-Ala)-Sar10-ALPP-(SEQ ID NO: 17) (herein referred to as Ac-(B-Ala)-Sar10-A-(17-120-09T03)HArg2 HArg9); ALPP-(SEQ ID NO: 17) (herein referred to as BCY3959); [Ac]LPP-(SEQ ID NO: 17) (herein referred to as BCY9933); [Ac]APP-(SEQ ID NO: 17) (herein referred to as BCY9934); [Ac]LAP-(SEQ ID NO: 17) (herein referred to as BCY9935); [Ac]LPA-(SEQ ID NO: 17) (herein referred to as BCY9936); [Ac]-(SEQ ID NO: 17) (herein referred to as BCY9968); [Ac]LYP-(SEQ ID NO: 17) (herein referred to as BCY11147); [Ac]LPY-(SEQ ID NO: 17) (herein referred to as BCY11148); [Ac][dA]PP-(SEQ ID NO: 17) (herein referred to as BCY11165); [Ac]L[dA]P-(SEQ ID NO: 17) (herein referred to as BCY11166); [Ac]LP[dA]-(SEQ ID NO: 17) (herein referred to as BCY11167); ALPP-(SEQ ID NO: 17)-A (herein referred to as BCY10288); (B-Ala)-Sar10-ALPP-(SEQ ID NO: 17) (referred to here as (B-Ala)-Sar10-A-(17-120-09-T03) HArg2 HArg9); (B-Ala)-Sar10-ALPP-(SEQ ID NO: 18) (referred to here as (B-Ala)-Sar10-A-(17-120-09-T03) HArg2 Ala9); (B-Ala)-Sar10-ALPP-(SEQ ID NO: 19) (referred to here as (B-Ala)-Sar10-A-(17-120-09-T03) Ala2 HArg9); - 33 046487 [Ac]LPP-(SEQ ID NO: 19) (herein referred to as BCY9938); APMP-(SEQ ID NO: 20)-A (referred to herein as 17-120-10-T01); APSP-(SEQ ID NO: 21)-A (referred to herein as 17-120-11-T01); AALP-(SEQ ID NO: 22)-A (referred to herein as 17-120-12-T01); ALDP-(SEQ ID NO: 23)-A (referred to herein as 17-120-13-T01); ADRP-(SEQ ID NO: 24)-A (referred to herein as 17-120-14-T01); ATQP-(SEQ ID NO: 25)-A (referred to herein as 17-120-15-T01); SPPP-(SEQ ID NO: 25)-A (referred to herein as 17-120-15-T02); ARHP-(SEQ ID NO: 26)-A (referred to herein as 17-120-16-T01); ALPP-(SEQ ID NO: 27)-A (referred to herein as 17-120-17-T01); A-(SEQ ID NO: 28)-A (referred to herein as 17-120-18); A-(SEQ ID NO: 29)-A (referred to herein as 17-120-19); A-(SEQ ID NO: 30)-A (referred to herein as 17-120-20); A-(SEQ ID NO: 31)-A (referred to herein as 17-120-21); APPP-(SEQ ID NO: 31)-A (referred to herein as 17-120-21-T01); APSP-(SEQ ID NO: 32)-A (referred to herein as 17-120-22-T01); PLPP-(SEQ ID NO: 32)-A (referred to herein as 17-120-22-T02); APAP-(SEQ ID NO: 33)-A (referred to herein as 17-120-23-T01); AVEP-(SEQ ID NO: 34)-A (referred to herein as 17-120-24-T01); AEPA-(SEQ ID NO: 35)-A (referred to herein as 17-120-25-T01); ASPP-(SEQ ID NO: 36)-A (referred to herein as 17-120-26-T01); AAPP-(SEQ ID NO: 37)-A (referred to herein as 17-120-27-T01); APPP-(SEQ ID NO: 38)-A (referred to herein as 17-120-28-T01); AVPP-(SEQ ID NO: 39)-A (referred to herein as 17-120-29-T01); SPPP-(SEQ ID NO: 40)-A (referred to herein as 17-120-30-T01); HLPP-(SEQ ID NO: 41)-A (referred to herein as 17-120-31-T01); RLPP-(SEQ ID NO: 41)-A (referred to herein as 17-120-31-T02); APPP-(SEQ ID NO: 41)-A (referred to herein as 17-120-31-T03); MPPP-(SEQ ID NO: 42)-A (referred to herein as 17-120-32-T01); SPPP-(SEQ ID NO: 43)-A (referred to herein as 17-120-33-T01); APPP-(SEQ ID NO: 44)-A (referred to herein as 17-120-34-T01); APPP-(SEQ ID NO: 45)-A (referred to herein as 17-120-35-T01); [Ac]LPP-(SEQ ID NO: 46) (herein referred to as BCY9937); [Ac]LPP-(SEQ ID NO: 47) (herein referred to as BCY9943); [Ac]LPP-(SEQ ID NO: 48) (herein referred to as BCY9945); [Ac]LPP-(SEQ ID NO: 49) (herein referred to as BCY9946); [Ac]LPP-(SEQ ID NO: 50) (herein referred to as BCY9949); [Ac]LPP-(SEQ ID NO: 51) (herein referred to as BCY9951); [Ac]LPP-(SEQ ID NO: 52) (herein referred to as BCY9952); [Ac]LPP-(SEQ ID NO: 53) (herein referred to as BCY9953); [Ac]LPP-(SEQ ID NO: 54) (herein referred to as BCY9954); [Ac]LPP-(SEQ ID NO: 55) (herein referred to as BCY9955); [Ac]LPP-(SEQ ID NO: 56) (herein referred to as BCY9957); [Ac]LPP-(SEQ ID NO: 57) (herein referred to as BCY9959); [Ac]LYP-(SEQ ID NO: 57) (herein referred to as BCY12401); [Ac]EYP-(SEQ ID NO: 57) (referred to here as BCY12405); [Ac]LPP-(SEQ ID NO: 58) (herein referred to as BCY9960); [Ac]LPP-(SEQ ID NO: 59) (herein referred to as BCY9961); [Ac]LPP-(SEQ ID NO: 60) (herein referred to as BCY9963); [Ac]LPP-(SEQ ID NO: 61) (herein referred to as BCY9964); [Ac]LPP-(SEQ ID NO: 62) (herein referred to as BCY9965); [Ac]LPP-(SEQ ID NO: 63) (herein referred to as BCY9966); [Ac]LPP-(SEQ ID NO: 64) (herein referred to as BCY10223); [Ac]LPP-(SEQ ID NO: 65) (herein referred to as BCY10224); [Ac]LPP-(SEQ ID NO: 66) (herein referred to as BCY11149); [Ac]LPP-(SEQ ID NO: 67) (herein referred to as BCY11150); [Ac]LPP-(SEQ ID NO: 68) (herein referred to as BCY11151); [Ac]LPP-(SEQ ID NO: 69) (herein referred to as BCY11152); [Ac]LPP-(SEQ ID NO: 70) (herein referred to as BCY11153); [Ac]LPP-(SEQ ID NO: 71) (herein referred to as BCY11154); [Ac]LPP-(SEQ ID NO: 72) (herein referred to as BCY11155); [Ac]LPP-(SEQ ID NO: 73) (herein referred to as BCY11163); - 34 046487 [Ac]LPP-(SEQ ID NO: 74) (herein referred to as BCY11158); [Ac]LPP-(SEQ ID NO: 75) (herein referred to as BCY11160); [Ac]LYP-(SEQ ID NO: 76) (herein referred to as BCY12402); [Ac]LYP-(SEQ ID NO: 77) (herein referred to as BCY12403); [Ac]LYP-(SEQ ID NO: 78) (herein referred to as BCY12404); A-(SEQ ID NO: 79)-A (referred to herein as 17-121-00); A-(SEQ ID NO: 80)-A (referred to herein as 17-127-01); A-(SEQ ID NO: 81)-A (referred to herein as 17-129-00); SQT-(SEQ ID NO: 82)-A (referred to herein as 17-129-01-T01); SMT-(SEQ ID NO: 82)-A (referred to herein as 17-129-01-T02); SLV-(SEQ ID NO: 82)-A (referred to herein as 17-129-01-T03); ISSYG-(SEQ ID NO: 82)-A (referred to herein as 17-129-01-T04); ENITT-(SEQ ID NO: 82)-A (referred to herein as 17-129-01-T05); A-(SEQ ID NO: 83)-A (referred to herein as 17-129-02); A-(SEQ ID NO: 84)-A (referred to herein as 17-129-03); A-(SEQ ID NO: 85)-A (referred to herein as 17-129-04); A-(SEQ ID NO: 86)-A (referred to herein as 17-129-05); A-(SEQ ID NO: 87)-A (referred to herein as 17-129-06); A-(SEQ ID NO: 88)-A (referred to herein as 17-129-07); A-(SEQ ID NO: 89)-A (referred to herein as 17-129-08); A-(SEQ ID NO: 90)-A (referred to herein as 17-129-09); A-(SEQ ID NO: 91)-A (referred to herein as 17-129-10); A-(SEQ ID NO: 92)-A (referred to herein as 17-129-11); L-(SEQ ID NO: 93)-HA (here designated as 17-129-12-T01); T-(SEQ ID NO: 94)-NA (referred to herein as 17-129-13-T01); Q-(SEQ ID NO: 95)-NA (referred to herein as 17-129-14-T01); A-(SEQ ID NO: 95)-NVI (referred to herein as 17-129-14-T02); N-(SEQ ID NO: 96)-NA (herein referred to as 17-129-15-T01); D-(SEQ ID NO: 97)-RA (referred to herein as 17-129-16-T01); SRM-(SEQ ID NO: 98)-A (referred to herein as 17-129-17-T01); SRS-(SEQ ID NO: 98)-A (referred to herein as 17-129-17-T02); RYMTR-(SEQ ID NO: 98)-A (referred to herein as 17-129-17-T03); REE-(SEQ ID NO: 99)-A (referred to herein as 17-129-18-T01); DNM-(SEQ ID NO: 99)-A (referred to herein as 17-129-18-T02); QES-(SEQ ID NO: 99)-A (referred to herein as 17-129-18-T03); ADY-(SEQ ID NO: 99)-A (referred to herein as 17-129-18-T04); MAN-(SEQ ID NO: 100)-A (referred to herein as 17-129-19-T01); SQN-(SEQ ID NO: 100)-A (referred to herein as 17-129-19-T02); A-(SEQ ID NO: 101)-TVL (referred to herein as 17-129-20-T01); A-(SEQ ID NO: 102)-SWL (referred to herein as 17-129-21-T01); A-(SEQ ID NO: 103)-LTE (referred to here as 17-129-22-T01); A-(SEQ ID NO: 104)-YSE (referred to herein as 17-129-23-T01); Ac-(SEQ ID NO: 104)-YSE (herein referred to as Ac (17-129-23-T01)); A-(SEQ ID NO: 105)-A (referred to herein as 17-126-01); A-(SEQ ID NO: 106)-A (referred to herein as 17-126-02); A-(SEQ ID NO: 107)-A (referred to herein as 17-126-03); A-(SEQ ID NO: 108)-A (referred to herein as 17-126-06); A-(SEQ ID NO: 109)-A (referred to herein as 17-126-07); A-(SEQ ID NO: 110)-A (referred to herein as 17-126-08); A-(SEQ ID NO: 111)-A (referred to herein as 17-126-09); A-(SEQ ID NO: 112)-A (referred to herein as 17-126-10); A-(SEQ ID NO: 113)-A (referred to herein as 17-126-18); A-(SEQ ID NO: 114)-A (referred to herein as 17-126-19); A-(SEQ ID NO: 115)-A (referred to herein as 17-126-20); A-(SEQ ID NO: 116)-A (referred to herein as 17-126-21); A-(SEQ ID NO: 117)-A (referred to herein as 17-126-22); A-(SEQ ID NO: 118)-A (referred to herein as 17-126-23); A-(SEQ ID NO: 119)-A (referred to herein as 17-126-24); A-(SEQ ID NO: 120)-A (referred to herein as 17-126-25); Ac-A-(SEQ ID NO: 120)-A (herein referred to as Ac-(17-126-25)); A-(SEQ ID NO: 121)-A (referred to herein as 17-126-26); A-(SEQ ID NO: 122)-A (referred to herein as 17-126-27); -