HK1156864A - Anti-angiogenic compounds - Google Patents

Description

Anti-angiogenic compounds

Technical Field

The present invention relates to novel compounds having anti-angiogenic activity and methods of making and using these compounds. In particular, the present invention relates to peptides that bind to Vascular Endothelial Growth Factor (VEGF) and macromolecules that bind these peptides, as well as methods and uses related thereto.

Background

Angiogenesis is a fundamental process by which a new blood vessel is formed, and it is very important for various normal physical activities such as reproduction, development, and wound repair. Although angiogenesis is a highly controlled process under normal circumstances, many diseases (characterized by "angiogenic diseases") are initiated or exacerbated by unregulated angiogenesis. For example, ocular neovascularization is considered to be the most common cause of blindness. In certain existing conditions, such as arthritis, newly formed capillaries invade the joints and destroy cartilage. In diabetes, new capillary blood vessels formed in the retina invade the vitreous, bleed and lead to blindness. Growth and metastasis of solid tumors are also dependent on angiogenesis (J.Folkman, Cancer Res., 46: 467-473(1986), J.Folkman, J.Natl.cancer Inst., 82: 4-6 (1989)). It has been shown, for example, that tumors that grow more than 2mm gain their own blood supply by inducing the growth of new capillary blood vessels. Once these new blood vessels are embedded in the tumor, they provide a pathway for tumor cells to enter the circulation and migrate to distant locations such as the liver, lungs, and bones (n.weidner, et. al., n.engl.j.med., 324: 1-8 (1991)).

Vascular Endothelial Growth Factor (VEGF) has been identified as an extremely powerful angiogenic factor and is required for the growth and metastasis of many human tumors. Many studies have focused on attempting to inhibit the VEGF pathway to limit or prevent angiogenesis or metastasis. Fairbrother et al: 'Novel peptides selected to bind a vascular endothelial growth factor-binding site' (Biochemistry, 1998, 37, 17754-17764) discloses peptides having different binding abilities to VEGF. Various peptides have been identified that bind the angiogenesis-related factor angiopoietin-2 ("Ang-2") (Oliner, J.et al., Cancer Cell, 204(6), 507-. Ang-2 binding peptides have been shown to be anti-angiogenic.

It would be desirable to provide compounds that exhibit improved properties, such as improved binding to VEGF, compared to known compounds. It would further be desirable to provide compounds that exhibit binding to both VEGF and Ang 2.

References in any field of the specification are not, and should not be taken as, any form of admission or suggestion that the reference forms part of the prior art.

Disclosure of Invention

[ VEGF-peptide ] of the present invention

The present invention provides peptides, compounds and pharmaceutical compositions that bind to human VEGF (exemplified by SEQ ID NO: 3). Thus, in some aspects, the invention provides sequences that are substantially homologous to: v¹-E²-P³-N⁴-C⁵-D⁶-I⁷-H⁸-V⁹-M¹⁰-W¹¹-V¹²-W¹³-X¹⁴-C¹⁵-F¹⁶-E¹⁷-R¹⁸-X¹⁹(SEQ ID NO: 122) in which X¹⁴Is E or V, and X¹⁹Is a natural or non-natural hydrophobic amino acid or a D-isomer thereof.

It has surprisingly been found that such peptides and derivatives thereof, as described in detail below, exhibit improved binding to VEGF compared to peptides having related structures. In particular, mutating position 12 to a neutral hydrophobic residue (especially valine) provides improved binding properties compared to peptides that are negatively charged groups at this position, such as glutamic acid. In some embodiments, the present invention provides a polypeptide comprising an amino acid sequence substantially identical to SEQ ID NO: 122, provided that V is a peptide of substantially homologous sequence¹²Not substituted by E. In some embodiments，X¹⁹Is L. X¹⁹May be D-Leu. X¹⁹May be conservative substitutions of L, such as I, or other hydrophobic residues, such as A or V, or the D-isomer of either.

Further derivatives of the invention provide for the addition of at least 1-4 residues at the C-terminus. Some embodiments provide for the addition of 2-4 residues at the C-terminus. Some embodiments provide for the addition of 2 residues at the C-terminus. Some embodiments provide for the addition of 3 residues at the C-terminus. Some embodiments provide for the addition of 4 residues at the C-terminus.

Accordingly, the present invention also provides a peptide comprising a sequence substantially homologous to: v¹-E²-P³-N⁴-C⁵-D⁶-I⁷-H⁸-V⁹-M¹⁰-W¹¹-V¹²-W¹³-X¹⁴-C¹⁵-F¹⁶-E¹⁷-R¹⁸-X¹⁹-X²⁰(SEQ ID NO: 123). Wherein, X²⁰Is any neutral, hydrophobic or aromatic amino acid. X²⁰May be any aromatic amino acid such as Y, F, W or its D-isomer. X²⁰Can be any neutral hydrophobic amino acid, such as M, I, L, Nle, A or D-isomer thereof.

In some aspects, the invention provides peptides comprising sequences substantially homologous to: v¹-E²-P³-N⁴-C⁵-D⁶-I⁷-H⁸-V⁹-M¹⁰-W¹¹-V¹²-W¹³-X¹⁴-C¹⁵-F¹⁶-E¹⁷-R¹⁸-X¹⁹-X²⁰-X²¹(SEQ ID NO: 124). Wherein X²¹Is any amino acid. In some aspects, X²¹May be any neutral or positively charged amino acid such as G, A, I, L, K, R or K (ac) or its D-isomer. For example, X²¹May be D-Ala.

In some aspects, the invention provides a composition comprising a sequence ofPeptides of this homologous sequence: v¹-E²-P³-N⁴-C⁵-D⁶-I⁷-H⁸-V⁹-M¹⁰-W¹¹-V¹²-W¹³-X¹⁴-C¹⁵-F¹⁶-E¹⁷-R¹⁸-X¹⁹-X²⁰-X²¹-X²²(SEQ ID NO: 125). Wherein X²²Is any aliphatic, polar or negatively charged amino acid, such as V, L, P, E, G, I, S, T, W, F, E or its D-isomer.

In some aspects, the invention provides peptides comprising sequences substantially homologous to: v¹-E²-P³-N⁴-C⁵-D⁶-I⁷-H⁸-V⁹-M¹⁰-W¹¹-V¹²-W¹³-X¹⁴-C¹⁵-F¹⁶-E¹⁷-R¹⁸-X¹⁹-X²⁰-X²¹-X²²-X²³(SEQ ID NO: 126), wherein X²³Is any amino acid. X²³Can be selected from G, A, I, L, Q, E, F, T, W, S and Y and its D-isomer. X²³Can be selected from G, A, I, L, Q, E, F, T, S and Y and its D-isomer. In some aspects, X²³Not including side chains with a bicyclic ring. In some aspects, X²³Is not W. In some aspects, X²³May be E, T, S, L or F or its D-isomer.

In some embodiments, it has been found to be preferred that at least one of the 5 residues at the carboxy terminus is the D-isomer. In some aspects, at least two of the five C-terminal residues are D-isomers. In some aspects, no more than three of the five C-terminal residues are D-isomers. In some aspects, one to three of the five C-terminal residues are D-isomers. In some aspects, at least one of the four residues at the carboxy terminus is the D-isomer. In some aspects, at least two of the four C-terminal residues are D-isomers. In some aspects, no more than three of the four C-terminal residues are D-isomers. In some aspectsOne to three of the five C-terminal residues are the D-isomers. For each of these, it is preferred that at X¹⁹Followed by at least two, preferably at least three other residues. It has been found that providing an optimized amount of the D-isomer at the C-terminus provides better resistance to enzymatic degradation at the C-terminus, especially of residue E¹⁷And R¹⁸With residues R¹⁸And X¹⁹The advantages of enzymatic degradation.

In some aspects of the invention, X¹⁹Can be L, X²⁰Exist and can be Y, X²¹And may be any aliphatic hydrophobic amino acid (selected from A, L, I, V, G), X²²And is a hydrophobic amino acid (which may be selected from L, P, V). In certain embodiments, X¹⁹、X²¹And X²²Two of which are the D-isomers. In some embodiments, X²³May or may not be present.

In certain aspects, particular peptides and compounds of the invention can include peptides having a sequence identical to SEQ id no: 36-106.

In other aspects, the invention provides peptides comprising sequences substantially homologous to: v¹-E²-P³-N⁴-C⁵-D⁶-I⁷-H⁸-V⁹-M¹⁰-W¹¹-E¹²-W¹³-E¹⁴-C¹⁵-F¹⁶-E¹⁷-R¹⁸-X¹⁹-X²⁰-X²¹-X²²-X²³(SEQ ID NO: 127), wherein X¹⁹Is a natural or non-natural hydrophobic amino acid or its D-isomer, X²⁰Is an aromatic amino acid, a neutral amino acid, a hydrophobic amino acid or a polar amino acid or its D-isomer, X²¹Is a hydrophobic or positively charged amino acid or its D-isomer, X²²Absent or a hydrophobic amino acid, an aromatic amino acid, a negatively charged amino acid or D-isomer thereof, X²³Absent or aromatic, neutral, hydrophobic amino acids or the likeThe D-isomer. X¹⁹May be L. In another embodiment, X¹⁹Is D-Leu. X¹⁹May be conservative substitutions for L (e.g., I) or other hydrophobic residues (e.g., A or V), or the D-isomer of any of the foregoing. X²⁰Can be selected from A, V, I, L, Y, W, F, M, S and T or its D-isomer. X²⁰May be selected from A, Y, F, M and S or its D-isomer. X²¹Can be selected from K, R, H, ornithine, Dap, Dab, G, A, V, I and L or D-isomer thereof. X²¹Can be selected from K, R and G or its D-isomer. At X²⁰When hydrophobic or aromatic, X²¹May be a positively charged amino acid. At X²⁰Is one of A, F or Y, X²¹May be either K or R or the D-isomer thereof. At X²¹When it is hydrophobic, X²⁰May be polar. X²²When present, may be selected from E, D, G, A, L, I, V, M, W, Y and F or the D-isomer thereof. X²²When present, may be selected from E, G and W or the D-isomer thereof. Multiple X²¹When it is a positively charged residue, X²²When present, may be a negatively charged residue. When X is present²¹When is K, X²²May be E. X²³When present, may be selected from W, Y, F, G, A, I, L, V and M or the D-isomer thereof. X²³When present, may be selected from W, F, G, A, L and M or the D-isomer thereof.

In certain aspects, particular peptides and compounds of the invention can include peptide sequences having substantial homology to one or more of the following sequences: SEQ ID NO: 108. SEQ ID NO: 109. SEQ ID NO: 110. SEQ ID NO: 111. SEQ ID NO: 112. SEQ ID NO: 113. SEQ ID NO: 114. SEQ ID NO: 115. SEQ ID NO: 116. SEQ ID NO: 117. SEQ ID NO: 118. SEQ ID NO: 119. SEQ ID NO: 120 and SEQ ID NO: 121.

the peptides of the invention are useful for various uses, including diagnostics, screening and therapy. The peptides and compounds of the invention may also be used directly for therapeutic use, or may also be used to conjugate, covalently or non-covalently, larger molecules that provide other therapeutic value, such as extended half-life (PK). Examples of molecules that can increase half-life include proteins, polypeptides, antibodies, antibody fragments, and in particular the Fc domain of an antibody.

The invention provides a compound of formula R¹- [ VEGF-peptide]-R²Wherein the [ VEGF-peptide ]]Is a peptide having the sequence: x¹-X²-P³-N⁴-C⁵-X⁶-X⁷-X⁸-V⁹-X¹⁰-X¹¹-X¹²-W¹³-X¹⁴-C¹⁵-F¹⁶-E¹⁷-R¹⁸-X¹⁹-X²⁰-X²¹-X²²-X²³(SEQ ID NO: 131) wherein R¹Is absent or is CH₃、C(O)CH₃、C(O)CH₃、C(O)CH₂CH₃、C(O)CH₂CH₂CH₃、C(O)CH(CH₃)CH₃、C(O)CH₂CH₂CH₂CH₃、C(O)CH(CH₃)CH₂CH₃、C(O)C₆H₅、C(O)CH₂CH₂(CH₂CH₂O)_1-5Me, amino-2-PEG, N-acyl or N-alkylamino protecting groups, fatty acid groups or hydrocarbon groups; r²Is absent or OH, NH₂、NH(CH₃)、NHCH₂CH₃、NHCH₂CH₂CH₃、NHCH(CH₃)CH₃、NHCH₂CH₂CH₂CH₃、NHCH(CH₃)CH₂CH₃、NHC₆H₅、NHCH₂CH₂OCH₃、NHOCH₃、NHOCH₂CH₃A carboxyl protecting group, a fatty acid group of a lipid or a hydrocarbon group, and X¹Is a hydrophobic amino acid residue, X²Is a negatively charged residue, X⁶Is a negatively charged residue, X⁷Is a hydrophobic amino acid residue, X⁸Is a residue containing a ring structure, X¹⁰Can be M or any hydrophobic amino acid, X¹¹Is an aromatic amino acid, X¹²Selected from V, E and Kac, X¹⁴Is E or V, X¹⁹Is of any hydrophobicityAmino acid or its D-isomer, X²⁰Absent or any neutral, hydrophobic or aromatic amino acid or D-isomer thereof, X²¹Absent or any positively charged residue or any aliphatic nonpolar residue or D-isomer thereof, X²²Absent or selected from G, V, L, I, P, S, T, W, F, E, Kac or its D-isomer, X²³Absent or selected from G, A, I, L, Q, E, F, T, W, S, Y and Kac and its D-isomer.

X¹May be V. X²May be E. X⁶May be D. X⁷May be I. X⁸May be H. X¹¹May be W. [ VEGF-peptide]May be a peptide comprising a sequence substantially homologous to: v¹-E²-P³-N⁴-C⁵-D⁶-I⁷-H⁸-V⁹-X¹⁰-W¹¹-X¹²-W¹³-X¹⁴-C¹⁵-F¹⁶-E¹⁷-R¹⁸-X¹⁹-X²⁰-X²¹-X²²-X²³(SEQ ID NO: 132) in which X¹⁰Is M or any hydrophobic amino acid, X¹²Selected from V, E and Kac, X¹⁴Is E or V, X¹⁹May be a hydrophobic amino acid residue or a D-isomer thereof, X²⁰Absent or any neutral, hydrophobic or aromatic amino acid or D-isomer thereof, X²¹Absent or any positively charged residue or any aliphatic nonpolar residue or D-isomer thereof, X²²Is absent or may be selected from G, V, L, I, P, S, T, W, F, E, Kac or its D-isomer, X²³Absent or selected from G, A, I, L, Q, E, F, T, W, S, Y and Kac and its D-isomer.

[ VEGF-peptide ] includes sequences substantially homologous to one or more of:

V-E-P-N-C-D-I-H-V-M-W-E-W-E-C-F-E-R-L (SEQ ID NO：34)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-Y-G-G-W (SEQ ID NO：35)

V-E-P-N-C-D-I-H-V-M-W-V-W-V-C-F-E-R-L-F-R-E-A (SEQ ID NO：36

V-E-P-N-C-D-I-H-V-M-W-V-W-V-C-F-E-R-L-F-K-E-A (SEQ ID NO：37)

V-E-P-N-C-D-I-H-V-M-W-V-W-V-C-F-E-R-L-M-K (SEQ ID NO：38)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-Y-R-E-L (SEQ ID NO：39)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-Y-G-I-F (SEQ ID NO：40)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-Y (SEQ ID NO：41)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-Y-G-W-G (SEQ ID NO：42)

V-E-P-N-C-D-I-H-V-M-W-V-W-V-C-F-E-R-L-Y-G-G-G (SEQ ID NO：43)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-Y-G-L-Y (SEQ ID NO：44)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-S-G-G-G (SEQ ID NO：45)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-M-R-L-T (SEQ ID NO：46)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-Y-G (SEQ ID NO：47)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-V-K (SEQ ID NO：48)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-M-R (SEQ ID NO：49)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-Y-G-I-L (SEQ ID NO：50)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-(Yome)-G-L-T (SEQ IDNO：51)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-Y-G-G (SEQ ID NO：52)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-F-K-E-A (SEQ ID NO：53)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-(Nle)-K (SEQ ID NO：54)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-Y-G-L-T (SEQ ID NO：55)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-Y-G-G-F (SEQ ID NO：56)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-I-K (SEQ ID NO：57)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-Y-K (SEQ ID NO：58)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-M-G-L-T (SEQ ID NO：59)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-(D-Leu)-(Kac)(SEQ IDNO：60)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-Y-G-G-G (SEQ ID NO：61)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-M-K (SEQ ID NO：62)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-M-R-E-L (SEQ ID NO：63)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-(SEQ ID NO：64)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-M-K-E-L (SEQ ID NO：65)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-Y-G-P-W (SEQ ID NO：66)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-L-K (SEQ ID NO：67)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-L-(Kac)(SEQ ID NO：68)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-Y-G-L-T (SEQ ID NO：69)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-Y-G-E-F (SEQ ID NO：70)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-Y-G-L-S (SEQ ID NO：71)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-Y-G-L (SEQ ID NO：72)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-Y-G-(D-Leu)-T (SEQ IDNO：73)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-Y-G-V-Q (SEQ ID NO：74)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-Y-G-L-E (SEQ ID NO：75)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-Y-G-P-L (SEQ ID NO：76)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-Y-G-P-F (SEQ ID NO：77)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-Y-(D-Ala)-(D-Leu)(SEQID NO：78)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-R-(D-Leu)-(Kac)(SEQ IDNO：79)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-(D-Tyr)-G-(D-Pro)-L (SEQID NO：80)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-(D-Tyr)-G-(D-Pro)-(D-Leu)(SEQ ID NO：81)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-(D-Tyr)-G-(D-Leu)-(Kac)(SEQ ID NO：82)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-(D-Leu)-Y-(Aib)-L-T (SEQ IDNO：83)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-(D-Leu)-Y-(D-Ala)-V-(D-Gln)(SEQ ID NO：84)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-(D-Leu)-Y-(D-Ala)-L-(D-Thr)(SEQ ID NO：85)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-(Cha)-G-(DLeu)-T (SEQ IDNO：86)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-Y-(Aib)-L-(D-Thr)(SEQ IDNO：87)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-Y-(Sar)-(D-Leu)-T (SEQ IDNO：88)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-Y-G-(D-Pro)-L(SEQ IDNO：89)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-Y-(D-Ala)-V-(D-Gln)(SEQID NO：90)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-Y-(Aib)-L-T(SEQ IDNO：91)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-(D-Tyr)-G-(D-Leu)-T (SEQID NO：92)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-(D-Tyr)-G-L-T (SEQ IDNO：93)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-(D-Tyr)-D-L-(D-Thr)(SEQID NO：94)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-(D-Leu)-(Aib)(SEQ IDNO：95)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-Y-G-(D-Leu)-(D-Thr)(SEQID NO：96)

V-E-P-N-C-D-I-H-V-M-W-V-W-V-C-F-E-R-L (SEQ ID NO：97)

V-E-P-N-C-D-I-H-V-M-W-V-W-V-C-F-E-R-L-Y-G-P-F (SEQ ID NO：98)

V-E-P-N-C-D-I-H-V-M-W-V-W-V-C-F-E-R-L-Y-G-P-(SEQ ID NO：99)

V-E-P-N-C-D-I-H-V-M-W-V-W-V-C-F-E-R-L-Y-G-P-E (SEQ ID NO：100)

V-E-P-N-C-D-I-H-V-M-W-V-W-V-C-F-E-R-L-Y-G-P-Q (SEQ ID NO：101)

V-E-P-N-C-D-I-H-V-M-W-V-W-V-C-F-E-R-L-Y-G-(D-Leu)-T (SEQ IDNO：102)

V-E-P-N-C-D-I-H-V-M-W-V-W-V-C-F-E-R-L-Y-G-L ((SEQ ID NO：103)

V-E-P-N-C-D-I-H-V-M-W-V-W-V-C-F-E-R-L-(D-Leu)-Kac (SEQ IDNO：104)

V-E-P-N-C-D-I-H-V-M-W-V-W-V-C-F-E-R-L-Y-G-L-T (SEQ ID NO：105)

V-E-P-N-C-D-I-H-V-M-W-V-W-V-C-F-E-R-L-Y-(D-Ala)-(D-Leu)(SEQID NO：106)

V-E-P-N-C-D-I-H-V-M-W-E-W-E-C-F-E-R-L-Y-A-K (SEQ ID NO：108)

V-E-P-N-C-D-I-H-V-M-W-E-W-E-C-F-E-R-L-Y-F-K-E-W (SEQ IDNO：109)

V-E-P-N-C-D-I-H-V-M-W-E-W-E-C-F-E-R-L-Y-A-R (SEQ ID NO：110)

V-E-P-N-C-D-I-H-V-M-W-E-W-E-C-F-E-R-L-Y-F-K (SEQ ID NO：111)

V-E-P-N-C-D-I-H-V-M-W-E-W-E-C-F-E-R-L-Y-A-K-E-F (SEQ IDNO：112)

V-E-P-N-C-D-I-H-V-M-W-E-W-E-C-F-E-R-L-Y-S-G-W-G (SEQ IDNO：113)

V-E-P-N-C-D-I-H-V-M-W-E-W-E-C-F-E-R-L-Y-S-G-W-F (SEQ IDNO：114)

V-E-P-N-C-D-I-H-V-M-W-E-W-E-C-F-E-R-L-Y-A-K-E-A (SEQ IDNO：115)

V-E-P-N-C-D-I-H-V-M-W-E-W-E-C-F-E-R-L-Y-A-K-E-M (SEQ IDNO：116)

V-E-P-N-C-D-I-H-V-M-W-E-W-E-C-F-E-R-L-Y-A-K-E-L (SEQ IDNO：117)

V-E-P-N-C-D-I-H-V-M-W-E-W-E-C-F-E-R-L-Y-F-K-E-L (SEQ IDNO：118)

V-E-P-N-C-D-I-H-V-M-W-E-W-E-C-F-E-R-L-Y-F-K-E-A (SEQ IDNO：119)

V-E-P-N-C-D-I-H-V-M-W-E-W-E-C-F-E-R-L-Y-Y-G-G-G (SEQ IDNO：120)

V-E-P-N-C-D-I-H-V-M-W-E-W-E-C-F-E-R-L-Y-M-K (SEQ ID NO：121)

V-E-P-N-C-D-I-H-V-(Nle)-W-V-W-E-C-F-E-R-L-Y-(Aib)-L-T (SEQ IDNO：128)

V-E-P-N-C-D-I-H-V-L-W-V-W-E-C-F-E-R-L-Y-(Aib)-L-T (SEQ IDNO：129)

V-E-P-N-C-D-I-H-V-V-W-V-W-E-C-F-E-R-L-Y-(Aib)-L-T (SEQ IDNO：130)

V-E-P-N-C-D-I-H-V-M-W-(Kac)-W-E-C-F-E-R-L-Y-(D-Ala)-V-(D-Gln)(SEQ ID NO：136)

V-E-P-N-C-D-I-H-V-K-W-V-W-E-C-F-E-R-L-(D-Leu)-(Kac)(SEQ IDNO：192)

V-E-P-N-C-D-I-H-V-K-W-V-W-E-C-F-E-R-L-Y-G-(D-Leu)-T (SEQ IDNO：193)

V-E-P-N-C-D-I-H-V-K-W-V-W-E-C-F-E-R-L-Y-G-P-L(SEQ ID NO：194)

V-E-P-N-C-D-I-H-V-K-W-V-W-E-C-F-E-R-L-Y-(D-Ala)-(D-Leu)(SEQ IDNO：195)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L(-D-Leu)-K (SEQ IDNO：196)

V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-Y-G-(D-Leu)-K (SEQID NO：197)

V-E-P-N-C-D-I-H-V-M-W-E-W-E-C-F-K-R-(D-Leu) (SEQ IDNO：198)

V-E-P-N-C-D-I-H-V-KW-E-W-E-C-F- (N-glutamic acid methyl ester) -R-L (SEQ ID NO: 199)

V-E-P-N-C-D-I-H-V-KW-V-E-E-C-F-(D-Glu)-R-L (SEQ ID NO：200)

V-E-P-N-C-D-I-H-V-KW-E-W-E-C-F-E-R (N-methyl arginine) (SEQ ID NO: 201)

V-E-P-N-C-D-I-H-V-KW-E-W-E-C-F-E-R-(D-Asn) (SEQ ID NO：202)

V-E-P-N-C-D-I-H-V-KW-E-W-E-C-F-E-R-(Aib) (SEQ ID NO：203)

V-E-P-N-C-D-I-H-V-KW-E-W-E-C-F-(Aib)-R-L (SEQ ID NO：204)

V-E-P-N-C-D-I-H-V-KW-E-W-E-C-(Napthaline)-E-R-L (SEQ IDNO：205)

V-E-P-N-C-D-I-H-V-M-W-V-E-C-F-K-R-L-Y-G-L-T (SEQ ID NO：206)

V-E-P-N-C-D-I-H-V-M-W-V-E-C-F-E-R-L-Y-K-L-E (SEQ ID NO：207)

V-E-P-N-C-D-I-H-V-M-W-E-W-E-C-F-E-R-(D-Leu)(SEQ ID NO：208)

V-E-P-N-C-D-I-H-V-E-W-E-W-E-C-F-K-R-L(SEQ ID NO：209)

V-E-P-N-C-D-I-H-V-K-W-V-W-E-C-F-E- (homoarginine) -L-Y- (D-Ala) - (D-Leu) (SEQ ID NO: 210)

V-E-P-N-C-D-I-H-V-M-W-K-W-E-C-F-E-R-Y-G-(D-Leu)-E (SEQ IDNO：211)

V-E-P-N-C-D-I-H-V-K-W-V-W-E-C-F-E-R-L-Y-G-(D-Leu)-E (SEQ IDNO：212)

V-E-P-N-C-D-I-H-V-K-W-V-W-E-C-F-E-R-L-Y-(Aib)-L-E (SEQ IDNO：213)

V-E-P-N-C-D-I-H-V-M-W-K-W-E-C-F-E-R-L-Y-(Aib)-L-E (SEQ IDNO：214)

V-E-P-N-C-D-I-H-V-M-W-K-W-E-C-F-E-R-(D-Leu)-K(SEQ ID NO：215)

V-E-P-N-C-D-I-H-V-K-W-V-W-E-C-F-E-(Kac)-L-Y-(D-Ala)-(D-Leu)(SEQ ID NO：216)

V-E-P-N-C-D-I-H-V-K-W-V-W-E-C-F-E-(Kac)-(cyclo-Leu)-Y-(D-Ala)-(D-Leu)(SEQ ID NO：217)

V-E-P-N-C-D-I-H-V-K-W-V-W-E-C-F-E-(Kac)-(Tle)-Y-(D-Ala)-(D-Leu)(SEQ ID NO：218)

In some embodiments, the [ VEGF-peptide ] of the invention is selected from SEQ ID NO: 60. 73, 76, 78, 192, 193, 194 or 195.

X¹⁰May be M. [ VEGF-peptide]May be a peptide comprising a sequence substantially homologous to: v¹-E²-P³-N⁴-C⁵-D⁶-I⁷-H⁸-V⁹-M¹⁰-W¹¹-X¹²-W¹³-X¹⁴-C¹⁵-F¹⁶-E¹⁷-R¹⁸-X¹⁹-X²⁰-X²¹-X²²-X²³(SEQ ID NO: 133), wherein X¹²Selected from V, E and Kac, X¹⁴Is E or V, X¹⁹Can be any hydrophobic amino acid residue or D-isomer thereof, X²⁰Absent or may be any neutral, hydrophobic or aromatic amino acid or D-isomer thereof, X²¹Is absent or may be any positively charged residue or any aliphatic nonpolar residue or D-isomer thereof, X²²Absent or selected from G, V, L, I, P, S, T, W, F, E, Kac or its D-isomer, X²³Absent or selected from G, A, I, L, Q, E, F, T, W, S, Y and Kac and its D-isomer.

X¹²May be E. [ VEGF-peptide]May be a peptide comprising a sequence substantially homologous to: v¹-E²-P³-N⁴-C⁵-D⁶-I⁷-H⁸-V⁹-M¹⁰-W¹¹-E¹²-W¹³-X¹⁴-C¹⁵-F¹⁶-E¹⁷-R¹⁸-X¹⁹-X²⁰-X²¹-X²²-X²³(SEQ ID NO: 134) wherein X¹⁴Is E or V, X¹⁹Can be any hydrophobic amino acid residue or D-isomer thereof, X²⁰Absent or may be any neutral, hydrophobic or aromatic amino acid or D-isomer thereof, X²¹Is absent or may be any tapePositively charged residue or any aliphatic nonpolar residue or D-isomer thereof, X²²Absent or selected from G, V, L, I, P, S, T, W, F, E, Kac or its D-isomer, X²³Absent or selected from G, A, I, L, Q, E, F, T, W, S, Y and Kac and its D-isomer.

The [ VEGF-peptide ] can include sequences substantially homologous to one or more of: SEQ ID NO: 34. SEQ ID NO: 108. SEQ ID NO: 109. SEQ ID NO: 110. SEQ ID NO: 111. SEQ ID NO: 112. SEQ ID NO: 113. SEQ ID NO: 114. SEQ ID NO: 115. SEQ ID NO: 116. SEQ ID NO: 117. ID NO: 118. SEQ ID NO: 119. SEQ ID NO: 120. SEQ ID NO: 121.

X¹²may be V. [ VEGF-peptide]May be a peptide comprising a sequence substantially homologous to: v¹-E²-P³-N⁴-C⁵-D⁶-I⁷-H⁸-V⁹-M¹⁰-W¹¹-V¹²-W¹³-X¹⁴-C¹⁵-F¹⁶-E¹⁷-R¹⁸-X¹⁹-X²⁰-X²¹-X²²-X²³(SEQ ID NO: 135) in which X¹⁴Is E or V, X¹⁹Can be any hydrophobic amino acid residue or D-isomer thereof, X²⁰Absent or may be any neutral, hydrophobic or aromatic amino acid or D-isomer thereof, X²¹Is absent or may be any positively charged residue or any aliphatic nonpolar residue or D-isomer thereof, X²²Absent or selected from G, V, L, I, P, S, T, W, F, E, Kac or its D-isomer, X²³Absent or selected from G, A, I, L, Q, E, F, T, W, S, Y and Kac and its D-isomer.

The [ VEGF-peptide ] can include sequences substantially homologous to one or more of: SEQ ID NO: 35. SEQ ID NO: 36. SEQ ID NO: 37. SEQ ID NO: 38. SEQ ID NO: 39. SEQ ID NO: 40. SEQ ID NO: 41. SEQ ID NO: 42. SEQ ID NO: 43. SEQ ID NO: 44. SEQ ID NO: 45. ID NO: 46. SEQ ID NO: 47. SEQ ID NO: 48. SEQ ID NO: 49. SEQ ID NO: 50. SEQ ID NO: 51. SEQ ID NO: 52. SEQ ID NO: 53. SEQ ID NO: 54. SEQ ID NO: 55. SEQ ID NO: 56. IDNO: 57. SEQ ID NO: 58. SEQ ID NO: 59. SEQ ID NO: 60. SEQ ID NO: 61. SEQ ID NO: 62. SEQ ID NO: 63. SEQ ID NO: 64. SEQ ID NO: 65. SEQ ID NO: 66. SEQ ID NO: 67. SEQ ID NO: 68. SEQ ID NO: 69. SEQ ID NO: 70. ID NO: 71. SEQ ID NO: 72) SEQ ID NO: 73. SEQ ID NO: 74. SEQ ID NO: 75. SEQ ID NO: 76. SEQ ID NO: 77. SEQ ID NO: 78. SEQ ID NO: 79. SEQ ID NO: 80. SEQ ID NO: 81. SEQ ID NO: 82. ID NO: 83. SEQ ID NO: 84. SEQ ID NO: 85. SEQ ID NO: 86. SEQ ID NO: 87. IDNO: 88. SEQ ID NO: 89. SEQ ID NO: 90. SEQ ID NO: 91. SEQ ID NO: 92. SEQ ID NO: 93. SEQ ID NO: 94. SEQ ID NO: 95. SEQ ID NO: 96. IDNO: 97. SEQ ID NO: 98. SEQ ID NO: 99. SEQ ID NO: 100. SEQ ID NO: 101. SEQ ID NO: 102. SEQ ID NO: 103. SEQ ID NO: 104. SEQ ID NO: 105. SEQ ID NO: 106. SEQ ID NO: 128. SEQ ID NO: 129. SEQ ID NO: 130.

reference to [ VEGF-peptides as described herein]In some aspects of the invention, R¹May be C (O) CH₃. In some aspects of the invention, R²Is NH₂。R¹And/or R²May not be present. X¹⁹May be selected from L, I, A, V, G or its D-isomer. X¹⁹May be L. X¹⁹May be D-Leu. X²⁰May be Y. X²⁰May be D-Tyr. X²¹Can be selected from D-Ala, G, Aib and Kac. X²¹May be D-Ala. X²¹May be G. X²²Can be selected from L, V, P, D-Leu and D-Pro. X²²May be D-Leu. X²²May be D-Pro. X²²May be L. X²³May not be present.

In some embodiments of the invention, [ VEGF-peptide]Is (SEQ ID NO:78). In some embodiments, R¹- [ VEGF-peptide]-R²Is { C (O) CH₃}-V-E-P-N-C-D-I-H-V-K-W-V-W-E-C-F-E-R-L-Y-(D-Ala)-(D-Leu)-{NH₂} (SEQ ID NO: 188), and K¹¹Is a linking residue.

Connection of

The presence of the linking residues (VEGF-linking residue and Ang 2-linking residue, respectively) provides the compounds of the invention with great flexibility for attachment to scaffolds, macromolecules and other moieties. In particular, the compounds of the invention can be reliably, strongly and efficiently covalently linked to a scaffold such as an antibody, antibody fragment, PEG molecule, albumin, and the like. Surprisingly, it has been found that placing the linking residues at certain key positions of the corresponding peptide leads to an increased stability and/or binding of the peptide. The linking residue may be selected to provide a side chain whose chemical properties enable it to form a specific, reliable, directed, and efficient chemical covalent bond at that position. In some aspects of the invention, the linking residue is covalently linked to the binding site of the antibody, either directly or through an intermediate linker. The bonding is irreversible.

The compounds of the invention may be covalently linked to the linker moiety L (or L', as described below) through a linking residue. A large number of linkers are possible; a number of suitable linkers are disclosed in US2006205670, the contents of which are incorporated herein by reference. In particular, as described herein specifically or generally, various aspects of US2006205670 relating to linkers, specific linker structures, linker syntheses, and different component parts X, Y and Z groups are included herein. The linker is straight or branched chain and optionally includes one or more carbocyclic or heterocyclic groups. Linker length can be calculated from the number of atoms in the linear chain, and a cyclic moiety such as an aromatic ring is calculated by taking the shortest path along the ring. In some embodiments, the linker has a linear chain length of 5-15 atoms, in other embodiments 15-30 atoms, in still other embodiments 30-50 atoms, in still other embodiments 50-100 atoms, and in still other embodiments 100-200 atoms. Other considerations for linkers include effects on the physical or pharmacokinetic properties of the resulting compound, such as solubility, lipophilicity, hydrophilicity, hydrophobicity, stability (more or less stable and designed degradation), rigidity, flexibility, immunogenicity, modulation of antibody binding, ability to be introduced into micelles or liposomes, and the like.

In some aspects of the invention, the compound comprises a linker (L) or (L') covalently attached to the linking residue. The linker may comprise the formula: - [ linker ] -X-Y-Z; - [ linker ] -X-Y-Z'; -X-Y-Z-; or-X-Y-Z' -, wherein [ linker ] is present when the linker is branched and, when present, is covalently linked to the linking residue and one or more other reactive molecules, X is a biocompatible linking chain comprising any atom selected from C, H, N, O, P, S, F, Cl, Br, and I, and comprises a polymer or block copolymer, and is covalently linked to the linker when the linker is linear, Y is an optionally present recognition group comprising at least one ring structure; and Z is a reactive group capable of forming a covalent bond with an amino acid side chain within the binding site of the antibody, and Z' is an attachment moiety comprising a covalent linkage with an amino acid side chain within the binding site of the antibody.

When present, Y may have an optionally substituted structure:

wherein a, b, c, d and e are independently carbon or nitrogen; f is carbon, nitrogen, oxygen or sulfur; y is independently attached to X and Z at any two ring positions of sufficient valency; and no more than four of a, b, c, d, e or f are simultaneously nitrogen, and preferably each of a, b, c, d and e in the ring structure is carbon. In some aspects, Y may be phenyl. While not wishing to be bound by any theory, it is believed that the Y group can help place the reactive group at the antibody binding site so that the Z group can react with the reactive amino acid side chain.

The linker may be designed to contain a reactive group capable of forming a bond, covalently or non-covalently, with a macromolecule such as an antibody, protein, or fragment thereof. The reactive group is selected to be a reactive residue for a particular binding site. For example, the chemical moiety for modification by an aminidase antibody can be a ketone, diketone, beta-lactam, active ester haloketone, lactone, anhydride, maleimide, alpha-haloacetamide, cyclohexyl diketone, epoxide, aldehyde, amidine, guanidine, imine, enamine, phosphate, phosphonate, epoxide, aziridine, thioepoxide, masked or protected diketone (e.g., ketal), lactam, haloketone, aldehyde, and the like. In embodiments of the invention where the peptide of the invention is linked using a linker L or L ', the moiety Z (or Z' when attached to a macromolecule) is a reactive group.

In some embodiments, Z comprises one or more C ═ O groups arranged to form azetidinone, diketones, acyllactams, active esters, haloketones, cyclohexyl dione groups, aldehydes, maleimides, activated alkenes, activated alkynes, or, in general, molecules comprising leaving groups susceptible to nucleophilic or electrophilic substitution. Other groups may include lactones, anhydrides, α -haloacetamides, imines, hydrazides, or epoxides. Exemplary linking electrophilic reactive groups capable of covalent bonding to a reactive nucleophilic group (e.g., lysine or cysteine side chains) within an antibody binding site include acyl β -lactams, simple diketones, succinimide active esters, maleimides, haloacetamides with linkers, haloketones, cyclohexyl diketones, aldehydes, amidines, guanidines, imines, enamines, phosphates, phosphonates, epoxides, aziridines, thioepoxides, masked or protected diketones (e.g., ketals), lactams, haloketones, aldehydes, and the like, masked C ═ O groups such as imines, ketals, acetals, and any other known electrophilic groups. In certain embodiments, the reactive group comprises one or more C ═ O groups arranged to form acyl β -lactams, simple diketones, succinimide active esters, maleimides, haloacetamides with linkers, haloketones, cyclohexyl diketones, or aldehydes. Z or Z', when present, can be a substituted alkyl, substituted cycloalkyl, substituted aryl, substituted arylalkyl, substituted heterocyclyl or substituted heterocyclylalkyl group, wherein at least one substituent is a 1, 3-diketone moiety, acyl beta-lactam, active ester, alpha-haloketone, aldehyde, maleimide, lactone, anhydride, alpha-haloacetamide, amine, hydrazide, or epoxide. In some aspects, the Z group, when present, is covalently attached to the binding site of the antibody. In other aspects, the Z group is covalently attached to a macromolecular backbone that provides the peptides of the invention with an extended half-life.

In some aspects, Z, when present, has the structure:

wherein q is 0 to 5. Q is 1 or 2. Q may be 1. In other aspects, q can be 2.

Z', when present, may have the structure:

wherein q is 0-5 and antibody-N-is a covalent bond to a side chain at the antibody binding site. Q may be 1 or 2. Q may be 1. In other aspects, q can be 2.

X may be a three component: Xp-Xs-Xy, wherein Xp is a group specifically adapted to be attached to a side chain of said attachment residue, Xs is a spacer region of said X group, and Xy is a group adapted to bind said Y group. In some aspects, Xy is selected from an amide linkage, an enamine linkage, or a guanidinium linkage. Xy is selected to provide a hydrogen molecule (within two atoms) adjacent to the Y group. While not wishing to be bound by theory, it is believed that the H atom contributes to the Y group recognition of hydrophobic pockets (hydrophobic pockets) by H-bond interactions, particularly hydrophobic pockets that catalyze the binding cleft of antibodies, such as H38C2 (e.g., fig. 2A). Thus, for example, the amide bond may be oriented such that the NH group is directly bonded to the Y group, thereby providing the H of the NH group for hydrogen bonding. Alternatively, the C ═ O group of the amide can be bonded to the Y group, and the H of the NH group is adjacent to the Y group by 2 atoms, but provision can also be made for an H-bond. In some aspects, Xs is selected such that Xs does not provide any over-reactive groups. Xs can be selected to provide a total length of X groups of 2 to 10 atoms. Xs can be chosen such that the total length of the X group is 2-10 atoms. Xs can be chosen such that the total length of the X group is 4-8 atoms. Xs can be chosen such that the total length of the X group is 5 atoms. Xs may be chosen such that the total length of the X group is 6 atoms. Xp may be ideally chosen to enable a specifically directed covalent attachment strategy to the attachment residue. For example, when the linking group comprises a nucleophilic group, Xp can be an electrophilic group, and vice versa. For example, if the linking residue side chain includes an amino group, such as K, H, Y, ornithine, Dap, or Dab, Xp can be COOH or other similar reactive electrophiles. If the linking residue is D or E, Xp can include a nucleophilic group, such as an amino group. Either of these strategies allows for the formation of covalent bonds between the Xp groups and the linking residues through an amide bond formation strategy. When the linking group is an C, C analog or other thiol group-containing residue, Xp can include a maleimide group, enabling a thiol-maleimide addition reaction to covalently attach the Xp group to the linking residue. In some aspects, Xp can also include a thiol group, enabling it to form a disulfide bridge between the linking residue and the Xp group.

The backbone length of X may be 3-15 atoms.

X may be:

and in some aspects, X-Y may be:

wherein v and w are selected such that the backbone length of X is 6-12 atoms, or 3-9 atoms, or 4-7 atoms, or 6 atoms, or 7 atoms or 8 atoms. V and W may each be 0-8. V may be 1 or 2. W may be 1 or 2. V and W may each be 1. R^bMay be hydrogen, substituted or unsubstituted C_1-10Alkyl, substituted or unsubstituted C_3-7cycloalkyl-C_0-6Alkyl or substituted or unsubstituted aryl-C_0-6An alkyl group. R^bMay be H. In some aspects, V and W are both 1, and R^bIs H.

Certain embodiments of the invention have the structure:

wherein the peptide is a [ VEGF-peptide of the invention]V is 1, 2,3, 4 or 5; w is 1, 2,3, 4 or 5; q is 0, 1, 2,3, 4 or 5; and R^bIs hydrogen, substituted or unsubstituted C_1-10Alkyl, substituted or unsubstituted C_3-7cycloalkyl-C_0-6Alkyl or substituted or unsubstituted aryl-C_0-6An alkyl group. In certain embodiments, 1, 2, or 3; w is 1, 2 or 3; and q is 0, 1, 2 or 3. In some embodiments, v is 1 or 2; w is 1 or 2; and q is 1 or 2 and R^bIs H.

In some aspects, the present invention provides a compound selected from the group consisting of compound 2001-2010, compound 2014-2016, compound 2018, compound 2020, compound 2022-2024, compound 2027-2033, compound 2036, compound 2038, compound 2041-2042, compound 2045-2046, compound 2048-2050, and compound 2052-2053. These compounds can be covalently linked to the binding site of a catalytic antibody, such as an aldolase antibody, such as h38C 2. In some aspects, the present invention provides a compound of the invention selected from compounds 2018, 2036, 2045 and 2050. These compounds can be covalently linked to the binding site of a catalytic antibody, such as an aldolase antibody, such as h38C 2. In some aspects, the present invention provides compound 2018.

The invention also provides nucleic acid sequences (including DNA sequences, RNA sequences and DNA-RNA sequences) encoding the peptides of the invention and precursors thereof.

Bifunctional molecules

In some aspects, the invention provides a compound of the formula:

wherein [ active molecule-1 ] is the [ VEGF-peptide ] of the present invention and [ linker ] is a moiety covalently linked to both [ VEGF-peptide ] and [ active molecule-2 ]. It will be appreciated that in some aspects of the invention, the [ linker ] may be covalently linked to more than one other [ active molecule ] in addition to the [ VEGF-peptide ].

R¹- [ VEGF-peptide]-R²May be { C (O) CH₃}-V-E-P-N-C-D-I-H-V-K-W-V-W-E-C-F-E-R-L-Y-(D-Ala)-(D-Leu)-{NH₂} (SEQ ID NO: 188), and K¹¹May be connected to said [ joint ]]Covalently linked VEGF-linking residues.

The active molecule may be any chemical, biochemical or biological entity capable of covalent bonding to the [ linker ] and interacting with the biological system. Examples include therapeutic agents, drugs, prodrugs, targeting agents, toxins, proteins, peptides, nucleic acid molecules, and lipids. In some aspects of the invention, the active molecule is a peptide, and may be an anti-angiogenic peptide. In some aspects, the active molecule is a [ VEGF-peptide ] of the invention and/or an [ Ang 2-peptide ] of the invention, as the case may be.

The [ VEGF-peptide]By attaching nucleophilic side chains of residues, or N-terminal amino or C-terminal carboxyl groups, to the [ linker ]]Covalently linked. Alternatively, when the linker has no branch, the [ VEGF-peptide ]]The X group of L or L' may be covalently attached through the nucleophilic side chain of a VEGF-linked residue, or the N-terminal amino group or C-terminal carboxyl group. The VEGF-linking residue may be selected from the group consisting of K, R, Y, C, T, S, lysine analogs, homocysteine, homoserine, Dap, Dab, the N-terminal residue and the C-terminal residue. The VEGF-linking residue may be selected from K, Y, T, Dap and Dab. In some aspects of the invention, the VEGF-linking residue is K. The linking residue may be K¹⁰. The linking residue may be K¹². In some aspects of the invention, V¹、E²、P³、N⁴、V⁹、M¹⁰、V¹²、X¹⁴、E¹⁷And the [ VEGF-peptide]Is replaced by a (VEGF-) linking residue containing a nucleophilic side chain, or by an N-terminal amino group or a C-terminal carboxyl group covalently linked to the binding site of the antibody, either directly or through an intermediate linker, said linking residue being selected from the group consisting of K, R, Y, C, T, S, lysine analogs, homocysteine, homoserine, Dap, Dab, N-terminal residue and C-terminal residue. In some aspects, when the linking residue is at the N-terminus or C-terminus, the linkage can be through the amino group at the N-terminus or the carboxyl group at the C-terminus, rather than through the corresponding amino acid side chain. In some aspects of the invention, V¹、N⁴、M¹⁰、V¹²、X¹⁴And E¹⁷One of which is replaced by said VEGF-linking residue. In some aspects of the invention, M¹⁰And V¹²One of which is replaced by the linking residue. In some aspects of the invention, M¹⁰Is replaced by the linking residue. In some aspects of the invention, V¹²Is replaced by the linking residue.

The [ linker ] comprises the formula: - [ AM 1-spacer ] - [ branched ] - [ AM 2-spacer ] -, wherein [ AM-1-spacer ] and [ AM-2-spacer ] are each independently a biocompatible polymer, block copolymer, C, H, N, O, P, S, halogen (F, Cl, Br, I) or a salt thereof alkyl, alkenyl, alkynyl, oxyalkyl, oxyalkenyl, oxyalkynyl, aminoalkyl, aminoalkenyl, aminoalkynyl, sulfoalkyl, sulfoalkenyl, sulfoalkynyl, phosphorylalkyl, phosphorylalkenyl, or phosphorylalkynyl covalently bonded to the [ branch ], and [ branched ] is a molecule having at least three reactive groups, [ AM-1-spacer ] covalently linked to [ branched ] and said [ active molecule-1 ] and [ AM-2-spacer ] covalently linked to [ branched ] and said [ active molecule-2 ]. Each of [ AM-1-spacer ] and [ AM-2-spacer ] is independently a neutral, water-soluble molecule that forms a covalent bond with their respective reactive molecule and [ branch ]. Wherein the active molecules include peptides, [ AM-1-spacer ] and [ AM-2-spacer ] capable of forming peptide bonds with corresponding active molecules.

[ AM-1-spacer ] and [ AM-2-spacer ] are each independently selected from the group consisting of aminopolyglycolic acid, polyethylene glycol diacid, amino alkanoic acid, polyglycine. [ AM-1-spacer ] and [ AM-2-spacer ] are each independently selected from (the exemplary molecules shown below are merely illustrative of certain suitable spacer species):

[ AM-1-spacer ] and [ AM-2-spacer ] may be independently selected from: 2-PEG, 4-PEG and 6-PEG:

the [ AM-1-spacer ] and [ AM-2-spacer ] may each independently be 6 to 15 atoms in length. The [ AM-1-spacer ] and [ AM-2-spacer ] may each independently be a 4-PEG spacer. The [ AM-1-spacer ] and [ AM-2-spacer ] may each independently be a 2-PEG spacer. The [ AM-1-spacer ] and [ AM-2-spacer ] may each independently be a 1-PEG spacer. The [ AM-1-spacer ] may be 6 to 15 atoms in length. [ AM-1-spacer ] may be 4-PEG. The [ AM-2-spacer ] can be 6 to 15 atoms in length. The [ AM-2-spacer ] may be 4-PEG.

[ branched ] may be a chemical moiety comprising three orthogonal reactive groups. [ branched ] may be selected from cysteine, diaminopropionic acid, diaminobutyric acid, ornithine, lysine, homocysteine, bismaleimide and maleimide-acids and derivatives and analogues thereof.

[ branched ] can be selected from:

and derivatives and analogs thereof. [ branched chain]May be cysteine.

The compounds of the invention may include the formula: [ branched ] -L or [ branched ] -L ', wherein L is a linker, L ' is a linker covalently linked to an amino acid side chain in the binding site of the antibody, and [ branched ] is covalently linked to L or L '. [ branched ] -L may be selected from:

[ Ang2 peptide ]

In some aspects of the invention, [ active molecule 2] is an Ang-2 binding peptide [ Ang 2-peptide ]. The [ Ang 2-peptide ] can be covalently attached to the [ linker ] through the nucleophilic side chain or N-terminus or C-terminus of an Ang 2-linking residue, the Ang 2-linking residue being selected from the group consisting of K, R, Y, C, T, S, lysine analog, homocysteine, homoserine, Dap, Dab, N-terminal residue, and C-terminal residue. When the Ang-2 linking residue is located at the N-terminus or C-terminus, the linkage may be through the amino group at the N-terminus or the carboxyl group at the C-terminus, rather than through the side chain of the particular amino acid located at that position. The Ang 2-linking residue may be selected from K, Y, T, Dap and Dab. The Ang 2-linking residue can be K.

In some aspects of the invention, the [ Ang 2-peptide]Comprising a sequence substantially homologous to: q¹X²Y³Q⁴X⁵L⁶D⁷E⁸X⁹D¹⁰X¹¹X¹²X¹³X¹⁴D¹⁵X¹⁶F¹⁷M¹⁸X¹⁹Q²⁰Q²¹G²²(SEQ ID NO: 107) in which X²Selected from K, N, R, H, Kac, Nick and CbcK, X⁵Selected from P, hP, dhP and BnHP, X⁹Selected from L, I, ThA and Kac, X¹¹Selected from Q, N, C, K, Kac, Dab and Dap, X¹²Selected from L, HL, Nva, I, HchA, HF and ThA, X¹³Selected from L, HL, Nva, I, HchA, HF and ThA, X¹⁴Selected from aromatic residues, X¹⁶Selected from Q and N, X¹⁹Is selected from L and I, wherein Q¹、E⁸、X⁹、X¹¹、X¹²、D¹⁵、X¹⁶、M¹⁸、X¹⁹Or G²²One of which is replaced by the Ang 2-linking residue.

In some aspects, X²Selected from K, N and Kac. X²May be N. X²May be Kac. In some aspects, X⁵Selected from P, hP and dhP. X⁵May be P. In some aspects, X⁹Is L. In some aspects, X⁹Is Kac. In some aspects, X¹¹Selected from K, Kac. In some aspects, X¹³Is selected from L, HL, Nva and I. X¹³May be L. In some aspects, X¹⁴Selected from F, Y, W, BPA, CF, NF. X¹⁴May be Y. In some aspects, X¹⁶Is Q. The Ang 2-linking residue may be replaced by X⁹、X¹¹、X¹²、D¹⁵、X¹⁶、M¹⁸And X¹⁹An alternative. X¹¹May be located at the position of the Ang 2-linking residue.

The [ Ang 2-peptide ] can include a peptide selected from the group consisting of: SEQ ID NO: 137-191 or a compound thereof. The Ang-2 peptide may include an amino acid sequence selected from SEQ ID NOs: 137-172 and SEQ ID NO: 182, or a pharmaceutically acceptable salt thereof. The [ Ang 2-peptide ] can include a peptide selected from the group consisting of: SEQ ID NO: 137. SEQ ID NO: 138. SEQ ID NO: 139. SEQ ID NO: 140. SEQ ID NO: 153. SEQ ID NO: 154. SEQ ID NO: 155. SEQ ID NO: 157. SEQ ID NO: 161. SEQ ID NO: 174 and SEQ ID NO: 181, or a sequence substantially homologous to one or more compounds of the group.

The [ Ang 2-peptide]Sequences substantially homologous to: q (Kac) YQPLDE (Kac) DKTLYDQFMLQQG (SEQ ID NO: 153). R¹May be C (O) CH₃。R²May be NH₂. In some aspects, R¹- [ Ang-peptide]-R²May be { C (O) CH₃}Q(Kac)YQPLDE(Kac)DKTLYDQFMLQQG-{NH₂} (SEQ ID NO: 153), and K¹¹Is connected with the [ joint ]]Covalently linked Ang 2-linking residues.

In some aspects of the invention, the compound is selected from: compounds 5001-5028 and 5031-5062. In some aspects, the compound is selected from the group consisting of compound 5001-5028 and compound 5031-5074. In some aspects, the compound is selected from 5053, 5060, 5061, and 5062. In some aspects, the compound is compound 5053. In some aspects, the compound is compound 5037. In some aspects of the invention, the compound is selected from the group consisting of compounds 6001-6028 and 6031-6062. In some aspects, the compound is selected from compounds 6001-6026, 6029, 6031-6069. In some aspects, the compound is selected from the group consisting of compounds 6001-6028 and compounds 6031-6074. In some aspects, the compound is selected from compounds 6053, 6060, 6061, and 6062. In some aspects, the compound is compound 6053. In some aspects, the compound is compound 6037.

Antibodies

The content of US2006205670 is incorporated herein by reference, especially paragraphs [0153] - [0233], which describe antibodies and useful fragments, variants and modifications thereof, binding sites and CDRs, antibody preparation, expression, humanization, aa modification, glycosylation, ADCC, CDC, increasing the serum half-life of the antibody, expression vectors, mammalian host systems and folding, and other elements in antibody technology.

As discussed, in certain embodiments, certain antibodies that can be used in combination with the compounds of the present invention require reactive side chains at the antibody binding site. The reactive side chain may be naturally occurring or placed in the antibody by mutation. Reactive residues of an antibody binding site can be attached to the antibody, such as when the residues are encoded by nucleic acids present in lymphocytes originally identified for use in making the antibody. Alternatively, the amino acid residues may be obtained by deliberate mutation of the DNA to encode the particular residue (see, e.g., WO 01/22922 to Meares et al). As discussed herein, the reactive residue can be, for example, an unnatural residue resulting from biosynthetic introduction of unique codons, trnas, and aminoacyl-trnas. In another approach, the amino acid residue or a reactive functional group thereof (e.g., a nucleophilic amino or thiol group) can be attached to an amino acid residue in the binding site of the antibody. Thus, as used herein, a covalent bond to the antibody "through a bond to an amino acid residue in the binding site of the antibody" means that the bond can be attached to an amino acid residue of the binding site of the antibody either directly or through a chemical moiety attached to an amino acid side chain of the binding site of the antibody. In some embodiments, the amino acid is cysteine and the reactive group of the side chain is a thiol group. In other embodiments, the amino acid residue is lysine and the reactive group of the side chain is an epsilon-amino group.

Catalytic antibodies are a source of antibodies having suitable binding sites comprising one or more amino acid side chains. Suitable antibodies include aldolase antibodies, beta lactamase antibodies, esterase antibodies, and the like.

One embodiment includes aldolase antibodies such as the mouse monoclonal antibodies mAb33F12 and mAb 38C2, as well as appropriately chimeric and humanized versions of these antibodies (e.g., h38C2, SEQ ID NOS: 1 and 2). Mouse mAb 38C2 (and h38C2) have reactive lysines close to but outside HCDR3 and are prototypes for a new class of catalytic antibodies produced by reactive immunization and that mechanically mimic the natural aldolase. See c.f. barbas 3^rdetal, Science 278: 2085-2092(1997)). Other useful aldolase catalytic antibodies include antibodies produced by the following hybridomas: hybridoma 85a2, having ATCC accession number PTA-1015; hybridoma 85C7, having ATCC accession number PTA-1014; hybridoma 92F9, having ATCC accession number PTA-1017; hybridoma 93F3, having ATCC accession number PTA-823; hybridoma 84G3, having ATCC accession number PTA-824; hybridoma 84G11, having ATCC accession number PTA-1018; hybridoma 84H9, having ATCC accession number PTA-1019; hybridoma 85H6, having ATCC accession number PTA-825; hybridoma 90G8, having ATCC accession number PTA-1016. Via reactive lysine, these antibodies catalyze aldol and retro-aldol reactions using the enamine mechanism of the native aldolase. See, e.g., j.wagner et al, Science 270: 1797 + 1800 (1995); barbas 3^rdet al, Science 278: 2085-2092 (1997); g.zhong et al, angelw.chem.int.ed.engl.38: 3738-3741 (1999); karlstrom et al, proc.natl.acad.sci.u.s.a., 97: 3878-3883(2000). Aldolase antibodies and methods of producing aldolase antibodies are disclosed in U.S. patents 6,210,938, 6,368,839, 6,326,176, 6,589,766, 5,985,626 and 5,733,75, which are incorporated herein by reference.

The compounds of the present invention may also be formed by linking the compounds of the present invention to reactive cysteines such as those found in the binding sites of thioesterase and esterase catalytic antibodies. Suitable thioesterase catalytic antibodies are described by k.d. janda et al, proc.natl.acad.sci.u.s.a.91: 2532, 2536 (1994). Suitable esterase catalytic antibodies are described by p.wiresching et al, Science 270: 1775-1782 (1995). Antibodies containing reactive amino acids can be prepared by means known in the art, including mutating the antibody binding site to encode the reactive amino acid, or chemically derivatizing the amino acid side chain in the antibody binding site with a linker containing the reactive group.

The antibody may be a humanized antibody. When the compound of the invention is covalently linked to the binding site of an antibody and the antibody is humanized, it is important that the antibody is humanized while retaining high binding affinity for the Z group. Various forms of humanized murine aldolase antibodies are contemplated. One embodiment uses the humanized aldolase catalytic antibody h38C2IgG1 or h38C 2Fab and human constant domain C_κAnd C_γ11. R. rader et al, j.mol.bio.332: 889-899(2003) discloses the gene sequences and vectors for making h38c 2Fab and h38c2IgG 1. Human germ cell line V_kGene DPK-9 and human J_kGene JK4 framework for humanization of the kappa light chain variable domain of m38c2, human germ cell line DP-47 and human J_HGene JH4 was used for the framework for humanization of the heavy chain variable domain of m38c 2. Fig. 2A depicts the sequence alignment between variable light and heavy chains of m38c2, h38c2, and a human germ cell line. h38c2 may employ IgG1, IgG2, IgG3 or IgG4 constant domains, including any allotype thereof. FIG. 2B depicts an embodiment of h38c2IgG1 employing the G1m (f) allotype, wherein the light and heavy chain amino acid sequences of this h38c2IgG1 are illustrated in this figure. In certain embodiments of the compounds of the present invention, wherein the antibody is h38c2IgG1 having the G1m (f) allotype, Z binds to the lysine residue at position 99 of the heavy chain. This residue is indicated in bold in FIG. 2B. Another embodiment uses a variable field (V) comprising h38c2_LAnd V_H) And a constant domain derived from IgG1, IgG2, IgG3, or IgG4The chimeric antibody of (4). The antibody may be a full-length antibody, Fab ', F (ab')₂、F_v、dsF_v、scF_v、V_H、V_LDiabodies or comprising V from h38c2_HAnd V_LMini-antibodies of the domain. The antibody may be a V comprising V from h38c2_HAnd V_LAn antibody comprising a domain and a constant domain selected from the group consisting of IgG1, IgG2, IgG3 and IgG 4. The antibody may be h38C2IgG 1. The antibody may be a humanized version of a murine aldolase antibody comprising constant domains from human IgG, IgA, IgM, IgD, and IgE antibodies. In another embodiment, the antibody is a chimeric antibody comprising a variable domain from a murine aldolase antibody and a constant domain from a human IgG, IgA, IgM, IgD, or IgE antibody. In still other embodiments, the antibody is a fully human version of a murine aldolase antibody comprising a polypeptide sequence from a native or native human IgG, IgA, IgM, IgD, or IgE antibody.

Different forms of humanized aldolase antibody fragments are also contemplated. One embodiment uses h38c2F (ab')₂。h38c2F(ab’)₂Can be produced by proteolytic digestion of h38c2IgG 1. Another embodiment uses a linker comprising (Gly) optionally via an intermediate linker₄Ser)₃Connected V from h38c2_LAnd V_HDomain h38c2 scFv. As an alternative to humanization, human antibodies may also be produced. For example, it has now been possible to produce transgenic animals (e.g., mice) that, upon immunization (or reactive immunization in the case of catalytic antibodies), are capable of producing fully functional human antibodies without the production of endogenous immunoglobulins.

Alternatively, phage display technology (see, e.g., J.McCafferty et al, Nature 348: 552-553 (1990); H.J.de Haard et al, J Biol Chem 274, 18218-18230 (1999); and A.Kanppik et al, J Mol Biol, 296, 57-86(2000)) can be used to generate human antibodies and human antibody fragments in vitro from non-immunized donors using immunoglobulin variable (V) domain gene function. As indicated above, human antibodies can also be produced by activating B cells in vitro. See, for example, U.S. patent nos. 5,567,610 and 5,229,275; and c.a.k.borrebaeck et al, proc.natl.acad.sci.u.s.a.85: 3995-3999(1988).

Amino acid sequence modifications of the antibodies described herein are contemplated. For example, it may be desirable to increase the binding affinity and/or other biological properties of an antibody. Amino acid sequence variants of an antibody can be prepared by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletions, insertions, and/or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions will occur in the final construct, provided that the final construct possesses the desired properties. The amino acid changes can alter post-translational processes of the antibody, such as changing the number or position of glycosylation sites. An efficient method for identifying certain residues or regions that are preferred mutagenesis positions in an antibody is referred to as "alanine mutagenesis scan", e.g., b.c. cunningham and j.a. wells, Science 244: 1081-1085 (1989).

Exemplary applications of the Compounds and compositions of the invention

The invention provides the use of a compound of the invention or a pharmaceutical composition of the invention for inhibiting or reducing angiogenesis or for the treatment or prevention of a disease or condition associated with a deregulated angiogenesis. The present invention provides methods of inhibiting or reducing angiogenesis or treating or preventing diseases or conditions associated with deregulated angiogenesis, comprising administering to a patient a therapeutically effective dose of the compounds and compositions of the present invention. Also provided are methods of delivering or administering the compounds and compositions of the invention, and methods of treatment using the compounds and compositions of the invention. As used herein, an angiogenesis-mediated condition is one that results from aberrant angiogenesis activity, or in which compounds capable of modulating angiogenesis activity have therapeutic applications. One aspect of the invention provides a method of modulating VEGF activity in vivo, comprising administering to a subject an effective amount of a compound or composition as described herein. Another aspect of the invention includes methods of using the compounds and compositions of the invention for diagnostic purposes. Diseases and conditions that may be treated and/or diagnosed with a compound or composition of the invention include cancer, arthritis, hypertension, nephropathy, psoriasis, ocular angiogenesis associated with ocular disease, infection or surgical intervention, macular degeneration, diabetic retinopathy, and the like.

More specifically, examples of "cancer" as used herein in connection with the present invention include cancer selected from lung cancer (NSCLC and SCLC), head or neck cancer, ovarian cancer, colon cancer, rectal cancer, prostate cancer, cancer of the anal region, gastric cancer, breast cancer, cancer of the kidney or ureter, renal cell carcinoma, cancer of the renal pelvis, Central Nervous System (CNS) tumors, primary CNS lymphoma, non-hodgkin's lymphoma, spinal axis tumors; oropharyngeal, hypopharyngeal, esophageal, pancreatic, liver, gallbladder and bile duct, small intestine, urethral tumors; or lymphoma; or a combination of one or more of the foregoing cancers. More specifically, examples of "cancer" as used herein in connection with the present invention include cancers selected from lung cancer (NSCLC and SCLC), breast cancer, ovarian cancer, colon cancer, rectal cancer, prostate cancer, anal region cancer or a combination of one or more of the foregoing cancers.

In some aspects, the compounds of the present invention are useful for the treatment and/or prophylaxis of ocular diseases such as age-related macular degeneration (wet and dry), glaucoma, diabetic retinopathy (including diabetic macular edema), choroidal neovascular membrane (CNV), uveitis, myopic degeneration, ocular tumors, central retinal vein occlusion, flushing, ocular neovascularization, central serous retinopathy, dry eye and other ocular surface diseases, central retinal artery occlusion, cystoid macular edema, and other retinal degenerative diseases. In some embodiments, it is advantageous to employ molecules with high binding affinity for the corresponding target but with lower PK or half-life, because there is less enzymatic degradation of the molecule when ocular, and once the molecule is cleared from the eye, it is desirable that the molecule is degraded as quickly as possible and cleared from the kidney to minimize potential direct or side effects outside the eye. In some aspects of the invention, compound 6037 is useful for the treatment of ocular diseases. In some aspects of the invention, compound 6044 is useful for the treatment of ocular diseases. In some aspects of the invention, compound 6053 is useful for the treatment of ocular diseases.

The compounds of the invention may be solvated, especially hydrated. Hydration may occur during the preparation of the compound or composition comprising the compound, or over time due to the hygroscopic nature of the compound.

In other aspects, the invention includes methods of altering at least one physical or biological property of a compound or composition. The methods comprise covalently linking a [ VEGF-peptide ] of the invention to a binding site of an antibody, either directly or through a linker. Properties of the compounds of the invention that can be altered include, but are not limited to, binding affinity, susceptibility to degradation (e.g., by proteases), pharmacokinetics, pharmacodynamics, immunogenicity, solubility, lipophilicity, hydrophilicity, hydrophobicity, stability (more or less stable, and planned degradation), rigidity, flexibility, modulation of antibody binding, and the like. In addition, the biological potency of a particular compound of the invention may be increased by the addition of effector functions provided by the antibody. For example, antibodies provide effector functions such as complementary mediating effector functions. Without wishing to be bound by any theory, the antibody portion of the compounds of the invention generally extends the half-life of the smaller size [ VEGF-peptide ] in vivo. Thus, in one aspect, the invention provides methods for extending the effective circulating half-life of a [ VEGF-peptide ].

In another aspect, the invention includes methods of altering the antibody binding site to create binding specificity for VEGF, or VEGF with one or more other active molecules, including Ang 2. The method comprises covalently attaching reactive amino acid side chains within the antibody binding site to a [ VEGF-peptide ] as described herein]-a chemical moiety on a linker of a linker compound. Chemical moieties of the linker are separated from the [ VEGF-peptide ]]Far enough so as to be in the [ VEGF-peptide ]]Linker Compounds and antibody conjugates(iv) said [ VEGF-peptide when covalently linked at the point of conjugation]Can bind to its homologue (with similar limitations for tethering to other half-life extending macromolecules). Generally, the antibodies are not considered to be specific for the target molecule. In certain embodiments, the affinity of the antibody to VEGF prior to covalent attachment is less than about 1x 10^-5Mol/l. However, in the case of the antibodies and the [ VEGF-peptide ]]Following covalent attachment, the altered antibody preferably has at least about 1X 10 to the target molecule^-6Affinity of at least about 1X 10 mol/l, and^-7mole/liter, otherwise at least about 1X 10^-8Mole/liter, otherwise at least about 1X 10^-9Moles/liter, or alternatively at least about 1X 10^-10Mol/l.

Administration of the [ VEGF-peptide ] of the invention to an immunocompetent subject will result in the formation of antibodies against the conjugate. Such antibodies will target different regions, including the antibody idiotype as well as the targeting agent or any linker used to conjugate the targeting agent to the antibody. The immunogenicity of the [ VEGF-peptide ] can be reduced by methods known in the art, such as by attaching a long-chain polyvinyl alcohol (PEG) -like spacer or the like to the [ VEGF-peptide ]. It is known that the ability of long-chain PEG and other polymers to mask foreign epitopes will lead to the immunogenicity of therapeutic proteins displaying foreign epitopes (N.V.Katre, J.Immunol.144: 209-213 (1990); G.E.Francis et al, int.J.Hematol.68: 1-18 (1998). alternatively or additionally, subjects administered antibody- [ VEGF-peptide ] conjugates may be further administered with immunosuppressive agents such as cyclosporin A, anti-CD 3 antibodies, etc. this also applies when other active molecules such as Ang2 are covalently linked to the compounds of the invention via a branched chain linker.

The present invention also provides stereoisomers, tautomers, solvates, prodrugs and pharmaceutically acceptable salts of the compounds of the invention.

Pharmaceutical compositions, methods of administration and combination therapies

The invention also provides pharmaceutical compositions comprising a therapeutically effective amount of one or more compounds of the invention. The invention also provides methods of treatment using such compositions, and methods of preparing the pharmaceutical compositions of the invention. The content of US2006205670 is incorporated herein by reference, especially paragraphs [0497] - [0510], describing administration ([0498], [0499 ]); preparations ([0499], [0500], [0501], [0502 ]); preparing ([0503], [504 ]); kit ([0505 ]); dose strategies ([0506], [0507], [0508], [0509 ]); according to the corresponding targeting agent/AA targeting agent/[ peptide ] of the invention and methods for visualizing or localizing biological targets to which the [ peptide ] of the invention is directed.

The invention also includes the administration of one or more compounds or compositions of the invention together with one or more tumor therapeutic agents, all according to a regimen appropriate for that therapeutic agent. The components of the combination therapy may be administered simultaneously or non-simultaneously. The components may be administered in the same composition or in different compositions, by the same or different routes of administration. Examples of suitable tumor therapeutic agents and combinations that can be used in combination with the peptides and compositions of the present invention are listed in tables 4-6 of US2006205670 (incorporated herein by reference). The pharmaceutical compositions of the present invention may further comprise a therapeutically effective amount of one or more chemotherapeutic agents, preferably a compound selected from the group consisting of 5-fluorouracil, irinotecan, taxotere, sunitinib, axitinib, oxaliplatin, bevacizumab, cetuximab or chemical equivalents thereof. In some aspects, the chemotherapeutic agent is selected from the group consisting of 5-fluorouracil, irinotecan, taxotere, sunitinib, and axitinib; or a chemical equivalent thereof.

Synthesis examples of the Compounds of the present invention

The compounds of the present invention may be prepared by techniques known in the art. Generally, the synthesis of the [ VEGF-peptide ] or [ Ang 2-peptide ] (or other active molecule) is a first step and is performed as described herein. The active molecule is then derivatized to attach to a linking member (the linker) and then to bind to the antibody. Those skilled in the art will readily appreciate that the particular synthetic steps used depend on the precise nature of the three components. Thus, the [ VEGF-peptide ] -linker conjugates and compounds of the invention described herein will be readily prepared. [ VEGF-peptide ] and [ Ang 2-peptide ] can be synthesized by a number of techniques known to those skilled in the art. For solid phase peptide Synthesis, an exemplary technical profile can be found in Chemical applications to the Synthesis of Peptides and Proteins (Williams et al, eds.), CRC Press, Boca Raton, FL (1997).

In general, the desired peptide active molecules are sequentially synthesized on a solid phase according to procedures known in the art. See, e.g., U.S. patent application No. 2003/0045477). The linker may be partially or fully attached to the peptide on a solid phase, or may be added using solution phase techniques after the peptide is removed from the resin (see fig. 1A and 1B). For example, the N-protected amino group and carboxylic acid containing linking moiety may be attached to a resin such as 4-hydroxymethyl-phenoxymethyl-poly (styrene-1% divinylbenzene). The N-protecting group can be removed by a suitable acid (e.g., TFA for Boc) or base (e.g., piperidine for Fmoc), and the peptide sequence is grown in the normal C-to N-terminal manner (see fig. 1A). Alternatively, the peptide sequence may be synthesized first and the linker added to the N-terminal amino acid residue last (see FIG. 1B). In another approach, suitable side chains are deprotected in the synthesis and derivatized with a suitable reactive linker. For example, the lysine side chain may be deprotected and reacted with a linker having an active ester. Alternatively, an amino acid derivative already having a suitably protected linker moiety attached to the side chain, or in some cases to the alpha-amino nitrogen (see FIG. 1B), may be added as part of the growth peptide sequence.

At the end of the solid phase synthesis, the targeting agent-linker conjugate is removed from the resin and deprotected, either in a continuous operation or in a single operation. Removal and deprotection of the targeting agent-linker conjugate can be accomplished in a single step by treating the resin-bound peptide-linker conjugate with a cleavage reagent, e.g., trifluoroacetic acid containing a scavenger such as thioanisole (thioanisole), water, or ethanedithiol. After deprotection and release of the targeting agent, further derivatization of the targeting agent peptide may be performed. The fully deprotected peptidyl active molecule-linker conjugates are purified by employing any or all of the following types of sequence of chromatographic steps: ion exchange on weak base resin in acetate form; hydrophobic adsorption chromatography on underivatized polystyrene-divinylbenzene (e.g., AMBERLITE XAD); silica gel adsorption chromatography; ion exchange chromatography on carboxymethyl cellulose; for example, distribution chromatography or countercurrent distribution on SEPHADEX G-25, LH-20; high Performance Liquid Chromatography (HPLC), especially reverse phase HPLC on octyl or octadecyl silyl-silica bound phase column packing.

Drawings

Fig. 1A and 1B depict solid phase synthesis of targeting agent-linker conjugates of the invention.

FIG. 2A depicts the amino acid sequence alignment of the variable domains of FIG. 2A m38c2, h38c2 and a human germ cell line. Framework domains (FR) and complementarity determining domains (CDRs) are as defined by Kabat et al. Asterisks mark differences between m38c2 and h38c2 or between h38c2 and the human germ cell line. FIG. 2B depicts the amino acid sequences of the light and heavy chains of one embodiment of a humanized 38c2IgG1 (SEQ ID NOS: 1 and 2, respectively).

FIG. 3 shows different structures that can be used as linker reactive groups. Structures a-C form reversible covalent bonds with surface accessible reactive nucleophilic groups (e.g., lysine or cysteine side chains) of the antibody binding site. R 'in structure A-C'₁、R’₂、R′₃And R₄Represents a substituent including C, H, N, O, P, S, halogen (F, Cl, Br, I) or a salt thereof. X is N, C or any other heteroatom. These substituents may also include, for example, alkyl, alkenyl, alkynyl, oxyalkyl, oxyalkenyl, oxyalkynyl, aminoalkyl, aminoalkenyl, aminoalkynyl, sulfoalkyl, sulfoalkenyl, sulfoalkynyl, phosphorylalkyl, phosphorusAn acylalkenyl group or a phosphorylalkynyl group. R'₂And R'₃There may be rings as exemplified in structures B and C, and X may be a heteroatom. For example, if X is N and R'₁And R₃Forming part of a ring structure, structure a may form an irreversible covalent bond with the reactive affinity agent. Structure D-G can form a non-reversible covalent bond with a reactive nucleophilic group in the antibody binding site. In these structures, R "₁And R "₂Denotes C, O, N, halogen or a leaving group such as methanesulfonyl or toluenesulfonyl.

Figure 4 shows various electrophiles suitable for reactive modification of the side chains of reactive amino acids in the binding site of an antibody and thus can serve as linker reactive groups. The key points are as follows: (A) acyl β -lactams; (B) simple diketones; (C) a succinimide active ester; (D) a maleimide; (E) a haloacetamide with a linker; (F) a halogenated ketone; (G) a cyclohexyl dione; and (H) an aldehyde. The wavy line indicates the point of attachment to other portions of the linker or targeting agent. X represents a halogen.

FIG. 5 shows the addition of nucleophilic ("nu") side chains in the antibody binding site to compounds A-G in FIG. 3. antibody-Nu-refers to a covalent bond to an amino acid side chain with a nucleophile in the binding site of the antibody.

FIG. 6 shows the addition of nucleophilic side chains in the binding site of an antibody to compounds A-H in FIG. 5. antibody-Nu-refers to a covalent bond to an amino acid side chain with a nucleophile in the binding site of the antibody.

FIG. 7 showsAnd (4) synthesizing.

FIG. 8: exemplary synthesis of [ Ang 2-peptide ] - [ Ang 2-spacer ] using SEQ ID NO: 153 is an example.

FIG. 9: exemplary synthesis of [ VEGF-peptide ] - [ VEGF-spacer ], using SEQ ID NO: 153 is an example.

FIG. 10: exemplary synthesis of compound 5053.

FIG. 11: tumor volume of Colo205 colon adenocarcinoma xenografts after treatment with vehicle or compound 6053(IP, 1 x/wk). Data are plotted as mean and SE for 0-35 days, n-20/group (n-10 for all groups over 35 days), and the vertical arrow indicates the first dosing day. At day 35, the amount of the carrier was comparable to the amount of the carrier,^*P＜0.05，^**p < 0.01 (one-way ANOVA and Dunnett's multiple comparative test).

FIG. 12: tumor microvascular density of Colo205 colon adenocarcinoma xenografts after treatment with vehicle or compound 6053(IP, 1 x/wk). Data are plotted as mean and SE for n-9-10/panel. Compared with the carrier, the carrier has the advantages that,^*p < 0.05 (one-way ANOVA and Dunnett's multiple comparison test).

FIG. 13: tumor volume of Colo205 colon adenocarcinoma xenografts after treatment with vehicle or compound 6053(IP, 1 x/wk). Data are plotted as mean and SE for n-10/group, with the vertical arrow indicating the first dosing day. Compared with the carrier, the carrier has the advantages that,^**p < 0.01 (one-way ANOVA and Dunnett's multiple comparative test).

FIG. 14: active tumor volume of Colo205 colon adenocarcinoma xenografts after treatment with vehicle or compound 6053(IP, 1 x/wk). Data are plotted as mean and SE for n-9-10/panel. Compared with the carrier, the carrier has the advantages that,^**p < 0.01 (one-way ANOVA and Dunnett's multiple comparative test).

FIG. 15: ang2 immunoreactivity of Colo205 colon adenocarcinoma xenografts after treatment with vehicle or compound 6053(IP, 1 x/wk). Data are plotted as mean and SE for n-9-10/panel. Compared with the carrier, the carrier has the advantages that,^**p < 0.01 (one-way ANOVA and Dunnett's multiple comparative test).

FIG. 16: phosphorylated VEGFR2 (pvgfr 2) immunoreactivity of Colo205 colon adenocarcinoma xenografts after treatment with vehicle or compound 6053(IP, 1x/wk) as a percentage of total VEGFR2 immunoreactivity. Data are plotted as mean and SE for n-9-10/panel. Compared with the carrier, the carrier has the advantages that,^**p < 0.01 (one-way ANOVA and Dunnett's multiple comparative test).

FIG. 17: tumor volumes of MDA-MB-435 breast cancer (A) and A431 skin cancer (B) after weekly treatment with vehicle or compound 6053(IP, 1 x/wk). Data are plotted as mean and SE for n-9-10/group, with the vertical arrow indicating the first dosing day. At day 68, the cells were compared to the vector,^*p is less than 0.05; comparing the vector at day 35 with the vector at day 35,^**p < 0.01 (one-way ANOVA and Dunnett's multiple comparative test).

FIG. 18: tumor volume of Colo205 colon adenocarcinoma xenografts after once weekly treatment with vehicle, compound 6053(IP) only, 5-FU (IP) only, or compound 6053 in combination with 5-FU (all IP). Data are plotted as mean and SE for n-9-10/group, with the vertical arrow indicating the first dosing day. At day 29, the cells were compared to the vector,^**p < 0.01 (one-way ANOVA and Dunnett's multiple comparative test).

FIG. 19: tumor volume of Colo205 colon adenocarcinoma xenografts after weekly treatment with vehicle, compound 6053(IP) only, Irinotecan (IP) only, or compound 6053 in combination with irinotecan (both IP). Data are plotted as mean and SE for n-9-10/group, with the vertical arrow indicating the first dosing day. At day 29, the cells were compared to the vector,^**p < 0.01 (one-way ANOVA and Bonferroni's multiple comparison test).

FIG. 20: tumor volume of Colo205 colon adenocarcinoma xenografts after once weekly treatment with vehicle, compound 6053(IP) only, taxotere (IP) only, or compound 6053 in combination with taxotere (both IP). Data are plotted as mean and SE for n-9-10/group, with the vertical arrow indicating the first dosing day. At day 29, the cells were compared to the vector,^**p < 0.01, compared to vehicle and compared to compound 6053 alone,^***p < 0.01 (one-way ANOVA and Bonferroni's multiple comparison test).

FIG. 21: tumor volume of Colo205 colon adenocarcinoma xenografts following treatment with vehicle, compound 6053(IP) only once weekly, sunitinib (PO) daily, or compound 6053(IP) in combination with sunitinib (PO). Data are plotted as mean and SE for n-9-10/panel, with the vertical arrow indicating the first doseThe day of administration. At day 28, the cells were compared to the vector,^*P＜0.05，^**p < 0.01 (one-way ANOVA and Bonferroni's multiple comparison test).

FIG. 22: tumor volume of Colo205 colon adenocarcinoma xenografts following treatment with vehicle, compound 6053(IP) only once weekly, axitinib (PO) daily, or compound 6053(IP) in combination with axitinib (PO). Data are plotted as mean and SE for n-9-10/group, with the vertical arrow indicating the first dosing day. At day 28, the cells were compared to the vector,^*P＜0.05，^**p < 0.01 (one-way ANOVA and Bonferroni's multiple comparison test).

FIG. 23: tumor volume of Colo205 colon adenocarcinoma xenografts after weekly treatment with vehicle, compound 2071, compound 2049, or compound 2046 (all IP). Data are plotted as mean and SE for n-10/group, with the vertical arrow indicating the first dosing day.

FIG. 24: tumor volume of Colo205 colon adenocarcinoma xenografts after once weekly treatment with vehicle, compound 2049, compound 6045, compound 6042, or compound 6053 (all IP). Data are plotted as mean and SE for n-10/group, with the vertical arrow indicating the first dosing day. At day 28, the cells were compared to the vector,^**p < 0.01 (Compounds 6045, 6042 and 6053)

FIG. 25: tumor volume of Colo205 colon adenocarcinoma xenografts after weekly treatment with vehicle, compound 4043, compound 2018, or compound 6053 (all IP). Data are plotted as mean and SE for n-10/group, with the vertical arrow indicating the first dosing day. At day 28, the cells were compared to the vector,^**p < 0.01 (compounds 4043 and 2018), on day 28, compared to vehicle,^***p < 0.001 (Compound 6053, 10 and 30mg/kg, weekly dose).

FIG. 26: tumor volume of Colo205 colon adenocarcinoma xenografts after once weekly treatment with vehicle, compound 2018, compound 4043, compound 6053, compound 6062, or a combination of compounds 2018 and 4043, all IP. Data are plotted as mean and SE for n-9/group, with the vertical arrow indicating the first dosing day.

FIG. 27 is a schematic view showing: tumor volume of Colo205 colon adenocarcinoma xenografts after once weekly treatment with vehicle, compound 4043, compound 2018, compound 6053, or a combination of compounds 4043 and 2018, all IP. Data are plotted as mean and SE for n-10/group, with the vertical arrow indicating the first dosing day. At day 42, the number of cells was compared to the vector,^**p < 0.01 (combination of compounds 4043 and 6053, compounds 4043 and 2018).

FIG. 28: tumor volume of Colo205 colon adenocarcinoma xenografts after once weekly treatment with vehicle, compound 4043, compound 2018, compound 6053, or a combination of compounds 4043 and 2018, all IP. Data are plotted as mean and SE for n-10/group, with the vertical arrow indicating the first dosing day. At day 32, the cells were compared to the vector,^*p < 0.05 (compound 2018, and the combination of compounds 4043 and 2018), at 32 days, compared to vehicle,^**p < 0.01 (Compound 6053).

FIG. 29: fluorescein angiographic scores in rabbit VEGF-induced retinal leakage 3 days after IVT injection and 48 hours after VEGF dosing. Compound 6037 response study.

FIG. 30: fluorescein angiographic scores in rabbit VEGF-induced retinal leakage 7 days after IVT injection and 48 hours after VEGF dosing.

Detailed Description

Definition of

The following abbreviations, terms and phrases are used herein as defined below.

Unnatural amino acid abbreviations

BnHP refers to (2S, 4R) -4-hydroxyproline Tle refers to tert-butylglycine

Unless otherwise indicated by the prefix "D", such as D-Ala or N-Me-D-Ile, or by lower case forms, such as a, i, L (D forms of Ala, Ile, Leu), the alpha carbons of the amino acids and aminoacyl residues are in the natural or "L" configuration in this specification and the appended claims. Cahn-Ingold-Prelog "R" and "S" indicate the stereochemistry used to designate chiral centers in certain acyl substituents at the N-terminus of a peptide. The designation "R, S" is used to designate the racemic mixture of the two enantiomeric forms. The nomenclature complies with r.s.cahn, et al, angelw.chem.int.ed.engl., 5: 385- > 415 (1966).

"polypeptide," "peptide," and "protein" are used interchangeably to refer to a polymer of amino acid residues. As used herein, these terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding natural amino acids. These terms also apply to natural amino acid polymers. The amino acids may be in the L or D form, as long as the binding function of the peptide is maintained. The peptide may be cyclic, having intramolecular bonds between two non-adjacent amino acids within the peptide, such as backbone to backbone, side chain to backbone, and side chain to side chain cyclization. The cyclic peptides can be prepared by methods known in the art. See, for example, U.S. patent No. 6,013,625.

All peptide sequences are written according to widely accepted practice, and thus the α -N-terminal amino acid residue is located on the left side and the α -C-terminal amino acid is located on the right side. As used herein, the term "N-terminus" refers to the free alpha-amino group of an amino acid in a peptide, and the term "C-terminus" refers to the free alpha-carboxylic acid terminus of an amino acid in a peptide. A peptide with a group blocking the N-terminus is a peptide with a group on the alpha-amino nitrogen of the N-terminal amino acid residue. Amino acids with a group blocking the N-terminus are those with a group on the alpha-amino nitrogen.

In general, the term "hydrophobic amino acid" or "non-polar amino acid" refers to an amino acid residue that does not contain an ionizing group at physiological pH. Examples of hydrophobic amino acid residues include, but are not limited to, Gly, allylglycine, cyclohexylglycine (Chg), Dpg, hydroxyproline, HoLeu, alloIle, Ala, Abu (aminobutyric acid), homocycloleucine, Acpc (1-aminocyclopropane-1-carboxylic acid), Aib, Aic, Val, Leu, Nle, Ile, Met, Phe, Cys, Pro, Gln, Asn, Cha (β -cyclohexylalanine), cyclopentylalanine, β -cyclopropylalanine, 3-diphenylalanine, β -2-furanylalanine, homocyclohexylalanine (Hocha), 3- (1-naphthylalanine, 2-furanylalanine, pyridylalanine, quinolinylalanine, thiazolylalanine, thienylalanine, Val, Nva, ring-substituted phenylalanine, Sar, HoSer, Tic (OH), Ring-substituted tryptophan, ring-substituted tyrosine and derivatives thereof.

In general, the terms "aliphatic amino acid" and "non-polar aliphatic amino acid" refer to an amino acid having a non-aromatic hydrophobic side chain. Examples include, but are not limited to, Ala, AlllylGly, Nva, Chg, Abu, Aib, Aic, Acpc, homocycloleucine, cyclopentylalanine, β -cyclopropylalanine, Cha, Hocha, Val, Ile, Leu, and Met, and derivatives thereof.

In general, the term "aromatic amino acid" refers to an amino acid having an aromatic side chain. Examples include Phe, Tyr, Trp, His, 2-furylalanine, Tyr (Me), and derivatives thereof.

In general, the term "polar amino acid" includes polar and uncharged amino acids, negatively charged amino acids, and positively charged amino acids. Examples include Arg, HoArg, Cit, Glu, Asp, Lys, Gln, Asn, Ser, Thr, His, Trp, Tyr, Lys (Ac) and derivatives thereof.

In general, the term "positively charged amino acid" (or basic amino acid) refers to an amino acid whose side chain is or can be protonated under physiological conditions. Examples include, but are not limited to, Lys, Ornithine (Orn), Arg, HoArg, Dab, Dap, Trp, His, and derivatives thereof.

In general, the term "negatively charged amino acid" (or acidic amino acid) refers to an amino acid whose side chain can be deprotonated under physiological conditions. Examples include, but are not limited to, Asp, Asu, Glu, Aad and derivatives thereof.

In general, "polar uncharged amino acid" refers to an amino acid having an uncharged side chain that is capable of forming an H-bond with water in a physiological environment. Examples include, but are not limited to, Gln, Asn, Lys (Ac), Ser, Thr, Cit and HoCit and derivatives thereof.

"substantially homologous" refers to sequences that are at least about 75% (preferably at least about 80%, more preferably at least about 90% or most preferably at least about 95%) of the amino acid residues that match a peptide sequence of defined length by comparing the sequences using standard software provided in sequence databases, such as the BLAST program provided by the national center for cancer biotechnology information located at ncbi.

In general, "substituted" refers to groups defined below wherein one or more of the bonds contained to a hydrogen atom are replaced with a bond to a non-hydrogen or non-carbon atom such as, but not limited to, a halogen atom, such as F, Cl, Br, and I; oxygen atoms such as in hydroxyl groups, alkoxy groups, aryloxy groups, and ester groups; sulfur atoms such as thiol groups, alkyl or aryl sulfides, sulfone groups, sulfonyl groups, and sulfoxide groups; nitrogen atoms such as amines, amides, alkylamines, dialkylamines, acylamines, alkylarylamines, diarylamines, N-oxides, imides, and enamines; silicon atoms such as trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl and triarylsilyl; and other heteroatoms in various other groups. Substituted alkyl, substituted cycloalkyl and other groups also include oxygen wherein one or more bonds to a carbon or hydrogen atom are linked to a heteroatom such as carbonyl, carboxyl and ester groups; such as nitrogen atoms in imines, oximes, hydrazones, and nitriles. As used herein, a group that is "optionally substituted" may be substituted or unsubstituted. Thus, for example, "optionally substituted alkyl" refers to both substituted alkyl and unsubstituted alkyl.

The phrase "unsubstituted alkyl" refers to an alkyl group that does not contain heteroatoms. Thus, the phrase includes straight chain alkyl groups such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, and the like. The phrase also includes branched isomers of straight chain alkyl groups, including but not limited to those exemplified below: -CH (CH)₃)₂、-CH(CH₃)(CH₂CH₃)、-CH(CH₂CH₃)₂、-C(CH₃)₃、-C(CH₂CH₃)₃、-CH₂CH(CH₃)₂、-CH₂CH(CH₃)(CH₂CH₃)、-CH₂CH(CH₂CH₃)₂、-CH₂C(CH₃)₃、-CH₂C(CH₂CH₃)₃、-CH(CH₃)CH(CH₃)(CH₂CH₃)、-CH₂CH₂CH(CH₃)₂、-CH₂CH₂CH(CH₃)(CH₂CH₃)、-CH₂CH₂CH(CH₂CH₃)₂、-CH₂CH₂C(CH₃)₃、-CH₂CH₂C(CH₂CH₃)₃、-CH(CH₃)CH₂CH(CH₃)₂、-CH(CH₃)CH(CH₃)CH(CH₃)₂、-CH(CH₂CH₃)CH(CH₃)CH(CH₃)(CH₂CH₃) And others. The phrase does not include cycloalkyl groups. Thus, the phrase unsubstituted alkyl group includesIncluding primary, secondary and tertiary alkyl groups. Unsubstituted alkyl groups may be attached to one or more carbon atoms in the parent compound. Oxygen atoms, nitrogen atoms and/or sulfur atoms. Possible unsubstituted alkyl groups include straight and branched chain alkyl groups having 1 to 20 carbon atoms. In addition, these unsubstituted alkyl groups have 1 to 10 carbon atoms or are lower alkyl groups having 1 to about 6 carbon atoms. Other unsubstituted alkyl groups include straight and branched chain alkyl groups having 1 to 3 carbon atoms and include methyl, ethyl, propyl and-CH (CH)₃)₂。

The term "substituted alkyl" refers to an alkyl group in which one or more bonds to a carbon or hydrogen atom contained therein are replaced by bonds to a non-hydrogen or non-carbon atom such as, but not limited to, halogen atoms, such as F, Cl, Br, and I; oxygen atoms such as in hydroxyl groups, alkoxy groups, aryloxy groups, and ester groups; sulfur atoms such as thiol groups, alkyl or aryl sulfides, sulfone groups, sulfonyl groups, and sulfoxide groups; nitrogen atoms such as amines, amides, alkylamines, dialkylamines, acylamines, alkylarylamines, diarylamines, N-oxides, imides, and enamines; silicon atoms such as trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl and triarylsilyl; and other heteroatoms in various other groups. Substituted alkyl groups also include those wherein one or more bonds to a carbon or hydrogen atom are replaced with heteroatoms such as carbonyl, carboxyl and oxygen in ester groups; such as nitrogen atoms in imines, oximes, hydrazones, and nitriles. Substituted alkyl groups include, among others, those in which one or more bonds to a carbon atom or a hydrogen atom are replaced by a bond to a fluorine atom. An example of a substituted alkyl group is trifluoromethyl and other groups containing trifluoromethyl. Other alkyl groups include those in which one or more bonds to a carbon atom or a hydrogen atom are replaced with a bond to an oxygen atom, such that the substituted alkyl group contains a hydroxyl, alkoxy, aryloxy, or heterocycloxy group. Still other alkyl groups include alkyl groups having amine, alkylamine, dialkylamine, arylamine, (alkyl) (aryl) amine, diarylamine, heterocyclylamine, (alkyl) (heterocyclyl) amine, (aryl) (heterocyclyl) amine, or diheterocyclylamine groups.

The phrase "unsubstituted alkylene" refers to a divalent unsubstituted alkyl group as defined above. Thus, methylene, ethylene and propylene are each examples of unsubstituted alkylene groups. The phrase "substituted alkylene" refers to a divalent substituted alkyl group as defined above. Substituted or unsubstituted lower alkylene has 1 to about 6 carbons.

The phrase "unsubstituted cycloalkyl" cycloalkyl groups, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl, and these rings are substituted with straight or branched chain alkyl groups as defined above. The phrase also includes polycyclic alkyl groups such as, but not limited to, adamantyl, bornyl, and bicyclo [2.2.2] octyl, and the like, and such rings are substituted with straight or branched chain alkyl groups as defined above. Thus, the phrase also includes, among other things, methylcyclohexyl. The phrase does not include cycloalkyl groups containing heteroatoms. Unsubstituted cycloalkyl groups may be bonded to one or more carbon, oxygen, nitrogen, and/or sulfur atoms in the parent compound. In some embodiments, unsubstituted cycloalkyl groups have 3 to 20 carbon atoms. In other embodiments, these unsubstituted alkyl groups have 3 to 8 carbon atoms and in other embodiments, these groups have 3 to 7 carbon atoms.

The phrase "substituted cycloalkyl" has the same meaning with respect to unsubstituted cycloalkyl as substituted alkyl has with respect to unsubstituted alkyl. Thus, the phrase includes, but is not limited to, oxycyclohexyl, chlorocyclohexyl, hydroxycyclopentyl, and chloromethylcyclohexyl.

The phrase "unsubstituted aryl" refers to aryl groups that do not contain heteroatoms. Thus, the phrase includes, but is not limited to, illustrative groups such as phenyl, biphenyl, anthracenyl, and naphthyl. Although the phrase "unsubstituted aryl" includes groups containing fused rings such as naphthyl, aryl groups having other groups attached to the ring atoms, such as alkyl or halogen groups, are not included because aryl groups such as tolyl are considered substituted aryl groups herein as described below. Typically, unsubstituted aryl groups may be lower aryl groups having from 6 to about 10 carbon atoms. One unsubstituted aryl group is phenyl. However, an unsubstituted aryl group may be bonded to one or more carbon, oxygen, nitrogen, and/or sulfur atoms in the parent compound.

The phrase "substituted aryl" has the same meaning with respect to unsubstituted aryl as substituted alkyl has with respect to unsubstituted alkyl. However, substituted aryl groups also include aryl groups in which one or more aromatic carbon atoms are bonded to one or more non-carbon or non-hydrogen atoms as described above, and also include aryl groups in which one or more aromatic carbons of the aryl group are bonded to a substituted and/or unsubstituted alkyl, alkenyl, or alkynyl group as defined herein. This includes bonding configurations in which two carbon atoms of an aryl group are bonded to two atoms of an alkyl, alkenyl, or alkynyl group to define a fused ring system (e.g., a dihydronaphthyl or tetrahydronaphthyl group). Thus, the phrase "substituted aryl" includes, but is not limited to, tolyl and hydroxyphenyl, among others.

The phrase "unsubstituted alkenyl" refers to straight, branched, and cyclic groups as described above with respect to unsubstituted alkyl groups, except that at least one double bond between two carbon atoms is present. Examples include, but are not limited to: vinyl, -CH ═ C (H) (CH)₃)、-CH＝C(CH₃)₂、-C(CH₃)＝C(H)₂、-C(CH₃)＝C(H)(CH₃)、-C(CH₂CH₃)＝CH₂Cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, hexadienyl, and the like. Lower unsubstituted alkenyl has 1 to about 6 carbons.

The phrase "substituted alkenyl" has the same meaning with respect to unsubstituted alkenyl as substituted alkyl has with respect to unsubstituted alkyl. Substituted alkenyl groups include alkenyl groups in which a non-carbon or non-hydrogen atom is bonded to a carbon that is double bonded to another carbon and alkenyl groups in which one of the non-carbon or non-hydrogen atoms is bonded to a carbon that is not double bonded to another carbon. For example,-CH＝CH-OCH₃and-CH ═ CH-CH₂-OH is a substituted alkenyl group. Wherein CH₂Oxoalkenyl groups in which the group is substituted by a carbonyl group, such as-CH ═ CH-c (o) -CH₃Also substituted alkenyl.

The phrase "unsubstituted alkenylene" refers to a divalent unsubstituted alkenyl group as defined above. For example, -CH ═ CH-is an exemplary unsubstituted alkenylene group. The phrase "substituted alkenylene" refers to a divalent substituted alkenyl group as defined above.

The phrase "unsubstituted alkynyl" refers to straight, branched, and cyclic groups as described above with respect to unsubstituted alkyl groups, except that at least one triple bond between two carbon atoms is present. Examples include, but are not limited to: -C ≡ C (H), -C ≡ C (CH)₃)、-C≡C(CH₂CH₃)、-C(H₂)C≡C(H)、-C(H)₂C≡C(CH₃) and-C (H)₂C≡C(CH₂CH₃) And the like. Unsubstituted lower alkynyl groups have 1 to about 6 carbons.

The phrase "substituted alkynyl" has the same meaning with respect to unsubstituted alkynyl as substituted alkyl has with respect to unsubstituted alkyl. Substituted alkenyl groups include alkenyl groups in which a non-carbon or non-hydrogen atom is bonded to a carbon that is bonded to another carbon triple bond and alkenyl groups in which one of the non-carbon or non-hydrogen atoms is bonded to a carbon that is not bonded to another carbon triple bond. Examples include, but are not limited to, those in which CH₂Oxoalkenyl radicals substituted by carbonyl groups, such as-C (O) -CH ≡ CH-CH₃and-C (O) -CH₂-CH≡CH。

The phrase "unsubstituted alkynylene" refers to a divalent unsubstituted alkynyl group as defined above. -C.ident.C-is an example of an unsubstituted alkynylene group. The phrase "substituted alkynylene" refers to a divalent substituted alkynyl group as defined above.

The phrase "unsubstituted aralkyl" means that a hydrogen or carbon bond of an unsubstituted alkyl group as defined above is replaced by a bond to an aryl group as defined above. For example, methyl (-CH)₃) Is not takenAnd (c) a substituted alkyl group. If the hydrogen atom of the methyl group is replaced by a bond to a phenyl group, such as the methyl carbon being bonded to a benzene carbon, the compound is an unsubstituted aralkyl group (i.e., benzyl). Thus, the term includes, but is not limited to, benzyl, diphenylmethyl, and 1-phenylethyl (-CH (C)₆H₅)(CH₃))。

The phrase "substituted aralkyl" has the same meaning with respect to unsubstituted aralkyl as substituted aryl with respect to unsubstituted aryl. However, substituted aralkyl groups also include groups in which the carbon or hydrogen bond of the alkyl portion of the group is replaced by a bond to a non-carbon or non-hydrogen atom. Examples of substituted aralkyl groups include, but are not limited to, -CH₂C(＝O)(C₆H₅) and-CH₂(2-methylphenyl).

The phrase "unsubstituted aralkenyl" means that a hydrogen or carbon bond of an unsubstituted alkenyl group as defined above is replaced by a bond to an aryl group as defined above. For example, a vinyl group is an unsubstituted alkenyl group. If the hydrogen atom of the vinyl group is replaced by a bond to a phenyl group, such as the carbon of the vinyl group being bonded to the carbon of a benzene, the compound is an unsubstituted aralkenyl group (i.e., styryl). Thus, the term includes, but is not limited to, styryl, diphenylvinyl, and 1-phenylvinyl (-C (C)₆H₅)(CH₂))。

The phrase "substituted aralkenyl" has the same meaning with respect to unsubstituted aralkenyl as substituted aryl has with respect to unsubstituted aryl. Substituted aralkenyl also includes groups in which a carbon or hydrogen bond of the alkenyl portion of the group is replaced by a bond to a non-carbon or non-hydrogen atom. Examples of substituted aralkenyl include, but are not limited to, -CH ═ C (cl) (C)₆H₅) and-CH ═ CH (2-methylphenyl).

The phrase "unsubstituted arylalkynyl" means that in an unsubstituted alkynyl group as defined above the hydrogen or carbon bond of said unsubstituted alkynyl group is attached to an aryl group as defined aboveIs replaced by a key of (1). For example, ethynyl is an unsubstituted alkenyl. The compound is an unsubstituted arynyl group if the hydrogen atom of the ethynyl group is replaced by a bond to a phenyl group, such as the carbon of the ethynyl group is bonded to the carbon of a benzene. Thus, the term includes, but is not limited to, -C ≡ C-phenyl-CH₂-C ≡ C-phenyl and the like.

The phrase "substituted arylalkynyl" has the same meaning with respect to unsubstituted arylalkynyl as substituted aryl has with respect to unsubstituted aryl. However, substituted aralkynyl groups also include groups in which a carbon or hydrogen bond of the alkynyl moiety of the group is replaced by a bond to a non-carbon or non-hydrogen atom. Examples of substituted aralkynyl groups include, but are not limited to, -C.ident.C-C (Br) (C)₆H₅) and-C ≡ C (2-methylphenyl).

The phrase "unsubstituted heteroalkyl" refers to an unsubstituted alkyl as described above wherein the carbon chain is interrupted by one or more heteroatoms selected from N, O and S. The N-containing unsubstituted heteroalkyl group may have NH or N (unsubstituted alkyl) in the carbon chain. For example, unsubstituted heteroalkyl groups include alkoxy, alkoxyalkyl, alkoxyalkoxy, thioether, alkylaminoalkyl, aminoalkoxy, and other such groups. Typically, unsubstituted heteroalkyl groups contain from 1 to 5 heteroatoms, and especially from 1 to 3 heteroatoms. In some embodiments, unsubstituted heteroalkyl groups include, for example, alkoxyalkoxyalkoxy groups, such as ethoxyethoxyethoxy.

The phrase "substituted heteroalkyl" has the same meaning with respect to unsubstituted heteroalkyl as substituted alkyl has with respect to unsubstituted alkyl.

The phrase "unsubstituted heteroalkylene" refers to a divalent unsubstituted heteroalkyl group as defined above. For example, -CH₂-O-CH₂-and-CH₂-NH-CH₂CH₂Each is an exemplary unsubstituted heteroalkylene. The phrase "substituted heteroalkylene" refers to a divalent substituted heteroalkyl group as defined above.

The phrase "unsubstituted heteroalkenyl" refers to an unsubstituted alkenyl group as described above wherein the carbon chain is interrupted by one or more heteroatoms selected from N, O and S. The N-containing unsubstituted heteroalkenyl group can have NH or N (unsubstituted alkyl or alkenyl) in the carbon chain. The phrase "substituted heteroalkenyl" has the same meaning with respect to unsubstituted heteroalkenyl as substituted heteroalkenyl has with respect to unsubstituted heteroalkenyl.

The phrase "unsubstituted heteroalkenylene" refers to a divalent unsubstituted heteroalkenylene group as defined above. Thus, -CH₂-O-CH ═ CH-is an example of an unsubstituted heteroalkenylene group. The phrase "substituted heteroalkenylene" refers to a divalent substituted heteroalkenyl group as defined above.

The phrase "unsubstituted heteroalkynyl" refers to an unsubstituted alkynyl group as described above in which the carbon chain is interrupted by one or more heteroatoms selected from N, O and S. The N-containing unsubstituted heteroalkenyl can have NH or N (unsubstituted alkyl, alkenyl, or alkynyl) in the carbon chain. The phrase "substituted heteroalkynyl" has the same meaning with respect to unsubstituted heteroalkynyl as substituted heteroalkynyl has with respect to unsubstituted heteroalkynyl.

The phrase "unsubstituted heteroalkynylene" refers to a divalent unsubstituted heteroalkynylene group as defined above and thus-CH₂-O-CH₂-C.ident.C-is an example of an unsubstituted heteroalkynylene group. The phrase "substituted heteroalkynylene" refers to a divalent substituted heteroalkynyl group as defined above.

The phrase "unsubstituted heterocyclyl" includes monocyclic, bicyclic, and polycyclic aromatic or nonaromatic compounds containing more than three ring atoms wherein one or more is a heteroatom selected from the group consisting of, but not limited to, N, O and S, such as, but not limited to, quinolinyl. While the phrase "unsubstituted heterocyclyl" includes condensed heterocyclic groups such as benzimidazolyl, it does not include heterocyclic groups having other groups such as alkyl or halogen bonded to one of the ring atoms, such as 2-methylbenzimidazolyl which is a substituted heterocyclic group. Examples of heterocyclyl groups include, but are not limited to: unsaturated 3-8 membered rings containing 1 to 4 nitrogen atoms such as, but not limited to, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, dihydropyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazolyl (e.g., 4H-1, 2, 4-triazolyl, 1H-1, 2, 3-triazolyl, 2H-1, 2, 3-triazolyl, etc.), tetrazolyl (e.g., 1H-tetrazolyl, 2H-tetrazolyl, etc.); saturated 3-8 membered rings containing 1 to 4 nitrogen atoms, such as but not limited to: pyrrolidinyl, imidazolidinyl, piperidinyl, piperazinyl; condensed unsaturated heterocyclylindolyl, isoindolyl, indolinyl, indolizinyl, benzimidazolyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl containing 1 to 4 nitrogen atoms; unsaturated 3 to 8 membered rings containing 1 to 2 oxygen atoms such as, but not limited to, oxazolyl, isoxazolyl, oxadiazolyl (e.g., 1, 2, 4-oxadiazolyl, 1, 3, 4-oxadiazolyl, 1, 2, 5-oxadiazolyl, etc.); saturated 3 to 8 membered rings containing 1 to 2 oxygen atoms such as but not limited to morpholinyl; unsaturated condensed heterocyclic groups containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, for example, benzoxazolyl, benzooxadiazolyl, benzoxazinyl (e.g., 2H-1, 4-benzoxazinyl, etc.); unsaturated 3-8 membered rings containing 1 to 3 sulfur atoms and 1 to 3 nitrogen atoms, such as but not limited to thiazolyl, isothiazolyl, thiadiazolyl (e.g., 1, 2, 3-thiadiazolyl, 1, 2, 4-thiadiazolyl, 1, 3, 4-thiadiazolyl, 1, 2, 5-thiadiazolyl, etc.); saturated 3 to 8 membered rings containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms, such as but not limited to thiazolidinyl; saturated and unsaturated 3 to 8 membered rings containing 1 to 2 sulfur atoms such as, but not limited to, thienyl, dihydrothienyl, dihydrodithioketonyl, tetrahydrothienyl, tetrahydrothiopyranyl; unsaturated condensed heterocyclic rings containing 1 to 2 sulfur atoms such as, but not limited to, benzothiazolyl, benzothiadiazolyl, benzothiazinyl (e.g., 2H-1, 4-benzothiazinyl, etc.), dihydrobenzothiinyl (e.g., 2H-3, 4-dihydrobenzothiinyl, etc.); oxygen-containing saturated 3 to 8 membered rings such as, but not limited to, furyl; unsaturated condensed heterocyclic rings containing 1 to 2 oxygens such as benzodioxanyl (e.g., 1, 3-benzodioxanyl, etc.); unsaturated 3 to 8 membered rings containing oxygen and 1 to 2 sulfur atoms such as, but not limited to, dihydrooxathienyl; saturated 3 to 8 membered rings containing 1 to 3 oxygen atoms and 1 to 2 sulfur atoms, such as 1, 4-oxathianyl; unsaturated condensed rings containing 1 to 2 sulfur atoms, such as benzothienyl, benzodithienyl; and unsaturated condensed heterocycles containing an oxygen atom and 1 to 3 oxygen atoms, such as a benzoxazolyl group. Heterocyclyl groups are also included in the above groups, where one or more S atoms in the ring are double bonded to one or two oxygen atoms (sulfoxides and sulfones). For example, heterocyclyl groups include tetrahydrothienyl, tetrahydrothienyl oxide, and tetrahydrothienyl 1, 1-dioxide. In some embodiments, heterocyclyl contains 5 or 6 ring atoms. In some embodiments, heterocyclyl includes morpholinyl, piperazinyl, piperidinyl, pyrrolidinyl, imidazolyl, pyrazolyl, 1, 2, 3-triazolyl, 1, 2, 4-triazolyl, tetrazolyl, thiomorpholinyl in which the S atom of the thiomorpholinyl is bonded to one or more O, pyrrolyl, homopiperazinyl, oxazolidin-2-one, pyrrolidin-2-one, oxazolyl, quinolinyl, thiazolyl, isoxazolyl, furyl, and tetrahydrofuranyl.

The phrase "substituted heterocyclyl" means that one of the ring atoms in the unsubstituted heterocyclyl group defined above is bonded to a non-hydrogen atom, as described above with respect to substituted alkyl and substituted aryl groups. Examples include, but are not limited to, 2-methylbenzimidazolyl, 5-chlorobenzothiazolyl, 1-methylpiperazinyl, and 2-chloropyridyl.

The phrase "unsubstituted heteroaryl" refers to an unsubstituted aromatic heterocyclic group as defined above. Thus, unsubstituted heteroaryl groups include, but are not limited to, furyl, imidazolyl, oxazolyl, isoxazolyl, pyridyl, benzimidazolyl, and benzothiazolyl. The phrase "substituted heteroaryl" refers to a substituted aromatic heterocyclic group as defined above.

The phrase "unsubstituted heterocycloalkyl" means that a hydrogen or carbon bond of the unsubstituted alkyl group as defined above is replaced by a bond to the heterocyclyl group as defined above. For example, methyl (-CH)₃) Is thatAn unsubstituted alkyl group. A compound is unsubstituted heterocycloalkyl if the hydrogen atom of the methyl group is replaced by a bond to a heterocycle, such as the carbon of the methyl group is bonded to carbon 2 of pyridine (one of the carbons bonded to the N of pyridine), or carbon 3 or 4 of the pyridine.

The phrase "substituted heterocycloalkyl" has the same meaning with respect to unsubstituted heterocycloalkyl as substituted aralkyl has with respect to unsubstituted aralkyl. Substituted heterocycloalkyl groups also include groups in which a non-hydrogen atom is bonded to a heteroatom of the heterocyclic group in the heterocycloalkyl group, such as, but not limited to, the piperidine ring nitrogen atom in piperidinoalkyl.

The phrase "unsubstituted heterocycloalkenyl" means that a hydrogen or carbon bond of the unsubstituted alkenyl group as defined above is replaced by a bond to the heterocyclyl group as defined above. For example, a vinyl group is an unsubstituted alkenyl group. A compound is an unsubstituted heterocycloalkenyl group if the hydrogen atom of the vinyl group is replaced by a bond to the heterocyclyl group, such as the carbon of the vinyl group is bonded to carbon 2 of a pyridine, or carbon 3 or 4 of the pyridine.

The phrase "substituted heterocycloalkenyl" has the same meaning with respect to unsubstituted heterocycloalkenyl as substituted aralkenyl has with respect to unsubstituted aralkenyl. However, substituted heterocycloalkenyl also includes groups in which a non-hydrogen atom is bonded to a heteroatom of a heterocyclyl group in the heterocycloalkenyl group, such as, but not limited to, a piperidine ring nitrogen atom in piperidinoalkenyl.

The phrase "unsubstituted heterocyclic alkynyl" means that a hydrogen or carbon bond of said unsubstituted alkynyl group is replaced in the unsubstituted alkynyl group as defined above by a bond to a heterocyclic group as defined above. For example, ethynyl is an unsubstituted alkenyl. A compound is unsubstituted heterocycloalkynyl if the hydrogen atom of the ethynyl group is replaced by a bond to the heterocyclyl group, such as the carbon of the ethynyl group is bonded to carbon 2 of a pyridine, or to carbon 3 or 4 of the pyridine.

The phrase "substituted heterocyclic alkynyl" has the same meaning with respect to unsubstituted heterocyclic alkynyl as substituted arylalkynyl with respect to unsubstituted arylalkynyl. Substituted heterocycloalkynyl also includes groups in which a non-hydrogen atom is bonded to a heteroatom of a heterocyclyl group in the heterocycloalkynyl, such as, but not limited to, a piperidine ring nitrogen atom in piperidinoalkynyl.

The phrase "unsubstituted alkoxy" means that the bond to a hydrogen atom in a hydroxyl (-OH) group is replaced by a bond to a carbon atom of an unsubstituted alkyl group as defined above.

The phrase "substituted alkoxy" means that the bond to a hydrogen atom in a hydroxyl (-OH) group is replaced by a bond to a carbon atom of a substituted alkyl group as defined above.

"pharmaceutically acceptable salts" include salts with inorganic bases, organic bases, inorganic acids, organic acids, or basic or acidic amino acids. Salts of inorganic bases include, for example, alkali metals such as sodium or potassium; alkaline earth metals such as calcium and magnesium or aluminum; and ammonium. Salts of organic bases include, for example, trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, and triethanolamine. Salts of inorganic acids include, for example, salts of hydrochloric, hydrobromic, nitric, sulfuric, and phosphoric acids. Salts of organic acids include, for example, salts of formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid. Salts of basic amino acids include, for example, salts of arginine, lysine, and ornithine. Acidic amino acids include, for example, aspartic acid and glutamic acid.

"tautomer" refers to isomeric forms of a compound that are in equilibrium with each other. The concentration of the isomeric forms depends on the environment in which the compound is placed and may vary depending on, for example, whether the compound is a solid or in an organic or aqueous solution. For example, in aqueous solution, the ketone is usually in equilibrium with its enol form. Thus, ketones and their enols are referred to as tautomers of each other. One skilled in the art can readily appreciate that a wide variety of functional groups and other structures can exhibit tautomerism and that all tautomers of the compounds of the invention are within the scope of the invention.

Certain embodiments are derivatives referred to as prodrugs. The expression "prodrug" refers to a derivative of a drug or therapeutically active drug, such as esters and amides, wherein said derivative has enhanced properties compared to said drug, such as better delivery and therapeutic value, and can be converted into said drug by enzymatic or chemical processes. See, e.g., r.e. notari, Methods enzymol.112: 309-323 (1985); bodor, Drugs, soft he Future 6: 165-182 (1981); H.Bundgaard, Chapter 1 in Design of Prodrugs (H.Bundgaard, ed.), Elsevier, New York (1985); and A.G.Gilman et al, Goodman And Gilman's The Pharmacological Basis of Therapeutics, 8^thed., McGraw-Hill (1990). Thus, the prodrug may be designed to alter the metabolic stability or transport properties of the drug, mask side effects or toxicity of the drug, improve the taste of the drug, or alter other properties or attributes of the drug. The compounds of the present invention include enriched or resolved optical isomers at any or all asymmetric atoms as is apparent from the description. Racemic and diastereomeric mixtures, as well as the individual optical isomers, may be separated or synthesized so as to be substantially free of their enantiomeric or diastereomeric partners. All such stereoisomers are within the scope of the present invention.

The term "carboxyl protecting group" as used herein refers to a carboxylic acid employed to protect an ester group thereby blocking or protecting the carboxylic acid functionality when carrying out reactions involving other functional sites of the compound. Carboxyl protecting Groups are described, for example, in Greene, Protective Groups in Organic Synthesis, pp.152-186, John Wiley&Sons, New York (1981), incorporated herein by reference. Furthermore, the carboxyl protecting group may be used as a prodrug, whereby the protecting group may be cleaved off in vivo by, for example, enzymatic hydrolysis to release the biologically active parent. T.Higuchi and V.Stella "Pro-drugs as Novel Delivery Systems", Vol.14 of the A.A discussion of the concept of prodrugs is provided in c.s.symposium Series, American Chemical Society (1975), which is incorporated herein by reference. These carboxyl protecting groups are well known to those skilled in the art and are widely used in the field of carboxyl protection in penicillins and cephalosporins, e.g. U.S. patents 3,840,556 and 3,719,667, s.kukolja, j.am.chem.soc.93: 6267-: 1779-1782(1970), the disclosure of which is incorporated herein by reference. Esters which have been found to be useful as prodrugs of compounds containing carboxyl groups, for example, pp.14-21 in Bioreversible Carriers in Drug Design: theory and Application (e.b. roche, ed.), Pergamon Press, New York (1987), incorporated herein by reference. An exemplary carboxyl protecting group is C₁-C₈Alkyl (e.g., methyl, ethyl, tert-butyl, and the like); a haloalkyl group; an alkenyl group; cycloalkyl and substituted derivatives thereof such as cyclohexyl, cyclopentyl, and the like; cycloalkylalkyl groups or substituted derivatives thereof such as cyclohexylmethyl, cyclopentylmethyl, and the like; arylalkyl groups, for example, phenethyl or benzyl or substituted derivatives thereof, such as alkoxybenzyl or nitrobenzyl and the like; arylalkenyl, e.g., phenylvinyl, and the like; aryl and substituted derivatives thereof, for example, 5-indanyl and the like; dialkylaminoalkyl (e.g., dimethylaminoethyl, and the like); alkanoyloxyalkyl groups such as acetoxymethyl, butyryloxymethyl, valeryloxymethyl, isobutyryloxymethyl, isovaleryloxymethyl, 1- (propionyloxy) -1-ethyl, 1- (pivaloyloxy) -1-ethyl, 1-methyl-1- (propionyloxy) -1-ethyl, pivaloyloxymethyl, propionyloxymethyl and the like; cycloalkanoyloxyalkyl groups such as cyclopropylcarbonyloxymethyl, cyclobutylcarbonyloxymethyl, cyclopentylcarbonyloxymethyl, cyclohexylcarbonyloxymethyl and the like; aroyloxyalkyl groups such as benzoyloxymethyl, benzoyloxyethyl, and the like; arylalkyl carbonyloxyalkyl such as benzylcarbonyloxymethyl, 2-benzylcarbonyloxyethyl and the like; alkoxycarbonylalkyl such as methoxycarbonylmethyl, cyclohexyloxycarbonylmethyl, 1-methoxycarbonyl-1-ethyl and the like; alkoxycarbonyloxyalkyl radicals such as methoxycarbonyloxymethyl, tert-butoxycarbonyloxymethyl,1-ethoxycarbonyloxy-1-ethyl, 1-cyclohexyloxycarbonyloxy-1-ethyl, etc.; alkoxycarbonylaminoalkyl such as tert-butoxycarbonylaminomethyl and the like; alkylaminocarbonylaminoalkyl groups such as methylaminocarbonylaminomethyl groups and the like; alkanoylaminoalkyl groups such as acetylaminomethyl and the like; heterocyclic carbonyloxyalkyl such as 4-methylpiperazinylcarbonyloxymethyl and the like; dialkylaminocarbonylalkyl groups such as dimethylaminocarbonylmethyl, diethylaminocarbonylmethyl, and the like; (5- (alkyl) -2-oxo-1, 3-dioxol-4-yl) alkyl such as (5-tert-butyl-2-oxo-1, 3-dioxol-4-yl) methyl and the like; and (5-phenyl-2-oxo-1, 3-dioxol-4-yl) alkyl groups such as (5-phenyl-2-oxo-1, 3-dioxol-4-yl) methyl and the like.

The term "N-protecting group" or "N-protection" as used herein refers to those intended to protect the N-terminus of an amino acid or peptide during synthesis or to protect the amino group from undesired reactions. Commonly used N-protecting Groups are disclosed, for example, in Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York (1981), incorporated herein by reference. For example, the nitrogen protecting group may include acyl groups such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthaloyl, o-nitrophenoxyacetyl, a-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl and the like; sulfonyl groups such as benzenesulfonyl, p-toluenesulfonyl, and the like; carbamate-forming groups such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3, 4-dimethoxybenzyloxycarbonyl, 3, 5-dimethoxybenzyloxycarbonyl, 2, 4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4, 5-dimethoxybenzyloxycarbonyl, 3, 4, 5-trimethoxybenzyloxycarbonyl, 1- (p-biphenylyl) -1-methylethoxycarbonyl, α -dimethyl-3, 5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl, t-butoxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, Methoxycarbonyl, allyloxycarbonyl, 2, 2, 2-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl and the like; alkyl groups such as benzyl, triphenylmethyl, benzyloxymethyl, and the like; and silyl groups such as trimethylsilyl and the like. In some embodiments, the N-protecting groups are formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, phenylsulfonyl, benzyl, 9-fluorenylmethyloxycarbonyl (Fmoc), t-butoxycarbonyl (Boc), and benzyloxycarbonyl (Cbz).

As used herein, "halo," "halogen," or "halide" refers to F, Cl, Br, or I.

As used herein, abbreviations for any protecting group, amino acid or other compound, unless otherwise indicated, are used with their commonly used accepted abbreviations or IUPAC-IUBCommission on Biochemical Nomenclature, biochem.11: 942-944 (1972).

As used herein, "substantially pure" refers to a substance that is sufficiently homogeneous to be free of readily detectable impurities in appearance, or that is sufficiently pure so that further purification does not detectably alter physical and chemical properties, such as enzyme activity and biological activity, as determined by standard analytical methods used by those skilled in the art to assess purity, such as Thin Layer Chromatography (TLC), gel electrophoresis, and High Performance Liquid Chromatography (HPLC). Substantially pure includes compositions in which the compounds of the invention constitute the major component of the composition, e.g., about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% or more of the materials in the composition. Methods for purifying compounds to obtain substantially chemically pure compounds are known to those skilled in the art. However, a substantially chemically pure compound may be a mixture of stereoisomers. In this case, further purification may enhance the specific activity of the compound. However, the compounds of the present invention need not often be provided in a particularly pure state. Partially pure compositions may also be employed in certain embodiments, depending on the desired use. For example, purification methods that can result in higher yields of the compounds of the invention may result in reduced levels of relative purity.

As used herein, "biological activity" refers to the in vivo activity of a compound, composition or other mixture, or the physiological response resulting from the in vivo administration of a compound, composition or other mixture. Biological activity thus encompasses the therapeutic, diagnostic and pharmaceutical activity of these compounds, compositions and mixtures. The term "biologically active" or "functional" when used as a modifier of a composition of the invention comprising a polypeptide or a composition thereof refers to a polypeptide that exhibits at least one activity characteristic of a compound of the invention, or an activity similar to a compound of the invention.

As used herein, "pharmacokinetics" refers to the concentration of an administered compound in the serum over time. Pharmacodynamics refers to the concentration of the administered compound in target and non-target tissues over time as well as the effect on the target tissue (e.g., efficacy) and the effect on non-target tissues (e.g., toxicity). The pharmacokinetic or pharmacodynamic improvements, such as the use of labile bonds or the alteration of the chemistry of any linker (e.g., altering solubility, charge, etc.), can be tailored for a particular targeted or biological agent.

As used herein, the phrases "effective amount" and "therapeutically effective amount" refer to an amount of a compound of the present invention that is useful in or capable of supporting an observable change in the level of one or more bioactive characteristics of the composition of the present invention, or an amount sufficient to impart a beneficial effect, e.g., alleviate the symptoms of its recipient. The specific therapeutically effective dose level for a particular subject will depend upon a variety of factors including the symptom or condition being treated, the severity of the symptom or condition, the activity of the specific compound, the route of administration, the rate of clearance of the compound, the duration of the treatment, the drug used in combination or concurrently with the compound, the age of the subjectWeight, sex, diet, and general health, among others, as well as other factors known in the medical arts and sciences. A therapeutically effective amount can be an amount of a compound of the invention sufficient to form a measurable inhibition of angiogenesis in the tissue being treated, i.e., an angiogenesis inhibiting amount. Inhibition of angiogenesis can be measured in situ by immunohistochemistry or other methods known to those skilled in the art. Various common factors to be considered in determining a "therapeutically effective amount" are known to those skilled in The art And are described, for example, in Gilman, A.G., et al, Goodman And Gilman's The Pharmacological Basis of Therapeutics, 8^th ed.，McGraw-Hill(1990)；and Remington′s Pharmaceutical Sciences，17^thed., Mack Publishing Co., Easton, Pa (1990).

As used herein, the terms "simultaneous administration" and "simultaneous administration" encompass the substantially simultaneous administration of one or more compounds of the present invention and one other tumor therapeutic agent.

As used herein, the term "non-simultaneous" administration encompasses administration of one or more compounds of the invention at different times, in any order, whether overlapping or not. This includes, but is not limited to, sequential treatment with the components of the composition (such as pretreatment, follow-up or overlap therapy), and regimens in which the drug is altered, or in which one component is administered chronically while the other component is administered intermittently.

Examples

The breadth of the present invention is described by the following examples, which describe common embodiments of the present invention and do not limit the claims and specification in any way.

Example 1

Synthesis of Compounds of the invention

The peptides of the invention can be linked to antibody 38C2 by: 1mL of 38C2 in phosphate buffered saline (10mg/mL) was added to 12 μ L of a 10mg/mL peptide stock solution 2 hours prior to use, and the resulting mixture was maintained at room temperature.

Example 2

Rader, et al, j.mol.biol.332: 889-899(2003) describe in detail the method of generating h38c 2. The results, materials and methods in this reference are described in detail below. Humanization, human V.kappa.Gene DPK-9 and human J.kappa.Gene JK4 used as a framework for humanization of the m38C2 kappa light chain variable domain, human VH Gene DP-47 and human J.kappa._HGene J_H4 was used as framework for humanization of the heavy chain variable domain of m38C 2. All Complementarity Determining Region (CDR) residues identified by Kabat et al, as well as established framework residues in the light and heavy chain variable domains, were grafted to a human framework by m38C 2. The choice of grafted framework residues may depend on the crystal structure of the mouse mAb33F12Fab (PDB 1 AXT). mAb33F12Fab shares 92% sequence homology with m38c2 in the variable domain and has the same CDR length. In addition, 33F12 and m38C2 have similar catalytic activity. The framework residues are composed of five residues in the light chain and seven residues in the heavy chain (fig. 7A), and encompass residues that may be involved directly or indirectly in the catalytic activity of m38C 2. These include the reactive lysine, Lys, of m38C2^H93It is located in the framework region 3 of the heavy chain (FR 3). Six residues, Ser^H35、Val^H37、Trp^H47、Trp^H103And Phe^L98They are conserved in mouse mAbs 33F12 and 38C2, and in Lys^H93Of epsilon-amino groupWithin the radius. These residues are also conserved in humanization. Lys^H93At the bottom of the highly hydrophobic substrate binding sites of mouse mAbs 33F12 and 38C 2. In addition to the CDR residues, a number of framework residues are arranged in this pocket. Wherein Leu^L37、Gln^L42、Ser^L43、Val^L85、Phe^L87、Val^H5、Ser^H40、Glu^H42、Gly^H88、Ile^H89And Thr^H94Transplantation into human frameworks.

Expression of

By combining the humanized variable domain with a human constant domain C_κAnd C_γ11 fusion, h38C2 was originally produced as Fab expressed in e. Thereafter, h38c2IgG was formed from h38c2 using a PIGG vector engineered for human IgG1 expression in mammalian cells. Supernatants from transiently transfected human 293T cells were injected onto affinity chromatography for recombinant protein A to give approximately 1mg/L h38C2IgG 1. Purity was determined by SDS-PAGE followed by Coomassie blue staining.

Beta-diketone compound-

Covalent addition of a beta-diketone to m38c2 to form an enaminone_maxCharacteristic UV absorption at 318 nm. Similar to m38C2IgG, h38C2IgG showed characteristic enaminone uptake after incubation with β -diketone. As a negative control, recombinant human anti-HIV-1 gp120mAb b12, having the same IgG1 isotype as h38C2, did not show enaminone uptake after incubation with β -diketone 2. For quantitative comparison of beta-diketone binding to m38C2 and h38C2, the authors used a competition ELISA. The antibodies were incubated with increasing concentrations of beta-diketone 2 and 3 and detected against immobilized BSA-coupled beta-diketone 1. The apparent equilibrium dissociation constants were 38 μ M (M38C2) and 7.6 μ M (h38C2) for β -diketone 2 and 0.43 μ M (M38C2) and 1.0 μ M (h38C2) for β -diketone 3, revealing similar β -diketone binding properties of the mouse and humanized antibodies. (FIG. 6)

Molecular modeling-A molecular model of h38C 2Fab was constructed by homology modeling using the crystal structure of the relevant aldolase antibody mouse 33F12Fab (protein database ID: 1AXT) as a template. Previously inHas determined the resolution ofCrystal structure of murine 33F12 Fab. Alignment of the amino acid sequences of mice 33F12 and 38C2 using the homo dogy module in INSIGHT II software (Accelrys) confirmed that the two sequences are highly homologous. The difference is 19 of the 226 amino acids in the two variable domains are different from each other and their CDRs share the same length. In addition to high sequence homology, the two structures also show comparable structural similarity as observed by the low resolution crystal structure of 38C 2. Residues in the model were mutated to conform to the h38C2 amino acid sequence, with side chains placed according to the standard rotamer. The model energy was minimized over 100 steps using the DISCOVER module in INSIGHT II, with each step being the steepest dip minimization followed by conjugate gradient minimization.

Construction of the H38C 2Fab

The sequences of the variable light and heavy domains of m38C2 (SEQ ID NOS: 4 and 5, respectively), and the human germ line sequences DPK-9(SEQ ID NO: 6), JK4(SEQ ID NO: 7), DP-47(SEQ ID NO: 8), and JH4(SEQ ID NOS: 9, 10, and 11) (V BASE;http://vbase.mrc-cpe.cam.ac.uk/) For designing humanized V_κAnd V_HThe synthetic assembled overlapping oligonucleotides of (1). N-glycosylation sites with the sequence NXS/T as well as restriction enzyme sites HindIII, Xbal, SacI, ApaI and SfiI were avoided. PCR was performed by using the Expand High Fidelity PCR System (Roche Molecular Systems). Humanized V_κThe oligonucleotides are: l flanking sense (L flash sense) (Rader, C., Ritter, G., Nathan, S., Elia, M., Gout, I., Junbluth, A.A., J.biol.chem.275: 13668-13676(2000)) (sense 5'-GAGGAGGAGGAGGAGGGCCCAGGCGGCCGAGCTCCAGATGACCCAGTCTCTCCA-3' SEQ ID NO: 12); h38C2L1 (antisense; 5'-GAGCTCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGTGACCGCGTCACCATCACTTG-3') (SEQ ID NO: 13); h38C2L2 (antisense; 5'-ATTCAGATATGGGCTGCCATAAGTGTGCAGGAGGCTCTGACTGGAGCGGCAAGTGATGGTGACGCGGTC-3') (SEQ ID NO: 14); h38C2L3 (sense; 5'-TATGGCAGCCCATATCTGAATTGGTATCTCCAGAAACCAGGCCAGTCTCCTAAGCTCCTGATCTAT-3') (SEQ ID NO: 15); h38C2L4 (antisense; 5' -CTGAAAC)GTGATGGGACACCACTGAAACGATTGGACACTTTATAGATCAGGAGCTTAGGAGACTG-3') (SEQ ID NO: 16) (ii) a h38C2L5 (sense 5'-AGTGGTGTCCCATCACGTTTCAGTGGCAGTGGTTCTGGCACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAGTG-3') (SEQ ID NO: 17);

h38C2L6 (antisense 5'-GATCTCCACCTTGGTCCCTCCGCCGAAAGTATAAGGGAGGTGGGTGCCCTGACTACAGAAGTACACTGCAAAATCTTCAGGTTGCAG-3') (SEQ ID NO: 18); l antisense flank (C.rader et al, J.biol.chem.275: 13668-13676(2000)) (antisense 5'-GACAGATGGTGCAGCCACAGTTCGTTTGATCTCCACCTTGGTCCTCC-3' SEQ ID NO: 19). Humanized V_HThe oligonucleotides are: h-flanking sense (C.rader et al, J.biol.chem.275: 13668-13676(2000)) (sense 5'-GCTGCCCAACCAGCCATGGCCGAGGTGCAGCTGGTGGAGTCTGGGGGA-3' SEQ ID NO: 20); H38C2H1 (sense 5'-GAGGTGCAGCTGGTGGAGTCTGGCGGTGGCTTGGTACAGCCTGGCGGTTCCCTGCGCCTCTCCTGTGCAGCCTCTGGCT-3') (SEQ ID NO: 21); H38C2H2 (antisense 5'-CTCCAGGCCCTTCTCTGGAGACTGGCGGACCCAGCTCATCCAATAGTTGCTAAAGGTGAAGCCAGAGGCTGCACAGGAGAG-3') (SEQ ID NO: 22); H38C2H3 (sense 5'-TCTCCAGAGAAGGGCCTGGAGTGGGTCTCAGAGATTCGTCTGCGCAGTGACAACTACGCCACGCACTATGCAGAGTCTGTC-3') (SEQ ID NO: 23); H38C2H4 (antisense 5'-CAGATACAGCGTGTTCTTGGAATTGTCACGGGAGATGGTGAAGCGGCCCTTGACAGACTCTGCATAGTGCGTG-3') (SEQ ID NO: 24); H38C2H5 (sense 5'-CAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGCGCCGAGGACACGGGCATTTATTACTGTAAAACG-3') (SEQ ID NO: 25); H38C2H6 (antisense 5'-TGAGGAGACGGTGACCAGGGTGCCCTGGCCCCAGTAGCTGAAACTGTAGAAGTACGTTTTACAGTAATAAATGCCCGTG-3') (SEQ ID NO: 26); h-flanking antisense (C.rader et al, J.biol.chem.275: 13668-13676(2000)) (antisense 5'-GACCGATGGGCCCTTGGTGGAGGCTGAGGAGACGGTGACCAGGGTGCC-3' SEQ ID NO: 27). After assembly, humanised V_κAnd V_HRespectively with human C_κAnd C_γ11 fusion, the resulting light and heavy chain fragments were fused as described and SfiI-cloned into the phagemid vector pComb3X (C.Rader et al, J.biol.chem.275: 13668-13676 (2000); C.F.Barbas 3^rd et al.，Phage Display：Alaboratory manual，Cold Spring Harbor Laboratory，Cold Spring Harbor N.(2001)). To enrich for clones with the correct h38C2 sequence, fabs were displayed on phage and selected by one round of screening against immobilized β -diketone 1(JW) conjugated to BSA. Soluble Fab was prepared from a single clone and tested for binding to immobilized JW-BSA by using ELISA using monkey-anti-human F (ab') conjugated with horseradish peroxidase (Jackson ImmunoResearch Laboratories)₂Polyclonal antibodies were used as secondary antibodies. Analysis of the light and heavy chain coding sequences of the clones was performed by DNA sequencing using primers OMPSEQ (5'-AAGACAGCTATCGCGATTGCAG-3' SEQ ID NO: 28) and PELSEQ (5'-CTATTGCCTACGGCAGCCGCTG-3' SEQ ID NO: 29), respectively (C.F. Barbas 3)^rdet al, Phage Display: a Laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor N.Y. (2001)) to confirm the V of the assembled h38C2_κAnd V_HAnd (4) sequencing.

Construction, Generation and purification of h38C2IgG 1-the recently described vector PIGG (C.Rader et al, FASEB J., 16: 2000-2002(2002)) was used for mammalian expression of h38C2IgG 1. Mammalian expression of PIGG-h38c2 is depicted in FIG. 23. The 9kb vector includes heavy chain γ 1 and light chain κ expression cassettes driven by a bidirectional CM promoter construct using primers PIGG-h38C2H (sense 5'-GAGGAGGAGGAGGAGGAGCTCACTCCGAGGTGCAGCTGGTGGAGTCTG-3') (SEQ ID NO: 30) and GBACK (5'-GCCCCCTTATTAGCGTTTGCCATC-3' SEQ ID NO: 31) (C.F.Barbas 3)^rdet al, Phage Display: a Laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor N.Y. (2001)), the VH coding sequence of the h38C 2Fab from phagemid vector pComb3X was amplified, digested with SacI and ApaI, and cloned into the appropriately digested vector PIGG. Primers PIGG-h38C2L (sense: 5'-GAGGAGGAGGAGGAGAAGCTTGTTGCTCTGGATCTCTGGTGCCTACGGGGAGCTCCAGATGACCCAGTCTCC-3') (SEQ ID NO: 32) and LEADB (5'-GCCATGGCTGGTTGGGCAGC-3' SEQ ID NO: 33) ((C.F. Barbas 3) were used^rdet al, Phage Display: a Laboratory, Cold Spring Harbor N.Y. (2001)), the light chain coding sequence of the h28C 2Fab from phagemid vector pComb3X was amplifiedSubsequently, it was digested with HindIII and XbaI and amplified into the appropriately digested vector PIGG which already contained the h38C2 heavy chain. Coli strain SURE (Stratagene) and intermediate and final PIGG vectors were prepared with the QIAGENPlasmid Maxi Kit. The h38C2IgG1 was produced from the final PIGG vector constructed by transient transfection of human 293T cells with Lipofectamine 2000 (Invitrogen). Transfected cells were maintained in GIBCO 10% ultra-low IgG (< 0.1%) FCS (Invitrogen) in RPMI 1640(Hyclone) for two weeks. During this time, the medium was collected and replaced three times. The collected medium was subjected to affinity chromatography on a recombinant protein AHiTrap column (Amersham Biosciences). The optical density at 280nm was measured using an Eppendorf BioPhotometer to determine that this purification procedure yielded 2.45mg of h38C2IgG1 from 2,300mL of collected media. After dialysis against PBS in Slide-A-Lyzer 10K dialysis cassettes (Pierce), the antibody was concentrated to 760. mu.g/mL using an Ultrafree-15 centrifugal filtration device (UFV2BTK 40; Millipore) and sterile filtered through a 0.2- μm Acrodisc 13MM S-200 syringe filter (Pall). The final yield was 2.13mg (87%). Purified h38C2IgG1 was confirmed by non-reducing SDS-PAGE followed by Coomassie blue staining.

Enaminone formation-antibody (h38C2IgG1 or b12IgG1) was added to beta-diketone 2 to a final concentration of 25 μ M antibody binding site and 125 μ M beta-diketone. The mixture was incubated at room temperature for 10 minutes before UV spectra were acquired using a SpectraMax Plus 384UV plate counter (Molecular Devices) using SOFTmax Pro software (version 3.1.2).

Binding test-unless otherwise noted, all solutions were phosphate buffered saline (pH 7.4). A2X solution of beta-diketone 2 or 3 (50. mu.L) was added to 50. mu.L of antibody (h38C2 or m38C2) and incubated at 37 ℃ for 1 hour. The solution was mixed with a pipette. Antibody binding sites at final concentrations of 0.4 to 8nM, and β -diketones 2 and 3 at final concentrations of 10^-9To 10^-2M and 10^-10To 10^-4And M. 100ng of the BSA conjugate of beta-diketone 1 in TBS was coated in each well of a Costar 369096-well plate (Corning). Each well was then blocked with 3% (w/v) BSA in TBS.Then, 50. mu.L of the antibody/beta-diketone mixture was added followed by 50. mu.L of a 1: 1,000 dilution of goat anti-human FcIgG polyclonal antibody conjugated with horseradish peroxidase (Pierce) or rabbit anti-mouse Fc IgG polyclonal antibody (Jackson ImmunoResearch Laboratories). Followed by 50 μ L ABTS substrate solution. Between each addition, the plates were covered, incubated at 37 ℃ for 1 hour, and then rinsed 5 times with deionized water. The absorption at 405nm was measured as described above until the reaction with the absence of beta-diketone reached the appropriate value (0.5 < A)₄₀₅< 1.0). Fractional inhibition of ELISA Signal for each well (v)_i) The calculation is performed using equation i:

v_i＝(A_o-A_i)/(A_o) (i) wherein A_oIs the ELISA uptake obtained in the absence of beta-diketone, A_iIs the absorption obtained in the presence of a beta-diketone. For monovalent binding proteins, the fraction of antibody binding to soluble β -diketone (f) is equal to v_i. However, IgG antibodies are bivalent, and ELISA signal is inhibited only by the bidentate antibody, not by monovalent binding. Therefore, Stevens correction using bivalent antibodies:

f_i＝(v_i)^1/2 (ii)

the apparent equilibrium dissociation constant ([ ref.37] modified) was determined using the following relationship:

f_i＝f_min+(f_max-f_min)(1+K_D/a₀)^-1(iii) wherein a is₀Corresponding to the total beta-diketone concentration, K_DIs the equilibrium dissociation constant, f_minAnd f_maxRepresents experimentally determined values when the antibody binding site is unoccupied or saturated, respectively. Since the equation is only at K_DThe value is at least 10X higher than the antibody concentration, therefore, the K determined by equation iii is verified_DThe value satisfies this condition. Nonlinear least squares fitting procedure using KaleidaGraph (version 3.0.5, Abelbick software) using K_D、f_maxAnd f_minThe data were fitted as adjustable parameters and normalized using equation iv:

f_norm＝(f_i-f_min)/(f_max-f_min) (iv)

example 3

Linking a peptide of the invention to a linker of the invention

The compounds of the present invention can be prepared by a variety of methods. In one approach, a [ peptide ] -linker compound is synthesized with a linker that includes one or more reactive groups designed to covalently react with an amino acid side chain of a binding site of an antibody. The targeting agent-linker compound and antibody are conjugated under conditions in which the linker reactive group forms a covalent bond with an amino acid side chain.

In another approach, linking may be achieved by synthesizing an antibody-linker compound comprising an antibody and a linker, wherein the linker comprises one or more reactive groups designed for covalent reaction with a suitable chemical moiety of the [ peptide ]. [ peptide ] may be modified to provide a suitable moiety for reacting with the linker reactive group. The antibody-linker and [ peptide ] are bound under conditions in which the linker-reactive group covalently links to the targeting agent and/or biological agent.

And a further antibody- [ peptide ] formation]The method of conjugate employs a dual linker design. In certain embodiments, a [ peptide ] is synthesized]A linker comprising [ peptide ]]-a linker and a linker having a reactive group. Antibody-linker compounds are synthesized, including antibodies and the [ peptides ] with facile interaction with the first step]-a linker of a chemical group for which the reactive group of the linker reacts. The two linker-containing compounds combine under conditions in which the linkers are covalently linked to form an antibody- [ peptide ]]A compound is provided. Table 9 shows a peptide comprising [ VEGF-peptide covalently linked to a linker]In accordance with the present inventionExemplary compounds of (a): d-amino acids are indicated with a lower case letter type label: for example, compound 2018 (comprising SEQ ID NO: 78, with K)¹⁰Substitutions as linking residues are also equivalent to SEQ ID NO: 195) including the residues "R-L-Y- (D-Ala) - (D-Leu)", written as "R-L-Y-a-L". Disulfide bonds are represented as the connecting line between two cysteine residues.

Z may be a group capable of forming a covalent bond, reversible or irreversible. In some embodiments, a reversible covalent bond may be formed with a diketone Z group as shown in fig. 3. Thus, structures a-C can form reversible covalent bonds with reactive nucleophilic groups (e.g., lysine or cysteine side chains) of the antibody binding site. R 'in structures A-C of FIG. 3'₁、R’₂、R′₃And R₄Represents a substituent which may be C, H, N, O, P, S, halogen (F, Cl, Br, I) or a salt thereof. These substituents may also include, for example, alkyl, alkenyl, alkynyl, oxyalkyl, oxyalkenyl, oxyalkynyl, aminoalkyl, aminoalkenyl, aminoalkynyl, sulfoalkyl, sulfoalkenyl, sulfoalkynyl, phosphorylalkyl, phosphorylalkenyl, or phosphorylalkynyl. R'₂And R'₃There may be rings as exemplified in structures B and C. X in fig. 3 may be a heteroatom. Other Z groups that form reversible covalent bonds include amidines, imines, and other reactive molecules encompassed by structure G of FIG. 3. Fig. 4 includes structures of other linker reactive groups, such as B, G, H, that form reversible covalent bonds, and E and F when X is not a leaving group.

The Z-reactive groups that form irreversible covalent bonds with the antibody binding site include structures D-G in fig. 3 (e.g., when G is an imido ester) and structures A, C and D of fig. 4. Structures E and F of fig. 4 may also form irreversible covalent bonds when X is a leaving group. These structures can be used to attach targeting agent-linkers to reactive nucleophilic groups of antibody binding sites. As used herein, L 'is a linker moiety linking the antibody to the targeting agent and has the formula-X-Y-Z' -. FIGS. 5 and 6 depict the mechanism of addition of reactive nucleophilic side chains in the antibody binding site to the Z moiety depicted in FIGS. 3 and 4, respectively.

The following shows the case where the linker has a diketone moiety as a reactive group and forms a bond with a side chain amino group of a lysine residue in the binding site of the antibody. The antibody is illustratively shown as bivalent, with one reactive amino acid side chain for each binding site indicated

Another embodiment shown below is directed to the case where the linker has a β lactam moiety as a reactive group and forms a bond with a side chain amino group of lysine of the antibody binding site. The antibody is illustratively shown as bivalent, with one reactive amino acid side chain for each binding site indicated

The compounds of the invention can also be readily synthesized by linking the [ peptide ] -linker compounds described herein to the binding site of a multivalent antibody. For example, [ peptide ] -linker conjugates, wherein the linker comprises a diketone reactive moiety, can be incubated with 0.5 equivalents of an aldolase antibody such as h38C2IgG1 to produce the compounds of the invention.

Another example of a reaction mechanism for binding [ linker ] - [ antibody ] to a peptide is shown below, using "click chemistry" to bind alkynes and azides (where R is attached to the [ peptide ] of the invention).

Example 4

Is provided in FIG. 7And (4) synthesizing.

Example 5

The ability of Ang 2-binding compounds to interact with Ang2 was measured by competition with Tie-2. These tests are described in US2008166364 (also in PCT/IE2007/000110), the content of which is incorporated herein in its entirety by reference. In particular, these sections of PCT/IE2007/000110 and US2008166364 describe in detail the advantages and disadvantages of various Ang 2-binding peptides, as well as the advantages and disadvantages of covalently linking certain Ang 2-binding peptides to catalytic antibodies (and those sections specifically associated with h38C2 linkages), and the advantages and disadvantages of using specific residues of the corresponding Ang 2-specific peptides as linking residues (in this section, linking residues refer to residues covalently linked to the linker (such as used in example 4 herein)). For use in competitive ELISA, human angiopoietin-2 protein and Tie-2-Fc (R & D Systems) were reconstituted without carrier proteins. Mouse anti-human Tie-2(Pharmingen) was used as the primary antibody, and goat anti-mouse-IgG 1-hrp (pierce) was used as the secondary antibody. TMB substrate from Pierce was used.

High binding half-well plates were coated with Ang-2 in 50. mu.l PBS (100 ng/well) and incubated overnight at 4 ℃. Plates were washed three times with wash buffer (0.1% Tween 20, PBS, pH 7.4) and blocked with superblock (Scytek) 100. mu.l/well for 1 hour at room temperature ("RT"). After removal of the blocking solution, 50. mu.l of Ang-2 binding peptide compound (1. mu.M and 5X serial dilutions) was added in the presence of 0.25nM hTie-2-Fc using Superblock as diluent and incubated for 2 hours at room temperature. Plates were washed 3 times with wash buffer. Then, 50ul of 0.1ug/ml mouse anti-human Tie-2 diluted in SuperBlock was added and incubated for 1 hour at room temperature. After incubation, 50ul of goat anti-mouse IgG-HRP diluted 1: 5,000 in SuperBlock was added and incubated for 1 hour at room temperature. After 3 hours of rinsing, 50. mu.l (25. mu.l TMB + 25. mu.l H) was added₂O₂) And incubated for 3-5 minutes.Color development was detected with 25. mu.l of 2M H₂SO₄And (5) stopping. OD450nm was measured using a correction wavelength of 540 nm. IC was calculated using a non-linear sigmoidal dose-response curve fitting function in Prism 4 software (GraphPad)₅₀Value (50% inhibition of Ang-2-Tie-2 binding).

For the anti-competition ELISA, human Tie-2-Fc, angiopoietin-2 protein, biotinylated anti-human Ang-2 antibody and streptavidin HRP (R)&D Systems) and TMB substrate from Pierce. High binding half-well plates were coated with Tie-2-Fc in 50. mu.l PBS (50 ng/well) and incubated overnight at 4 ℃. Plates were washed three times with wash buffer (0.1% Tween 20, PBS, pH 7.4) and blocked with Superblock, 100. mu.l/well for 1 hour at room temperature. Plates were rinsed three times. After washing, 50. mu.l of Ang-2 binding peptide compound (50. mu.M and 5X serial dilutions) was added in SuperBlock in the presence of 50ng/ml (0.83nM) Ang-2 and incubated for 1 hour at room temperature. Plates were washed three times, 50. mu.l of 1. mu.g/ml biotinylated anti-Ang-2 detection antibody in Superblock was added and incubated for 2 hours at room temperature. The plate was washed 3 times and 50. mu.l streptavidin HRP (1: 200 dilution in Superblock) was added at room temperature for 20 minutes. The plate was washed 3 times and 50. mu.l (25. mu.l TMB + 25. mu. l H) was added₂O₂) Substrate solution and incubation for 20-30 minutes. Using 25. mu.l of 2M H₂SO₄The color development was stopped. OD450nm was measured using a correction wavelength of 540 nm. IC was calculated using a non-linear sigmoidal dose-response curve fitting function in Prism 4 software (GraphPad)₅₀Value (50% inhibition of Ang-2-Tie-2 binding).

The IC of exemplary Ang-2 binding peptide compounds as determined by competition ELISA is listed in Table 5₅₀The value is obtained. IC is provided for an Ang 2-binding peptide (T) covalently linked to a linker as shown in example 4 via the side chain of the Ang 2-linking residue, and an Ang 2-binding peptide covalently linked to a linker of example 4 via the side chain of the Ang 2-linking residue, wherein the Z group of the linker is covalently linked (P) to the binding site of h38C2₅₀The value is obtained.

In table 5, compounds 4021, 4022, and 4023 are only Ang-2 binding peptides, the two being conjugated to a linker or linker-antibody; compounds 4024 and 4025 are Ang-2 binding peptides conjugated to a linker-antibody, wherein the linker is 4P ("4" PEG); while compound 4026-4063 is the Ang-2 binding peptide conjugated to a linker-antibody, wherein the linker is OP ("O" PEG) and has the structure shown in example 4. When the data were obtained as shown below, compounds 4024-4063 were conjugated to the humanized aldolase antibody unless otherwise indicated. The compounds of the invention shown in the tables are blocked at the N-terminus with an acyl group and at the C-terminus with an amino group unless otherwise indicated (e.g., compounds 4026, 4049, 4050, 4051 and 4052). In table 5, the amino acid sequence of the peptide compound is shown, with the position of the linker OP or 4P indicated in parentheses after the internal amino acid residue to which the linker is attached. For compound 4026, the N-terminal "OP" linker is indicated at the beginning of the peptide sequence.

For example, compound 4024 in table 5 has the following sequence: q (Kac) Y QPL DELDK (4P) T LYD QFM LQQ G (parent SEQ ID is SEQ ID NO: 138). In this example, the second amino acid residue is epsilon acyl lysine followed by tyrosine, and the attachment position (in this case the 4P linker) is lysine residue 11 followed by threonine. Likewise, the compounds have the following sequence: (amino 2-PEG) QKacY QPL DEL DK (0P) T LYDQFMLQQ G (SEQ ID NO: 162). In this case, the N-terminal glutamine residue is blocked by an amino-2-PEG group, the second amino acid residue is epsilon-acyl lysine, and the OP linker is attached to lysine residue 11.

Tables 5 and 6 also show half-lives (T1/2) and "screening" half-lives (structures in parentheses), another method for determining T1/2, based on shorter test times. For "screening" T1/2, test compounds were administered intravenously into male Swiss Webster mice. At the following time points: blood samples were taken from 4 mice at each time point by reverse orbital sinus blood collection at 0.08, 5 and 32 hours. Blood levels of test compounds were determined by ELISA. The data is reported as the percentage of test compound at 32 hours compared to 5 minutes. After additional data analysis using WinNonlin version 4.1(Pharsight corporation), normal T1/2 was calculated in the same manner. The data is fit to one model according to the shape of the curve (i.e., a two-exponential drop will be fit to a two-chamber model, etc.). The criterion for the best fit (i.e. lowest% CV) will be based on iterative reweighted least squares.

The IC of exemplary Ang-2 binding peptide compounds determined by an anti-competition ELISA is listed in Table 5₅₀The value is obtained. IC is provided for targeting peptide plus linker and targeting peptide (P) linked to antibody by linker of example 4₅₀The value is obtained. In table 5, the compounds were conjugated to the humanized aldolase antibody h38c2 and the linker structure (OP) shown in example 4.

Xenograft study

Colo205 cells were cultured in 10% FBS RPMI medium and 3X 10 cells in 0.1ml Hank's Balanced Salt Solution (HBSS)⁶Cells were injected subcutaneously into the right upper flank of nude mice. After 7-9 days, animals were randomized to an average tumor size of 200-³An appropriate number of groups. Mice were then treated with the necessary amount of the compound of the invention and tumor volume was measured twice weekly. Once their mean tumor volume reached 2000mm³The animals were sacrificed. At sacrifice, tumors were weighed and stored for further histological studies. Treatment efficacy was assessed by measuring the difference in tumor volume in the treated group compared to the control group. Results are reported as% T/C, where% T/C is calculated as follows: % T/C ═ V_t-V₀)/(C_t-C₀) X 100, wherein V₀And V_tIs the mean tumor volume at the beginning and end of the treatment group. C₀And C_tIs the mean tumor volume at the beginning and end of the control group (Table 7).

Example 6

Exemplary [ VEGF-peptide ] - [ linker ] -antibody compounds are shown in table 9 and exemplary [ VEGF-peptide ] - [ linker ] -antibody compounds are shown in table 10.

Example 7

VEGF peptide binding assays

High binding half well 96-well plates (Costar #3690) were coated with recombinant human VEGFR2/Fc in 50. mu.l PBS (50 ng/well) and incubated overnight at 4 ℃. After washing the plates three times with 1 Xflush buffer (KPLCat #50-65-00), Superblock (Scytek # AAA500) was used for blocking at Room Temperature (RT) for 1 hour, and the VEGF-peptides of the invention (concentration: 0.128-10,000nM) were diluted sequentially in 50. mu.l Superblock in the presence of 25ng/ml recombinant human VEGF165, then added to the 96-well plates and incubated at room temperature for 1 hour. The peptides of the invention tested in this way were all C (O) CH at the amino terminus₃Blocked by a group at the carboxyl end with NH₂The groups are closed. The plate was then washed three times with wash buffer and diluted (1: 500) biotinylated anti-human VEGF antibody (R) was added&D systems, Cat # BAF293) and incubated at room temperature for 2 hours. After washing the plates, streptavidin-HRP conjugate (1: 200 in Superblock) (R) was added&D systems, Cat # DY998) to detect bound anti-VEGF antibody, followed by development with the addition of Tetramethylbenzidine (TMB) substrate (Pierce, Cat # 34021). With 2M H₂SO₄The reaction was stopped. OD450 was measured using SpectraMax (molecular Devices) using a correction wavelength of 540 nM. Data were analyzed using Prism software. OD450 values are plotted as a function of VEGF peptide concentration. IC was obtained using sigmoidal dose-response curve fitting in Prism₅₀Value, indication of the efficacy of VEGF peptides to inhibit VEGF-VEGFR2 interaction. The results of the VEGF binding assays are shown in tables 1 and 2 (Table 1 as VEGF peptide with V)¹²Predominantly (except for SEQ ID NO: 34), Table 2 shows VEGF peptides vs E¹²Dominant). Tables 1 and 2 indicate the compounds of the invention tested by the parent SEQ id no, although the N 'and C' ends of the tested compounds were blocked as described above. Not all compounds were tested in the same example: IC in parentheses₅₀The values have been normalized.

Example 8

Peptide mooring

A solution of linker (as in example 4) (0.1g, 0.3mmol), HBTU (O-benzotriazole-N, N, N ', N' -tetramethyl-uronium-hexafluoro-phosphate) (0.11g, 0.3mmol) in anhydrous DMF (dimethylformamide) (2mL) was cooled to 2-5 ℃ in an ice-water bath under argon atmosphere. (N-methylmorpholine) (0.12g, 0.9mmol) was added via syringe and stirred for 5 min. A cold solution (2ml) of the other peptide (SEQ ID NO: 64) (0.24g, 0.1mmol) in anhydrous DMF was added to the first reaction vessel and stirred for 30 minutes. After completion of the reaction was monitored by LCMS (liquid chromatography/mass spectrometry), water (1ml) was added and the reaction compound was purified by HPLC (high performance liquid chromatography) to give a pure product (145mg) with a mass of 2785. In the following figures, the complete structure of the unnatural amino acids and residues involved in any linkage other than peptide binding is depicted. Thus, the N of the two cysteines is shown. Other compounds of the invention are formed by covalent bonds.

Example 9

Covalently linking the [ peptide ] - [ linker ] moiety to the [ antibody ]

Programmed preparation and purification of [ peptide ] - [ linker ] - [ antibody ] complex

The tethered peptides were prepared programmed at room temperature at a ratio of 3: 1 to h38C2 overnight. The resulting [ peptide ] - [ linker ] - [ antibody ] macromolecule was purified using a PD-10 desalting column. Protein concentration was measured via UV a 280. Characterization of [ peptide ] - [ linker ] - [ antibody ] macromolecular assay programmed preparation efficiency by LCMS analysis.

Example 10

Mouse PK test (reverse ELISA format)

PK studies were performed using male Swiss Webster mice (CFW, Charles Rivers Hollister, CA) weighing approximately 20-22 grams at the start of dosing. Administration of [ VEGF-peptides by tail vein IV administration]- [ linker]- [ antibody]Complexes, at the following time points: blood samples were collected from 3 mice at each time point by retro-orbital sinus blood collection at 0.08, 0.5, 1, 3, 5,7 and 24 hours. For 32 hoursAt intermediate points, mice were anesthetized with isoflurane and the volume of blood drawn did not exceed 0.1 mL/blood. For the remaining time points (32-120hrs), CO was used₂Mice were killed by inhalation and terminal heart samples were drawn. The protease inhibitor cocktail was added to all blood tubes prior to sample collection. The blood was frozen on ice for 30 minutes, then centrifuged at 12000rpm at 4 ℃ for 5-10 minutes to collect serum, and immediately frozen at-80 ℃ until analysis by ELISA. Dosing solutions were used to establish standard curves for VEGF reverse ELISA (also described herein as IgG coated ELISA) or VEGF ELISA (VEGF coated ELISA) analysis of serum samples.

VEGF reverse ELISA

High binding half well 96 well plates (Costar #3690) were coated with 1: 100 goat anti-human IgG in coating buffer (Bethy Laboratories ELISA kit, Cat # E80-104) at 4 ℃ overnight. After washing the plate three times with 1 Xwash buffer (KPL Cat #50-65-00), blocking with Superblock (Scytek # AAA500) at Room Temperature (RT) for 1 hour, prepared dosing solution standards (range: 3.91-500ng/ml) and serum samples were added to the plate and incubated on a plate shaker for 1 hour to allow for [ VEGF-peptide ]]- [ linker]- [ antibody]The composite is bonded to the plate. A fixed concentration of VEGF (3nM) was then added and incubated for 1 hour on a plate shaker. The plate was then washed three times with washing buffer and diluted (1: 500) biotinylated anti-human VEGF antibody (R)&D systems, Cat # BAF293) was added and incubated at room temperature for 2 hours. After washing the plates, streptavidin-HRP conjugate (1: 200 in Superblock) (R) was added&D systems, Cat # DY998) to detect bound anti-VEGF antibody, followed by development with the addition of Tetramethylbenzidine (TMB) substrate (Pierce, Cat # 34021). With 2M H₂SO₄The reaction was stopped. OD450 was measured using SpectraMax (molecular device) with a correction wavelength of 540 nM. Calculation of the [ VEGF-peptide Using a Standard Curve]- [ linker]- [ antibody]Serum concentration of the complex. Serum compound concentrations determined by ELISA were plotted as a function of time. Further data analysis was performed using WinNonlin version 4.1(Pharsight Corporation) to determine the beta half-life (T)_1/2) And against [ peptide ]]- [ linker]- [ antibody]Area under the curve (AUC) of the complex.

VEGF ELISA

High binding half well 96 well plates (Costar #3690) were coated with 50. mu.l of rhVEGF165 in PBS/well (6.25 ng/well) and incubated overnight at 4 ℃. After washing the plate three times with 1 Xflush buffer (KPLCat #50-65-00), blocking with Superblock (Scytek # AAA500) at Room Temperature (RT) for 1 hour, prepared dosing solution standards (range: 3.91-500ng/ml) and serum samples were added to the plate and incubated on a plate shaker for 1 hour to allow for [ VEGF-peptide ]]- [ linker]- [ antibody]The complexes bind to the VEGF-coated plate. The plate was washed three times with washing buffer, and diluted goat anti-human IgG-HRP (1: 5000) (Bethyyl A80-104P-52) was added and incubated at room temperature for 1 hour. Streptavidin HRP conjugate (1: 200 in Superblock) was then added (R)&D systems, Cat # DY998) to detect bound anti-VEGF antibody, followed by development with the addition of Tetramethylbenzidine (TMB) substrate (Pierce, Cat # 34021). With 2M H₂SO₄The reaction was stopped. OD450 was measured using SpectraMax (molecular device) with a correction wavelength of 540 nM. Calculation of the [ VEGF-peptide Using a Standard Curve]- [ linker]- [ antibody]Serum concentration of the complex. Serum compound concentrations determined by ELISA were plotted as a function of time. Further data analysis was performed using WinNonlin version 4.1(Pharsight Corporation) to determine the beta half-life (T)_1/2) And against [ peptide ]]- [ linker]- [ antibody]Area under the curve (AUC) of the complex.

Table 3 shows a series of [ VEGF-peptides ]]- [ linker]- [ antibody]And (4) binding of the complex. VEGF-peptide per complex]All based in part on SEQ ID NO: 34, X¹To X¹⁹One of which is replaced by K as a linking residue, C⁵And C¹⁵Are excluded. In each case, the [ VEGF-peptide]At the amino terminus with C (O) CH₃Blocked by a group at the carboxyl end with NH₂The groups are closed. Linker of each Complex]Partially depicted in example 4. Of each complex [ antibody]The moiety is h38C 2: comprises the amino acid sequence of SEQ ID NO: 1 and SEQ ID NO: 2. table 6 also showsShow some [ VEGF-peptides]- [ linker]- [ antibody]Half-life data for the complexes, and data representing the area under the curve (AUC). AUC data represent the total area under the curve, taking both the α -half-life and the β -half-life into account.

Table 4 shows a series of [ VEGF-peptides ]]- [ linker]- [ antibody]And (4) binding of the complex. VEGF-peptide per complex]All parts are based on the indicated SEQ ID NO and the indicated residue is replaced by K as the linking residue. In each case, the [ VEGF-peptide]At the amino terminus with C (O) CH₃Blocked by a group at the carboxyl end with NH₂The groups are closed. Residues 1-18 of the peptide are not described in table 4, but have been described elsewhere herein, except for the corresponding substitution of the linking residue (which in each of these embodiments is K). Linker of each Complex]Partially depicted in example 4. Of each complex [ antibody]The moiety is h38C 2: comprises the amino acid sequence of SEQ ID NO: 1 and SEQ ID NO: 2. table 4 also shows some of the [ peptides ]]- [ linker]- [ antibody]Some half-life data for the complexes. Results were obtained from VEGFELISA and VEGF inverse ELISA (results in parentheses).

Example 11

The formulas of the present invention are shown in FIGS. 8-10: synthesis of compounds [ Ang 2-peptide ] - [ linker ] - [ VEGF-peptide ].

Example 12

Table 11 shows the formula of the present invention: exemplary compounds of [ Ang 2-peptide ] - [ linker ] - [ VEGF-peptide ].

Example 13

Table 12 shows exemplary compounds of the present invention of the following formula:

([ Ang 2-peptide)]- [ linker ]]- [ VEGF-peptide])_{1 or 2}

|

[ antibody ].

Example 14

Testing of the Compounds of example 13 for VEGF binding (IC)₅₀) VEGF T1/2, Ang2 binding (IC)₅₀) And Ang2T 1/2. The test method is as described above. The results are shown in Table 8. The compounds of the invention exemplified in example 13 contain a fixed ratio of [ Ang-2-peptide]And [ VEGF-peptide]. It was reported that circulating levels of Ang-2 were about 10-fold higher than circulating levels of VEGF in prostate cancer patients (2.5ng/ml Ang-2 and 0.2ng/ml VEGF) and in breast cancer patients (2ng/ml Ang-2 and 0.3ng/ml VEGF) (Ref. European Journal of Clinical Investigation (2003)33, 883-. It is clear that some compounds of the invention (see, e.g., example 14) exhibit binding IC of Ang2 and VEGF relative to each other in the same molecule₅₀And greater variation in vivo half-life. Thus, for certain applications, it is desirable to use compounds whose binding affinity for Ang2 is about 3 to about 15 times higher than its binding affinity for VEGF, in order to obtain greater circulating amounts of the corresponding target. Naturally, different therapeutic uses require or allow or favor different compounds of the invention.

For example, compound 6053 shows a K of 3E-10 for VEGF in the Biacore test_DK of 9E-11 for Ang-2 binding_D. Higher affinity for Ang-2 binding would be advantageous when higher plasma concentrations of Ang-2 are reported in certain cancer patients. Compound 6053 shows similar VEGF inhibition IC₅₀(0.7nM) and Ang-2 inhibit IC₅₀(0.6 nM). Furthermore, the [ Ang 2-peptide of compound 6053]And [ VEGF-peptide]The stability of the fractions in mice (T1/2) was very similar. Comparative compound 2018, compound 4043 and compound 6053 were subsequently tested (table 8).

In vivo pharmacology

The antitumor activity of compound 6053 was evaluated in a human xenograft model. Induction of xenografts by Subcutaneous (SC) implantation of tumor cells into 5-7 week old female nu/nu mice and formation of 200-400 mm prior to initiation of treatment³The volume of (a). Once tumors had formed, mice were randomized into treatment groups of the same tumor volume (n-9-10/group) and injected Intraperitoneally (IP) weeklyCompound 6053 is administered once for treatment. In combination studies, treatment was initiated concomitantly with compound 6053, and other anti-cancer agents were administered either once weekly by IP injection or once daily by oral gavage (PO). During treatment, tumor volume was measured once or twice weekly using calipers and body weight was weighed once weekly. In some studies, tumor volume in the control group reached 2000mm once vehicle treatment was performed³All mice were passed over CO₂Asphyxia kills and excises the tumor. In the pseudo-survival study, the mean tumor volume once per treatment group exceeded 2000mm³By CO₂Mice were asphyxiated and tumors were excised, weighed and processed for further histological and/or immunochemical evaluation.

Example 15

Single reagent study

The experiments performed in the Colo205 (human colon adenocarcinoma) xenograft model are depicted in fig. 11. Compound 6053 administered once a week inhibited Colo205 tumor growth dose-dependently. At 35 days, 3mg/kg (IP, 1x/wk) of compound 6053 resulted in a significant-30% reduction in tumor growth, whereby at 10 or 30mg/kg (IP, 1x/wk), a significant-50% reduction in tumor growth was seen when compared to vehicle-treated controls. Treatment with compound 6053 at 10 or 30mg/kg also resulted in sustained tumor inhibition compared to the control group. Compound 6053 treatment did not affect weight gain (data not shown) and mice also showed good health status in the study.

At day 35, half of each group in both vehicle-treated and compound 6053-treated groups were sacrificed and tumors were excised and immediately frozen. To evaluate the anti-angiogenic effect of compound 6053, tumor microvascular density was evaluated immunohistochemically on frozen sections of Colo205 colon adenocarcinoma xenograft tumors treated with vehicle or compound 6053. Tumors were stained with a mouse-specific monoclonal antibody against CD31 and immunoreactivity was quantified from 5 regions of 3 sections of each tumor. Compound 6053 treatment (30mg/kg, 1x/wk) significantly reduced tumor microvascular density by-44% compared to vehicle treated group (figure 12), confirming the anti-angiogenic activity of the molecule.

Compound 6053 was administered once a week to again dose-dependently inhibit Colo205 tumor growth in a separate Colo205 xenograft study. Compound 6053 at a 30mg/kg weekly dose resulted in a significant-70% reduction in tumor growth on day 28 (using CO) compared to vehicle-treated controls₂Asphyxia kill) (fig. 13). Excised tumors were sectioned and treated with hematoxylin and eosin (H)&E) Staining was performed and the live tumor area was assessed using standard image analysis calculation methods. Live tumor area was determined from 4 slices from each tumor, and the mean of these measurements was multiplied by the tumor volume at the end of the study to obtain an estimate of the live tumor volume for each tumor. Live tumor volume data from experimental Colo205 colon adenocarcinoma xenografts are shown in fig. 14. Compared with the vehicle treatment group, the compound 6053-treated mice treated at the doses of 10mg/kg and 30mg/kg have the tumor volumes respectively and remarkably reduced by 60 percent and 76 percent.

To investigate whether compound 6053 targets Ang2 and VEGF simultaneously in vivo, the effect of compound 6053 on Ang2 expression and levels of phosphorylated VEGFR2 (pvgfr 2) was assessed using immunofluorescence of Colo205 xenograft tumors treated with vehicle or compound 6053. Frozen sections of these tumors were stained with FITC-labeled anti-Ang 2 monoclonal antibody and Ang2 immunoreactivity was quantified from 3 images of 1 section from each tumor. Ang2 immunoreactivity was significantly reduced by-70% in the compound 6053 treated group (10 and 30mg/kg) compared to the vehicle treated group (fig. 15). Frozen sections of these tumors were also double stained with FITC-labeled VEGFR antibody and rhodamine-labeled pVEGFR2 antibody, and pVEGFR2 immunoreactivity was quantified from 3 image pairs of 1 section from each tumor and expressed as a percentage of total VEGFR2 immunocompetence (pVEGFR2/VEGFR 2). Compound 6053 treatment significantly reduced pvgfr 2/VEGFR2 in a dose-dependent manner compared to the vehicle treated group by-43% and-70% for 10 and 30mg/kg, respectively (fig. 16). These data demonstrate that compound 6053 affects both Ang2 and the VEGF pathway in the Colo205 xenograft model.

The antitumor efficacy of compound 6053 was also evaluated in an MDA-MB-435 breast cancer xenograft model and an a431 skin cancer xenograft model. Weekly administration of compound 6053(30mg/kg IP) resulted in a significant 45% reduction in tumor growth in the MDA-MB-435 model (day 68) (fig. 17A) and a significant 54% reduction in tumor growth in the a431 model (day 35) model (fig. 17B). Thus, compound 6053 demonstrates significant anti-tumor efficacy in three human xenograft tumor models.

Additional single reagent studies were performed with compounds 2018, 2036, 2049, 2071, 4043 and 6053 and are shown in figures 23-28.

Example 16

Combinatorial study

The antitumor efficacy of compound 6053 was further evaluated in a combination therapy study conducted in a Colo205 xenograft model, comparing the antitumor efficacy of compound 6053 administered alone (10mg/kg IP, once per week) and in combination with 5-fluorouracil (5-FU, fig. 18), irinotecan (fig. 19), taxotere (fig. 20), sunitinib (fig. 21), and axitinib (fig. 22). The combination of compound 6053 with these chemotherapeutic agents (5-FU, irinotecan, taxotere) or the tested multiple tyrosine kinase inhibitors (sunitinib or axitinib) resulted in significantly stronger tumor growth inhibition (p < 0.05, two-way ANOVA and Bonferroni multiple contrast test) than either single agent treatment alone. In the compound 6053+ taxotere combination study (FIG. 17), one tumor was the knock-out value (tumor volume > 2000mm was reached on day 57)³Requiring euthanasia) and are therefore removed from the analysis. Taken together, these data provide an excellent rationale for the pre-evidence for the widespread use of compound 6053 in combination with a variety of anti-cancer therapeutics to target tumor and angiogenesis mechanisms of action.

Summary of efficacy in the early stage of clinical syndrome

Compound 6053 binds to its cognate receptor with sub-nanomolar potency to inhibit human Ang2 and human VEGF and exhibits a prolonged, balanced pharmacokinetic profile in various pre-symptomatic species. Compound 6053 is effective in a variety of human xenograft models and provides additional benefits for use in combination with a variety of standard therapeutic agents, including anti-angiogenic agents and anti-tumor cell therapeutic agents, as evidenced by delayed tumor growth and time to progression. Taken together, these data demonstrate that the high potency of compound 6053 on Ang2 and VEGF, alone or in combination with standard chemotherapeutic agents, significantly affects tumor growth and activity.

Example 17

Anti-angiogenic activity in VEGF-induced rabbit eye retinal leakage assay

The anti-angiogenic activity of compounds 6054, 6037 and 6044 was evaluated in a VEGF-induced rabbit ocular retinal leak test. The test is performed in 3-day or 7-day format. In the 3-day test format, the right eye of each rabbit (3/group) was Injected (IVT) with compound (1 mg/eye) or vehicle on day 0, followed by VEGF (1 μ g/eye) on the next or 1 day into the same eye. On day 3, an IV injection of fluorescein and an angiogram of the eye was taken. In the 7-day assay format, compound (1 mg/eye) was injected on day 0, VEGF (1 μ g/eye) was injected on day 5, and fluorescein was injected IV on day 7. In addition, binding and half-life were examined as described above and shown in table 14. For reference, the VEGF half-life of compound 6037 is indicated as about 36 hours in table 14, which is a similar molecule, the VEGF half-life of compound 2053 (see table 4).

After the fluorescent angiogram was acquired, each eye was given a rating of 0-4 based on the fluorescence density between retinal vessels compared to background. A rating of 0 indicates that the fluorescein density between retinal vessels is equivalent to the background not induced by VEGF and represents minimal or no retinal leakage. A rating of 4 indicates a fluorescein density equivalent to the VEGF-induced retinal leak background and severe fluorescein retinal leaks.

Compound 6037 showed grade 1 retinal leaks at a dose of 1 mg/eye in the 3-day VEGF-induced retinal leak test (fig. 29). The 0.1mg and 0.01mg doses showed no effect.

Compounds 6037, 6044 and 6054 were evaluated in a 7-day rabbit leakage test. Compound 6037 showed a classification of 2 at 1mg dose (fig. 30).

The present invention has thus been fully disclosed and described in accordance with the exemplary embodiments set forth above. Those skilled in the art will recognize that various modifications may be made to the present invention without departing from the spirit and scope of the invention. All publications, patent applications, and issued patents are herein incorporated by reference to the same extent as if each individual publication, patent application, or issued patent was specifically and individually indicated to be incorporated by reference in its entirety. Definitions contained in the contents incorporated by reference are to be excluded if they contradict the definitions in the present disclosure.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-group.

It is specifically contemplated that any of the limitations discussed with respect to one embodiment of the present invention may be applicable to any other embodiment of the present invention. Further, any of the compositions of the present invention can be used in any of the methods of the present invention, and any of the methods of the present invention can be used to make or utilize the compositions of the present invention. In particular, any aspect described in the claims, alone or in combination with one or more claim and/or specification aspects, is to be understood as being combinable with other aspects of the invention set forth elsewhere in the claims and/or specification.

The use of the term "or" in the claims means "and/or" unless explicitly stated otherwise to refer only to or exclude alternatives, even though the description supports a definition that refers only to alternatives and "and/or".

As used in this specification, "a" or "an" may refer to one or more, unless expressly specified otherwise. As used in the claims, the term "a" or "an" when used in conjunction with the word "comprising" may mean one or more than one. As used herein, "another" may mean at least a second or more.

The words "comprise," "comprising," "include," "including," "have," "including," and "having/including," when used herein in connection with the present invention, are intended to specify the presence of stated features, integers, steps, or components, but do not preclude the presence or addition of one or more other features, integers, steps, components, or groups thereof.

Table 4: [ VEGF-peptide]-an antibody complex:

bonding of&Half-life-linking residue and X ¹⁹ -X ²³ Decoration

Table 4 continues: [ VEGF-peptide]-an antibody complex:

bonding of&Half-life-linking residue and X ¹⁹ -X ²³ Decoration

Table 5:

[ Ang 2-peptide ] and [ Ang 2-peptide ] -antibody-complex: binding and half-life

Table 5 continues:

[ Ang 2-peptide ] and [ Ang 2-peptide ] -antibody-complex: binding and half-life

Table 6:

[ Ang 2-peptide ] -antibody-complex: binding and half-life

Table 6 continues:

[ Ang 2-peptide ] -antibody-complex: binding and half-life

4074

Q(Kac)Y QPL DEL D(Kac)T LK(0P)D QFM LQQ G(SEQ ID NO：182)

32.82

4075	Q(Kac)Y QPL DEL D(Kac)T LYK(0P)QFM LQQ G(SEQ ID NO：183)	0.19	65
				4076	Q(Kac)Y QPL DEL D(Kac)T LYD K(0P)FM LQQ G(SEQ ID NO：184)	0.27	94
4077	Q(Kac)Y QPL DEL D(Kac)T LYD QK(0P)M LQQ G(SEQ ID NO：185)	19.53
				4078	Q(Kac)Y QPL DEL D(Kac)T LYD QFK(0P)LQQ G(SEQ ID NO：186)	0.74	72
4079	Q(Kac)Y QPL DEL D(Kac)T LYD QFM K(0P)QQ G(SEQ ID N0：187)	0.077	65
				4080	Q(Kac)Y QPL DEL D(Kac)T LYD QFM LK(0P)Q G(SEQ ID NO：188)	0.11	35
4081	Q(Kac)Y QPL DEL D(Kac)T LYD QFM LQK(0P)G(SEQ ID NO：189)	0.27	27
				4082	Q(Kac)Y QPL DEL D(Kac)T LYD QFM LQQ K(0P)(SEQ ID NO：190)	0.21	20

Table 7:

TABLE 8

Table 9:

[ VEGF-peptide ] -linker compounds

Table 10:

[ VEGF-peptide ] - [ linker ] -antibody compounds

Table 11:

formula (II): [ Ang 2-peptide ] - [ linker ] - [ VEGF-peptide ] compounds of the invention

TABLE 12

{ ([ Ang 2-peptide]- [ linker ]]- [ VEGF-peptide])_{1 or 2}} - [ antibodies]

Watch 13

Comparison of Compounds 2018, 4043 and 6053

TABLE 14

Comparison of Compounds 2018, 4043 and 6053

Claims (25)

1. Formula (II): r¹- [ VEGF-peptide]-R²Or a pharmaceutically acceptable salt thereof, wherein the [ VEGF-peptide ]]Is a peptide having the sequence: x¹-X²-P³-N⁴-C⁵-X⁶-X⁷-X⁸-V⁹-X¹⁰-X¹¹-X¹²-W¹³-X¹⁴-C¹⁵-F¹⁶-E¹⁷-R¹⁸-X¹⁹-X²⁰-X²¹-X²²-X²³(SEQ ID NO: 131) wherein R¹Is absent or is CH₃、C(O)CH₃、C(O)CH₃、C(O)CH₂CH₃、C(O)CH₂CH₂CH₃、C(O)CH(CH₃)CH₃、C(O)CH₂CH₂CH₂CH₃、C(O)CH(CH₃)CH₂CH₃、C(O)C₆H₅、C(O)CH₂CH₂(CH₂CH₂O)_1-5Me, amino-2-PEG or N-acyl or N-alkylamino protecting groups, fatty acid groups or hydrocarbon groups; r²Is absent or OH, NH₂、NH(CH₃)、NHCH₂CH₃、NHCH₂CH₂CH₃、NHCH(CH₃)CH₃、NHCH₂CH₂CH₂CH₃、NHCH(CH₃)CH₂CH₃、NHC₆H₅、NHCH₂CH₂OCH₃、NHOCH₃、NHOCH₂CH₃A carboxyl protecting group, a fatty acid group of a lipid or a hydrocarbon group, and X¹Is a hydrophobic amino acid residue, X²Is a negatively charged residue, X⁶Is a negatively charged residue, X⁷Is a hydrophobic amino acid residue, X⁸Is a residue containing a ring structure, X¹⁰Is a hydrophobic amino acid, X¹¹Is an aromatic amino acid, X¹²Selected from V, E and Kac, X¹⁴Is E or V, X¹⁹Is any hydrophobic amino acid or its D-isomer, X²⁰Absent or any neutral, hydrophobic or aromatic amino acid or D-isomer thereof, X²¹Absent or any positively charged residue or any aliphatic nonpolar residue or D-isomer thereof, X²²Absent or selected from G, V, L, I, P, S, T, W, F, E, Kac or its D-isomer, X²³Is absent or selected from G, A, I, L, Q, E, F, T, W, S, Y and Kac and its D-isomer, and wherein X¹、X²、P³、N⁴、V⁹、X¹⁰、X¹²、X¹⁴、E¹⁷And one of the carbon-terminal residues can be linked to a linking residue comprising a nucleophilic side chain, either directly or via an intermediate linkerThe binding site of the antibody is replaced by a covalently linked N-terminal amino group or C-terminal carboxyl group, and the linking residue is selected from the group consisting of K, R, Y, C, T, S, lysine homologs, homocysteine, homoserine, Dap, Dab, the N-terminal residue, and the C-terminal residue.

2. The compound of claim 1, wherein the [ VEGF-peptide ] comprises a sequence that is identical to a sequence selected from SEQ ID NOs: 34-136 and SEQ ID NO: 192- & ltSUB & gt 195.

3. The compound of any one of the preceding claims, wherein the [ VEGF-peptide ] comprises a sequence substantially homologous to V-E-P-N-C-D-I-H-V-M-W-V-W-E-C-F-E-R-L-Y- (D-Ala) - (D-Leu) (SEQ ID NO: 78).

4. The compound of claim 3, wherein M¹⁰Substituted with a linking residue K, the compound comprising the formula (SEQ ID NO: 195): { C (O) CH₃}-V-E-P-N-C-D-I-H-V-K-W-V-W-E-C-F-E-R-L-Y-(D-Ala)-(D-Leu)-{NH₂}。

5. The compound of any one of the preceding claims, wherein the linking residue is present and the compound further comprises a linear or branched linker covalently attached to a side chain of the linking residue.

6. The compound of claim 5, wherein the linker comprises formula L or L', L being formula (la): - [ linker ] -X-Y-Z or-X-Y-Z-, and L' is of formula: - [ linker ] -X-Y-Z 'or-X-Y-Z' -, wherein: [ linker ] is present when the linker is branched and, when present, is covalently linked to the linking residue and one or more other reactive molecules, X is a biocompatible linking chain comprising any atom selected from the group consisting of C, H, N, O, P, S, F, Cl, Br, and I, and comprises a polymer or block copolymer, and is covalently linked to the linker when the linker is linear, Y is present and has an optionally substituted structure:

wherein a, b, c, d and e are independently carbon or nitrogen; f is carbon, nitrogen, oxygen or sulfur; y is independently attached to X and Z at any two ring positions of sufficient valency; and no more than four of a, b, c, d, e or f are simultaneously nitrogen, and preferably each of a, b, c, d and e in the ring structure is carbon, and Z, when present, has the structure:

and wherein Z', when present, has the structure:

wherein q is 0-5 and antibody-N-when present is a covalent bond with a side chain in the antibody binding site.

7. The compound of claim 6, wherein

X is:

and X-Y is

Wherein v and w are selected such that X has a backbone length of 6 to 12 atoms, R^bIs hydrogen, substituted or unsubstituted C_1-10Alkyl, substituted or unsubstituted C_3-7cycloalkyl-C_0-6Alkyl, or substituted or unsubstitutedAryl of (A) to (C)_0-6Alkyl, v ═ 1 or 2; w is 1 or 2; and R is^bHydrogen is preferred.

8. The compound of any one of claims 6-7, wherein the linker is linear and the [ linker ] is when: absent, when Z is present, from compounds 1001-1080 and when Z' is present, from compounds 2001-2080.

9. The compound of claim 10, wherein Z' is present, said compound comprising the structural formula of compound 2018.

10. A compound comprising the formula:

wherein [ active molecule-1 ] is the [ VEGF-peptide ] according to any one of claims 5 to 11, [ linker ] is covalently linked to both [ VEGF-peptide ] and [ active molecule-2 ], and [ linker ] is further covalently linked to the linker L or L'.

11. The compound of claim 10, wherein [ linker ] comprises formula (la): - [ AM 1-spacer ] - [ branched ] - [ AM 2-spacer ] -; and wherein [ AM-1-spacer ] and [ AM-2-spacer ] are each independently a biocompatible polymer, a block copolymer, C, H, N, O, P, S, a halogen (F, Cl, Br, I) or a salt thereof, an alkyl, alkenyl, alkynyl, oxyalkyl, oxyalkenyl, oxyalkynyl, aminoalkyl, aminoalkenyl, aminoalkynyl, sulfoalkyl, sulfoalkenyl, sulfoalkynyl, phosphorylalkyl, phosphorylalkenyl, or phosphorylalkynyl; and [ branched ] is a molecule having at least three reactive groups; and [ AM-1-spacer ] is covalently linked to [ branch ] and said [ active molecule-1 ], and [ AM-2-spacer ] is covalently linked to [ branch ] and said [ active molecule-2 ].

12. The compound of claim 11, wherein the [ AM-1-spacer ] and [ AM-2-spacer ] are each independently neutral, water-soluble molecules capable of forming covalent bonds with their respective reactive molecules and [ branches ], and are selected from amino polyethylene glycol acids, polyethylene glycol diacids, amino alkanoic acids, polyglycine, 2-PEG, 4-PEG, and 6-PEG, and can each be 4-PEG.

13. The compound of any one of claims 10-12, wherein the [ branch ] is a chemical moiety comprising three orthogonal reactive groups and is selected from the group consisting of a cysteine branch, a diaminopropionic acid-based branch, a diaminobutyric acid-based branch, an ornithine-based branch, a lysine-based branch, a homocysteine branch, a bismaleimide branch, and a maleimide-acid branch, and derivatives and analogs thereof, and the following structure:

14. the compound of claim 13, wherein [ branched ] -L is selected from:

15. the compound of any one of claims 10-14, wherein the [ active molecule 2]]Is of the formula: r³- [ Ang 2-peptide]-R⁴Wherein R is³Is absent or is CH₃、C(O)CH₃、C(O)CH₃、C(O)CH₂CH₃、C(O)CH₂CH₂CH₃、C(O)CH(CH₃)CH₃、C(O)CH₂CH₂CH₂CH₃、C(O)CH(CH₃)CH₂CH₃、C(O)C₆H₅、C(O)CH₂CH₂(CH₂CH₂O)_1-5Me, amino-2-PEG, N-acyl and N-alkylamino protecting groups, fatty acid groups or hydrocarbon groups; and R is⁴Is absent or OH, NH₂、NH(CH₃)、NHCH₂CH₃、NHCH₂CH₂CH₃、NHCH(CH₃)CH₃、NHCH₂CH₂CH₂CH₃、NHCH(CH₃)CH₂CH₃、NHC₆H₅、NHCH₂CH₂OCH₃、NHOCH₃、NHOCH₂CH₃A carboxyl protecting group, a lipid fatty acid group or a hydrocarbon group, and wherein the [ Ang 2-peptide]Comprises and Q¹X²Y³Q⁴X⁵L⁶D⁷E⁸X⁹D¹⁰X¹¹X¹²X¹³X¹⁴D¹⁵X¹⁶F¹⁷M¹⁸X¹⁹Q²⁰Q²¹G²²(SEQ ID NO: 107) substantially homologous sequence, wherein X²Selected from K, N, R, H, Kac, Nick and CbcK, X⁵Selected from P, hP, dhP and BnHP, X⁹Selected from L, I, ThA and Kac, X¹¹Selected from Q, N, C, K, Kac, Dab and Dap, X¹²Selected from L, HL, Nva, I, HchA, HF and ThA, X¹³Selected from L, HL, Nva, I, HchA, HF and ThA, X¹⁴Selected from aromatic residues, X¹⁶Selected from Q and N, X¹⁹Selected from L and I, and [ Ang-2-peptide]By attaching the N-terminal amino group or C-terminal carboxyl group of a residue via a nucleophilic side chain or Ang2-]Covalently linked, said Ang 2-linking residue is selected from the group consisting of K, R, Y, C, T, S, lysine analogs, homocysteine, homoserine, Dap, Dab, the N-terminal residue and the C-terminal residue, and wherein Q¹、E⁸、X⁹、X¹¹、X¹²、D¹⁵、X¹⁶、M¹⁸、X¹⁹Or G²²One of which is replaced by the Ang 2-linking residue.

16. The compound of claim 15, wherein the [ Ang 2-peptide ] comprises an amino acid sequence identical to a sequence selected from SEQ ID NOs: 137-191 or a compound thereof.

17. The compound of claim 16, wherein R¹Is C (O) CH₃，R²Is NH₂And R is¹- [ Ang-peptide]-R²Is (SEQ ID NO: 153): { C (O) CH₃}-Q-(Kac)-Y-Q-P-L-D-E-(Kac)-D-K-T-L-Y-D-Q-F-M-L-Q-Q-G-{NH₂And K at position 11 is with [ junction ]]Covalently linked Ang 2-linking residues.

18. The compound of any one of claims 10-17, wherein the [ VEGF-peptide ] is covalently linked to the [ linker ] through a nucleophilic residue or an N-terminal amino group or a C-terminal carboxyl group of a VEGF-linking residue selected from the group consisting of K, R, Y, C, T, S, lysine analogs, homocysteine, homoserine, Dap, Dab, N-terminal residue, and C-terminal residue.

19. The compound of any one of claims 10-18, selected from: compound 5001-compound 5028 and compound 5031-compound 5074, and can be selected from compound 5053, 5060, 5061 and 5062.

20. The compound of any one of claims 10-19, wherein the Z group, when present, is covalently attached to the binding site of the antibody.

21. The compound of any one of claims 10-20, wherein the antibody is present and the compound is selected from the group consisting of: 6001-6026, 6028, 6029 and 6031-6074 and can be selected from the group consisting of compounds 6053, 6060, 6061 and 6062.

22. The compound of claim 21, comprising the structural formula of compound 6053.

23. The compound of any one of claims 6-22, wherein the antibody is present and is a catalytic antibody and can be selected from the group consisting of an aldolase antibody, a beta-lactamase, an esterase antibody and an amidase antibody.

24. The compound of any one of claims 6-23, wherein the antibody is present and is a full length antibody, Fab ', F (ab')₂、F_v、dsF_v、scF_v、V_H、V_LBifunctional antibody comprising V starting from h38c2_HAnd V_LMinibodies of domains or V containing starting from h38c2_HAnd V_LA full length antibody of the domain, and a constant domain selected from IgG1, IgG2, IgG3, and IgG4, and can be h38C2IgG 1.

25. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any of the preceding claims, and optionally comprising a therapeutically effective amount of one or more chemotherapeutic agents, preferably a compound selected from the group consisting of: 5-fluorouracil, irinotecan, taxotere, sunitinib, axitinib, oxaliplatin, bevacizumab and cetuximab.