Conjugates comprising peptide groups and methods related thereto

1. A ligand-active agent conjugate, comprising a ligand, a branched linker, at least two cleavage groups, and at least two active agents, wherein:

the ligand is covalently coupled to the branched linker;

each cleavage group is covalently coupled to the branched linker;

each active agent is covalently coupled to one cleavage group;

the branched linker comprises a peptide sequence of a plurality of amino acids wherein the cleavage groups are covalently coupled to the side chains of the amino acids; and each cleavage group has a structure consisting of formula I:

wherein:

G is a sugar, sugar acid, or modified sugar;

W is —C(O)—, —C(O)NR′—, —C(O)O—, —S(O) 2 NR′-, —P(O)R″NR′—, —S(O)NR′—, or -PO 2 NR′-, in each case wherein the C, S, or P is directly bound to the phenyl ring, and the N or O is directly bound to the branched linker;

R′ and R″ are each independently hydrogen, (C 1 -C 8 )alkyl, mono- or di-carboxyl(C 1 -C 8 )alkyl, (C 3 -C 8 )cycloalkyl, (C 1 -C 8 )alkoxy, (C 1 -C 8 )alkylthio, mono- or di-(C 1 -C 8 )alkylamino, (C 3 -C 20 )heteroaryl, or (C 6 -C 20 )aryl;

each Z independently is hydrogen, (C 1 -C 8 )alkyl, or an electron-withdrawing group;

n is an integer from 1 to 3;

m is 0 or 1;

R 1 and R 2 are each independently hydrogen, (C 1 -C 8 )alkyl, or (C 3 -C 8 )cycloalkyl, or R 1 and R 2 taken together with the carbon atom to which they are attached form a (C 3 -C 8 )cycloalkyl ring;

is a connection to the branched linker; and

is a connection to the active agent.

2. The conjugate of claim 1 , wherein the ligand is an antibody.

3. The conjugate of claim 1 , wherein at least two of the active agents are covalently coupled via the cleavage groups to side chains of lysine, 5-hydroxylysine, 4-oxalysine, 4-thialysine, 4-selenalysine, 4-thiahomolysine, 5,5-dimethyllysine, 5,5-difluorolysine, trans-4-dehydrolysine, 2,6-diamino-4-hexynoic acid, cis-4-dehydrolysine, 6-N-methyllysine, diaminopimelic acid, ornithine, 3-methylornithine, a-methylornithine, citrulline, and/or homocitrulline.

4. The conjugate of claim 1 , wherein the plurality of amino acids comprises alanine, aspartate, asparagine, glutamate, glutamine, glycine, histidine, lysine, ornithine, proline, serine, or threonine.

5. The conjugate of claim 1 , wherein:

the branched linker is covalently bound to the ligand by a thioether bond;

the ligand comprises a C-terminal amino acid motif that is recognized by an isoprenoid transferase; and

the thioether bond comprises a sulfur atom of a cysteine of the C-terminal amino acid motif.

6. The conjugate of claim 5 , wherein:

the C-terminal amino acid motif is a sequence CYYX;

C is cysteine;

Y, independently for each occurrence, is an aliphatic amino acid;

X, independently for each occurrence, is glutamine, glutamate, serine, cysteine, methionine, alanine, or leucine; and

the thioether bond is formed from a sulfur atom of a cysteine of the C-terminal amino acid motif.

7. The conjugate of claim 5 , wherein the thioether bond comprises a carbon atom of at least one isoprenyl moiety.

8. The conjugate of claim 1 , wherein the branched linker further comprises a connection unit selected from the group consisting of—(CH 2 ) r (V(CH 2 ) p ) q -, -((CH 2 ) p V) q -, -(CH 2 ) r (V(CH 2 ) p ) q Y 1 -, -((CH 2 ) p V) q (CH 2 ) r -, -Y 1 (((CH 2 ) p V) q - and -(CH 2 ) r (V(CH 2 ) p ) q Y 1 CH 2 — wherein:

r is an integer from 0 to 10;

p is an integer from 1 to 10;

q is an integer from 1 to 20;

V and Y 1 are each independently a single bond, —O—, —S—, —NR 21 —, —C(O)NR 22 —, —NR 23 C(O)—, —NR 24 SO 2 —, or —SO 2 NR 25 —; and

R 21 to R 25 are each independently hydrogen, (C 1 -C 6 )alkyl, (C 1 -C 6 )alkyl(C 6 -C 20 )aryl or (C 1 -C 6 )alkyl(C 3 -C 20 )heteroaryl.

9. The conjugate of claim 1 , wherein the branched linker further comprises a binding unit, and the binding unit has a structure according to Formula A, B, C, or D:

wherein:

L 1 is a single bond or alkylene having 1 to 30 carbon atoms;

R 11 is hydrogen or alkyl having 1 to 10 carbon atoms; and

L 2 is alkylene having 1 to 30 carbon atoms.

10. The conjugate of claim 1 , wherein each branched linker comprises a branching unit, and each active agent is coupled to the branching unit through the cleavage group and a secondary linker; and the branching unit is coupled to the peptide by a primary linker.

11. The conjugate of claim 10 , wherein at least one branching unit has the structure

wherein L 11 , L 22 , L 33 is each independently a bond or —C n H 2n — where n is a integer of 1 to 30;

wherein G 1 , G 2 , G 3 is each independently a bond,

wherein R 33 is hydrogen or C 1 -C 30 alkyl;

wherein R 44 is hydrogen or L 4 -COOR 55 , wherein L 4 is a bond or —C n H 2n — wherein n is a integer of 1 to 10, and R 55 is hydrogen or C 1 -C 30 alkyl.

12. The conjugate of claim 1 , wherein the ligand is selected from the group consisting of muromonab-CD3 abciximab, rituximab, daclizumab, palivizumab, infliximab, trastuzumab, etanercept, basiliximab, gemtuzumab, alemtuzumab, ibritumomab, adalimumab, alefacept, omalizumab, efalizumab, tositumomab, cetuximab, ABT-806, bevacizumab, natalizumab, ranibizumab, panitumumab, eculizumab, rilonacept, certolizumab, romiplostim, AMG-531, golimumab, ustekinumab, ABT-874, belatacept, belimumab, atacicept, an anti-CD20 antibody, canakinumab, tocilizumab, atlizumab, mepolizumab, pertuzumab, HuMax CD20, tremelimumab, ticilimumab, ipilimumab, IDEC-114, inotuzumab, HuMax EGFR, aflibercept, HuMax-CD4, teplizumab, otelixizumab, catumaxomab, the anti-EpCAM antibody IGN101, adecatumomab, oregovomab, dinutuximab, girentuximab, denosumab, bapineuzumab, motavizumab, efumgumab, raxibacumab, LY2469298, and veltuzumab.

13. The conjugate of claim 1 , wherein:

G is

R 3 is hydrogen or a carboxyl protecting group; and

each R 4 is independently hydrogen or a hydroxyl protecting group.

14. The conjugate of claim 1 , wherein W is —C(O)NR′—, where C is bonded to the phenyl ring and NR′ is bonded to the branched linker.

15. A pharmaceutical composition comprising the conjugate of claim 1 and a pharmaceutically acceptable excipient.

16. A method of inhibiting cancer cell growth or killing cancer cell(s) in a subject in need thereof comprising administering the conjugate of claim 1 to the subject, wherein the conjugate comprises a ligand which targets the cancer cell(s).

17. A method for making the ligand-active agent conjugate of claim 1 , comprising

providing a biomolecule comprising a ligand, a branched linker, at least 2 cleavage groups, and a reactive group selected from the group consisting of a ketone and an aldehyde to react with a prodrug comprising an active agent and an alkoxyamine; and

the reaction produces an oxime, thereby covalently linking the biomolecule to the prodrug to produce the conjugate of claim 1 .

18. The conjugate of claim 1 , wherein Z is an amide, carboxylic acid, carboxylic acid ester, halogen, cyano, or nitro.

19. The conjugate of claim 1 , wherein the peptide sequence of the branched linker comprises 2 to 20 amino acids.

20. The conjugate of clam 63, wherein:

the ligand is an antibody;

G is

R 3 and each R 4 is hydrogen;

W is —C(O)NR′—, where C(O) is bonded to the phenyl ring and NR′ is bonded to the branched linker;

R′ is hydrogen;

R 1 and R 2 are each hydrogen;

m is 1;

Z is halogen, cyano, or nitro;

the conjugate comprises two, three, or four active agents and cleavage groups;

the peptide sequence of the branched linker comprises 2 to 20 amino acids selected from the group consisting of alanine, aspartate, asparagine, glutamate, glutamine, glycine, histidine, lysine, ornithine, proline, serine, and threonine; and

the branched linker further comprises a binding unit with a structure according to Formula A, B, C, or D:

wherein:

L 1 is a single bond or alkylene having 1 to 30 carbon atoms;

R 11 is hydrogen or alkyl having 1 to 10 carbon atoms; and

L 2 is alkylene having 1 to 30 carbon atoms.

21. The conjugate of claim 1 , wherein the conjugate comprises:

and

MMAE is monomethyl auristatin E;

MMAF is monomethyl auristatin F; and

is a connection to the remaining portion of the branched linker which connects to the ligand.