D-peptide compounds against VEGF

Abstract

D-peptide compounds that specifically bind to VEGF are provided. Also provided are multivalent D-peptide compounds comprising two or more domains linked via a linking component. The multivalent (e.g., divalent, trivalent, tetravalent, etc.) compounds may include a plurality of distinct domains that specifically bind to different binding sites on a protein of interest to provide high affinity binding to and potent activity against a VEGF protein of interest. Also provided are D-peptide GA and Z domains for the multivalent compounds, the polypeptides having a specific determinant motif (SDM) for specific binding to VEGF (e.g., VEGF-A). Since the protein of interest is homodimeric (e.g., VEGF-A), the D-peptide compounds may be similarly dimeric and include dimers of multivalent (e.g., divalent) D-peptide compounds. Also provided are methods for treating a disease or condition associated with VEGF or angiogenesis in a subject, such as age-related macular degeneration (AMD) or cancer.

Description

[0001] This application is a divisional application of the patent application with application number 202080038052.1, application date March 20, 2020, and name “D-peptide compounds targeting VEGF”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 822,241, filed March 22, 2019, and U.S. Provisional Patent Application No. 62 / 865,469, filed June 24, 2019, which are incorporated herein by reference in their entireties. Technical Field Background Art

[0004] Vascular endothelial growth factor (VEGF-A) is a key regulator of normal and abnormal or pathological angiogenesis. In addition to being an angiogenic factor in angiogenesis and vasculogenesis, VEGF is also a pleiotropic growth factor that exhibits multiple biological effects in other physiological processes, such as endothelial cell survival, vascular permeability and vasodilation, monocyte chemotaxis, and calcium influx. Angiogenesis is an important cellular event in which vascular endothelial cells proliferate to form new blood vessels from the existing vascular network. Angiogenesis is associated with the pathogenesis of a variety of conditions, such as tumors, proliferative retinopathy, age-related macular degeneration (AMD), rheumatoid arthritis (RA), and psoriasis. Angiogenesis is crucial for the growth of most primary tumors and their subsequent metastasis in a variety of cancers.

[0005] In patients with diabetes and other ischemic retinopathy, the concentration of VEGF-A in the eye fluid is relevant to the presence of active blood vessel proliferation. In addition, in patients with AMD, VEGF is located in the choroidal neovascular membrane. Dry AMD occurs before wet AMD. The feature of dry AMD is the occurrence of yellow-white deposits under the retina, as well as varying degrees of thinning and dysfunction of retinal tissue, but lacks any abnormal new blood vessel growth. When new and abnormal blood vessels invade the retina, dry AMD changes to wet AMD. This abnormal new blood vessel growth is called choroidal neovascularization (CNV). Anti-VEGF-A drugs are used to treat wet AMD.

[0006] VEGF-A-targeted therapies are used to treat a variety of cancers. However, in some cases, patients eventually develop resistance to such therapies. Currently, combination therapies targeting VEGF-A and one or more additional cancer targets, such as programmed cell death protein 1 (PD-1) or programmed death ligand 1 (PD-L1), are gaining attention. For example, combination therapy targeting VEGF-A and PD-L1 using bevacizumab and atezolizumab has shown a reduced risk of disease progression or death in patients with PD-L1-positive metastatic renal cell carcinoma.

[0007] The ability to manipulate the interactions of proteins such as VEGF-A is of interest both for basic biological research and for the development of therapeutics and diagnostics. Protein ligands can form large binding surfaces with multiple contacts with target molecules, resulting in binding events with high specificity and affinity. For example, antibodies are a class of proteins that generate specific and tightly binding ligands for various target proteins. Additionally, Mandal et al. (“Chemical synthesis and X-ray structure of a heterochiral {D-protein antagonist plus VEGF} protein complex by racemic crystallography,” Proc. Natl. Acad. Sci. USA 109, 14779-14784 (2012)) and Uppalapati et al. (“A potent D-protein antagonist of VEGF-A is nonimmunogenic, metabolically stable and longer-circulating in vivo,” ACS Chem Biol (2016)) describe D-protein antagonists of VEGF-A. Due to the diversity of target molecules and the binding properties of protein ligands, the preparation of binding proteins with applicable functions has attracted attention. Summary of the Invention

[0008] D-peptide compounds that specifically bind to vascular endothelial growth factor (VEGF) are provided. The compounds of the present invention may include a VEGF-A binding GA domain. The compounds of the present invention may include a VEGF-A binding Z domain motif. Also provided are multivalent compounds comprising two or more D-peptide domains of the present invention connected via a linking component. Multivalent (e.g., bivalent, trivalent, tetravalent, etc.) D-peptide compounds may include multiple distinct domains that specifically bind to different binding sites on a target protein to provide high-affinity binding to the VEGF target protein and potent activity against the VEGF target protein. Also provided are D-peptide GA and Z domains for use in the multivalent compounds, the polypeptides having a specificity determining motif (SDM) for specific binding to VEGF (e.g., VEGF-A). Since the target protein is homodimeric (e.g., VEGF-A), the D-peptide compounds may similarly be dimeric and include dimers of multivalent (e.g., bivalent) D-peptide compounds. The D-peptide compounds of the present invention are useful in a variety of applications requiring specific binding to the VEGF-A target. Methods of using the compounds are provided, including methods for treating a disease or condition associated with VEGF in a subject or associated with angiogenesis in a subject, such as methods for treating age-related macular degeneration (AMD) or cancer in a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 Shown is a view of the X-ray crystal structure of exemplary compound 1.1.1 (c21a) (white sticks) in complex with VEGF-A (space filling diagram). The binding site residues for VEGF-A are depicted in pink. The VEGF-A (8-109) binding site residues are indicated in bold: GQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMR CGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCEC RPKKD (SEQ ID NO: 88).

[0010] Figure 2 Shown is an overlay of the X-ray crystal structure of exemplary compound 1.1.1(c21a) (white sticks) in complex with VEGF-A (space-filling diagram) and the structure of a D-protein antagonist as described by Mandal et al. (PNAS 109, 14779-14784 (2012)) (magenta sticks). The VEGF-A binding site residues are depicted in pink. The structure shows that compound 1.1.1(c21a) binds to the same antagonist site as the compound of Mandal et al.

[0011] Figures 3A-3BShown is a side-by-side comparison of the L-protein GA domain and the three-helix bundle structure of an exemplary D-peptide compound that specifically binds to VEGF-A. Figure 3A Shown is a view of the X-ray crystal structure of the L-protein GA domain (Protein Data Bank structure 1tf0) and a schematic indicating the arrangement of helices 1-3. Figure 3B A similar view of the X-ray crystal structure of compound 1.1.1 (c21a) in complex with VEGF-A (not shown in this view) is shown, along with a schematic diagram indicating the arrangement of helices 1-3.

[0012] Figure 4 A view of the X-ray crystal structure of compound 1.1.1 (c21a) in complex with VEGF-A (not shown in this view) is shown. Helix 1 (201), helix 2 (202), and helix 3 (203) are α-helical regions of the D-peptide compound that correspond to those of the native GA domain. 206 is a phenylalanine residue at position 31 (f31). 205, 207, and 210 are histidine residues at positions 27 (h27), 34 (h34), and 40 (h40), respectively. 209 is a tyrosine residue at position 37 (y37). 204 and 208 are the terminating proline residues of helix 2 at positions 26 (p26) and 36 (p36), respectively.

[0013] Figure 5 The binding interface between an exemplary D-peptide compound (1.1.1 (c21a); ​​stick diagram) and VEGF-A (space-filling diagram) obtained from the X-ray crystal structure of the complex is depicted. Residue f31 (206) of the compound protrudes into the binding pocket of VEGF-A at the binding interface of the complex. Histidine residues at positions 27 (205), 34 (207), and 40 (210) make additional contacts with VEGF-A at the binding interface. The side chain of residue y37 (209) protrudes toward the surface of VEGF-A but does not make close contacts.

[0014] Figures 6A-6D Structural models of compounds of the present invention based on a three-helix bundle structure are depicted. Figure 6A A schematic diagram showing the arrangement of the three helices in the native GA domain is shown. Figure 6B Schematic diagram showing the arrangement of the three helices in the D-peptide GA domain motif. Figure 6C The Degrado structural model of an antiparallel triple helix based on the hydrophobic packing of heptad repeat units is shown; the seven-residue motif (abcdefg)n forming the helical segments has characteristic residues at specific positions of the motif. Figure 6DAdaptation of the DeLaduo heptad repeat model to the D-peptide triple helical domain motif is shown.

[0015] Figures 7A-7B The three-helix bundle structural model of the D-peptide compound of the present invention is depicted. Figure 7A A first arrangement of helices 1-3 as found in the GA domain motif is depicted. Figure 7B The structural model of the three-helix bundle of the compound of the present invention is shown.

[0016] Figures 8A-8C Structural models of the compounds of the present invention based on the two-helical complex structure are depicted. Figure 8A A first arrangement of helix AB consistent with the helix AB found in the GA domain motif is depicted in side and top views, where N and C represent the N- and C-termini of the peptide compound. Figure 8B Shown is a heptad repeat model of the structure of the two-helical complex of the compound of the invention, including the gg face that contacts VEGF-A. Figure 8C The variant motif is depicted, which includes a heptad repeat model located in a two-helical complex bounded by helices A and B (see Figure 8B ) of the solvent-exposed c and g positions (blue) of the selected VEGF-A contact residues, where h* is histidine or its analog, f* is phenylalanine or its analog, and u is a non-polar amino acid residue. Figure 8C In the , "_" indicates the position of the basic scaffold domain, and the dotted lines indicate the positions of possible inter-helical contacts or connections of the residues.

[0017] Figures 9A-9C Structural models of compounds of the invention relating the compound sequence to a three-helix bundle structure are depicted. Figure 9A A three-dimensional image of a portion of a heptad repeat model for an exemplary compound is shown. Selected residues of compound 1.1.1 (c21a) are assigned to positions within the heptad repeat unit model consistent with the X-ray crystal structure of the compound in complex with VEGF-A. The VEGF-A binding face of the compound, defined by helices 2 and 3, corresponds to the gg face of the heptad repeat model. Figure 9B Shown is a view of the X-ray crystal structure of compound 1.1.1 (c21a), with the a and d residues of the heptapeptide register shown in red, packed in the core of the three-helix bundle structure. Figure 9C A linear alignment of the sequence to the heptad repeat model of the tertiary structure is shown (H1 = Helix 1; H2 = Helix 2; H3 = Helix 3), with core residues indicated in red and selected VEGF-A contact residues in blue. Figure 9A The structural model described in the Figure 9CThe registration shown in shows all residues in each of helices 1 to 3. For simplicity, only a portion of the structure is depicted.

[0018] Figures 10A-10B Further depictions of specific and general heptad repeat models for compounds of the invention are provided. Figure 10A An alignment of the sequence of exemplary compound 1.1.1 (c21a) with the heptad repeat model of tertiary structure is shown, with arrows depicting hydrophobic contacts of core residues between helices of the three-helix bundle. Figure 10B The variant motif is depicted, which includes solvent-exposed c and g positions located on the gg face defined by helices 2 and 3 (see Figure 7B and 8A ) wherein h* is histidine or an analog thereof, f* is phenylalanine or an analog thereof, and u is a non-polar amino acid residue. Figure 10B In FIG, “_” indicates the position of the basic scaffold domain, and the dashed lines indicate hydrophobic contacts of core residues between the helices of the possible three-helix bundle.

[0019] Figure 11 Shown is an enlarged stick view of a portion of the X-ray crystal structure of an exemplary D-peptide compound (1.1.1 (c21a)) obtained from a binding complex with VEGF-A (not shown). The fragment corresponds to a portion of the helix 2-linker 2-helix 3 region spanning positions 26-45. 202 indicates helix 2 and 203 indicates helix 3, which is joined by linker 2. Hydrophobic residues at positions 32, 35, 41, and 44 are included in the helix 2-helix 3 intramolecular contacts.

[0020] Figure 12 An enlarged ribbon view of a portion of the X-ray crystal structure of the L-protein GA domain (1tf0) is shown. The view corresponds to a portion of the helix 2 to helix 3 region spanning positions 31-44. 102 and 103 are α-helical regions of the native GA domain structure corresponding to the helix 2 (202) and helix 3 (203) regions, respectively. Linker 2 is the linker region. Residues at positions 32, 35, 41, and 44 are shown, which are part of the intramolecular hydrophobic contacts between helix 2 and helix 3, with Figure 12 Similar to the one shown.

[0021] Figure 13 Shown are a structural depiction and base sequence (SEQ ID NO: 2) of the scaffolded library SCF32 ​​based on the GA domain of protein G (e.g., Protein Data Bank (PDB) structure 1tf0), including sequence positions (bold) randomized for mirror-image phage display screening against VEGF-A.

[0022] Figure 14 An alignment of a series of GA scaffold domains of interest (SEQ ID NOs: 6-21) and the GA domain consensus sequence (SEQ ID NO: 1) is shown (Johansson et al. ("Structure, Specificity, and Mode of Interaction for Bacterial Albumin-binding Modules", J. Biol. Chem., Vol. 277, No. 10, pp. 8114-8120, 2002)). Figure 1 , which may be suitable for use as a scaffold domain for the compounds of the present invention.

[0023] Figure 15 Shown is an alignment of the GA scaffold domain (SEQ ID NO: 2) with the following exemplary VEGF-A binding compounds: 1 (SEQ ID NO: 106), 1.1 (SEQ ID NO: 22), 1.1.1 (SEQ ID NO: 23), and 1.1.1 (c21a) (SEQ ID NO: 24).

[0024] Figure 16 Melting and refolding curves are shown for exemplary compound 1.1.1. The melting temperature was determined to be approximately 50°C.

[0025] Figure 17 Shown are views of the X-ray crystal structure of the dimeric complex between an exemplary D-peptide compound (1.1.1(c21a); ​​stick diagram) and VEGF-A (space-filling diagram).

[0026] Figures 18A-18B Depicted is the design of an exemplary compound ((-)-TIDQW) with a truncated N-terminus relative to compound 1.1.1 (c21a). Figure 18A Shown is a magnified view of the X-ray crystal structure of the complex between an exemplary D-peptide compound (1.1.1(c21a); ​​stick diagram) and VEGF-A (space-filling diagram), demonstrating that the N-terminal residues of helix 1 do not contact helix 2 or helix 3. In some cases, selected N-terminal residues can be truncated from helix 1 without significant loss of stability or binding affinity. Figure 18B Shown is a side-by-side comparison of the structures of truncated (-)TIDQW and non-truncated (+)-TIDQW compound 1.1.1 (c21a).

[0027] Figures 19A-19C Shown are a series of positions in the compounds where affinity maturation or the incorporation of optionally selected point mutations was performed. Figure 19A and19B Depicts the separation obtained from the X-ray crystal structure ( Figure 19A ) or in combination with VEGF-A ( Figure 19B ) of compound 1.1.1 (c21a). Figure 19C The sequence of compound 1.1.1 (c21a) is shown (SEQ ID NO: 24) and mutations of interest are annotated.

[0028] Figure 20 Shown is a magnified view of the X-ray crystal structure of compound 1.1.1(c21a) (stick diagram) in complex with VEGF-A (space-filling diagram), in which the phenylalanine (f) residue at position 31, shown in yellow, protrudes into the binding pocket of VEGF-A at the binding interface of the complex.

[0029] Figure 21 A magnified view of the side chain of the f31 residue protruding into the binding pocket of the VEGF-A binding interface is shown, with selected distances between the phenyl ring and adjacent VEGF-A residues shown in angstroms. Analysis of the complex structure suggests that various phenylalanine analogs can be tolerated at position 31, such as analogs that include substituents at positions 3, 4, and / or 5 of the phenyl ring that can occupy the available space (4.6 to 5.3 angstroms) of the VEGF-A binding pocket.

[0030] Figure 22 A magnified view of the X-ray crystal structure of compound 1.1.1 (c21a) (stick diagram) in complex with VEGF-A (space-filling diagram) is shown, showing selected helix 2 contacts. 205 and 207 are histidine residues at positions 27 and 34, respectively. The structure shows a weak hydrogen bond (approximately 4.6 angstroms) between the nitrogen atom of histidine 34 (h34; 207) and the adjacent Asp90 of VEGF-A. 209 is the tyrosine residue at position 37 of the compound, which protrudes toward the VEGF-A surface. Analysis of the complex structure suggests that various histidine analogs are tolerated at positions 27 and 34, including, for example, substituted or unsubstituted aryl or heterocyclic analogs that can occupy available space on the VEGF-A surface and / or form stronger hydrogen bonds (e.g., lengths < 4.6 angstroms) with adjacent VEGF-A residues.

[0031] Figure 23A magnified view of the X-ray crystal structure of compound 1.1.1 (c21a) (stick diagram) in complex with VEGF-A (space-filling diagram) is shown, showing selected helix 3 contacts. The structure shows a moderately strong hydrogen bond (2.9 angstroms) between the nitrogen atom of histidine 40 (h40; 210) and the adjacent residue Tyr48 of VEGF-A. Analysis of the complex structure suggests that a variety of histidine analogs can be tolerated at position 40, including analogs that can occupy the available space and maintain or strengthen hydrogen bonding with VEGF-A.

[0032] Figure 24 A magnified view of the X-ray crystal structure of compound 1.1.1 (c21a) (pink and green sticks) in complex with VEGF-A (cyan ribbon) is shown, focusing on the tyrosine (y) residue (209) at position 37 of linker 2. The distances between the y37 oxygen and the oxygen or nitrogen atoms of adjacent residues on the surface of VEGF-A are shown, e.g., 6.5 and 7.2 angstroms, suggesting that a variety of tyrosine analogs may be tolerated at position 37, including, for example, substituted or unsubstituted alkyl-aryl or alkyl-heteroaryl extended side chain groups that can form closer contacts (e.g., hydrophobic contacts and / or hydrogen bonds) with adjacent VEGF-A residues.

[0033] Figure 25 A magnified view of the X-ray crystal structure of compound 1.1.1 (c21a) (stick diagram) in complex with VEGF-A (space filling diagram) is shown, focusing on the histidine residue (h) (205) at position 27. Analysis of the structure suggests that a variety of aromatic residues or histidine analogs can be utilized at position 27 to contact the same pocket on the surface of VEGF-A and, in some cases, increase the desired hydrophobic contacts. Also shown is the glutamic acid residue at position 25 (e25, 211) of the [Linker 1] region, which makes contacts with VEGF-A, including a hydrogen bond (2.5 angstroms) to the backbone carbonyl group of the peptide backbone of VEGF-A.

[0034] Figure 26 Shown are sequence logos for selected positions of all clones identified during the phage display mirror image screening for D-VEGF-A binders, aligned compared to the corresponding residues of the compound 1 sequence and the native GA domain (GA-wt).

[0035] Figures 27A-27B The L-protein GA domain is shown ( Figure 27A ) and D-compound 1.1.1(c21a)( Figure 27B ) show that the alignment angle between helices 2 and 3 is increased in the VEGF-A binding compound.

[0036] Figures 28A-28B Two depictions of the X-ray crystal structure of the D-peptide compound 11055 bound to a VEGF-A homodimer are shown. Figure 28A It was shown that D-peptide compound 11055 binds to VEGF-A primarily through binding contacts of helix 2 (H2) of the variant GA domain of compound 11055. Figure 28B Shows Figure 28A Figure 1 shows the structure of D-peptide compound 11055, shown as a space-filling model, superimposed on the structure of VEGFR2 (domains 2 and 3) bound to VEGF-A. The overlay shows that D-peptide compound 11055 blocks the binding of domain 2 (D2) of VEGFR2 to VEGF-A.

[0037] Figures 29A-29B The structure of the affinity maturation library is shown ( Figure 29A ) and sequence( Figure 29B ), the affinity maturation library was designed to screen and identify residues at specific positions that stabilize the folding of the variant GA domain of compound 11055. A total of seven residues were selected for mutations at the packing interface between helix 1 (H1) and the loop connecting helix 2 (H2) and helix 3 (H3).

[0038] Figures 30A-30C Shown are the results of a screen for high affinity VEGF-A binding compounds comprising a consensus sequence signature having cysteine ​​residues at positions 7 and 38 ( Figure 30A ), and the selected variant sequence of interest ( Figure 30B ) (SEQ ID NO: 108-113) and their binding affinity to VEGF-A relative to the parent compound 11055. Figure 30C The structure of the parent compound 11055 is shown ( Figure 29A ), in which the identified variant amino acid residue positions l7c and v38c, shown in yellow, are close to each other (βC to βC interhelical distance of 5.9 angstroms), such that inclusion of the l7c and v38c variants will result in the formation of a stabilizing disulfide bond between those residues.

[0039] Figures 31A-31B Shown are graphs of data demonstrating the activity of VEGF-A D-peptide. Figure 31A VEGF-A antagonistic activity of selected compounds in VEGFR1 binding ELISA is shown. Figure 31B Inhibition of cell proliferation in response to VEGF signaling by selected compounds relative to a bevacizumab control is shown.

[0040] Figures 32A-32B The structure of the phage display library based on the parental Z domain scaffold is shown ( Figure 32A) and sequence( Figure 32B Ten positions (X) were selected within helix 1 to helix 2 of the Z domain to be randomized using Kunkel mutagenesis with three nucleotide codons representing all amino acids except cysteine ​​( Figure 32B ).

[0041] Figures 33A-33B Shown is the structure of a mirror-image phage-displayed Z domain phage display library screened for binding to VEGF-A. Figure 33A Consensus sequence logos providing binding to VEGF-A are shown. Figure 33B Selected variant Z domain sequences of interest (SEQ ID NOs: 114-118) and their binding affinities to native L-VEGF-A are shown. NB means nonbinding.

[0042] Figure 34 Shown are surface plasmon resonance (SPR) sensorgrams showing additive binding of compounds 978336 and 11055, indicating that compound 978336 (variant Z domain compound) binds to a non-overlapping and independent binding site on VEGF-A from that of compound 11055 (variant GA domain compound).

[0043] Figures 35A-35G Three depictions of the X-ray crystal structure of the D-peptide compound 978336 bound to a VEGF-A homodimer are shown. Figure 35A Two monomeric D-peptide compounds 978336 are shown bound to their VEGF-A binding site. Figure 35B Shows Figure 35A Figure 1 shows the structure of D-peptide compound 978336, shown as a space-filling model, superimposed on the structure of VEGFR2 (domains 2 and 3) bound to VEGF-A. The overlay shows that D-peptide compound 978336 blocks the binding of domain 3 (D3) of VEGFR2 to VEGF-A. Figure 35C The structure of isolated 978336 is shown, focusing on the VEGF-A binding face of the compound, with variant amino acid residues selected from the Z domain library shown in red. Figure 35D Shown are protein-protein contacts (upper panel) and a magnified view of the binding site on VEGF-A (lower panel) for compound 978336 (SEQ ID NO: 117), including the configuration of variant amino acids that contact the binding site (upper panel). Figures 35E-35G Shown are exemplary VEGF-A binding compound 978336 (SEQ ID NO: 117), the identified consensus sequence (SEQ ID NO: 158) ( Figure 35F) and affinity maturation studies of the sequence of exemplary compound 980181 (SEQ ID NO: 119).

[0044] Figures 36A-36B Shown are structure-based designs of exemplary bivalent compound conjugates, including compounds 11055 and 978336 ( Figure 36A ). Figure 36B Shown is the sequence of the bivalent compound 979111 including N-terminal to N-terminal linkage via bifunctional conjugation with bismaleimide PEG8, which exhibited a binding affinity of 1.7 nM for L-VEGF-A as measured by SPR.

[0045] Figures 37A-37B The structure of the phage display library (SEQ ID NO: 159) based on the parental GA domain scaffold (SEQ ID NO: 2) is shown ( Figure 37A ) and sequence( Figure 37B Eleven positions (X) were selected within helix 2 to helix 3 of the GA domain scaffold to be randomized using Kunkel mutagenesis with three nucleotide codons representing all amino acids except cysteine.

[0046] Figures 38A-38E The design, synthesis, and sequence of exemplary dimeric bivalent (ie, tetradomain-containing) compounds 980870 and 980871 are shown. Figure 38A Shown are depictions of the X-ray crystal structures of exemplary compounds 11055 and 978336 bound to VEGF-A, and designs of linkers used to generate exemplary dimeric, bivalent VEGF-A binding compounds. Residue K19 of compound 11055 and residue K7 of compound 978336 can be linked via their side chain amino groups via a linker, for example, of about 23 angstroms or greater in length. Figure 38B Shown is a synthetic scheme for preparing linked tetradomain compounds 980870 and 980871. D-Pra is a D-propargylglycine residue linked via a -NH-PEG2-CO-linker to the amine side chain of k7 of compound 980181. An azido-CH2CONH-PEG2 / 3-CO- group is attached to the amine side chain of k19 of compound 979110 and subsequently conjugated to the propargyl group using click chemistry to form the interdomain linker. Figure 38C Shows the Figure 38B A depiction of the sequences of exemplary four-domain compounds prepared by the scheme. Figure 38D is a schematic diagram of an exemplary bivalent compound including a linker L between residue x19 of the GA domain and residue x7 of the Z domain. 1 . Figure 38Eis a schematic diagram of an exemplary dimeric bivalent compound including a second linker L between the C-terminal residues of the GA and Z domains. 2 .

[0047] Figures 39A-39B Shown are measurements of the in vitro ( Figure 39A ) and cell-based ( Figure 39B ) is a graph showing the analysis results of the antagonistic activity of .

[0048] Figures 40A-40C Activity data for a D-protein VEGF-A antagonist developed using mirror-image phage display are shown. Figure 40A ) Phage titration ELISA against GA domain and Z domain hits of D-VEGF-A target, showing titratable binding. ( Figure 40B ) Phage competition ELISA using synthetic L-enantiomers corresponding to GA domain hits as soluble competitors for phage binding to D-VEGF-A. ( Figure 40C ) Titration of synthetic D-proteins RFX-11055 and RFX-978336 in a VEGF-A blocking ELISA showing antagonistic activity relative to bevacizumab.

[0049] Figures 41A-41F The structures of D-proteins RFX-11055 and RFX-978336 in complex with VEGF-A are shown. ( Figure 41A and 41B ) Overview of RFX-11055 (purple) and RFX-978336 (blue) binding to distinct, non-overlapping epitopes at the distal end of the VEGF-A homodimer (grey). ( Figure 41C and 41D ) Interface D-amino acid side chains contacting VEGF-A depicted for RFX-11055 and RFX-978336, with selected library residues (orange) and native scaffold residues (blue) within helices 2 and 3 of RFX-11055 and helices 1 and 2 of RFX-978336. VEGF-A is shown with electrostatic surface potential to highlight positive (blue), negative (red), and neutral hydrophobic (white) contact sites. ( Figure 41E ) Previously reported crystal structure of VEGF-A (grey) in complex with the VEGFR-1 receptor (light orange). Ig domains 2 and 3 (D2 and D3) of VEGFR-1 were isolated to highlight the molecular interactions involved in VEGF-A (PDB code: 5T89) receptor engagement (24). Figure 41F) Overlay of RFX-11055 and RFX-978336 on the VEGF-A / VEGFR-1 complex to demonstrate direct competition with D2 and D3 as the mechanism of VEGF-A blockade.

[0050] Figures 42A-42C Structure-guided affinity maturation of RFX-11055 and RFX-978336 is shown. Figure 42A ) Structure of RFX-11055 (purple) bound to VEGF-A (grey), showing seven residues (orange) that were targets of the affinity maturation library to stabilize the packing between helix 1 and the helix 2-3 binding interface. ( Figure 42B ) Structure of RFX-978336 (blue) bound to VEGF-A (grey), showing the helix 1-2 binding interface and the four residues selected for the soft randomization library. ( Figure 42C ) Titration of affinity matured D-proteins RFX-979110 and RFX-980181 in a VEGF blocking ELISA showing antagonistic activity relative to bevacizumab.

[0051] Figures 43A-43B The D-protein heterodimeric VEGF-A antagonist activity was shown in vitro. Figure 43A Titration of affinity-matured D-protein RFX-979110 and high-affinity heterodimer RFX-980869 compared to bevacizumab and VEGFR1-Fc soluble decoy receptor in a VEGF-A blocking ELISA. Figure 43B ) Cell viability assays showed that RFX-980869 potently blocked VEGF-A signaling through VEGFR2, with efficacy comparable to that of bevacizumab.

[0052] Figures 44A-44B In vivo activity of D-protein RFX-980869 was demonstrated in a rabbit eye model of wet AMD. Figure 44A ) Representative fluorescein angiography (FA) images depicting the extent of VEGF-A165-induced vascular leakage at day 5 and day 26 after administration of the corresponding drug (control = no drug treatment). ( Figure 44B ) Graphs of individual FA scores at day 5 and day 26. Scoring is as follows: 0 = straight large vessels, some tortuosity of small vessels, and no dilatation; 1 = increased tortuosity of large vessels and some dilatation; 2 = leakage and marked dilatation between large vessels; 3 = leakage between large and small vessels, with still visible small vessels; 4 = leakage between large and small vessels, with poor or no visibility of small vessels. N = 5 rabbits (10 eyes) per group. All data are plotted as mean ± SEM. (****p < 0.0001, Mann-Whitney test)

[0053] Figures 45A-45D RFX-980869 demonstrated tumor growth inhibitory activity and lack of immunogenicity. Figure 45A ) MC38 tumor growth curves in C57BL6 mice, showing dose-dependent efficacy of both RFX-980869 and nivolumab. N=6 mice per group. ( Figure 45B ) Tumor volume on day 15 (*p<0.05, Mann-Whitney test) ( Figure 45C ) Anti-drug antibodies from the MC38 tumor study measured in day 22 serum samples using ELISA for antigen-specific serum IgG. ( Figure 45D ) Anti-drug antibody titers measured from serum on day 42 after subcutaneous immunization of BALB / c mice with the corresponding drugs. N = 5 mice per group. All data are plotted as mean ± SEM.

[0054] Figures 46A-46C The sequences of the phage display library and D-protein are shown. Figure 46A ) GA domain scaffold sequences and libraries used for panning. The underlined residues in the GA library were hard randomized using NNK codons to obtain full amino acid diversity. The underlined residues in the AM library were hard randomized using NNC codons to obtain a diversity of 15 amino acids including cysteine. The lowercase amino acids of RFX-11055 and RFX-979110 represent D-amino acids. Sequences from top to bottom: (SEQ ID NO: 2; SEQ ID NO: 108; SEQ ID NO: 108; SEQ ID NO: 108; SEQ ID NO: 113) ( Figure 46B ) Z domain scaffold sequences and libraries used for panning. The underlined residues in the GA library were hard randomized using trinucleotide codons for each amino acid except cysteine ​​for full amino acid diversity. The underlined residues in the AM library were soft randomized using codons to incorporate a 30% mutation rate at each amino acid. Lowercase amino acids for RFX-978336 and RFX-980181 represent D-amino acids. Sequences from top to bottom: SEQ ID NO: 163; SEQ ID NO: 117; SEQ ID NO: 117; SEQ ID NO: 117; SEQ ID NO: 119). ( Figure 46C ) Full D-amino acid sequence of heterodimeric antagonist 980869.

[0055] Figure 47 Shown are SPR sensorgrams of kinetic binding parameters measured for D-protein and bevacizumab.

[0056] Figure 48 SPR-based epitope mapping of RFX-978336 and RFX-11055 is shown. In the first association step, 5 μM RFX-978336 was used to saturate VEGF-A on the chip surface. In the second association step, 1 μM RFX-11055 was included along with 5 μM RFX-978336 and exhibited additive binding to VEGF-A, indicating that the site of RFX-11055 was not blocked by RFX-978336. Both D-proteins showed complete dissociation from VEGF-A.

[0057] Figures 49A-49B Structural representation of the VEGF-A / VEGFR-1 contact is shown. ( Figure 49A ) A previous structure solved for VEGF-A (grey) in complex with VEGFR-1 (light orange), depicting the epitope on VEGF-A contacted by the D2 and D3 Ig domains of VEGFR-1, colored by element (white carbon, red oxygen, blue nitrogen, and yellow sulfur) (PDB ID: 5T89, 24). ( Figure 49B )( Figure 49A ) in which the D2 and D3 domains are rotated 180 degrees away from VEGF-A and the electrostatic surface potential of the two molecules is shown. The D2 and D3 binding sites are circled, highlighting the primarily non-polar, hydrophobic nature of the D2 interaction and the polar, hydrophilic nature of the D3 interaction.

[0058] Figures 50A-50B Shown is the design and synthesis of the heterodimeric D-protein RFX-980869. ( Figure 50A ) Overlay of the structures of RFX-11055 (purple) and RFX-978336 (blue) bound to VEGF-A (grey), showing the lysine residues (K19 on RFX-11055 and K7 on RFX-978336) represented as spheres and distance measurements for the proposed PEG linkage. Figure 50B ) Synthetic scheme for the generation of the D-protein heterodimer RFX-980869 using solid phase peptide synthesis with peptides equipped with 'click' chemistry functionalities and PEG moieties.

[0059] Figure 51 Shown is a table with a summary of SPR-derived kinetic binding parameters for protein D and bevacizumab.

[0060] Figure 52 Shown is a table with a summary of IC50 values ​​for protein D and bevacizumab blocking VEGF-A121 binding to VEGFR1-Fc in a non-equilibrium ELISA.

[0061] Figure 53Shown is a table with data collection and optimization statistics for the VEGF / D-protein complex.

[0062] Figure 54 Shown is a table summarizing the IC50 values ​​of Protein D and Bevacizumab for blocking VEGF-121A binding to VEGFR1-Fc in an equilibrium binding ELISA and for inhibiting VEGF-A signaling in a cell signaling assay.

[0063] Figure 55 Shown are sequence logos for selected positions of all clones identified during the phage display mirror image screening for D-peptide Z domain VEGF-A binders, aligned compared to the corresponding residues of the native Z domain (Z-wt). DETAILED DESCRIPTION

[0064] Multivalent D-peptide binding compounds

[0065] As outlined above, aspects of the present disclosure include multivalent D-peptide compounds that specifically bind to VEGF with high affinity. The present disclosure provides a class of multivalent compounds that are capable of specifically binding to a VEGF target protein at two or more distinct binding sites on the target protein. The term "multivalent" refers to an interaction between a compound and a target protein that can occur at two or more separate and distinct sites on a target protein molecule. Multivalent D-peptide compounds are capable of a variety of binding interactions that can occur collaboratively to provide high-affinity binders for the target protein and potent biological effects on the function of the target protein. The term "multimeric" refers to a compound comprising two (i.e., dimerization), three (i.e., trimerization) or more monomeric peptide units (e.g., domains). When the multimeric compound is homologous, each peptide unit may have the same binding properties, i.e., each monomeric unit is capable of binding to the same binding site on the VEGF target protein molecule. Such multimeric compounds are used to bind to target proteins that naturally exist in the form of homodimers or are capable of multimerization. The dimeric compound can simultaneously bind to two identical binding sites on two molecules of the VEGF target protein homodimer. In some cases, depending on the target protein, the multivalent D-peptide compounds of the present disclosure can be multimerized, for example, a dimeric bivalent D-peptide compound can include a dimer of two bivalent D-peptide compounds. In some cases, the multimeric compound is heterologous, and each peptide unit (e.g., a domain or bivalent unit) specifically binds to a different target site or protein.

[0066] The multivalent peptide compound comprises at least two peptide domains, wherein each domain has a specificity determining motif composed of variant amino acids, and the variant amino acids are configured to provide an interface for a specific protein-protein interaction at the binding site. When multiple peptide domains are linked together, they can contact the target protein at the same time and provide multiple interfaces at multiple binding sites. Multiple protein-protein binding interactions can occur collaboratively via an avidity effect to provide an effective affinity significantly higher than that achieved by any one D-peptide domain alone. The present disclosure discloses the use of mirror phage display screening using a scaffolded small protein domain library to produce multiple peptide domains that bind to multiple target binding sites, and such domains can be successfully linked to produce high-affinity binders that exhibit strong avidity effects. The multimeric compounds presented by the present inventors have an affinity comparable to or better than that of the corresponding antibody agent in vivo and provide effective biological activity against the VEGF target protein.

[0067] Generally, the VEGF target protein is a naturally occurring L-protein, and the compound is a D-peptide compound. It should be understood that for any of the D-peptide compounds described herein, the L-peptide form of the compound is also encompassed by the present disclosure, which L-peptide form specifically binds to the D-VEGF target protein. The peptide compounds of the present invention were identified, in part, by using a method of mirror-screening a variety of scaffolded domain phage display libraries for binding to a synthetic D-VEGF target protein.

[0068] Compared to corresponding L-polypeptides, D-peptide compounds can offer many desirable properties for therapeutic applications, such as proteolytic stability, significantly reduced immunogenicity, and long in vivo half-life. Compared to antibody agents targeting VEGF, the D-peptide compounds of the present disclosure are generally significantly smaller in size. In some cases, the smaller size and properties of the compounds of the present invention provide advantages over antibody-based therapeutics in terms of route of administration, tissue distribution and penetration, and dosage regimens.

[0069] The present disclosure provides a multivalent D-peptide compound comprising at least a first and a second D-peptide domain. The first and second D-peptide domains can specifically bind to different non-overlapping binding sites of a target protein and can be connected to each other via a connecting component (e.g., as described herein). The connecting component can be configured to allow simultaneous or sequential binding to the target protein. "Sequential binding" means that binding of the first D-peptide domain to the target can increase the likelihood of binding to the second D-peptide domain, even if the binding does not occur simultaneously.

[0070] The first and second D-peptide domains can be heterologous to each other, i.e., the domains are of different domain types. For example, the first D-peptide domain can be a variant GA domain and the second D-peptide domain can be a variant Z domain, or vice versa. Mirror-image phage display screening of VEGF using two different scaffolded domain libraries provides variant domain binders to two different binding sites on VEGF.

[0071] When a multivalent D-peptide compound includes only two such domains, it can be referred to as bivalent. Trivalent, tetravalent and higher multivalents are also possible. A trivalent D-peptide compound may include three D-peptide domains connected via two linking components in a linear manner or via a single trivalent linking component. A trivalent D-peptide compound may include two identical D-peptide compounds connected via a disulfide connection between two cysteine ​​residues on each D-peptide compound, and a linking component between one of the disulfide-linked D-peptide compounds and a third D-peptide compound. Tetravalent and higher multivalent compounds can similarly be connected in a linear manner via a bivalent linking component, or connected in a branched configuration via one or more multivalent or branched linking components.

[0072] Connecting components

[0073] The term "linking component" is intended to encompass a multivalent moiety capable of establishing a covalent connection between two or more D-peptide domains of a compound of the invention. Sometimes, the linking component is bivalent. Alternatively, the linking component is trivalent or dendritic. The linking component can be placed during or after synthesis of the D-peptide domain polypeptide, for example, via conjugation of two or more folded D-peptide domains. The linking component can be placed in the compound of the invention via conjugation of two D-peptide domains using a bifunctional linker. The linking component can also be designed so that it can be incorporated during synthesis of the D-peptide domain polypeptide, for example, where the linking component itself is a peptide and is prepared via solid phase peptide synthesis (SPPS) of an amino acid residue sequence. In addition, chemically selective functional groups and / or linkers can be placed during polypeptide synthesis so that the D-peptide domain can be easily conjugated after SPPS.

[0074] Any convenient linking group or linker may be suitable for use as the linking component of the multivalent compounds of the present invention. Linking groups and linker units of interest include, but are not limited to, amino acid residues, polypeptides, PEG units, (PEG)n linkers (e.g., where n is 2-50, such as 2-40, 2-30, 2-20, or 2-10), terminally modified PEGs (e.g., -NH(CH2) m O[(CH2)2O] n (CH2) p CO-, or -NH(CH2) mO[(CH2)2O] n (CH2) m NH-, or -CO(CH2) p O[(CH2)2O] n (CH2) p CO-linker, wherein m is 2-6, p is 1-6, and n is 1-50, such as 1-20, 1-12 or 1-6), C1-C6 alkyl or substituted C1-C6 alkyl linker, C2-C12 alkyl or substituted C2-C12 alkyl linker, succinyl (e.g., -COCH2CH2CO-) unit, diaminoethylene unit (e.g., -NRCH2CH2NR-, wherein R is H, alkyl or substituted alkyl), -CO(CH2) m CO-, -NR(CH2) p NR-, -CO(CH2) m NR-, -CO(CH2) m O-, -CO(CH2) m S- (wherein m is 1 to 6, p is 2-6, and each R is independently H, C(1-6) alkyl or substituted C(1-6) alkyl) and combinations thereof, for example, via a linking functional group such as an amide (e.g., -CONH- or -CONR-, where R is C1-C6 alkyl), sulfonamide, carbamate, carbonyl (-CO-), ether, thioether, ester, thioester, amino (-NH-) and the like. The linking component can be a peptide, for example, a linker comprising an amino acid residue sequence. The linking component can be of the formula -(L 1 ) a -(L 2 ) b -(L 3 ) c -(L 4 ) d -(L 5 ) e -linker, where L 1 to L 5 Each is independently a linker unit, and a, b, c, d, and e are each independently 0 or 1, wherein the sum of a, b, c, d, and e is 1 to 5. As shown in the polymeric compounds described herein, other linkers are possible.

[0075] The linking component may include a terminally modified PEG linker that is linked to the D-peptide compound using any convenient linking chemistry. PEG is polyethylene glycol. The term "terminally modified PEG" refers to a polyethylene glycol of any convenient length, wherein one or both ends are modified to include a chemoselective functional group suitable for conjugation to, for example, another linking group portion, or the end or side chain of the peptide compound. The Examples section describes the use of several exemplary terminally modified PEG bifunctional linkers with terminal maleimide functional groups for chemoselective conjugation to thiol groups, such as cysteine ​​residues placed in the D-peptide domain sequence. The D-peptide compound may be modified at the N and / or C termini of the GA domain motif to include one or more additional amino acid residues that can provide a specific connection or connection chemistry to connect to a multivalent linking group, such as cysteine ​​or lysine.

[0076] Chemoselective reactive functional groups that can be used to attach the peptide compounds of the invention via a linker include, but are not limited to, amino (e.g., an N-terminal amino group or a lysine side chain group), azide, alkynyl, phosphine, thiol (e.g., a cysteine ​​residue), C-terminal thioester, aryl azide, maleimide, carbodiimide, N-hydroxysuccinimide (NHS)-ester, hydrazide, PFP-ester, hydroxymethylphosphine, psoralen, imidoester, pyridyl disulfide, isocyanate, aminooxy-, aldehyde, ketone, chloroacetyl, bromoacetyl, and vinyl sulfone.

[0077] Any convenient multivalent linker can be utilized in the multimers of the present invention. Multivalent means that the linker includes two or more terminal or side chain groups suitable for connection to a component of the compound of the present invention, such as a peptide domain, as described herein. In some cases, the multivalent linker is divalent or trivalent. In some cases, the multivalent linker is a dendrimer scaffold. Any convenient dendrimer scaffold can be used in the multimers of the present invention. The dendrimer scaffold is a branched molecule that includes at least one branch point and two or more ends suitable for connection to the N-terminus or C-terminus of the domain via an optionally selected linker. The dendrimer scaffold can be selected to provide the desired spatial arrangement of two or more domains. In some cases, the spatial arrangement of two or more domains is selected to provide the desired binding affinity and avidity for the VEGF target protein.

[0078] In some cases, the multivalent linker group is derived from / includes a chemoselective reactive functional group that can be conjugated to a compatible functional group on the second peptide domain. In some cases, the multivalent linker group is a specific binding moiety (e.g., biotin or a peptide tag) that can specifically bind to a multivalent binding moiety (e.g., streptavidin or an antibody). In some cases, the multivalent linker group is a specific binding moiety that can directly form a homodimer or heterodimer with the second specific binding moiety of the second compound. Therefore, in some cases, when the compound includes a molecule of interest comprising a multivalent linker group, the compound can be a part of a multimer. Alternatively, the compound can be a monomer that can directly multimerize with one or more other compounds or indirectly multimerize via binding to a multivalent binding moiety.

[0079] Exemplary Multivalent D-Peptide Compounds

[0080] The present disclosure provides multivalent compounds that bind to VEGF-A. The multivalent VEGF-A binding compounds can be bivalent and include two distinct variant domains connected via a linking component (e.g., as described herein). Disclosed herein are exemplary single D-peptide domains that specifically bind to VEGF-A, binding to one of two different binding sites on a target protein. Figure 36A The crystal structures of two such single domains simultaneously bound to the target VEGF-A are shown. VEGF-A specific variant GA domain polypeptides that bind at the first binding site of VEGF-A are described herein. In some cases, the first binding site is defined by the amino acid side chains F43, M44, Y47, Y51, N88, D89, L92, I72, K74, M107, I109, Q115, and I117 of VEGF-A. In some cases, the VEGF-A specific polypeptide is a locked variant GA domain (e.g., as described herein). Any of the VEGF-A specific D-peptide variant GA domain polypeptides of the present invention can be connected to a second D-peptide domain via a connecting component that specifically binds to a second and different binding site of the target VEGF-A. In some cases, the second binding site is defined by amino acid side chains E90, F62, D67, I69, E70, K110, P111, H112, and Q113 of VEGF-A. Figure 36A , which shows that the exemplary Z domain polypeptide binds at a different site than the exemplary GA domain polypeptide compound 11055. At least one or both of the target binding sites should partially overlap with the VEGFR2 binding site on the VEGF-A target protein in order to provide antagonistic activity. See, e.g. Figure 35B .

[0081] D-peptide variant GA domain polypeptides that can be linked to a D-peptide variant Z domain polypeptide to provide a VEGF-A binding bivalent compound include, but are not limited to, compounds 11055, 979102, and 979107-979110, and variants thereof (e.g., as described herein).

[0082] D-peptide variant Z domain polypeptides that can be linked to a D-peptide variant GA domain polypeptide to provide a VEGF-A binding bivalent compound include, but are not limited to, compounds 978333 to 978337, 980181, 980174-980180, and 981188-981190, and variants thereof (e.g., as described herein).

[0083] exist Figure 36A In , a schematic diagram of a possible linking component connecting the N-termini of two D-peptide domains is shown. In some cases, the N-terminal to N-terminal linker is a (PEG)n bifunctional linker, wherein n is 2-20, such as 4-20 or 8-20 (e.g., n is 5, 6, 7, 8, 9, 10, 11 or 12). Any convenient chemoselective functional group can be incorporated into the connected D-peptide domain to provide conjugation. Interdomain connection can be achieved after peptide synthesis using compatible chemoselective functional groups (e.g., as described herein). The linking component can also be incorporated into the D-peptide polypeptide of the multivalent compound of the present invention during solid phase peptide synthesis (SPPS). See, e.g. Figure 50B .

[0084] In some cases, an N-terminal to N-terminal linker can be placed by extending the polypeptide sequence of the domain to incorporate a cysteine ​​residue that provides for conjugation to a homobifunctional PEG linker comprising a maleimide. For example, compounds 11055 and 978336 were both chemically synthesized with an additional N-terminal cysteine ​​residue that was conjugated to a bismaleimide PEG8 linker using conventional methods to provide an N-terminal to N-terminal linkage ( Figure 36A ). For example, Table 5 provides details of an exemplary bivalent compound, compound 979111, that binds VEGF-A with high affinity. Figure 50A Shown are views of the crystal structures of D-peptide domains 11055 and 978336 bound to VEGF-A, and the location of an alternative interdomain linker, from k19 of the variant GA domain to k7 of the variant Z domain, which can be used to prepare bivalent compounds from a variety of variant GA domain and Z domain polypeptides that bind to VEGF-A.

[0085] Figure 38D The general structure of an exemplary bivalent compound is shown, which includes a linker L between residue x19 of the GA domain and residue x7 of the Z domain.1 Any of the exemplary D-peptide GA domains (e.g., as described herein) and D-peptide Z domains (e.g., as described herein) can be configured as follows Figure 38D The linking component L shown in 1 In some embodiments, the X19 and X7 residues are each independently lysine and ornithine, and the linker has one of the following structures:

[0086]

[0087] wherein n and m are independently 1-12, such as 1-6; and p, q and r are each independently 0-3, such as 0 or 1; and s is 1-6, such as 1-3. 1 In some cases, n+m is 2-6, such as 3, 4 or 5. 1 In some cases, n and m are each 2. 1 In some cases, n and m are each 3. 1 In some cases, p, q, and r are each 1. 1 In some cases, p is 0. 1 In some cases, q is 0. 1 In some cases, r is 0. 1 In some cases, s is 2. 1 In some cases, s is 3.

[0088] Figure 38E is a schematic diagram of an exemplary dimeric bivalent compound including a second linker L between the C-terminal residues of the GA and Z domains. 2 . Figure 38B An exemplary linker L is shown 2 , which is used to connect the C-terminal residues of the Z domains of the two bivalent compounds and can be placed during SPPS. The C-terminal to C-terminal linker can include one or more amino acid residues and one or more linking units (e.g., as described herein). It includes at least one residue that provides branching of the amino acid (e.g., lysine) and coupling, such as coupling to an amino side chain and an α-amino group. The C-terminal to C-terminal linker can include one or more amino acid residues and one or more linking units (e.g., as described herein). In some cases, one or more residues can be placed at the C-terminus of the domain during SPPS, which provides a covalent link so that the protein domains can simultaneously bind to the VEGF target.

[0089] Exemplary Polymeric Multivalent D-Peptide Compounds

[0090] Aspects of the present disclosure include multimeric (e.g., dimeric, trimeric, or tetrameric, etc.) D-peptide compounds that include any two or more of the variable domain polypeptides and / or bivalent compounds of the present invention described herein. A multimer of the present disclosure may refer to a compound having two or more homologous domains or two or more homologous bivalent compounds. Thus, a dimer of a bivalent compound may include two molecules of any of the bivalent compounds described herein connected via a linking component. The target molecule VEGF-A may be a homodimer, and the homodimeric compound may provide binding to similar sites on each VEGF-A target monomer. For example, Figure 36A Overlays of the crystal structures of two molecules of domain 11055 and two molecules of domain 978336 bound to a VEGF-A dimer are shown. Exemplary sites for incorporating chemical linkages to connect the four domains are indicated. Figure 38B and 38C In some cases, dimerization of the bivalent compound is achieved using a peptide linker between the C-termini of the domains (11055+978336). For example, Tables 5 and Figure 38C Shown are the sequences and configurations of exemplary VEGF-A binding dimeric bivalent compounds 980870 and 980871, which were presented during SPPS (see Figure 38B ) or after SPPS (e.g., as described herein), any convenient linking group can be attached to the C-terminus of the polypeptide domain to introduce the dimerization linking component.

[0091] Peptide domain

[0092] Any suitable peptide domain can be used in the compounds of the present invention. In phage display screening, a variety of small protein domains have been utilized, which can be applicable to use in the method for mirror-image screening for target proteins as described herein. The small peptide domain of interest can be composed of 25 to 80 amino acid residues, such as 30 to 70 residues, 40 to 70 residues, 40 to 60 residues, 45 to 60 residues, 50 to 60 residues or 52 to 58 residues of single-chain polypeptide sequence composition. The molecular weight (MW) of the peptide domain can be 1 to 20 kilodaltons (kDa), such as 2 to 15kDa, 2 to 10kDa, 2 to 8kDa, 3 to 8kDa or 4 to 6kDa.

[0093] The peptide domain may be a three-helix bundle domain. A three-helix bundle domain has a structure consisting of two parallel helices and one antiparallel helix joined by a loop region. Three-helix bundle domains of interest include, but are not limited to, GA domains, Z domains, and albumin binding domain (ABD) domains.

[0094] Based on the present disclosure, it should be understood that several amino acid residues that are not located at the target binding surface of the structure of the peptide domain motif can be modified without significantly adversely affecting the three-dimensional structure or target binding activity of the modified compound obtained. Therefore, several amino acid modifications / mutations can be incorporated into the compounds of the present invention as needed to impart desired properties to the compound, including but not limited to improved water solubility, ease of chemical synthesis, synthesis cost, conjugation site, helical connection site, stability, isoelectric point (pI), anti-aggregation and / or reduced non-specific binding. The position of the mutation can be selected to avoid or minimize any destruction of the basic three-dimensional structure of the target binding domain motif for specific binding to the target protein. For example, the solvent-exposed position on the opposite side of the domain structure to the binding surface can be mutated to introduce desired variant amino acid residues, such as to improve solubility or provide desired protein pI, or to incorporate into conjugation or connection sites. In some cases, based on the three-dimensional structure of the target binding domain motif, the position of the mutation can be selected to provide improved stability (e.g., by introducing variant amino acids into core stacking residues of the structure), or improved binding affinity (e.g., by introducing variant amino acids into the SDM). In some cases, the compound includes two or more, e.g., 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more surface mutations at positions that are not part of the binding surface to the VEGF target protein.

[0095] VEGF-binding Z domain

[0096] The present disclosure provides a D-peptide Z domain that specifically binds to VEGF. The Z domain can include a VEGF specificity determining motif (SDM) defined by 5 or more variant amino acid residues (e.g., 5, 6, 7, 8, 9, or 10 variant amino acid residues) located at positions 9, 10, 13, 14, 17, 24, 27, 28, 32, and / or 35 of the Z domain. It will be appreciated that a variety of basic Z domain scaffolds or peptide framework sequences can be used to provide the characteristic three-dimensional structure of the Z domain.

[0097] The term "Z domain" refers to a peptide domain having a three-helix bundle tertiary structure related to the immunoglobulin G binding domain of protein A. An exemplary Z domain structure is provided in the Protein Data Bank (PDB) as structure 2spz. See also Figure 32A and Figure 32B, which includes a depiction of a native Z domain structure and an exemplary sequence of an unmodified native Z domain. The term "Z domain scaffold" refers to a basic Z domain sequence that provides a characteristic 3-helix bundle structure and is suitable for use in the compounds of the present invention. A "variant Z domain" is a Z domain that includes variant amino acids at selected positions in the tertiary structure of the three-helix bundle, which provides specific binding to the target protein. The Z domain motif can generally be described by the following formula:

[0098] [Helix 3]-[Linker 1]-[Helix 2]-[Linker 2]-[Helix 1]

[0099] wherein [Linker 1] and [Linker 2] are independently peptide linker sequences of between 1 and 10 residues, and [Helix 1], [Helix 2], and [Helix 3] are as described above for the GA domain.

[0100] Z domains of interest include, but are not limited to, those described by Nygren (“Alternative binding proteins: Affibody binding proteins developed from a small three-helix bundle scaffold”, FEBS Journal 275 (2008) 2668-2676), US20160200772, US9,469,670, and the 33-residue minimized Z domain of protein A, described by Tjhung et al. (Front. Microbiol., April 28, 2015), the disclosures of which are incorporated herein by reference in their entirety.

[0101] For the purpose of describing some exemplary VEGF-A-specific Z domains of the present disclosure, Figure 36B In some embodiments, the D-peptide Z domain is a three-helix bundle of the following structure: [helix 1 (#8-18) ]-[Linker 1 (#19-24) ]-[Spiral 2 (#25-36) ]-[Linker 2 (#37-40) ]-[Spiral 3 (#41-54) ]

[0102] Wherein: # represents the reference position of the amino acid residues contained in the D-peptide GA domain. It should be understood that helices 1-3 can be defined to include one or more additional residues extending from the termini of the helices, and the residues located at such termini can have a partial helical configuration and / or be at the beginning of a turn or loop region. In some cases, helix 1 of the Z domain can further include one or more additional amino acid residues at the N-terminus, such as helix residues at position 7 and, optionally, position 6. In some cases, helix 1 of the Z domain can further include an amino acid residue at position 7. In some cases, the Z domain includes an N-terminal residue at position 8, which can provide desirable properties, such as stabilization of helix 1, stabilization of the three-helix bundle, additional VEGF binding contacts, extension of helix 1, and connection to a second domain or portion of interest (e.g., as described herein). In some cases, the Z domain includes a C-terminal residue at position 54, which can provide desirable properties, such as stabilization of helix 3, stabilization of the three-helix bundle, additional VEGF binding contacts, extension of helix 3, and connection to a second domain or portion of interest (e.g., as described herein).

[0103] The D-peptide Z domain compound can specifically bind to VEGF-A at a binding site defined by amino acid side chains E90, F62, D67, I69, E70, K110, P111, H112, and Q113 of VEGF.

[0104] Exemplary VEGF-A binding D-peptide Z domains include the domains described in Table 4, and the domains described by the sequences of the following compounds: 978333 to 978337 and 980181 (SEQ ID NOs: 114-119), 980174-980180, and 981188-981190 (SEQ ID NOs: 120-129). In view of the structural and sequence variants described in this disclosure, it will be appreciated that numerous amino acid substitutions can be made to the sequences of the exemplary compounds while retaining specific binding to VEGF-A. By selecting positions in the variant Z domain that tolerate variation without adversely affecting the three-dimensional architecture of the Z domain, numerous amino acid substitutions can be incorporated.

[0105] Thus, the present disclosure includes sequences of 978333 to 978337 and 980181 (SEQ ID NOs: 114-119), 980174-980180, and 981188-981190 (SEQ ID NOs: 120-129) having 1-10 amino acid substitutions (e.g., 1-8, 1-6, or 1-5 substitutions, such as 1, 2, 3, 4, or 5 amino acid substitutions). The 1-10 amino acid substitutions can be based on the physical properties of the amino acid side chains, for example, according to Table 6. Sometimes, an amino acid in the sequences of 978333 to 978337 and 980181 (SEQ ID NOs: 114-119), 980174-980180, and 981188-981190 (SEQ ID NOs: 120-129) is substituted with a similar amino acid according to Table 6. In some cases, the substitution is with respect to a conservative amino acid substitution or a highly conservative amino acid substitution according to Table 6.

[0106] The present disclosure includes VEGF-A binding D-peptide Z domains described by sequences having 80% or greater sequence identity, e.g., 85% or greater, 87% or greater, 89% or greater, 91% or greater, 93% or greater, 94% or greater, 96% or greater, 98% or greater sequence identity, to the sequences of 978333 to 978337 and 980181 (SEQ ID NOs: 114-119), 980174-980180, and 981188-981190 (SEQ ID NOs: 120-129).

[0107] The VEGF-A binding D-peptide Z domain may have amino acid residues at positions 9, 10, 13, 14, 17, 24, 27, 28, 32, and 35 of the Z domain scaffold, said positions being defined by Figure 33A and / or Figure 35F In some cases, the specificity determining motif (SDM) is defined by the following sequence motifs:

[0108] w 9 d 10 --w 13 x 14 --r 17 ------x 24 --k 27 x 28 ---x 32 --y 35 (SEQ ID NO: 160)

[0109] Where: x 14 、x 24 、x 28 and x32 Each independently represents any amino acid residue. In certain instances of SDM: x 14 Selected from l, r and t; x 24 selected from h, i, l, r, and v; x 28 is selected from G, r, and v; and x 32 is selected from a, r, h, s and t. In some cases, the specificity determining motif (SDM) is:

[0110] w 9 d 10 --w 13 r 14 --r 17 ------l 24 --k 27 r 28 ---s 32 --y 35 (SEQ ID NO: 161); or

[0111] w 9 d 10 --w 13 r 14 --r 17 ------v 24 --k 27 r 28 ---r 32 --y 35 (SEQ ID NO: 162).

[0112] In some embodiments, a D-peptide compound that specifically binds to VEGF comprises a D-peptide Z domain comprising a VEGF specificity determining motif (SDM) defined by the following amino acid residues:

[0113] w 9 d 10 --w 13 x 14 --r 17 ------x 24 --k 27 x 28 ---x 32 --y 35 (SEQ ID NO: 160)

[0114] in:

[0115] x 14 Selected from l, r and t;

[0116] x 24 selected from h, i, l, r and v;

[0117] x 28 selected from G, r and v;

[0118] x 32 is selected from a, r, h, s, and t; and

[0119] x 35 Selected from k or y.

[0120] In some embodiments of VEGF SDM, x 14 In some embodiments of VEGF SDM, x 14 In some embodiments of VEGFSDM, x 14 is t.

[0121] In some embodiments of VEGF SDM, x 24 In some embodiments of VEGF SDM, x 24 In some embodiments of VEGFSDM, x 24 In some embodiments of VEGF SDM, x 24 In some embodiments of VEGF SDM, x 24 For v.

[0122] In some embodiments of VEGF SDM, x 28 is G. In some embodiments of VEGF SDM, x 28 In some embodiments of VEGFSDM, x 28 For v.

[0123] In some embodiments of VEGF SDM, x 32 In some embodiments of VEGF SDM, x 32 In some embodiments of VEGFSDM, x 32 In some embodiments of VEGF SDM, x 32 In some embodiments of VEGF SDM, x 32 is t.

[0124] In some embodiments of VEGF SDM, x 35 In some embodiments of VEGF SDM, x 35 is y.

[0125] In some embodiments, the VEGF SDM is defined by the following residues:

[0126] w 9 d 10 --w 13 r 14 --r17 ------l 24 --k 27 r 28 ---s 32 --y 35 (SEQ ID NO: 161)

[0127] or

[0128] w 9 d 10 --w 13 r 14 --r 17 ------v 24 --k 27 r 28 ---r 32 --y 35 (SEQ ID NO: 162).

[0129] In some embodiments of the GA domain, the SDM residues are contained within a peptide framework sequence comprising peptide framework residues bounded by the following amino acid residues: 11 a--e 15 ih 18 lpnln-e 25 q--a 29 fi-s 33 l-.

[0130] In some embodiments, the GA domain comprises an SDM-containing sequence that is 80% or greater (e.g., 85% or greater, 90% or greater, or 95% or greater) identical to the following amino acid sequence:

[0131] w 9 d 10 naw 13 x 14 eir 17 hlpnlnx 24 eqk 27 x 28 afix 32 sly 35 (SEQ ID NO: 133)

[0132] in:

[0133] x 14 Selected from l, r and t;

[0134] x 24 selected from h, i, l, r and v;

[0135] x 28 selected from G, r and v;

[0136] x 32 is selected from a, r, h, s, and t; and

[0137] x 35 Selected from k or y.

[0138] In some embodiments comprising an SDM sequence, x 14 In some embodiments comprising an SDM sequence, x 14 In some embodiments comprising SDM sequences, x 14 is t.

[0139] In some embodiments comprising an SDM sequence, x 24 In some embodiments comprising SDM sequences, x 24 In some embodiments comprising SDM sequences, x 24 In some embodiments comprising an SDM sequence, x 24 In some embodiments comprising SDM sequences, x 24 For v.

[0140] In some embodiments comprising an SDM sequence, x 28 is G. In some embodiments comprising an SDM sequence, x 28 In some embodiments comprising SDM sequences, x 28 For v.

[0141] In some embodiments comprising an SDM sequence, x 32 In some embodiments comprising an SDM sequence, x 32 In some embodiments comprising SDM sequences, x 32 In some embodiments comprising SDM sequences, x 32 In some embodiments comprising an SDM sequence, x 32 is t.

[0142] In some embodiments comprising an SDM sequence, x 35 In some embodiments comprising SDM sequences, x 35 is y.

[0143] In some embodiments of the compound, helix 3 of the Z domain (#41-54) Contains peptide framework sequences 41 anllaeakklnda 54 (SEQ ID NO: 134).

[0144] In some embodiments, the D-peptide Z domain comprises a C-terminal peptide framework sequence: d 36dpsqsanllaeakklndaqapk 58 (SEQ ID NO: 135).

[0145] In some embodiments, the D-peptide Z domain comprises an N-terminal peptide framework sequence: 1 dnkfnke 8 (SEQ ID NO: 136).

[0146] VEGF-binding GA domain

[0147] The terms "GA domain" and "GA domain motif" refer to a peptide domain having a three-helix bundle tertiary structure related to the albumin binding domain of protein G. In the Protein Data Bank (PDB), structure 1tf0 provides an exemplary GA domain structure. Figures 3, 7A-7B, 10A and Figure 10B Included is a depiction of a native GA domain structure and an exemplary sequence of an unmodified native GA domain. The term "GA domain scaffold" refers to a basic peptide framework sequence that provides a characteristic 3-helix bundle structure and is suitable for use in the compounds of the present invention. In some cases, the GA domain scaffold or peptide framework sequence has a consensus sequence as defined in Table 3. Table 3 provides a list of exemplary GA domain scaffold sequences that are suitable for use in the compounds of the present invention. The terms "variant GA domain," "VEGF-binding GA domain," and "VEGF-binding GA domain" are used interchangeably and refer to a GA domain that includes variant amino acids at selected positions in the three-helix bundle tertiary structure that, together, provide specific binding to the VEGF target protein.

[0148] The GA domain can be described by the following structural formula:

[0149] [Helix 1]-[Linker 1]-[Helix 2]-[Linker 2]-[Helix 3]

[0150] Wherein [helix 1], [helix 2] and [helix 3] are helical regions of a characteristic three-helix bundle connected via peptide linkers [linker 1] and [linker 2]. In the three-helix bundle, [helix 1], [helix 2] and [helix 3] are connected peptide regions, wherein [helix 2] is arranged substantially antiparallel to the two-helix complex of parallel α-helices [helix 1] and [helix 3]. [Linker 1] and [Linker 3] can each independently comprise a sequence of 1 to 10 amino acid residues. In some cases, [Linker 1] is longer than [Linker 3]. The GA domain can be a peptide sequence between 30 and 90 residues, e.g., between 30 and 80 residues, between 40 and 70 residues, between 45 and 60 residues, between 45 and 60 residues, or between 45 and 55 residues. In some cases, the GA domain motif is a peptide sequence of between 35 and 55 residues, e.g., between 40 and 55 residues, or between 45 and 55 residues. In certain embodiments, the GA domain motif is a peptide sequence of 45, 46, 47, 48, 49, 50, 51, 52, or 53 residues.

[0151] In some embodiments, the D-peptide GA domain is a three-helix bundle of the following formula:

[0152] [Spiral 1 (#6-21) ]-[Linker 1 (#22-26) ]-[Spiral 2 (#27-35) ]-[Linker 2 (#36-37) ]-[Spiral 3 (#38-51) ]

[0153] Wherein: # represents the reference position of the amino acid residues contained in the D-peptide GA domain, for example, according to Figure 9C The numbering scheme shown in .

[0154] GA domains of interest include those described by Jonsson et al. (Engineering of a femtomolar affinity binding protein to human serum albumin, Protein Engineering, Design & Selection, 21(8), 2008, 515-527), the disclosure of which is incorporated herein by reference in its entirety, and include GA domains and phage display libraries having a scaffold sequence (G148-GA3) wherein the library mutations are at scaffold positions 25, 27, 31, 34, 36, 37, 39, 40, 43, 44, and 47. Other GA domains of interest include, but are not limited to, those described in US 6,534,628 and US 6,740,734, the disclosures of which are incorporated herein by reference in their entirety.

[0155] The variant GA domains of the present disclosure may have a specificity determining motif (SDM) comprising 5 or more variant amino acid residues at positions selected from 25, 27, 30, 31, 34, 36, 37, 39, 40, and 42-48. In some cases, the specificity determining motif (SDM) further comprises a variant amino acid at position 28 of the GA domain.

[0156] Locked GA domain

[0157] The present disclosure includes variant GA domain compounds having an interhelical linker or bridge between adjacent residues of helix 1 and helix 3. The terms "locked variant GA domain" and "locked GA domain" refer to variant GA domains that include a structure-stabilizing linker between any two helices of the GA domain. Sometimes, the linked adjacent residues are located at the ends of helices 1 and 3. Figure 29A and 37A The structure of the GA scaffold domain is shown, which shows the configuration of helices 1-3 in the three-helix bundle. The interhelical linker can be located between the amino acid residues at positions 7 (helix 1) and 38 (helix 3) of the domain, which are close to each other in the three-dimensional structure of the domain. Positions 7 and 38 can be regarded as core-facing residues located at the ends of the helices, which can make stable contacts with the hydrophobic core of the structure. As measured between the α-carbons of the connected amino acid residues, the interhelical linker can have a main chain of 3 to 7 atoms long. For example, a disulfide connection between two cysteine ​​residues provides a main chain of 4 atoms (-CH2-SS-CH2-) between the α-carbons of the two cysteine ​​amino acid residues.

[0158] A variety of compatible naturally occurring and non-naturally occurring amino acid residues can be incorporated at positions 7 and 38 of the GA domain and can be conjugated to each other to provide an interhelical linker. Compatible residues include, but are not limited to, aspartic acid or glutamic acid linked to serine or cysteine ​​via an ester or thioester linkage, aspartic acid or glutamic acid linked to ornithine or lysine via an amide linkage. Thus, the interhelical linker may include one or more residues selected from C (1-6) Alkyl, substituted C (1-6) Alkyl, -(CHR) n -CONH-(CHR) m -and-(CHR) n -SS-(CHR) m -, wherein each R is independently H, C (1-6) Alkyl or substituted C (1-6) alkyl, and n + m = 2, 3, 4 or 5. Any convenient non-naturally occurring residue may be used to incorporate compatible chemoselective tags at the side chains of the amino acid residues at positions 7 and 38, such as click chemistry tags, such as azide and alkyne tags, which can be conjugated to each other after polypeptide synthesis.

[0159] Incorporation of an intradomain linker can provide stability and / or increase binding affinity to a VEGF target protein. In some cases, the binding affinity (K) of a D-peptide compound to VEGF is D ) is 3-fold or more potent than a control polypeptide lacking an intradomain linker (i.e., K D fold lower), such as 5-fold or more potent, 10-fold or more potent, 30-fold or more potent, or even more potent. Exemplary locked variant GA domain compounds that specifically bind VEGF-A are described in more detail below.

[0160] Variant GA domain polypeptides may include an N-terminal region from position 1 to approximately position 6 that can be considered non-overlapping with helix 2 and helix 3 because this region does not directly participate in contacting the adjacent helix 2-loop-helix 3 region of the folded three-helix bundle structure (see, e.g., Figure 32A). In the D-peptide compounds of the present invention, the N-terminal region of positions 1-5 of the GA domain may be retained in the sequence as appropriate and optimized to provide desired properties, such as increased water solubility, stability or affinity. It should be understood that the N-terminal region of the variant D-peptide compound may be substituted, modified or truncated without significantly adversely affecting the activity of the compound. The N-terminal region may be modified to provide conjugation or connection to a molecule of interest (e.g., as described herein) or another D-peptide domain or multivalent compound (e.g., as described herein). In some cases, the N-terminal residue has a helical tendency to provide an extended helical structure of helix 1. Alternatively, the N-terminal region may incorporate a helical capping residue that stabilizes the N-terminus of helix 1. In some cases, by removing residues relative to, for example, Figure 32A 1, 2, 3, 4 or 5 residues of the parent GA domain structure shown in (i.e., truncation of positions 1-5), the variant GA domain compound is truncated at the N-terminus. In such cases, as Figure 32B Similarly, one, two, or three C-terminal residues at the end of helix 3 can be truncated without adversely affecting the stability of the three-helix bundle structure and target binding ability.

[0161] Figures 29A-29B Shown is the design of an exemplary affinity maturation library focused on positions 1-3, 6, 7, and 37-38 of variant GA domain compounds. Figures 30A-30B Screening results and variant GA domain compounds with a c7-c38 disulfide bridge and improved binding affinity for VEGF-A are shown. A variety of variant amino acid residues are tolerated at positions 1-3 of the N-terminal region of the compounds.

[0162] In some embodiments, the D-peptide GA domain includes one or more (e.g., two) of the following segments (I)-(II):

[0163] x 1 x 2 x 3 qwx 6 x 7 (I) (SEQ ID NO: 142)

[0164] x 37 x 38 (II)

[0165] in:

[0166] x 1 to x 3 independently selected from any D-amino acid residue;

[0167] x 6 Selected from i and v;

[0168] x 37 is selected from s and n; and

[0169] x 7 and x 38 are amino acid residues connected via intradomain / interhelical linkers, such as in amino acid residue x 7 and x 38 The backbone length of the linker is 3 to 7 atoms, measured between the α-carbons of the linker. 1 to x 3 are independently selected from f, h, i, p, r, y, n, s, and v. In some embodiments of Formula (I), x 6 is v. In some embodiments of formula (II), x 37 is n.

[0170] Intradomain / interhelical linkers can be represented by x 7 and x 38 Any suitable naturally or non-naturally occurring thiol-containing amino acid can be used to provide an intradomain linker. 7 and x 38 Contains: Cysteine 7 -Cysteine 38 disulfide; homocysteine 7 -Cysteine 38 disulfide; cysteine 7 -Homocysteine 38 disulfide; and homocysteine 7 -Homocysteine 38 Alternatively, intradomain / interhelical linkers may be included in x 7 and x 38 Amide bonds between the side chains of the amino acid residues. Any convenient naturally or non-naturally occurring amine and carboxylic acid containing amino acid can be used to provide an intradomain linker. Amino acid residues x that can be linked via an amide linkage 7 and x 38 These include: Asp7-Dap38, Asp7-Dab38, Asp7-Orn38, Glu7-Dap38, Glu7-Dap38, and Glu7-Orn38, wherein Dap is α,β-diaminopropionic acid, Dab is α,γ-diaminobutyric acid, and Orn is ornithine. 7 and x 38The residue pairs may be D-amino acid residues. Any convenient chemoselective functional groups and conjugates thereof may be used to achieve intradomain / interhelical linkages, including but not limited to azide-alkyne, thiol-maleimide, thiol-haloacetyl, thiol-vinyl sulfone, ester, thioester, amide, ether, and thioether.

[0171] Figure 13 A depiction of a GA domain library is shown, comprising a basic 53-residue scaffold sequence (SEQ ID NO: 2) and mutant positions shown in bold at positions 25, 27, 28, 31, 34, 36, 37, 39, 40, 43, 44, and 47 of the scaffold, which define one of the phage display libraries used for screening. Selected hit compounds derived from the scaffold domain library screen were identified. Compounds of the invention include a basic scaffold domain that presents a VEGF-A binding surface that contacts the target protein and provides specific binding to VEGF-A. Selected compounds from the selected GA domain library hits were subjected to additional affinity maturation and point mutation studies (e.g., as described herein) to assess variant amino acids at several additional positions of the GA domain motif, such as positions 26, 29, and 30. Described herein are the X-ray crystal structures of exemplary D-peptide compounds having a GA domain scaffold in complex with VEGF-A, which provide structural models for VEGF-A binding compounds of the invention.

[0172] The D-peptide variant GA domain compounds can specifically bind to VEGF-A at the binding site defined by the amino acid side chains F43, M44, Y47, Y51, N88, D89, L92, I72, K74, M107, I109, Q115, and I117 of VEGF-A (see Figures 28A-28B ).

[0173] In some cases, the VEGF-A binding motif includes at least two antiparallel helical regions [helix A] and [helix B] that contact each other and together define the VEGF-A binding surface. That portion of the VEGF-A binding motif that includes the antiparallel complex of [helix A] and [helix B] can be referred to as a "two-helical complex" structure. Figures 8A-8B A model of the heptad repeat structure is depicted for the two-helical complex structure. In some cases, the VEGF-A contact residues of interest may be located at surface or boundary mutation positions of the two-helical complex, such as the c or g positions of the heptad repeat. Figure 8CAn exemplary arrangement of VEGF-A contact residues on the gg face of the two-helix complex structure is shown. The VEGF-A binding surface may include 4 or more residues, such as 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more VEGF-A contact residues, wherein the residues include residues from both [helix A] and [helix B]. In some cases, the VEGF-A contact residues are independently selected from non-polar, aromatic, heterocyclic, and carbocyclic residues (e.g., as described herein). The two helices of the two-helix complex can be connected via any convenient connection that retains the generally antiparallel configuration of [helix A] and [helix B]. In some cases, [helix A] and [helix B] are connected via a C(helix A) to N(helix B) peptide linker. In some cases, [helix A] and [helix B] are connected via a C(helix A) to N(helix B) peptide linker. Figure 8A Possible end-joining of the two-helical complex structure is depicted (solid blue line).

[0174] The two-helical complex can be further stabilized by any convenient means, including but not limited to the incorporation of residues that provide the desired helix-helix packing interactions or hydrophilicity at solvent-exposed positions, the incorporation of interhelical linkages, the incorporation of intrahelical linkages, the incorporation of constrained turns or linkers connecting helices, and the attachment of a third peptide region capable of stabilizing contacts with both [Helix A] and [Helix B]. Figures 8B-8C Various interhelical side chain to side chain connections (e.g., dotted lines) that can be placed between any two convenient residues are depicted. Similarly, stable intrahelical side chain to side chain or side chain to terminal connections can be placed to provide the desired stability for the structure of the compound. Interhelical and intrahelical connections of interest for the compounds of the present invention include, but are not limited to, Cys-Cys disulfide connections, stapled peptide connections, and non-natural cross-links, such as those prepared by ring-closing metathesis and connections described by Douse et al. (ACS Chem Biol. 2014 Oct 17; 9(10): 2204-9).

[0175] In some embodiments, the two-helix complex can be stabilized by a third helix (helix C) that contacts both [helix A] and [helix B] on the opposite side of the VEGF-A binding surface of the compound and together define a three-helix bundle. As used herein, the terms "three-helix bundle" and "three-helix bundle motif" are used interchangeably to refer to a three-helix bundle, which is a small protein tertiary structure comprising three generally parallel or antiparallel alpha helices. The three helices are based on a linear sequence of connected helical regions arranged in a parallel-antiparallel-parallel configuration in the three-helix bundle structure.

[0176] DeGrado et al. (Analysis and design of three-stranded coiled coils and three-helix bundles, Folding & Design 1998, 3: R29-R40) provides an assembly model for three-stranded coiled coils and three-helix bundles, the disclosure of which is incorporated herein by reference in its entirety. A three-helix bundle can be a single-stranded structure having loops connecting helices that make regular contacts with each other in a nonpolar core. The three helices of the structure can exhibit an approximately seven-residue repeat motif, designated by italic letters ag, i.e., (abcdefg) n . The heptapeptide designations a, c, d, e, f, and g do not correspond to the single-letter codes for specific amino acids, but rather to positions in the heptapeptide sequence. Non-polar residues may occur at positions a and d of the heptapeptide, including side chain groups that are stacked to the center of the structure to provide hydrophobic stabilization. The non-polar a and d residues may be stacked in layers. In some cases, charged side chains may appear at interfacial e and g positions, where the non-polar portion of their side chains may shield the hydrophobic core and the polar portion may participate in electrostatic or hydrogen bonding interactions. In some cases, solvent-exposed positions b and c may be occupied by polar residues. In some cases, position f is highly solvent-exposed and may be occupied by a polar or charged residue. Figure 6D A D-peptide heptad repeat model of a three-helix bundle is shown, showing two parallel helices and one antiparallel helix. In some cases, residues at the gg face formed by the combined surfaces of helices 2 and 3 are modified to include VEGF-A contact residues configured to interact with the surface of VEGF-A and provide specific binding. It is understood that Figure 6D The two-helical complex form depicted in the structural model is possible, as Figure 8B Any convenient stabilizing element may be used in the compounds of the invention (e.g., as described herein) to maintain the desired arrangement of the two helices and provide for the presentation of VEGF-A binding residues that specifically bind to VEGF-A. The compounds of the invention may have a VEGF-A binding GA domain motif having a three-helix bundle tertiary structure into which variant amino acid residues are incorporated to provide a binding surface capable of specific binding to VEGF-A. Figure 1-2 The binding interface between exemplary peptide compounds and VEGF-A is depicted. Figure 3A and Figure 3B Shown is a side-by-side comparison of the three-helix bundle X-ray crystal structures of the L-protein GA domain and an exemplary D-peptide compound. Figure 3A and Figure 3BComparison of the peptide compounds with the parent GA domains indicates that the peptide compounds retain the essential three-helix bundle motif of the parent GA domain. In some cases, the α-helical structure of the compounds is substantially identical to that of the native GA scaffold domain. Modified variant amino acids may include a helix-terminating residue at the end of the helix 2 region, which is not present in the GA scaffold domain. Variant amino acids in the helix 2 region may also include three or more VEGF-A contact residues, such as aromatic amino acid residues. Figure 4 Depicted are the helix-terminating proline residues at positions 26 and 36 (p26; 204 and p36; 208), the VEGF-A contacting phenylalanine at position 31 (f31; 206), and the histidine residues at positions 27 and 34 (h27; 205 and h34; 207) of the helix 2 region of an exemplary VEGF-A binding compound.

[0177] In certain embodiments of the compounds described herein, a numbering scheme is used for convenience and simplicity to refer to specific positions in the structure and / or sequence of the compound, such as the position where a specific variant amino acid residue of interest is incorporated into the GA scaffold domain. This numbering scheme is based on the Figure 13 It will be appreciated that any convenient alignment method may be used to compare specific embodiments of the compounds of the invention with the Figure 15 The reference numbering schemes of the sequences described herein are used for comparison to assign numbered positions to amino acid residues of interest, e.g., positions in motifs or structural models as described herein. Figure 14 Shown is an exemplary alignment of various GA scaffold domain sequences of interest, any of which can serve as the basic parent sequence for compounds of the invention. Figure 14 Also refer to the sequence Figure 13 It should also be understood that Figure 13 The 1-53 numbering scheme is not meant to be limiting with respect to determining the total number of amino acid residues or the length of a linear compound sequence, or with respect to delineating each residue in a specific compound.

[0178] In some cases, the compounds of the invention include one or more variations relative to the numbered parent sequence, such as N-terminal truncation (e.g., from position 1), C-terminal truncation (e.g., from position 53), deletions (e.g., deletions of single residue positions at any convenient position of the parent sequence), insertions (e.g., insertions of 1, 2, 3 or more consecutive residues between two specific numbered positions of the parent sequence). In some cases, such variations incorporated into the compounds of the invention substantially retain the three-dimensional structure of the three-helix bundle, which provides specific binding to the target. The compounds of the invention may further include variant amino acids at one or more positions of the parent structure or sequence, such as 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or 15 or more positions, as described in the Examples below.

[0179] As described herein, the compounds of the invention may have a three-helix bundle structure, wherein specific solvent-exposed variant amino acids located at specific positions of [Helix 2] and [Helix 3] may form contacts with VEGF-A. In some cases, additional contacts may occur at specific residues of [Linker 2] and / or [Linker 1]. Figure 1 The binding interface between an exemplary peptide compound and VEGF-A as obtained from the X-ray crystal structure of the complex is depicted. In some cases, variant amino acids located at additional positions of [Helix 2], [Helix 3], [Linker 2], and / or [Linker 1] provide the desired stabilization of the modified three-helix bundle structure. For example, in Figure 4 , exemplary [helix 2] terminator residues (e.g., proline residues 204 and 208) are shown, which in some cases can confer desired increased stability to [helix 2]. In some cases, the hydrophobic core of the modified three-helix bundle is defined by amino acid residues that are substantially identical to those of the parent GA scaffold domain. For example, Figure 11 Shown is a magnified view of a portion of the [helix 2]-[linker 2]-[helix 3] structure of an exemplary D-peptide compound, including adjacent hydrophobic residues i32 (isoleucine, position 32) and a35 (alanine, position 35) of [helix 2], and adjacent hydrophobic residues v41 (valine, position 41) and l44 (leucine, position 44) of [helix 3], which provide the required intramolecular hydrophobic contacts. Figure 12A magnified view of the analogous region of the native L-peptide GA domain is shown in , where analogous residues I32 (isoleucine, position 32), A35 (alanine, position 35), V41 (valine, position 41), and L44 (leucine, position 44) provide similar required intramolecular hydrophobic contacts that are characteristic of the three-helix bundle structure of the GA scaffold domain.

[0180] Figure 6C The De Lado model of an antiparallel triple helix structure is depicted. The De Lado model of an antiparallel triple helix based on repeating heptapeptide units is adapted herein to provide a structural model relating the sequence motifs of the compounds of the invention to the modified three-helix bundle structure of the compounds of the invention, which includes the VEGF-A binding surface. This structural model of the three-helix bundle is consistent with the native GA domain (e.g., Figure 3A ) and exemplary VEGF-A binding compounds ( Figure 3B ) is consistent with the X-ray crystal structure. Figure 9A and 9C The model applied to the exemplary compound 1.1.1 (c21a) is shown, where the compound sequence ( Figure 9C ) are related to the various positions of the heptad repeat model and are structurally aligned, consistent with the X-ray structure ( Figure 9B ) are consistent. Figure 9A The model in the same manner as the X-ray structure of the compound in complex with VEGF-A (see e.g. Figure 5 and Figure 20 Comparison of the views of FIG5 ) shows that the VEGF-A binding surface of the exemplary compound is located at the gg plane defined by helix 2 and helix 3 ( Figure 9A The selected amino acid residues may be located at the VEGF-A binding surface of the compounds of the invention and configured to interact with VEGF-A (e.g., at the solvent exposed c and / or g positions of the gg face defined by helix 2 and helix 3).

[0181] The hydrophobic core of the compounds of the invention may include a and d residues of [helix 2] in contact with corresponding d and a residues of [helix 3]. Figure 6B and Figure 10A An alignment of exemplary compound 1.1.1 (c21a) with the heptad repeat model is shown, depicting the hydrophobic contacts of the core residues between the helices of the three-helix bundle. Figure 11 The structure of the portion of the [helix 2]-[linker 2]-[helix 3] region shown in FIG is consistent, including adjacent hydrophobic residues i32 (isoleucine, position 32) and a35 (alanine, position 35) of [helix 2], and adjacent hydrophobic residues v41 (valine, position 41) and l44 (leucine, position 44) of [helix 3], which provide the required intramolecular hydrophobic contacts. It should be understood that the model (e.g., as Figure 9A ) allows alignment of helices 2 and 3 that are not perfectly parallel (ie, inter-helix angle > 0 degrees, such as described herein and as depicted in FIG. 27 ).

[0182] like Figure 5 27, in some cases, although helices 2 and 3 may have a generally antiparallel configuration relative to the direction of the helices, and these helices do contact each other several times over the length of the helices, the axes of the helices may be aligned at an angle >0 degrees, e.g., about 10 degrees or greater, about 15 degrees or greater, about 20 degrees or greater, about 25 degrees or greater, or about 30 degrees or greater. Thus, in some cases of the compounds of the invention, Linker 2 is shorter than Linker 1, such that the angle between Helices 2 and 3 is measured with respect to the Linker 2 connection of the helices. In some cases, the "a" and "d" residues farthest from the Linker 2 end of the helices are more likely to be partially solvent exposed and / or available for contact with VEGF-A.

[0183] In some cases, the compounds of the invention include helix-terminating residues that provide an increase in the angle between helices 2 and 3, such as an increase of about 5 degrees or greater, such as about 10 degrees or greater, or about 15 degrees or greater. See, e.g., Figure 27B and Figure 27A .

[0184] In some embodiments, [helix 2] comprises a heptad repeat sequence [c 1 d 1 e 1 f 1 g 1 a 2 b 2 c 2 d 2 ], and [helix 3] contains a heptad repeat sequence [e 1 f 1 g 1 a 2 b 2 c 2 d 2 e 2 f 2 g 2 a 3 b 3 c 3 d 3 e 3 ], where individual heptad repeat residues can be numbered. In some cases of this arrangement of [helix 2] and [helix 3], residue d of [helix 2] 2 、a 2 and d 1 Residue a of [helix 3] 2 d 2 and a3 interact with each other, forming a network of structurally stable interactions. In some cases, residues c of [helix 2] 2 、g 1 and c 1 and residue g of [helix 3] 1 Each is independently an aromatic, heterocyclic, or carbocyclic residue that is configured to contact VEGF-A.

[0185] The VEGF-A binding surface of the compounds of the invention can be defined by the arrangement of aromatic amino acid residues located at the c and g positions of the heptad repeat model, which residues are arranged on the surface to interact with VEGF-A. In some cases, the VEGF-A binding surface includes two or more, three or more, e.g., four or more, or five or more aromatic amino acid residues located at the c and g positions of the heptad repeat sequence. Figure 10A and Figure 10B Depicted are examples of variant domain motifs comprising configurations of c and g residues capable of binding to [helix 2] and [helix 3] of VEGF-A. In some cases, the VEGF-A binding surface includes additional non-aromatic amino acid residues at the c and g positions of the heptad repeat sequence, e.g., Figure 10B In some cases, the VEGF-A binding surface includes additional non-aromatic amino acid residues, which are polar amino acid residues capable of hydrogen bonding interactions at the c and g positions of the heptad repeat sequence, for example, at residues c and / or g of helix 3. Based on the present disclosure, it will be understood that several amino acid residues in the GA domain motif that are not located at the VEGF-A binding surface of the structure can be modified without adversely affecting the VEGF-A binding activity of the resulting modified compound.

[0186] In some embodiments of Formula (I), [Helix 2] comprises a sequence of the formula:

[0187] ΛjxxΛjxΛj (SEQ ID NO: 143)

[0188] (II)

[0189] wherein: each "Λ" is independently a D-aromatic amino acid; each j is independently a hydrophobic residue; and each x is independently an amino acid residue. Aromatic amino acids of interest for use in formula (II) include, but are not limited to, h, f, y, and w, and substituted forms thereof. In some cases of formula (II), the first Λ is h, f, or y. The second Λ residue may be an aromatic residue comprising an aryl, heteroaryl, substituted aryl, or substituted heteroaryl ring (e.g., a residue having a side chain of the formula -CH2-Ar, wherein Ar is an aryl or substituted aryl). In some cases of formula (II), the second Λ is f or y, or a substituted form thereof. The second Λ residue may be configured on the binding surface of the GA domain motif structure to interact with the VEGF-A protein, for example, protruding onto Figure 20 and 21 In some instances of Formula (II), the second Λ is f or a substituted form thereof. In some instances of Formula (II), the third Λ is an aromatic residue comprising a heteroaryl or substituted heteroaryl ring (e.g., an aromatic residue comprising a side chain group capable of hydrogen bonding with VEGF-A). In some instances of Formula (II), each j is independently selected from v, i, a, and l. In some instances of Formula (II), the first j residue is valine. In some embodiments of Formula (II), [Helix 2] comprises a sequence of the formula: hv xxΛjxΛj.

[0190] In some embodiments of Formulas (I) and (II), [Helix 2] comprises a sequence of Formula (III):

[0191] h*jxxf*jxh*j (SEQ ID NO: 151)

[0192] (III)

[0193] in:

[0194] Each h* is independently histidine or an analog thereof;

[0195] f* is phenylalanine or its analogue;

[0196] Each j is independently a hydrophobic residue; and

[0197] Each x is independently an amino acid residue.

[0198] In some embodiments of Formula (III), [Helix 2] comprises a sequence of the formula: hvxxf*jxh*j. Residue f* of Formula (III) can be configured on the binding surface of the GA domain motif structure to interact with the VEGF-A protein, for example, by protruding to Figure 21 VEGF-A is depicted in the deep pockets on the surface. Figure 20A wide view of the X-ray structure of the complex is shown, in which residue f31 (phenylalanine, position 31) in helix 2 of exemplary compound 1.1.1 (c21a) is labeled and shown to protrude into a pocket on the surface of VEGF-A. Figure 21 A magnified view of f31 is shown, configured to protrude into a pocket at the VEGF-A binding interface. Selected distances between atoms of the phenylalanine ring and adjacent VEGF-A residues are shown in angstroms. Analysis of the crystal structure indicates that a variety of aromatic residues can be utilized at the positions described on the three-helix bundle structure to protrude into the same deep pocket as f31 and, in some cases, increase the desired hydrophobic contact with the VEGF-A pocket. In some cases, the phenylalanine analog includes (multiple) substituents on the phenyl ring. In some cases of formula (III), f* is phenylalanine. In some cases of formula (III), f* is a substituted derivative of phenylalanine. Phenylalanine derivatives of interest include, but are not limited to, 4-halogen-substituted phenylalanines (e.g., 4-chloro or 4-fluoro), 3-halogen-substituted phenylalanines (e.g., chloro, bromo or fluoro), 3,5-halogen-disubstituted phenylalanines (e.g., chloro or fluoro), 3,4-halogen-disubstituted phenylalanines (e.g., chloro or fluoro), 4-methyl-substituted phenylalanines, 4-trifluoromethyl-phenylalanine, and 4-ethyl-substituted phenylalanine. Various compounds including phenylalanine analogs at position 31 were prepared and shown to be active.

[0199] Figure 22 and Figure 25 A magnified view of residue h27 (205) of exemplary compound 1.1.1 (c21a) in contact with the VEGF-A surface is shown. Analysis of the crystal structure suggests that a variety of aromatic residues or histidine analogs can be utilized at position 27 on the three-helix bundle to contact the same surface pocket as h27 and, in some cases, increase the desired contact with the VEGF-A surface. In some cases of Formula (III), the first h* is a histidine, such as the residue at position 27. In some cases of Formula (III), the first and / or second h* is a histidine analog (e.g., a residue having a side chain comprising an alkyl-cycloalkyl group, such as an alkyl-cyclopentyl group or an alkyl-cyclohexyl group, or a substituted version thereof). In some cases of Formula (III), the first h* is an aromatic residue capable of making primarily hydrophobic contacts with VEGF. In some cases of Formula (III), the first h* is f or y.

[0200] Figure 22A magnified view of residue h34 (207) of exemplary compound 1.1.1 (c21a) in contact with the surface of VEGF-A is shown. Analysis of the complex structure indicates that various histidine analogs can be tolerated at position 34, including, for example, analogs that can occupy available space on the surface of VEGF-A and / or form stronger hydrogen bonds (e.g., length <4.6 angstroms) with adjacent VEGF-A residues, whether substituted or unsubstituted aryl or heterocyclic. In some instances of Formula (III), the second h* is a histidine, such as the residue at position 34. In some instances of Formula (III), the second h* is an aromatic residue capable of hydrogen bonding to VEGF. In some embodiments of Formula (III), the second h* is an aromatic residue comprising a heteroaryl or substituted heteroaryl ring (e.g., an aromatic residue comprising a side chain group capable of hydrogen bonding to VEGF-A).

[0201] In certain embodiments of Formulas (II) and (III), h* 27 、f* 31 and h* 34 Each is a variant residue. In certain embodiments of formula (II) and (III), j 28 and x 29 Each is a variant residue. In certain embodiments of formula (II) and (III), j 28 、x 29 and x 30 Each is a variable residue. In some instances of Formulas (II) and (III), each j is independently selected from a, i, l, and v. In some instances of Formulas (II) and (III), the first j residue is valine. In some instances, the heptad repeat alignment of Formulas (II) and (III) is b'a'gfedcba.

[0202] In some embodiments of Formula (III), [Helix 2] is described by the following helical motif from positions 26 to 36 of the three-helix bundle:

[0203] z 26 h*jxxf*jxh*jz 36 (SEQ ID NO: 144)

[0204] (IV)

[0205] wherein: each h*, f*, each j and each x are as defined above; and z 26 and 36Each is independently a helix-terminating residue. It is understood that in some cases, the helix-terminating residue is not considered a helix residue of the structure, but only defines the end of the [helix 2] region and the beginning of a turn or loop structure. The f* residue and each h* residue can be configured on the binding surface of the GA domain motif structure to specifically contact a target VEGF-A protein, such as described herein. In some embodiments of Formula (IV), [helix 2] comprises a sequence of the formula: 26 hvxxf*jxh*jp 36 (SEQ ID NO: 145).

[0206] The term "helix termination residue" refers to an amino acid residue that has a high free energy loss for forming a helical structure relative to a similar alanine residue. In some cases, the high free energy helix loss is referred to as a helical propensity value and is defined as 0.5 kcal / mol or greater as defined by the method of Pace and Scholtz, with higher values ​​indicating increased losses ("A Helix Propensity Scale Based on Experimental Studies of Peptides and Proteins", Biophysical Journal, Vol. 75, July 1998, 422-427). In some cases, a helix termination residue is a naturally occurring residue with a helical propensity value of 0.5 or greater (kcal / mol), such as 0.55 or greater, 0.60 or greater, 0.65 or greater, or 0.70 or greater. For example, the helical propensity value of proline is 3.16 kcal / mol, and the helical propensity value of glycine is 1.00 kcal / mol, as shown in Table 1. The helix propensity value of a non-naturally occurring helix-terminating residue can be estimated by using the value of the closest naturally occurring residue having side chain groups that are structural analogs. 26 and z 36 In some cases of Formula (IV), the helix-terminating residue is independently selected from d, n, G, and p. In some cases of Formula (IV), the helix-terminating residue is independently selected from d, G, and p. In some cases of Formula (IV), the helix-terminating residue is independently selected from G and p. In some cases of Formula (IV), the helix-terminating residue is independently selected from 26 and z 36 Each is p. In some cases of Formula (IV), z 36 For p.

[0207] Table 1: Naturally occurring amino acid α-helical propensity

[0208]

[0209]

[0210] *Estimated differences in free energy, relative to alanine, which was arbitrarily set to zero, are estimated in kcal / mol per residue in the α-helical configuration. Higher values ​​(more positive free energy) are less favorable. In some cases, deviations from these average values ​​may occur depending on the identities of neighboring residues.

[0211] In certain embodiments of Formula (IV), z 26 is a framework residue, e.g., a residue corresponding to a residue of a scaffold domain motif. In certain instances of Formula (IV), z 26 is a variant residue, e.g., a residue that differs from the corresponding residue of a scaffold domain motif, e.g., one or more of SEQ ID NOs: 1-21. In certain instances of Formula (IV), z 36 In certain embodiments of Formula (IV), h* 27 、f *31 and h *34 Each is a variant residue. In some embodiments of Formula (IV), j 28 and x 29 Each is a variant residue. In some cases of Formula (IV), j 28 、x 29 and x 30 In certain embodiments of Formula (IV), h* 27 is selected from h, y, and f. In certain embodiments of formula (IV), h *34 Selected from h, y and f.

[0212] In some embodiments of the compound, [Helix 2] is defined by the sequence of the formula:

[0213] p 26 hjjxfjxhjp 37 (SEQ ID NO:93)

[0214] (V)

[0215] wherein: each j is independently a hydrophobic residue; and each x is an amino acid residue. In certain instances, each j is independently selected from a, i, f, l, and v. In certain instances, each j is independently selected from a, i, l, and v. In certain instances, each j is independently selected from a, i, and v. In certain instances of formula (V), j 28 In certain cases of formula (V), j 29 is a, l or v. In some embodiments of formula (V), j 29 In some cases of Formula (V), j32 In certain cases of formula (V), j 36 In certain cases of formula (V), x 30 is a polar residue. In some cases of Formula (V), x 33 is a polar residue. In certain embodiments of Formula (V), x 30 and x 33 are independently selected from d, e, k, n, r, s, t, and q. In certain instances of Formula (V), x 30 and x 33 are independently selected from s and n. In certain instances of Formula (V), x 30 In certain cases of formula (V), x 33 In some embodiments of Formula (V), [Helix 2] comprises a sequence of the formula: 26 hvjxfjxhjp 37 (SEQ ID NO: 137).

[0216] In some embodiments of the compound, [Helix 2] is defined by the sequence of Formula (VI):

[0217] z 26 hvj 29 x 30 fix 33 haz 37 (SEQ ID NO:94)

[0218] (VI)

[0219] in:

[0220] z 26 selected from d, p and G;

[0221] j 29 Selected from f and i;

[0222] x 30 Selected from n and s;

[0223] x 33 is selected from n and s; and

[0224] z 37 Selected from p and G.

[0225] In some cases of Formula (VI), z 26 In some cases of formula (VI), j 29 In certain cases of formula (VI), x 30 In some embodiments of Formula (VI), x 33 is n. In some cases of Formula (VI), z 37 For p.

[0226] In some cases of the compound, [Helix 2] is defined by a sequence selected from:

[0227] a)phvj 29 x 30 fix 33 hap(VII) (SEQ ID NO:95) wherein: j 29 is selected from f and i; and x 30 and x 33 are independently polar amino acid residues; and

[0228] b) an amino acid sequence that is 80% or more identical to the sequence of formula (VII) as defined in a), for example 90% or more identical to the sequence as defined in a).

[0229] In some cases of the sequence of formula (VII) defined in a), x 30 and x 33 are independently selected from n, s, d, e, and k. In some cases of the sequence of Formula (VII) defined in a), j 29 In some cases of the sequence of formula (VII) defined in a), x 30 is s or n. In some cases of the sequence of formula (VII) defined in a), x 33 In some cases of the sequence of formula (VII) defined in a), j 29 is i; x 30 is s or n; and x 33 is n.

[0230] In some embodiments of the compound, [Helix 2] is 66% identical or higher to the sequence of SEQ ID NO: 74, for example, 77% identical or higher, or 88% identical or higher to the sequence of SEQ ID NO: 74.

[0231] In some embodiments of Formula (I), [Helix 3] comprises a sequence of the formula:

[0232] Λjxujxxuj (SEQ ID NO: 146)

[0233] (VIII)

[0234] wherein: each "Λ" is independently a D-aromatic amino acid; each j is independently a hydrophobic residue; each u is independently a nonpolar amino acid residue; and each x is independently an amino acid residue. In some cases, the heptad repeat alignment of Formula (VIII) is edcbag'f'e'd'. In some cases of Formula (VIII), Λ is an aromatic residue comprising a heteroaryl or substituted heteroaryl ring (e.g., an aromatic residue comprising a side chain group capable of hydrogen bonding with VEGF-A). In some cases, Λ is histidine or a substituted form thereof. Figure 23 A moderate strength hydrogen bond (2.9 angstroms) is shown between the nitrogen atom of h40 (210) of the exemplary compound and the adjacent Tyr48 of VEGF-A. Analysis of the structure of the complex indicates that a variety of histidine analogs are tolerated at position 40, including analogs that can occupy the available space and retain or strengthen hydrogen bonding with VEGF-A. In some cases of Formula (VIII), each u is independently a non-polar residue having a side chain selected from H, a lower alkyl group, and a substituted lower alkyl group. In some cases of Formula (VIII), each u is independently selected from G and a. In some cases of Formula (VIII), the first u is G. In some cases of Formula (VIII), the second u is a. In some cases, each j is a residue independently selected from a, i, f, l, and v. In some cases, each j is a residue independently selected from a, i, l, and v. In certain embodiments of Formula (VIII), j 28 is v. In certain embodiments of Formula (VIII), j 29 It is a, l or v.

[0235] In some embodiments of Formula (I) or (VIII), [Helix 3] comprises a sequence of Formula (IX):

[0236] x 38 xh*jxujxxujx 49 (SEQ ID NO:96)

[0237] (IX)

[0238] wherein j, x, and u are as defined above, and h* is histidine or an analog thereof. In some cases, the heptad repeat alignment of Formula (IX) is gfedcbag′f′e′d′c′. In some cases of Formula (IX), h* is histidine. In some cases of Formula (IX), h* is a histidine analog (e.g., a residue having a side chain comprising an alkyl-cycloalkyl group, such as an alkyl-cyclopentyl group or an alkyl-cyclohexyl group, or a substituted version thereof). In some cases of Formula (IX), h* is a substituted histidine. In some cases of Formula (XI), u 43 is G. In some cases of Formula (IX), u 47In some cases of Formula (IX), x 38 is v. In some cases of Formula (IX), x 39 In certain instances of Formula (IX), each j is a residue independently selected from a, i, f, l, and v. In certain embodiments of Formula (IX), j 41 is v. In some cases of Formula (IX), j 44 In some cases of Formula (IX), j 48 In some cases of Formula (IX), x 51 is a hydrophobic residue. In some cases of Formula (IX), x 51 In some cases of Formula (IX), x 42 In some cases of Formula (IX), x 45 is k or r. In some cases of Formula (IX), x 45 In some cases of Formula (IX), x 46 In some cases of Formula (IX), x 49 In some cases of Formula (IX), helix 3 is terminated with a C-terminal residue sequence. In some cases, helix 3 of Formula (IX) includes additional residues x 50 x 51 , wherein x is an amino acid residue. In some cases, x 50 is k or r. In some cases of Formula (IX), x 50 is k and x 51 In some cases of Formula (IX), x 50 is e and x 51 In some cases of Formula (IX), x 50 is G and x 51 is r. In some cases, helix 3 of Formula (IX) comprises a C-terminal region selected from one of SEQ ID NOs: 85-87. In some cases, [helix 3] comprises the heptad repeat alignment gfedcbag′f′e′d′c′b′a′. It will be appreciated that a variety of truncations (e.g., truncation of 1, 2, or 3 residues) and extensions (e.g., extensions of 1, 2, 3, or more residues) can be utilized at the C-terminus of [helix 3] without significantly disrupting, for example, Figure 9B The three-helix bundle structure or variant domain depicted in .

[0239] In some instances of Formula (IX), [Helix 3] is defined by a sequence selected from:

[0240] a)x 38 x 39 hvx 42 Glx 45 x 46aix 49 (X) (SEQ ID NO: 97) wherein: x 38 Select from v, e, k, r; x 39 、x 42 and x 46 are independently selected from polar amino acid residues; and x 45 and x 49 are independently selected from l, k, r, and e; and

[0241] b) an amino acid sequence that is 75% or more identical to the sequence of formula (X) defined in a), for example, 83% or more identical, or 91% or more identical to the sequence defined in a).

[0242] In some instances of Formula (IX), [Helix 3] is defined by a sequence selected from:

[0243] a)x 38 x 39 hvx 42 Glx 45 x 46 aix 49 x 50 a(XI) (SEQ ID NO: 98) wherein: x 38 Select from v, e, k, r; x 39 、x 42 、x 46 and x 50 are independently selected from polar amino acid residues; and x 45 and x 49 are independently selected from l, k, r, and e; and

[0244] b) an amino acid sequence that is 78% or more identical to the sequence of formula (XI) defined in a), for example, 85% or more identical, or 92% or more identical to the sequence defined in a).

[0245] In some cases of Formulas (X)-(XI), x 39 、x 42 、x 46 and x 50 are independently selected from n, s, d, e, and k. In some instances of Formulas (X)-(XI), x 38 In some cases of formulas (X)-(XI), x 45 In some cases of formulas (X)-(XI), x 49 In some cases of Formulas (X)-(XI), x 39 In some cases of formulas (X)-(XI), x 42In some cases of Formulas (X)-(XI), x 46 In some cases of Formula (XI), x 50 is k.

[0246] In some embodiments of the compound, [Helix 3] has 65% identity or greater identity to the sequence of SEQ ID NO: 79, such as 75% identity or greater identity, 83% identity or greater identity, or 91% identity or greater identity to the sequence of SEQ ID NO: 79. In some embodiments of the compound, [Helix 3] has 70% identity or greater identity to the sequence of SEQ ID NO: 82, such as 78% identity or greater identity, 85% identity or greater identity, or 92% identity or greater identity to the sequence of SEQ ID NO: 82.

[0247] In Formula (I), [Linker 2] is a peptide linker connecting [Helix 2] and [Helix 3], and optionally makes additional contacts with the surface of VEGF-A. [Linker 2] can have any convenient length. In some cases, [Linker 2] is a linker shorter than [Linker 1]. The N-terminal residue of [Linker 2] adjacent to [Helix 2] can be considered, for example, as a helix-terminating residue as described herein. In some cases, the C-terminal residue of [Linker 2] adjacent to [Helix 3] can be considered, for example, as a helix-terminating residue as described herein. In some cases, [Linker 2] can include 4 amino acid residues or fewer, such as 3 or fewer, or 2 or fewer. In some cases, [Linker 2] has the same number of residues as the corresponding helix-connecting loop region of the native GA scaffold domain. In certain embodiments of Formula (I), [Linker 2] is zx, wherein z is the helix 2-terminating residue and x is an amino acid residue. In some instances of [Linker 2], z is p or G. In some instances of [Linker 2], z is p. In some instances of [Linker 2], x is a VEGF-A contact residue. In some instances of [Linker 2], x is an aromatic residue. In some instances of [Linker 2], x is a w or h residue or a substituted form thereof. In some instances of [Linker 2], x is tyrosine or an analog thereof. In some instances, [Linker 2] includes a helix-terminating proline residue that provides a modified helix 2 to helix 3 interhelical angle (i.e., the angle between the axes of the helices), e.g., as described herein. See Figure 27.

[0248] A tyrosine analog can be incorporated at position 37 in Linker 2, such as an analog that includes a substituted or unsubstituted alkyl-aryl or alkyl-heteroaryl extended side chain group that can form closer contacts (e.g., hydrophobic contacts and / or hydrogen bonds) with adjacent residues of VEGF-A. Figure 23 The binding interface between compound (1.1.1(c21a)) and VEGF-A is depicted, showing that the phenolic oxygen of residue y37(209), which protrudes toward the surface of VEGF-A, is 6.5 to 7.2 angstroms from the adjacent VEGF-A residue. In some cases, x is a tyrosine analog having a side chain of the formula: -(CH2) n -Ar, wherein n is 1, 2, 3, or 4; and Ar is aryl, substituted aryl, heteroaryl, or substituted heteroaryl. In certain instances of x, Ar is substituted phenyl. In certain instances of x, Ar is substituted phenyl, and n is 2 or 3. In certain instances of x, Ar is phenyl substituted with a hydrogen bond donor or acceptor group that is configured to hydrogen bond to an adjacent VEGF-A residue.

[0249] In some embodiments of Formula (I), [Helix 2]-[Linker 2]-[Helix 3] comprises a sequence of Formula (XII) that defines a VEGF-A binding surface:

[0250] z 26 h*jxxf*jxh*jzy*xxh*jxujxxujx 49 (SEQ ID NO:99)

[0251] (XII)

[0252] in:

[0253] Each z is a helix-terminating residue;

[0254] y* is tyrosine or its analogue;

[0255] Each h* is independently histidine or an analog thereof;

[0256] f* is phenylalanine or its analogue;

[0257] Each u is independently a non-polar residue.

[0258] Each j is independently a hydrophobic residue; and

[0259] Each x is independently an amino acid residue.

[0260] In some cases, helix 3 of formula (XII) includes additional residues x 50 x 51 , wherein x is an amino acid residue. In some cases, x 50 is k or r. In some cases of Extended Formula (XII), x 50 is k and x 51 In some cases of extended formula (XII), x 50 is e and x 51In certain cases of formula (XII), x 50 is G and x 51 In some cases, helix 3 of formula (XII) comprises a C-terminal region selected from one of SEQ ID NOs: 85-87. In some embodiments of extended formula (XII), x 51 is a framework residue. In some embodiments of extended formula (XII), x 51 is a non-polar residue (u). In some embodiments of extended formula (XII), x 51 A hydrophobic residue.

[0261] In some embodiments of the compound, [helix 2]-[linker 2]-[helix 3] has 70% identity or higher with the sequence of SEQ ID NO: 80, for example, 75% identity or higher, 83% identity or higher, 87% identity or higher, 91% identity or higher, or 95% identity or higher with the sequence of SEQ ID NO: 80. In some embodiments of the compound, [helix 2]-[linker 2]-[helix 3] has 70% identity or higher with the sequence of SEQ ID NO: 83, for example, 80% identity or higher, 84% identity or higher, 88% identity or higher, 92% identity or higher, or 96% identity or higher with the sequence of SEQ ID NO: 83.

[0262] In certain instances of Formula (I), [Linker 1] has a sequence of the formula:

[0263] z(x) n x′z (SEQ ID NO: 147)

[0264] (XIII)

[0265] wherein: x′ is a polar residue; each x is an amino acid and n is an integer from 1 to 6; and each z is independently a helix-terminating residue, e.g., the first z is a helix-1 terminating residue and the second z is a helix-2 terminating residue. In some cases, x′ is a polar residue capable of hydrogen bonding to VEGF-A. In some cases, x′ is selected from d, e, n, q, ornithine, 2-amino-3-guanidinopropionic acid, and citrulline. In some cases, n is 1, 2, or 3. In some cases of Formula (XIII), [Linker 1] has the sequence of Formula (XIV):

[0266] z(x) n e*z (SEQ ID NO: 148)

[0267] (XIV)

[0268] wherein: each x is an amino acid and n is 1, 2, or 3; each z is independently a helix-terminating residue; and e* is glutamic acid or an analog thereof. In some instances of Formulas (XIII) and (XIV), each z is selected from G and p. In some instances of Formulas (XIII) and (XIV), n is 2.

[0269] In certain instances of Formula (I), [Linker 1]-[Helix 2]-[Linker 2]-[Helix 3] comprises a sequence of the formula:

[0270] z 22 xxe*zh*jxxf*jxh*jzy*xxh*jxujxxujxxx 51 (SEQ ID NO: 100)

[0271] (XV)

[0272] in:

[0273] e* is glutamic acid or its analogue;

[0274] Each z is independently a helix-terminating residue;

[0275] y* is tyrosine or its analogue;

[0276] Each j is independently a hydrophobic residue;

[0277] Each u is independently a non-polar amino acid residue; and

[0278] Each x is independently an amino acid residue.

[0279] In some cases of Formulas (I), (XII), and (XV), [Helix 2] is defined by the sequence of Formula (XVI):

[0280] z 26 hj 28 xxfj 32 xhj 35 z 36 (SEQ ID NO: 101)

[0281] (XVI)

[0282] in:

[0283] z 26 selected from d, p and G;

[0284] z 36 Selected from p and G;

[0285] j 28 、j 32 and j 35 are each independently a hydrophobic residue; and

[0286] Each x is independently an amino acid residue.

[0287] In some cases, 28 、j 32 and j 35 is the corresponding residue of the GA scaffold domain selected from SEQ ID NOs: 1-21. In some cases, j 28 、j 32 and j 35 Independently selected from a, i, l and v.

[0288] In some cases of Formulas (I), (XII), (XV), and (XVI), [Helix 2] is defined by a sequence selected from the group consisting of: a) phvx 29 x 30 fix 33 hap(XVII) (SEQ ID NO: 102)

[0289] Where: x 29 is selected from f and i; and x 30 and x 33 are independently selected from polar amino acid residues; and

[0290] b) an amino acid sequence having 80% or greater identity (eg, 90% or greater identity) to the sequence of formula (XVII) defined in a).

[0291] In some cases of Formulas (XVI)-(XVII), x 30 and x 33 is independently selected from n, s, d, e, and k. In some instances of Formulas (XVI)-(XVII), x 29 In some cases of Formulas (XVI)-(XVII), x 30 is s or n. In some cases of Formulas (XVI)-(XVII), x 33 In some cases of Formulas (XVI)-(XVII), x 29 is i; x 30 is s or n; and x 33 is n.

[0292] In some cases of Formulas (I), (XII), and (XV), [Helix 3] is defined by the sequence of Formula (XVIII):

[0293] xxhj 41 xuj 44 xxuj 48 xxx 51 (SEQ ID NO: 103)

[0294] (XVIII)

[0295] in:

[0296] j 41 、j 44 and j 48 Each is independently a hydrophobic residue;

[0297] Each u is independently a non-polar amino acid residue; and

[0298] Each x is independently an amino acid residue.

[0299] In some cases, x 50 is k or r. In some cases of Formula (XVIII), x 50 is k and x 51 In some cases of Formula (XVIII), x 50 is e and x 51 In some cases of Formula (XVIII), x 50 is G and x 51 In some cases, helix 3 of formula (XVIII) comprises a C-terminal region selected from one of SEQ ID NOs: 85-87. In some embodiments of formula (XVIII), x 51 is a framework residue. In some embodiments of Formula (XVIII), x 51 is a non-polar residue (u). In some embodiments of Formula (XVIII), x 51 Is a hydrophobic residue. In some embodiments of Formula (XVIII), j 41 、j 44 and j 48 are independently selected from a, i, l, and v. In some embodiments of Formula (XVIII), j 41 、j 44 and j 48 is the corresponding residue of the GA scaffold domain selected from SEQ ID NOs: 1-21.

[0300] In some cases of Formulas (I), (XII), and (XV), [Helix 3] is defined by a sequence selected from the group consisting of: a) x 38 x 39 hvx 42 Glx 45 x 46 aix 49 x 50 a(XIX) (SEQ ID NO: 104)

[0301] in:

[0302] x 38Select from v, e, k, r;

[0303] x 39 、x 42 、x 46 and x 50 are independently selected from polar amino acid residues; and

[0304] x 45 and x 49 are independently selected from l, k, r, and e; and

[0305] b) an amino acid sequence having 80% or greater identity (eg, 90% or greater identity) to the sequence of formula (XIX) defined in a).

[0306] In some cases of Formula (XIX), x 39 、x 42 、x 46 and x 50 are independently selected from n, s, d, e, and k. In some instances of Formula (XIX), x 38 is v. In some cases of Formula (XIX), x 45 In some cases of Formula (XIX), x 49 In some cases of Formula (XIX), x 39 In some cases of Formula (XIX), x 42 In some cases of Formula (XIX), x 46 In some cases of Formula (XIX), x 50 is k.

[0307] In some cases, [helix 1] comprises the following consensus sequence: l 7 ..a 10 ke.ai.elk.. 21 , wherein the residues at positions 8, 9, 13, 16, 20, and 21 are defined by any of the corresponding residues of the sequence of the GA domain of Table 3. In some cases, [helix 1] comprises a 15-residue sequence having 66% or greater percent identity, e.g., 73% or greater, 80% or greater, 86% or greater, or 93% or greater percent identity, to the following sequence: 6 lknakedaiaelkk 20 .

[0308] In some embodiments of the compound, [Linker 1]-[Helix 2]-[Linker 2]-[Helix 3] has 70% identity or higher with the sequence of SEQ ID NO: 81, for example, 78% identity or higher, 82% identity or higher, 85% identity or higher, 89% identity or higher, 92% identity or higher, or 96% identity or higher with the sequence of SEQ ID NO: 81. In some embodiments of the compound, [Linker 1]-[Helix 2]-[Linker 2]-[Helix 3] has 70% identity or higher with the sequence of SEQ ID NO: 84, for example, 80% identity or higher, 83% identity or higher, 86% identity or higher, 90% identity or higher, 93% identity or higher, or 96% identity or higher with the sequence of SEQ ID NO: 84.

[0309] Any convenient N-terminal α-helical segment of the GA domain of interest may be suitable for use in the compounds of the invention. In some cases, [Helix 1] includes the sequence of the N-terminal residues from about position 6 to about position 20. Figure 18B N-terminally truncated derivatives of exemplary compounds are shown, where residues 1-5 can be removed from the compound without significantly adversely affecting the intramolecular hydrophobic contacts of the compound that stabilize the three-helix bundle. In some cases, the compounds of the invention are truncated at the N-terminus by 6 or fewer residues, e.g., 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 residue, relative to the numbering system 1-53 described herein. In some cases, one or more of the residues in positions 1-5 of the compounds of the invention are deleted or modified, e.g., to impart desired properties to the resulting compound, e.g., helix capping, increased water solubility, or attachment to a molecule of interest (e.g., as described herein).

[0310] In some cases, [helix 1] comprises the following consensus sequence: l 7 ..a 10 ke.ai.elk.. 21 (SEQ ID NO: 105), wherein the residues at positions 8, 9, 13, 16, 20, and 21 are defined by any of the corresponding residues of the sequences of SEQ ID NOs: 2-21. In some cases, [helix 1] comprises a 15-residue sequence having 66% or greater percent identity, e.g., 73% or greater, 80% or greater, 86% or greater, or 93% or greater percent identity, to the following sequence: 6 lknakedaiaelkk 20 (SEQ ID NO:74).

[0311] Described herein are D-peptide GA domains having a VEGF specificity determining motif (SDM) defined by the configuration of variant amino acid residues contained within a base sequence of peptide framework residues. Based on the present disclosure, it should be understood that variations in either the SDM or the peptide framework residues / sequences are also encompassed. In some embodiments, the GA domain comprises a VEGF SDM that is 50% or greater, 60% or greater, 65% or greater, 70% or greater, such as 75% or greater, 80% or greater, 85% or greater, 90% or greater, or 95% or greater identical to any of the embodiments of the SDM residues and / or peptide framework residues defined herein. In some embodiments, the GA domain comprises a VEGF SDM having 1 to 5, such as 1 to 4, or 1 to 3 amino acid residue substitutions (e.g., 1, 2, 3, 4, or 5 substitutions) relative to any of the embodiments of the SDM residues and / or peptide framework residues defined herein. In certain embodiments, the 1 to 3 amino acid residue substitutions are selected from similar, conservative, or highly conservative amino acid residue substitutions according to Table 6.

[0312] In some embodiments of the D-peptide compound that specifically binds to VEGF, the D-peptide GA domain comprises a VEGF specificity determining motif (SDM) defined by the following amino acid residues:

[0313] e 25 phvisf--h 34 -p 36 x 37 -s 39 h--G 43 ---a 47 (SEQ ID NO: 149)

[0314] where x 37 In some embodiments of VEGF SDM, x 37 In some embodiments of VEGF SDM, x 37 In some embodiments of VEGF SDM, x 37 is y.

[0315] In some embodiments, the VEGF SDM is further defined by the following residues:

[0316] c 7 -----------------e 25 phvisf--h 34 -p 36 x 37 c 38 sh--G 43 ---a 47(SEQ ID NO: 150)

[0317] where x 37 In some embodiments of VEGF SDM, x 37 In some embodiments of VEGF SDM, x 37 is n.

[0318] In some embodiments of the GA domain, helix 1 (#6-21) Contains peptide framework sequence: x 6 x 7 knakedaiaelkka 21 (SEQ ID NO: 138)

[0319] Where: x 6 is selected from l, v, and i; and x 7 Selected from l and c.

[0320] In some embodiments of Helix 1, x 6 In some embodiments of helix 1, x 6 In some embodiments of Helix 1, x 6 For i.

[0321] In some embodiments, the GA domain comprises the N-terminal peptide framework sequence:

[0322] x 1 x 2 x 3 qwx 6 x 7 knakedaiaelkkaGit 24 (SEQ ID NO: 139)

[0323] in:

[0324] x 1 selected from t, y, f, i, p and r;

[0325] x 2 selected from i, h, n, p and s;

[0326] x 3 Selected from d, i and v;

[0327] x 6 selected from l, v, and i; and

[0328] x 7 Selected from l and c.

[0329] In some embodiments of the peptide framework sequence, x 1 In some embodiments of the peptide framework sequence, x1 In some embodiments of the peptide framework sequence, x 1 In some embodiments of the peptide framework sequence, x 1 In some embodiments of the peptide framework sequence, x 1 In some embodiments of the peptide framework sequence, x 1 For r.

[0330] In some embodiments of the peptide framework sequence, x 2 In some embodiments of the peptide framework sequence, x 2 In some embodiments of the peptide framework sequence, x 2 In some embodiments of the peptide framework sequence, x 2 In some embodiments of the peptide framework sequence, x 2 For s.

[0331] In some embodiments of the peptide framework sequence, x 3 In some embodiments of the peptide framework sequence, x 3 In some embodiments of the peptide framework sequence, x 3 For v.

[0332] In some embodiments of the peptide framework sequence, x 6 In some embodiments of the peptide framework sequence, x 6 In some embodiments of the peptide framework sequence, x 6 For i.

[0333] In some embodiments of the peptide framework sequence, x 7 In some embodiments of the peptide framework sequence, x 7 is c.

[0334] In some embodiments, the D-peptide GA domain comprises the C-terminal peptide framework sequence: ilkaha (SEQ ID NO: 140).

[0335] In some embodiments, the D-peptide GA domain comprises the sequence:

[0336] x 1 x 2 x 3 qwx 6 x 7 knakedaiaelkkagitephvisfinhapx 37 x 38 shvnGlknailkaha 53 (SEQ ID

[0337] NO:141)

[0338] in:

[0339] x 1 selected from t, y, f, i, p and r;

[0340] x 2 selected from i, h, n, p and s;

[0341] x 3 Selected from d, i and v;

[0342] x 6 Selected from l, v and i;

[0343] x 7 Selected from l and c;

[0344] x 37 Selected from t, y, n and s;

[0345] x 38 Select from v and c;

[0346] x 39 Selected from e and s;

[0347] x 40 Selected from h and e;

[0348] x 43 selected from g and a; and

[0349] x 47 Select from a and e.

[0350] In some embodiments, x 1 In some embodiments, x 1 In some embodiments, x 1 In some embodiments, x 1 In some embodiments, x 1 In some embodiments, x 1 In some embodiments, x 2 In some embodiments, x 2 In some embodiments, x 2 In some embodiments, x 2 In some embodiments, x 2 In some embodiments, x 3 In some embodiments, x 3 In some embodiments, x 3 In some embodiments, x 6 In some embodiments, x 6 In some embodiments, x6 In some embodiments, x 7 In some embodiments, x 7 In some embodiments, x 37 In some embodiments, x 37 In some embodiments, x 37 In some embodiments, x 37 In some embodiments, x 38 In some embodiments, x 38 In some embodiments, x 39 In some embodiments, x 39 In some embodiments, x 40 In some embodiments, x 40 In some embodiments, x 43 In some embodiments, x 43 In some embodiments, x 47 In some embodiments, x 47 For e.

[0351] In some embodiments, the D-peptide compound comprises a sequence selected from one of compounds 11055, 979102, and 979107-979110 (SEQ ID NOs: 108-113).

[0352] In some embodiments, the D-peptide compound comprises a sequence that is 80% or more (eg, 90% or more) identical to one of compounds 11055, 979102, and 979107-979110 (SEQ ID NOs: 108-113).

[0353] In some embodiments, the D-peptide compound comprises a sequence having 1 to 10 amino acid residue substitutions (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, e.g., 1 or 2 amino acid residue substitutions) relative to one of compounds 11055, 979102, and 979107-979110 (SEQ ID NOs: 108-113). In certain embodiments, the 1 to 10 amino acid residue substitutions are selected from, e.g., similar, conservative, and highly conservative amino acid residue substitutions according to Table 6.

[0354] GA scaffold domain

[0355] Based on this disclosure, it will be appreciated that several amino acid residues in the GA domain motif that are not located at the VEGF-A binding surface of the structure can be modified without adversely affecting the VEGF-A binding activity of the resulting modified compound. Thus, any convenient amino acid can be incorporated into the compounds of the invention to impart desired properties, including but not limited to improved water solubility, ease of chemical synthesis, cost, bioconjugation sites, stability, pI, aggregation, reduced nonspecific binding, and / or specific binding to a second target protein. The positions of the mutations can be selected to minimize any disruption to the structure of the VEGF-A binding GA domain motif or its specific binding to the target VEGF-A protein, for example, by selecting a position on the side of the structure opposite the VEGF-A binding surface. In some cases, the compound includes two or more, e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more, surface mutations at positions that are not part of the binding surface for the target VEGF-A protein.

[0356] For example, in some cases, one or more of the c, f, and b residues of helix 1 and the c and f residues of helices 2 and 3 may be modified because those residues are not directly involved in VEGF-A binding and solvent exposure (see Figure 3B In some cases, variant amino acid residues can be selected for incorporation into the compounds of the invention at specific heptapeptide repeat positions based on the percentage of occurrence of known amino acids at similar positions, such as in known naturally occurring proteins. Table 2 provides a list of the percentage of occurrence of amino acids at triple-stranded coiled-coil heptapeptide positions that can be used to select variant amino acid residues, such as amino acid residues with a percentage of occurrence of 2% or more, such as 5% or more, 10% or more, or even more. In some cases, surface mutations include mutating residues to polar residues, such as residues that confer the desired solubility to the compound. In some cases, surface mutations include mutating residues to charged residues, such as residues that confer the desired solubility to the compound. In some cases, surface mutations include mutating residues to basic residues (e.g., k or h). In some cases, surface mutations include mutating residues to acidic residues (e.g., d or e), such as residues that confer the desired pI to the compound.

[0357] Table 2: Percentage of amino acids at positions in triple-stranded coiled-coil heptads*

[0358]

[0359] *M is the total number of times a particular amino acid was found at a heptapeptide position. N is the total number of residues counted at that heptapeptide position. See Table 3 of DeGrado et al.

[0360] In some cases, peptide compounds of the invention are selected from phage display libraries based on the GA scaffold domain and further developed (e.g., via additional affinity maturation and / or point mutations) to include several variant amino acids integrated into the GA scaffold domain. The variant motif comprises the variant amino acids and can define the VEGF-A binding surface of the compounds of the invention. SEQ ID NO: 25 shows the variant motif of exemplary compound 1.1.1 (c21a). Various aspects of the VEGF-A binding surface of the compounds of the invention are described above. It will be understood that a variety of basic GA scaffold domain sequences can be utilized in the compounds of the invention to provide a three-helix bundle scaffold structure into which the variant domains are incorporated. The structure of the compounds of the invention can be defined by a combination of variant and framework domains. The sequence of the compounds of the invention can be defined by a combination of variant and framework residues. Thus, in some cases, the framework residues of a structural or sequence motif can be defined by corresponding residues of the scaffold domain structure or sequence.

[0361] For example, comparison of scaffold SCF32 ​​(SEQ ID NO: 2) with compound 1.1.1 (c21a) (SEQ ID NO: 24) provides variant motifs (SEQ ID NO: 25) and framework domains (SEQ ID NO: 26). Various aspects of the variant motifs are described herein. It will be appreciated that a variety of modifications can be incorporated into the framework domains without significant adverse effects on the three-helix bundle structure or the VEGF-A binding surface. Figures 3 and 4 show an alignment of exemplary sequences and motifs with a heptad repeat structural model of a compound of the invention. The residues of helix 1 that are solvent exposed and not involved in hydrophobic core interactions can be any convenient amino acid residues, including but not limited to polar residues. In some cases, the b, c, and / or f residues of helix 1 of the compounds of the invention may be altered (see, e.g., Figure 6B ) without adversely affecting the VEGF-A binding activity of the compound, and in some cases providing desired properties. In some cases, the e and g residues of helix 1 may also be altered. In certain embodiments, the f residues of helix 2 and / or helix 3 may be altered without adversely affecting the VEGF-A binding activity of the compound, and in some cases providing desired properties. In some cases, C-terminal modifications may be included in helix 3, such as truncations or extensions (e.g., residues at positions 50-53 of helix 3, see Figure 10A ). The compounds of the present invention may have a framework domain motif as defined by one of SEQ ID NOs: 2-21. In some cases, the framework domain motif of the compound is defined by SEQ ID NO: 1.

[0362] In some cases, it is less desirable to have residues that make contact with the hydrophobic core of the GA scaffold domain (e.g. Figure 7B) are modified as these residues are involved in the helix-to-helix hydrophobic contacts that stabilize the three-helix bundle. However, a variety of non-polar or hydrophobic residues may be used in the hydrophobic core of the three-helix bundle of the compounds of the invention. Figures 9A-9C The sequences and structures of exemplary compounds are shown, with the configuration of the a and d residues of the heptad repeat model that can form hydrophobic interactions between the helices indicated in red. In some cases, the C-terminal e residue of the helix 3 heptad repeat located at the end of the helical region can be modified, for example, to provide helix capping, helix truncation, or extension to a linker. In some cases, the N-terminal residue of the helix 1 heptad repeat located at the end of the helical region can be modified (e.g., Figure 10A In certain embodiments, the a and d residues of the compounds of the invention may be selected from the corresponding hydrophobic core residues of any one of SEQ ID NOs: 1-21.

[0363] In some cases, each of the a and d residues of [helix 2] is a residue that can confer structural stability to the modified three-helix bundle of the compounds of the invention. In some cases, one or more of the a and d residues of the compounds of the invention, such as the residues at positions 28, 32, and 35 of [helix 2], provide intramolecular contacts that partially define the hydrophobic core of the compound. In certain embodiments of [helix 2], each of the a and d residues is independently a hydrophobic residue. In some cases of [helix 2], each of the a and d residues is selected from a, i, f, m, l, and v. In some embodiments of [helix 2], each of the a and d residues is selected from a, i, f, l, and v. In some cases of [helix 2], each of the a and d residues is selected from a, i, l, and v. In some cases of [helix 2], the a and d residues at positions 32 and 35 are part of a scaffold domain (e.g., framework residues having the same identity as corresponding residues of a scaffold domain motif).

[0364] In some cases, the "d" residues of [helix 2] and [helix 3] that are closest to the gg face of the structure that contacts VEGF-A may contact the protein. In such cases, the "d" residues that contact VEGF-A may be referred to as boundary residues. It is understood that

[0365] Table 3 lists a list of sequences of exemplary scaffold domains, exemplary compounds, and exemplary compound domains of interest. In some embodiments of Formulas (I)-(XIX), the residues correspond to residues located at the same position of one of SEQ ID NOs: 22-71 listed in Table 3. In certain embodiments of Formula (I), the compound comprises a residue sequence having 85% or greater percent identity, e.g., 88% or greater, 90% or greater, 92% or greater, 94% or greater, 96% or greater, or 98% or greater percent identity to one of SEQ ID NOs: 22-71. In some cases, the sequence identity comparison is based on a sequence region having the same length, e.g., 48 residues, 49 residues, 50 residues, 51 residues, 52 residues, or 53 residues in length. These compounds of the invention can be further mutated to incorporate residues at surface positions that are not involved in contacting the GA domain motif of the target VEGF-A protein. The residues can be selected to confer desired properties (e.g., as described herein) on the resulting modified compound.

[0366] Table 3: Sequences of scaffolds and compounds of interest

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373] Table 4: Exemplary D-peptide Z and GA domains that bind VEGF

[0374]

[0375]

[0376] Table 5: Exemplary Multivalent VEGF Binding D-Peptide Compounds

[0377]

[0378] Aspects of the present disclosure include compounds (e.g., as described herein), salts thereof (e.g., pharmaceutically acceptable salts) and / or solvates or hydrates thereof. It should be understood that the present disclosure is intended to encompass all permutations of salts, solvates, and hydrates. In some embodiments, the compounds of the present invention are provided in the form of pharmaceutically acceptable salts. Compounds containing amines and / or nitrogen-containing heteroaryl groups may be basic in nature and therefore react with any number of inorganic and organic acids to form pharmaceutically acceptable acid addition salts. Acids commonly used to form such salts include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, and phosphoric acid; and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, and acetic acid; and related inorganic and organic acids. Such pharmaceutically acceptable salts thus include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, octanoates, acrylates, formates, isobutyrates, decanoates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4-dioate, hexyne-1, Pharmaceutically acceptable acid addition salts include, but are not limited to, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, hippurate, gluconate, lactobionate, and similar salts. In certain specific embodiments, pharmaceutically acceptable acid addition salts include those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and those formed with organic acids such as fumaric acid and maleic acid.

[0379] Compound characteristics

[0380] The variant D-peptide domains of the multivalent compounds of the present invention can define a binding surface area of ​​suitable size to form a high functional affinity (e.g., equilibrium dissociation constant (K D )) and specificity (e.g., 300 nM or less, such as 100 nM or less, 30 nM or less, 10 nM or less, 3 nM or less, 1 nM or less, 300 pM or less or even less) of protein-protein interactions. The variant D-peptide domains can each comprise 600 and Between, for example, 800 and Between 1000 and Between 1100 and Between or about surface area.

[0381] In some cases, the multivalent D-peptide compound binds with a binding affinity (K D ) specifically binds to the target protein with an affinity that is 10 times greater than or greater than the binding affinity of the first and second D-peptide domains alone for the target protein, such as 30 times greater, 100 times greater, 300 times greater, 1000 times greater, or even greater. The affinity of the peptide compound for the target protein can be determined by any convenient method, such as using an SPR binding assay or an ELISA binding assay (e.g., as described herein). In some cases, the binding affinity (K) of the multivalent D-peptide compound for the target protein is 10 times greater than or greater than the binding affinity of the first and second D-peptide domains alone for the target protein. D ) is 3 nM or less, such as 1 nM or less, 300 pM or less, or 100 pM or less, and the binding affinity of the first and second D-peptide domains for the target protein is each independently 100 nM or greater, such as 200 nM or greater, 300 nM or greater, 400 nM or greater, 500 nM or greater, or 1 μM or greater. The effective binding affinity of the multivalent D-peptide compound as a whole can be optimized to provide desired biological efficacy and / or other properties, such as in vivo half-life. By selecting individual D-peptide domains with specific individual affinities for the target binding sites of the individual D-peptide domains, the overall functional affinity of the multivalent D-peptide compound can be optimized as desired.

[0382] The potency of the compound can be assessed using any convenient assay, for example, via an ELISA assay measuring IC50 as described in the Experimental section herein. In some cases, the multivalent compounds of the invention have in vitro antagonistic activity against the target protein that is at least 10-fold more potent than the potency of each of the first and second D-peptide domains alone, e.g., at least 30-fold, at least 100-fold, at least 300-fold, at least 1000-fold more potent.

[0383] In certain embodiments, the peptide compounds of the invention bind specifically to the VEGF-A target protein with high affinity, e.g., as determined by SPR binding assay or ELISA assay. The compounds of the invention may exhibit an affinity for VEGF-A of 1 μM or less, e.g., 300 nM or less, 100 nM or less, 30 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, 1 nM or less, 600 pM or less, 300 pM or less, or even less.

[0384] The D-peptide compounds of the present invention can exhibit specificity for VEGF-A, e.g., as determined by comparing the affinity of the compound for VEGF-A protein to the affinity for a reference protein (e.g., albumin) of 5:1 or greater, 10:1 or greater, e.g., 30:1 or greater, 100:1 or greater, 300:1 or greater, 1000:1 or greater, or even greater. In some cases, the specificity can be a coefficient of difference in binding affinity of 10 3 or higher, such as 10 4 or higher, 10 5 or higher, 10 6 or higher, or even higher. In some cases, the peptide compound can be optimized for any desired property, such as protein folding, protease stability, thermal stability, compatibility with pharmaceutical formulations, etc. Any convenient method can be used to select D-peptide compounds, for example, structure-activity relationship (SAR) analysis, affinity maturation methods, or phage display methods.

[0385] Also provided are D-peptide compounds with high thermal stability. In some cases, the melting temperature of the compound with high thermal stability is 50°C or higher, such as 60°C or higher, 70°C or higher, 80°C or higher, or even 90°C or higher. Also provided are D-peptide compounds with high protease stability. The D-peptide compounds of the present invention are resistant to proteases and may have long serum and / or saliva half-lives. Also provided are D-peptide compounds with long in vivo half-lives. As used herein, "half-life" refers to the time required for a measured parameter of a compound, such as potency, activity, and effective concentration, to drop to half of its original level, such as half of its original potency, activity, or effective concentration at time zero. Thus, parameters of polypeptide molecules, such as potency, activity, or effective concentration, are generally measured over time. For the purposes of this article, half-life can be measured in vitro or in vivo. In some cases, the half-life of the peptide compound is 1 hour or longer, e.g., 2 hours or longer, 6 hours or longer, 12 hours or longer, 1 day or longer, 2 days or longer, 7 days or longer, or even longer. Stability in human blood can be measured by any convenient method, e.g., by incubating the compound in human EDTA blood or serum for a specified time, quenching a sample of the mixture, and analyzing the sample for the amount and / or activity of the compound, e.g., by HPLC-MS, by an activity assay, e.g., as described herein.

[0386] Also provided are D-peptide compounds having low immunogenicity, e.g., non-immunogenicity. In certain embodiments, the D-peptide compounds have low immunogenicity compared to the L-peptide compounds. In certain embodiments, the immunogenicity of the D-peptide compounds is 10% or less, 20% or less, 30% or less, 40% or less, 50% or less, 70% or less, or 90% or less compared to the L-peptide compounds in an immunogenicity assay, such as that described in Dintzis et al., "A Comparison of the Immunogenicity of a Pair of Enantiomeric Proteins," Proteins: Structure, Function, and Genetics 16:306-308 (1993).

[0387] Also provided are D-peptide compounds optimized for binding affinity and specificity to VEGF-A by affinity maturation, such as second-generation D-peptide compounds based on parent compounds that bind to VEGF-A. In some embodiments, affinity maturation of the compounds of the present invention may include maintaining a portion of the variant amino acid positions as fixed positions while varying the remaining variant amino acid positions to select the optimal amino acid at each position. A parent D-peptide compound may be selected as a scaffold for affinity matured compounds. In some cases, a number of affinity matured compounds are prepared that include mutations at a limited subset of the parent's variant amino acid positions, while the remaining variant positions remain fixed positions. The mutant positions can be laid across the scaffold sequence to generate a series of compounds, thereby representing mutations at each variant position, with a different range of amino acids (e.g., all 20 naturally occurring amino acids) being substituted at each position. Mutations including deletions or insertions of one or more amino acids may also be included at the variant positions of the affinity matured compounds. Affinity matured compounds can be prepared and screened using any convenient method, such as phage display library screening, to identify second generation compounds with improved properties, such as increased binding affinity for the target molecule, protein folding, protease stability, thermal stability, compatibility with pharmaceutical formulations, etc.

[0388] In some embodiments, affinity maturation of the compounds of the invention may include maintaining most or all of the variant amino acid positions in the variable regions of the parent compound as fixed positions and introducing sequential mutations at positions adjacent to these variable regions. Such mutations may be introduced at positions in the parent compound that were previously considered fixed positions in the original GA scaffold domain. Such mutations can be used to optimize compound variants for any desired properties, such as protein folding, protease stability, thermal stability, compatibility with pharmaceutical formulations, and the like.

[0389] Aspects of the present disclosure include compounds (e.g., as described herein), salts thereof (e.g., pharmaceutically acceptable salts), and / or solvates, hydrates, and / or prodrug forms thereof. It will be understood that all permutations of salts, solvates, hydrates, and prodrugs are intended to be encompassed by the present disclosure.

[0390] In some embodiments, the compounds of the present invention or their prodrug forms are provided in the form of pharmaceutically acceptable salts. Compounds containing amines or nitrogen-containing heteroaryl groups may be basic in nature and, therefore, react with any number of inorganic and organic acids to form pharmaceutically acceptable acid addition salts. Acids commonly used to form such salts include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, and phosphoric acid; and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, and acetic acid; as well as related inorganic and organic acids. Such pharmaceutically acceptable salts thus include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, octanoates, acrylates, formates, isobutyrates, decanoates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4-dioate, hexyne-1, Pharmaceutically acceptable acid addition salts include, but are not limited to, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, hippurate, gluconate, lactobionate, and similar salts. In certain specific embodiments, pharmaceutically acceptable acid addition salts include those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and those formed with organic acids such as fumaric acid and maleic acid.

[0391] Polymeric compounds

[0392] Any suitable D-peptide compound (e.g., as described herein) can be polymerized to provide a multimer of the D-peptide compound. In certain embodiments, the multimer comprises two or more D-peptide compounds, such as two (e.g., dimers), three (e.g., trimers), or four or more compounds (e.g., tetramers or dendrimers, etc.). In some cases, the multimer is described by the following formula:

[0393] Y-(GA) n

[0394] wherein: Y is a multivalent linking group; n is an integer greater than one; and GA is a D-peptide compound comprising a GA domain motif (e.g., as described herein). In some cases, n is 2. In some cases, n is 3.

[0395] In certain instances, the multimer is a dimer of one of the following formulae:

[0396]

[0397] wherein each GA is independently a D-peptide compound (e.g., as described herein); and Y is a linker attached to the N-terminus (N-GA) or C-terminus (GA-C) of the compound. In some cases, the dimer is a homodimer of two identical GA domain motifs that each specifically bind to VEGF-A. In some cases, the dimer is a heterodimer. A heterodimer can be a dimer of two different GA domain motifs that each specifically bind to VEGF-A, or a dimer of a D-peptide compound of the invention and a second D-peptide binding domain.

[0398] Any suitable linking group can be used in the polymers of the present invention. The terms "linker," "connection," and "linking group" are used interchangeably and refer to a linking portion that covalently links two or more compounds. In some cases, the linker is divalent. In some cases, the linker is a branched or trivalent linking group. In some cases, the length of the straight or branched chain of the linker is 200 atoms or less (e.g., 100 atoms or less, 80 atoms or less, 60 atoms or less, 50 atoms or less, 40 atoms or less, 30 atoms or less, or even 20 atoms or less). The linking moiety can be a covalent bond connecting two groups or a length between 1 and 200 atoms, for example, a straight or branched chain of about 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 100, 150 or 200 carbon atoms in length, wherein the linker can be a straight chain, branched chain, cyclic or single atom. In some cases, one, two, three, four or five or more carbon atoms of the linker backbone can be optionally substituted by sulfur, nitrogen or oxygen heteroatoms. In some cases, when the linker includes a PEG group, the segment of the linker backbone is replaced by oxygen every two atoms. The key between the backbone atoms can be saturated or unsaturated, and typically no more than one, two or three unsaturated bonds will be present in the linker backbone. The linker can include one or more substituents, such as alkyl, aryl or alkenyl. The linker may include, but is not limited to, oligo(ethylene glycol), ethers, thioethers, disulfides, amides, carbonates, carbamates, tertiary amines, alkyl groups, which may be linear or branched, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), and the like. The linker backbone may include a cyclic group, such as an aryl, heterocyclic, or cycloalkyl group, wherein the backbone includes two or more atoms, such as two, three, or four atoms, of the cyclic group. The linker may be cleavable or non-cleavable. The linker may be a peptide, such as a linked sequence of residues.

[0399] Y may comprise any convenient group or linker unit, including but not limited to an amino acid residue, PEG, a modified PEG (e.g., -NH(CH2) m O[(CH2)2O] n (CH2) p In some cases, Y is a peptide.

[0400] In some embodiments, Y is a linker comprising -(L1)a-(L2)b-(L3)c-(L4)d-(L5)e-, wherein L1, L2, L3, L4, and L5 are each a linker unit, and a, b, c, d, and e are each independently 0 or 1, wherein the sum of a, b, c, d, and e is 1 to 5. Other linkers are possible, as shown in the multimeric compounds described herein.

[0401] In some cases, Y comprises a modified PEG linker that is connected to the D-peptide compound using any convenient connection chemistry. PEG is polyethylene glycol or modified polyethylene glycol. Modified PEG means a polyethylene glycol of any convenient length, wherein one or both of the ends are modified to include a chemoselective functional group suitable for conjugation to, for example, another linker moiety or to the end or side chain of the peptide compound. Table 9 and the Examples section describe several exemplary homodimers of compound 1.1.1 (c21a) connected via the N-terminus or C-terminus of the compound. The D-peptide compound can be modified at the N and / or C-terminus of the GA domain motif to include one or more additional amino acid residues that can provide a specific bond or connection chemistry to connect to the Y group, such as cysteine ​​or lysine.

[0402] Chemoselective reactive functional groups that can be used to attach the peptide compounds of the invention via a linker include, but are not limited to, amino (e.g., an N-terminal amino group or a lysine side chain group), azide, alkynyl, phosphine, thiol (e.g., a cysteine ​​residue), C-terminal thioester, aryl azide, maleimide, carbodiimide, N-hydroxysuccinimide (NHS)-ester, hydrazide, PFP-ester, hydroxymethylphosphine, psoralen, imidoester, pyridyl disulfide, isocyanate, aminooxy-, aldehyde, ketone, chloroacetyl, bromoacetyl, and vinyl sulfone.

[0403] Any convenient multivalent linker can be utilized in the multimers of the present invention. Multivalent means that the linker includes two or more terminal groups suitable for attachment to, for example, a compound of the present invention as described herein. In some cases, the multivalent linker is divalent or trivalent. In some cases, the multivalent linker Y is a dendrimer scaffold. Any convenient dendrimer scaffold can be used in the multimers of the present invention. A dendrimer scaffold is a branched molecule comprising at least one branch point and two or more termini suitable for attachment to the N-terminus or C-terminus of a GA domain motif via an optional linker. The dendrimer scaffold can be selected to provide a desired spatial arrangement of two or more GA domain motifs. In some cases, the spatial arrangement of two or more GA domain motifs is selected to provide a desired binding affinity and avidity for a target protein. Figure 17The X-ray crystal structure of compound 1.1.1 (c21a) is shown, which includes a complex comprising two VEGF-A molecules and two compounds. In the view of the depicted structure, the distance between the N-terminus (about 60 angstroms) and the C-terminus (about 70 angstroms) is marked by the dotted line. In some cases, the dimer includes N-N linked Y groups with a length of about 60 angstroms or longer. In some cases, the dimer includes C-C linked Y groups with a length of about 70 angstroms or longer.

[0404] In some cases, the D-peptide compounds each independently include a specific binding moiety (e.g., biotin or a peptide tag), wherein the D-peptide compounds can bind to each other via a multivalent binding moiety (e.g., streptavidin, avidin, or an antibody) that specifically binds to a particular binding moiety. In some embodiments, for example, two or more D-peptide compounds as described above each include a specific binding moiety that is a biotin moiety. In certain embodiments, the specific binding moiety is a terminal biotin moiety attached to the N-terminus or C-terminus of the compound via an optional linker. In some cases, the terminal biotin moiety is biotin-(Gly) n -, wherein n is 1 to 6, or biotin-Ahx- (Ahx = 6-aminohexanoic acid residue).

[0405] Modified compounds

[0406] Any suitable molecule or moiety of interest can be attached to the D-peptide compounds of the present invention. The molecule of interest can be peptide or non-peptide, naturally occurring or synthetic. Molecules of interest suitable for use in conjunction with the compounds of the present invention include, but are not limited to, additional protein domains; polypeptides or amino acid residues; peptide tags; specific binding moieties; polymeric moieties, such as polyethylene glycol (PEG), carbohydrates, dextran, or polyacrylates; linkers; half-life extending moieties; drugs; toxins; detectable labels; and solid supports. In some cases, the molecule of interest can impart enhanced and / or modified properties and functions to the resulting peptide compound, including but not limited to improved water solubility, ease of chemical synthesis, cost, bioconjugation sites, stability, isoelectric point (pI), aggregation, reduced nonspecific binding, and / or specific binding to, for example, a second target protein as described herein.

[0407] In some embodiments of any of the VEGF-A binding GA domain motif sequences described herein, the motif can be extended to include one or more additional residues at the N-terminus and / or C-terminus of the sequence, for example, two or more, three or more, four or more, five or more, six or more, or even more additional residues. Even if such additional residues do not provide for VEGF-A binding interactions, they can be considered part of the GA domain motif. Any convenient residue can be included at the N-terminus and / or C-terminus of the VEGF-A binding GA domain motif to provide desired properties or groups, such as increased solubility via a water-soluble group, a bond for dimerization or multimerization, or a bond for attachment to a label or specific binding moiety.

[0408] In some cases, the modified compounds of the invention are described by the following formula:

[0409] XLZ

[0410] wherein X is a VEGF-A binding GA domain motif (e.g., as described herein); L is an optional linking group; and Z is a molecule of interest, wherein L is linked to X at any convenient position (e.g., N-terminus, C-terminus, or via the side chain of a surface residue not involved in target binding).

[0411] D-peptide compounds can include one or more molecules of interest, such as an N-terminal portion and / or a C-terminal portion. In some cases, the molecules of interest are covalently linked via the α-amino group of the N-terminal residue, or covalently linked to the α-carboxylic acid group of the C-terminal residue. In other cases, the molecules of interest are linked to the motif via a side chain group of the residue (e.g., via a C, K, D, or E residue).

[0412] The molecule of interest may comprise a polypeptide or protein domain. Polypeptides and protein domains of interest include, but are not limited to, a gD tag, a c-Myc epitope, a FLAG tag, a His tag, a fluorescent protein (e.g., GFP), a β-galactosidase protein, GST, albumin, an immunoglobulin, an Fc domain or similar antibody-like fragment, a leucine zipper motif, a coiled-coil domain, a hydrophobic region, a hydrophilic region, a polypeptide comprising free thiol groups that form intermolecular disulfide bonds between two or more multimerization domains, a "protuberance-into-cavity" domain, β-lactoglobulin, or a fragment thereof.

[0413] The molecule of interest may include a half-life extending moiety. The term "half-life extending moiety" refers to a pharmaceutically acceptable portion, domain, or "vehicle" covalently linked or conjugated to a compound of the invention that prevents or reduces in vivo proteolytic degradation or other activity-reducing chemical modifications of the compound of the invention, extends half-life or improves other pharmacokinetic properties (e.g., absorption rate), reduces toxicity, improves solubility, increases the biological activity and / or target selectivity of the compound of the invention relative to a target of interest, improves manufacturability, and / or reduces the immunogenicity of the compound of the invention compared to the unconjugated form of the compound of the invention.

[0414] In certain embodiments, the half-life extending moiety is a polypeptide that binds a serum protein, such as an immunoglobulin (eg, IgG) or serum albumin (eg, human serum albumin (HSA)). Polyethylene glycol is an example of a suitable half-life extending moiety. Exemplary half-life extending moieties include polyalkylene glycol moieties (e.g., PEG), serum albumin or a fragment thereof, transferrin receptor or transferrin binding portion thereof, and moieties comprising a binding site for a polypeptide that extends in vivo half-life, ethylene glycol copolymers, propylene glycol copolymers, carboxymethylcellulose, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (e.g., polylysine), dextran N-vinyl pyrrolidone, poly N-vinyl pyrrolidone, propylene glycol homopolymers, propylene oxide polymers, ethylene oxide polymers, polyoxyethylene polyols, polyvinyl alcohol, linear or branched glycosylated chains, polysialic acid, polyacetals, long chain fatty acids, long chain hydrophobic aliphatic groups, immunoglobulin Fc domains (see, e.g., U.S. Pat. No. 6,660,843), albumin (e.g., human serum albumin; see, e.g., U.S. Pat. Nos. 6,926,898 and US Pat. Nos. 6,926,898 and 6,926,898). 2005 / 0054051; U.S. Patent No. 6,887,470), transthyretin (TTR; see, e.g., US 2003 / 0195154; 2003 / 0191056), or thyroxine-binding globulin (TBG).

[0415] Extended half-life can also be achieved through controlled-release or sustained-release dosage forms of the compounds of the invention, as described, for example, in Gilbert S. Banker and Christopher T. Rhodes, Sustained and controlled release drug delivery systems, Modern Pharmaceutics, 4th ed., Revised and Expanded, Marcel Dekker, New York, 2002, 11. This can be achieved through a variety of formulations, including liposomes and drug-polymer conjugates.

[0416] In certain embodiments, the half-life extending moiety is a fatty acid. Any suitable fatty acid can be used in the modified compounds of the present invention. See, for example, Chae et al., "The fatty acid conjugated exendin-4 analogs for type 2 antidiabetic therapeutics," J. Control Release. 2010 May 21; 144(1): 10-6.

[0417] In certain embodiments, the compound is modified to include a specific binding moiety. A specific binding moiety is a moiety that is capable of specifically binding to a second moiety that is complementary to it. In some cases, the specific binding moiety is present in an amount of at least 10 -7 Affinity of 100 nM or less (e.g., as determined by a K of 100 nM or less, such as 30 nM or less, 10 nM or less, 3 nM or less, 1 nM or less, 300 pM or less, or 100 pM or even less) D The specific binding moiety is a moiety that binds to a complementary second moiety (measured). Complementary binding moiety pairs of specific binding moieties include, but are not limited to, ligands and receptors, antibodies and antigens, complementary polynucleotides, complementary protein homodimers or heterodimers, aptamers and small molecules, polyhistidine tags and nickel, as well as chemoselective reactive groups (e.g., thiols) and electrophilic groups (e.g., reactive thiol groups can undergo Michael addition). Specific binding pairs can include analogs, derivatives, and fragments of the original specific binding members. For example, antibodies against protein antigens can also recognize peptide fragments, chemically synthesized labeled proteins, derivatized proteins, etc., as long as the epitope is present. Protein domains of interest that can be used as specific binding moieties include, but are not limited to, Fc domains or similar antibody-like fragments, leucine zipper motifs, coiled-coil domains, hydrophobic regions, hydrophilic regions, polypeptides containing free thiols that form intermolecular disulfide bonds between two or more multimerization domains, or "intraluminal protuberance" domains (see, e.g., WO 94 / 10308; U.S. Pat. No. 5,731,168, Lovejoy et al. (1993), Science 259: 1288-1293; Harbury et al. (1993), Science 262: 1401-05; Harbury et al. (1994), Nature 371: 80-83; Hakansson et al. (1999), Structure 7: 255-64).

[0418] In certain embodiments, the molecule of interest is a linked specific binding portion that specifically binds to a target protein. The linked specific binding portion may be an antibody, an antibody fragment, an aptamer, or a second D-peptide binding domain. The linked specific binding portion may specifically bind to any suitable target protein, such as a target protein that is targeted to VEGF-A in the therapeutic methods of the present invention. The target proteins of interest include, but are not limited to, PDGF (e.g., PDGF-B), VEGF-B, VEGF-C, VEGF-D, EGF, EGFR, Her2, PD-1, PD-L1, OX-40, and LAG3. In some cases, the linked specific binding portion is a second D-peptide binding domain that targets PDGF-B.

[0419] In certain embodiments, the specific binding moiety is an affinity tag, such as a biotin moiety. Exemplary biotin moieties include biotin, desthiobiotin, oxybiotin, 2'-iminobiotin, diaminobiotin, biotin sulfoxide, cytosine, and the like. In some cases, the biotin moiety can specifically bind with high affinity to a chromatography support containing immobilized avidin, neutravidin, or streptavidin. The biotin moiety can be at least 10 -8 In some cases, the biotin moiety can be combined with avidin, neutravidin or streptavidin in solution to form a multimeric compound, such as a dimeric or tetrameric complex of a D-peptide compound and avidin, neutravidin or streptavidin. The biotin moiety can also include a linker, such as -LC-biotin, -LC-LC-biotin, -SLC-biotin or -PEG. n - Biotin, wherein n is 3-12 (commercially available from Pierce Biotechnology).

[0420] In certain embodiments, the compound is modified to include a detectable label. Examples of detectable labels include labels that allow direct and indirect measurement of the presence of the peptide compounds of the invention. Examples of labels that allow direct measurement of the compound include radiolabels, fluorophores, dyes, beads, nanoparticles (e.g., quantum dots), chemiluminescent agents, colloidal particles, paramagnetic labels, and the like. Radiolabels can include radioisotopes, such as 35 S. 14 C. 125 I. 3 H. 64 Cu and 131I. The compounds of the present invention can be labeled with radioactive isotopes using any suitable technique, such as the techniques described in Current Protocols in Immunology, Volumes 1 and 2, edited by Coligen et al., Wiley-Interscience, New York, NY (1991), and radioactivity can be measured using scintillation counting or positron emission. Examples of detectable labels that allow indirect measurement of the presence of modified compounds include enzymes, wherein the substrate can provide a colored or fluorescent product. For example, the compound can include a covalently bound enzyme that can provide a detectable product signal after adding a suitable substrate. Instead of covalently binding the enzyme to the compound, the compound can include a first member of a specific binding pair that specifically binds to a second member of a specific binding pair that is conjugated to the enzyme, such as a compound that can be covalently bound to biotin and an enzyme conjugated to streptavidin. Examples of enzymes suitable for conjugates include horseradish peroxidase, alkaline phosphatase, malate dehydrogenase, etc. In the case of non-commercial use, such enzyme conjugates can be easily produced by any suitable technique.

[0421] In certain embodiments, the detectable label is a fluorophore. The term "fluorophore" refers to a molecule that emits light of a different wavelength when excited with light of a selected wavelength, and the molecule can emit light immediately or delayed after excitation. Fluorophores include, but are not limited to, fluorescein dyes such as 5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM), 2',4',1,4,-tetrachlorofluorescein (TET), 2',4',5',7',1,4-hexachlorofluorescein (HEX) and 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE); cyanine dyes such as Cy3, CY5, Cy5.5, QUASARTM dyes, etc.; dansyl derivatives; rhodamine dyes such as 6-carboxytetramethylrhodamine (TAMRA), CAL FLUOR dyes, tetrapropyl-6-carboxyrhodamine (ROX). BODIPY fluorophores, ALEXA dyes, Oregon Green, pyrene, perylene, benzopyrene, squarylium dyes, coumarin dyes, luminescent transition metal and lanthanide complexes, etc. The term fluorophore includes excimers and exciplexes of such dyes.

[0422] In some embodiments, the compound includes a detectable label, such as a radiolabel. In certain embodiments, the radiolabel is suitable for use in PET, SPECT, and / or MR imaging. In certain embodiments, the radiolabel is a PET imaging label. In some cases, the compound is 18 F. 64 Cu,68 Ga, 111 In, 99 mTc or 86 Y radiolabeled.

[0423] The detectable label can be attached to the peptide compound at any convenient position and via any convenient chemical method. Methods and materials of interest include, but are not limited to, those described in: USP 8,545,809; Meares et al., 1984, Acc Chem Res 17:202-209; Scheinberg et al., 1982, Science 215:1511-13; Miller et al., 2008, Angew Chem Int Ed 47:8998-9033; Shirrmacher et al., 2007, Bioconj Chem 18:2085-89; Hohne et al., 2008, Bioconj Chem 19:1871-79; Ting et al., 2008, Fluorine Chem 21:1871-1879; Chem 129:349-58, the labeling method of Poethko et al. (J. Nucl. Med. 2004; 45:892-902), in which 4-[18F]fluorobenzaldehyde is first synthesized and purified (Wilson et al., J. Labeled Compounds and Radiopharm. 1990; XXVIII:1189-1199), and then conjugated to a peptide and labeled with [18F]fluorobenzoic acid succinimidyl ester (SFB) (e.g., Vaidyanathan et al., 1992, Int. J. Rad. Appl. Instrum. B). 19:275); other acyl compounds (Tada et al., 1989, Journal of Labeled Compounds and Radiopharmaceuticals XXVII:1317; Wester et al., 1996, Nucl. Med. Biol. 23:365; Guhlke et al., 1994, Nucl. Med. Biol. 21:819); or click chemistry adducts (Li et al., 2007, Bioconjugate Chem. 18:1987).

[0424] Any convenient synthetic or bioconjugation method can be used to prepare the modified D-peptide compounds of the present invention. In some cases, the detectable label is attached to the compound via an optional linker. In certain embodiments, the detectable label is attached to the N-terminus of the compound. In certain embodiments, the detectable label is attached to the C-terminus of the compound. In certain embodiments, the detectable label is attached to a non-terminal residue of the compound, for example, via a side chain moiety. In certain embodiments, the detectable label is attached to the N-terminal peptide extension of the compound via an optional linker. In some cases, the N-terminal peptide extension is modified to include a reactive functional group capable of reacting with a compatible functional group of the moiety containing the radiolabel. Detectable labels can be attached to the compounds using any convenient reactive functional groups, chemistries, and radiolabel-containing moieties, including but not limited to click chemistry, azides, alkynes, cyclooctynes, copper-free click chemistry, nitrones, chelating groups (e.g., selected from DOTA, TETA, NOTA, NODA, (tert-butyl)2NODA, NETA, C-NETA, L-NETA, S-NETA, NODA-MPAA, and NODA-MPAEM), propargyl-glycine residues, and the like.

[0425] In some cases, the molecule of interest is a second active agent, such as an active agent or drug that can be used in combination with targeted VEGF-A in the treatment methods of the present invention. In some cases, the molecule of interest is a small molecule, a chemotherapeutic agent, an antibody, an antibody fragment, an aptamer, or an L-protein. In some embodiments, the compound is modified to include a moiety suitable for use as a drug (e.g., a protein, a nucleic acid, a small organic molecule, etc.). Exemplary pharmaceutical proteins include, for example, cytokines, antibodies, chemokines, growth factors, interleukins, cell surface proteins, extracellular domains, cell surface receptors, cytotoxins, and the like. Exemplary small molecule drugs include small molecule toxins or therapeutic agents.

[0426] Any convenient therapeutic or diagnostic agent (e.g., as described herein) may be conjugated to the D-peptide compound. A variety of therapeutic agents, including but not limited to anticancer agents, antiproliferative agents, cytotoxic agents, and chemotherapeutic agents, are described below in the section entitled [Combination Therapies], any of which may be suitable for use in the modified compounds of the present invention. Exemplary chemotherapeutic agents of interest include, for example, gemcitabine, docetaxel, bleomycin, erlotinib, gefitinib, lapatinib, imatinib, dasatinib, nilotinib, bosutinib, crizotinib, ceritinib, trametinib, bevacizumab, sunitinib, sorafenib, trastuzumab, trastuzumab-emtacin conjugate, and trastuzumab-emtacin.

[0014] The invention further includes but is not limited to: emtansine, rituximab, ipilimumab, rapamycin, temsirolimus, everolimus, methotrexate, doxorubicin, abraxane, folfirinox, cisplatin, carboplatin, 5-fluorouracil, teysumo, paclitaxel, prednisone, levothyroxine, pemetrexed, navitoclax, and ABT-199. Any exemplary cytotoxic agent used for ADCs may be suitable for use with the modified D-peptide compounds of the present invention.Cytotoxic agents of interest include, but are not limited to, auristatins (e.g., MMAE, MMAF), maytansine, dolastatin, calicheamicin, duocarmycin, pyrrolobenzodiazepine (PBD), centanamycin (ML-970; indolecarboxamide), doxorubicin, α-Amanitin, and derivatives and analogs thereof. In certain embodiments, the compound may include a cell penetrating peptide (e.g., tat). Cell penetrating peptides can promote cellular uptake of molecules. Any suitable tag polypeptide and its corresponding antibody can be used. Examples include polyhistidine (poly-his) or polyhistidine-glycine (poly-his-gly) tags; the influenza HA tag polypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol. 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al., Mol. Cell. Biol. 5:3610-3616 (1985)]; and the herpes simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al., Protein Engineering 3(6):547-553 (1990)]. Other tag polypeptides include Flag-peptide [Hopp et al., BioTechnology 6:1204-1210 (1988)]; KT3 epitope peptide [Martin et al., Science 255:192-194 (1992)]; tubulin epitope peptide [Skinner et al., J. Biol. Chem. 266:15163-15166 (1991)]; and T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA 87:6393-6397 (1990)].

[0427] In certain embodiments, the compound may include a cell penetrating peptide (e.g., tat). The cell penetrating peptide can promote cellular uptake of the molecule. Any suitable tag polypeptide and its corresponding antibody can be used. Examples include polyhistidine (poly-his) or polyhistidine-glycine (poly-his-gly) tags; influenza HA tag polypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol. 8:2159-2165 (1988)]; c-myc tag and 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al., Mol. Cell. Biol. 5:3610-3616 (1985)]; and herpes simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al., Protein Eng. 3(6):547-553 (1990)]. Other tag polypeptides include Flag-peptide [Hopp et al., Biotechnology 6:1204-1210 (1988)]; KT3 epitope peptide [Martin et al., Science 255:192-194 (1992)]; tubulin epitope peptide [Skinner et al., J. Biol. Chem. 266:15163-15166 (1991)]; and T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA 87:6393-6397 (1990)].

[0428] The molecule of interest can be connected to the modified compound of the present invention via any suitable method. In some cases, the molecule of interest is connected to the terminal amino acid residue via covalent conjugation, for example, at the amino terminal or at the carboxylic acid terminal. The molecule of interest can be connected to the peptide GA domain motif via a single bond or a suitable linker, such as a PEG linker, a peptide linker including one or more amino acids, or a saturated hydrocarbon linker. A variety of linkers (for example, as described herein) are used for the modified compound of the present invention. Any suitable reagent and method can be used to include the molecule of interest in the GA domain motif of the present invention, such as the conjugation method, solid phase peptide synthesis method, or fusion protein expression method described in G.T. Hermanson, "Bioconjugate Techniques" Academic Press, 2nd edition, 2008. Functional groups that can be used to produce modified compounds via optional linker covalent binding domains include: hydroxyl, thiol, amino, etc. Certain portions of the molecule of interest and / or the GA domain motif can be protected using convenient blocking groups, see, for example, Green and Wuts, Protective Groups in Organic Synthesis (John Wiley & Sons), 3rd ed. (1999). The particular molecule of interest and the site of attachment to the GA domain motif can be selected so as not to substantially adversely interfere with the desired binding activity, for example, to the target VEGF-A protein.

[0429] The molecule of interest may be a peptide. It should be understood that the molecule of interest may further include one or more non-peptide groups, including but not limited to a biotin moiety and / or a linker. Any suitable protein domain may be applicable to the modified peptide compounds of the present invention and used as the molecule of interest therein. The protein domain of interest includes but is not limited to any suitable serum protein, serum albumin (e.g., human serum albumin; see, e.g., U.S. Patent Nos. 6,926,898 and US2005 / 0054051; U.S. Patent No. 6,887,470), transferrin receptor or its transferrin binding portion, immunoglobulin (e.g., IgG), immunoglobulin Fc domain (see, e.g., U.S. Patent No. 6,660,843), transthyretin (TTR; see, e.g., US 2003 / 0195154; 2003 / 0191056), thyroxine binding globulin (TBG) or a fragment thereof.

[0430] A multimerizing group is any convenient group capable of forming a multimer (e.g., a dimer, trimer, or dendrimer), for example, by mediating binding between two or more compounds (e.g., directly or indirectly via a multivalent binding moiety), or by linking two or more compounds via covalent bonds. In some cases, the multimerizing group Z is a chemoselective reactive functional group that is conjugated to a compatible functional group on a second D-peptide compound. In other cases, the multimerizing group is a specific binding moiety (e.g., biotin or a peptide tag) that specifically binds to a multivalent binding moiety (e.g., streptavidin or an antibody). In some cases, the compound includes a multimerizing group and is a monomer that has not yet been multimerized.

[0431] Chemoselective reactive functional groups for inclusion in the peptide compounds of the invention include, but are not limited to, azide, alkynyl, phosphino, cysteine ​​residues, C-terminal thioesters, aryl azide, maleimide, carbodiimide, N-hydroxysuccinimide (NHS)-ester, hydrazide, PFP-ester, hydroxymethylphosphine, psoralen, imidoester, pyridyl disulfide, isocyanate, aminooxy-, aldehyde, ketone, chloroacetyl, bromoacetyl, and vinyl sulfone.

[0432] polynucleotides

[0433] Also provided are polynucleotides encoding sequences corresponding to the peptide compounds of the invention as described herein. The polynucleotides may encode L-peptide compounds that specifically bind to the D-VEGF-A target protein.

[0434] In some embodiments, the polynucleotide encodes a peptide compound comprising between 30 and 80 residues, between 40 and 70 residues, between 45 and 60 residues, or between 45 and 55 residues. In some cases, the polynucleotide encodes a peptide compound sequence of 35 and 55 residues, such as between 40 and 55 residues or between 45 and 55 residues. In certain embodiments, the polynucleotide encodes a peptide compound sequence of 45, 46, 47, 48, 49, 50, 51, 52, or 53 residues.

[0435] In certain embodiments, the polynucleotide is a replicable expression vector comprising a nucleic acid sequence encoding an L-peptide compound that can be expressed in a protein expression system. In certain embodiments, the polynucleotide is a replicable expression vector comprising a nucleic acid sequence encoding a gene fusion, wherein the gene fusion encodes a fusion protein comprising an L-peptide compound fused to all or a portion of a viral coat protein.

[0436] In certain embodiments, polynucleotides of the present invention can be expressed and displayed in a cell-based or cell-free display system. Any suitable display method can be used to display the L-peptide compounds encoded by the polynucleotides of the present invention, such as cell-based display technology and cell-free display technology. In certain embodiments, cell-based display technology includes phage display, bacterial display, yeast display and mammalian cell display. In certain embodiments, cell-free display technology includes mRNA display and ribosome display.

[0437] method

[0438] The compounds described herein can be used in a variety of methods. One such method comprises contacting a compound of the invention with a VEGF-A target protein under conditions suitable for binding of VEGF-A to produce a complex. In some embodiments, the method comprises administering a D-peptide compound to a subject, wherein the compound binds to VEGF-A in the subject.

[0439] The compounds of the invention may inhibit at least one activity of their VEGF-A target by 10% to 100%, for example, by 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. In certain assays, the compounds of the invention may inhibit at least one activity of their VEGF-A target by 10% to 100%, for example, by 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. -5 M or less (e.g., 1×10 -6 M or less, 1×10 -7 M or less, 1×10 -8 M or less, 1×10 -9 M or less, 1×10 -10 M or lower, or 1×10 -11 M or lower) 50 In some assays, the compounds of the present invention can inhibit VEGF-A. -6 IC of 500 nM or less (e.g., 500 nM or less, 200 nM or less, 100 nM or less, 30 nM or less, 10 nM or less, 3 nM or less, or 1 nM or less) 20 In some assays, the compounds of the present invention can inhibit VEGF-A. -6 IC of 500 nM or less (e.g., 500 nM or less, 200 nM or less, 100 nM or less, 30 nM or less, 10 nM or less, 3 nM or less, or 1 nM or less) 10 Inhibits its VEGF-A target. In an assay using mice, the compounds of the invention may have an ED of less than 1 μg / mouse (e.g., 1 ng / mouse to about 1 μg / mouse). 50 .

[0440] In some embodiments, the methods of the present invention are in vitro methods comprising contacting a sample with a compound of the present invention that specifically binds to a target molecule with high affinity. In certain embodiments, the sample is suspected of containing the target molecule, and the methods of the present invention further comprise assessing whether the compound specifically binds to the target molecule. In certain embodiments, the target molecule is a naturally occurring L-protein, and the compound is a D-peptide. In certain embodiments, the compound of the present invention is a modified compound that includes a label, such as a fluorescent label, and the methods of the present invention further comprise detecting the label present in the sample, for example, using optical detection. In certain embodiments, the compound is modified with a support so that any sample not bound to the compound can be removed (e.g., by washing). The presence of specifically bound target protein can then be detected using any convenient means, such as using binding of a labeled target-specific probe or using a fluorescent protein-reactive reagent. In another embodiment of the methods of the present invention, the sample is known to contain the target protein. In certain embodiments, the target VEGF-A protein is a synthetic D-protein, and the compound is an L-peptide. In certain embodiments, the target VEGF-A protein is an L-protein, and the compound is a D-peptide.

[0441] In certain embodiments, the compounds of the invention can be contacted with cells in the presence of VEGF-A, and the VEGF-A response phenotype of the cells can be monitored. Exemplary VEGF-A assays include assays using isolated proteins in cell-free systems, assays using cultured cells in vitro, or in vivo assays. Exemplary VEGF-A assays include, but are not limited to, receptor tyrosine kinase inhibition assays (see, e.g., Cancer Research, June 15, 2006; 66: 6025-6032), in vitro HUVEC proliferation assays (FASEB Journal, 2006; 20: 2027-2035; Wells et al., Biochemistry, 1998, 37, 17754-17764), in vivo solid tumor disease assays (USPN 6,811,779), and in vivo angiogenesis assays (FASEB Journal, 2006; 20: 2027-2035). The descriptions of these assays are incorporated herein by reference. There are many protocols that can be used in these methods and include, but are not limited to, cell-free assays, such as binding assays; cell-based assays that measure cell phenotypes, such as gene expression assays; and in vivo assays involving specific animals (which, in certain embodiments, may be animal models of conditions associated with the target). In some cases, the assay may be an angiogenesis assay. In certain embodiments, the target protein is VEGF-A, and the compounds of the present invention inhibit VEGF-A-dependent angiogenesis. In certain embodiments, the target protein is VEGF-A, and the compounds of the present invention inhibit VEGF-A-dependent cell proliferation. In some cases, the target protein is VEGF-A, and the compounds inhibit VEGFR2 phosphorylation.

[0442] In some embodiments, the method of the present invention is in vivo and includes administering to a subject a D-peptide compound that specifically binds to a target molecule with high affinity. In certain embodiments, the compound is administered in the form of a pharmaceutical formulation. Various subjects can be treated according to the methods of the present invention. Generally speaking, such subjects are "mammals" or "mammals," wherein these terms are widely used to describe organisms within the class mammalia, including carnivores (e.g., dogs and cats), rodents (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In some embodiments, the subject is human. The subject may be a subject (e.g., as described herein) who is in need of preventing or treating a disease or condition associated with angiogenesis.

[0443] The compounds of the present invention can bind to and inhibit VEGF-A and are therefore suitable for the treatment, in vivo diagnosis and imaging of diseases and conditions associated with angiogenesis. The term "diseases and conditions associated with angiogenesis" includes, but is not limited to, those diseases and conditions mentioned herein. Reference is also made to WO 98 / 47541 in this regard. Diseases and conditions associated with angiogenesis include various forms of cancer and metastasis, such as breast cancer, skin cancer, colorectal cancer, pancreatic cancer, prostate cancer, lung cancer or ovarian cancer. Other diseases and conditions associated with angiogenesis are inflammation (e.g., chronic inflammation), atherosclerosis, rheumatoid arthritis and gingivitis. Other diseases and conditions associated with angiogenesis are arteriovenous malformations, astrocytomas, choriocarcinomas, glioblastomas, gliomas, hemangiomas (children, capillary), liver cancer, proliferative endometrium, myocardial ischemia, endometriosis, Kaposi's sarcoma, macular degeneration, melanoma, neuroblastoma, obstructive peripheral arterial disease, osteoarthritis, psoriasis, retinopathy (diabetic, proliferative), scleroderma, seminoma, and ulcerative colitis. In some cases, the disease or condition associated with angiogenesis is cancer (e.g., breast cancer, skin cancer, colorectal cancer, pancreatic cancer, prostate cancer, lung cancer, or ovarian cancer), inflammatory diseases, atherosclerosis, rheumatoid arthritis, macular degeneration, and retinopathy. Treatment of diabetic macular edema (DME) or age-related macular degeneration (AMD) is of particular interest.

[0444] The compounds of the invention that bind VEGF-A are useful in treating a variety of neoplastic and non-neoplastic diseases and conditions. Tumors and related conditions suitable for treatment include breast cancer, lung cancer, stomach cancer, esophageal cancer, colorectal cancer, liver cancer, ovarian cancer, thecoma, arrhenoblastoma, cervical cancer, endometrial cancer, endometrial hyperplasia, endometriosis, fibrosarcoma, choriocarcinoma, head and neck cancer, nasopharyngeal cancer, laryngeal cancer, hepatoblastoma, Kaposi's sarcoma, melanoma, skin cancer, hemangioma, cavernous hemangioma, hemangioblastoma, pancreatic cancer, retinoblastoma, astrocytoma, glioblastoma, schwannoma, oligodendroglioma, medulloblastoma, neuroblastoma, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcoma, urinary tract cancer, thyroid cancer, Wilm's tumor, renal cell carcinoma, prostate cancer, abnormal blood vessel proliferation associated with phakomatose, edema (e.g., edema associated with brain tumors), and Meigs' syndrome.

[0445] Non-neoplastic conditions suitable for treatment include rheumatoid arthritis, psoriasis, atherosclerosis, diabetes and other proliferative retinopathies, including retinopathy of prematurity, retrolental fibroplasia, neovascular glaucoma, age-related macular degeneration, thyroid hyperplasia (including Graves' disease), corneal and other tissue transplants, chronic inflammation, lung inflammation, nephrotic syndrome, pre-eclampsia, ascites, pericardial effusions (e.g., those associated with pericarditis), and pleural effusions.

[0446] As used herein, the term "treating" or "treatment" means the treatment of a disease or medical condition in a patient, such as a mammal (e.g., a human), including: (a) preventing the disease or medical condition from occurring, such as prophylactic treatment of a subject; (b) ameliorating the disease or medical condition, such as eliminating the patient's disease or medical condition or causing the patient's disease or medical condition to regress; (c) inhibiting the disease or medical condition, such as by slowing or curbing the development of the patient's disease or medical condition; or (d) alleviating the symptoms of the patient's disease or medical condition. Thus, treatment also includes situations in which the pathological condition or at least the symptoms associated therewith are completely inhibited, such as preventing the occurrence or stopping, such as terminating, so that the subject no longer suffers from the pathological condition or at least the symptoms characteristic of the pathological condition. Treatment can also modulate the form of surrogate markers of the disease condition, such as those described above.

[0447] Aspects of the present disclosure include methods for preventing or treating AMD, such as wet age-related macular degeneration (AMD). Age-related macular degeneration (AMD) is a major cause of severe vision loss in the elderly population. The exudative form of AMD is characterized by choroidal angiogenesis and detachment of retinal pigment epithelial cells. Because choroidal angiogenesis is associated with a sharp deterioration in prognosis, VEGF-binding compounds of the present invention are used to reduce the severity of AMD. In some cases, the subject is a patient suffering from dry AMD, and the compound is administered according to the methods of the invention to prevent the occurrence of wet AMD in the subject or to reduce its severity.

[0448] In certain embodiments, the methods of the invention comprise administering a compound, such as a VEGF-A binding compound, and then detecting the compound after it binds to the target protein. In some methods, the same compound can serve as both a therapeutic compound and a diagnostic compound.

[0449] The VEGF-A binding compounds of the present disclosure are therapeutically useful in treating any disease or condition that is improved, ameliorated, inhibited, or prevented by removal, inhibition, or reduction of VEGF-A protein or fragments thereof.

[0450] In some embodiments, the methods of the present invention are methods of regulating angiogenesis in a subject, comprising administering to the subject an effective amount of a compound of the present invention that specifically binds to a VEGF-A protein with high affinity. In certain embodiments, the methods further comprise diagnosing the presence of a disease condition in the subject. In certain embodiments, the disease condition is a condition that can be treated by enhancing angiogenesis. In certain embodiments, the disease condition is a condition that can be treated by reducing angiogenesis. In certain embodiments, the methods of the present invention are methods of inhibiting angiogenesis, and the compound is a VEGF-A antagonist.

[0451] In some embodiments, the method of the present invention is a method of treating a subject having a cell proliferative disease condition, comprising administering to the subject an effective amount of a compound of the present invention that specifically binds to a VEGF-A protein with high affinity, thereby treating the cell proliferative disease condition in the subject.

[0452] In some embodiments, the method of the present invention is a method of inhibiting tumor growth in a subject, comprising administering to the subject an effective amount of a compound of the present invention that specifically binds to a VEGF-A protein with high affinity. In certain embodiments, the tumor is a solid tumor. In certain embodiments, the tumor is a non-solid tumor.

[0453] Any convenient method can be used to determine that the amount of compound administered is an amount sufficient to produce the desired effect in combination with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications of the unit dosage forms of the present disclosure will depend on the specific compound employed and the effect to be achieved, as well as the pharmacodynamics associated with each compound in the subject.

[0454] In some embodiments, an effective amount of a compound of the invention is from about 50 ng / ml to about 50 μg / ml (e.g., from about 50 ng / ml to about 40 μg / ml, from about 30 ng / ml to about 20 μg / ml, from about 50 ng / ml to about 10 μg / ml, from about 50 ng / ml to about 1 μg / ml, from about 50 ng / ml to about 800 ng / ml, from about 50 ng / ml to about 700 ng / ml, from about 50 ng / ml to about 600 ng / ml, from about 50 ng / ml to about 500 ng / ml, from about 50 ng / ml to about 400 ng / ml, from about 60 ng / ml to about 400 ng / ml, In some embodiments, the present invention provides an amount within the range of about 100 ng / ml, about 70 ng / ml to about 300 ng / ml, about 60 ng / ml to about 100 ng / ml, about 65 ng / ml to about 85 ng / ml, about 70 ng / ml to about 90 ng / ml, about 200 ng / ml to about 900 ng / ml, about 200 ng / ml to about 800 ng / ml, about 200 ng / ml to about 700 ng / ml, about 200 ng / ml to about 600 ng / ml, about 200 ng / ml to about 500 ng / ml, about 200 ng / ml to about 400 ng / ml, or about 200 ng / ml to about 300 ng / ml).

[0455] In some embodiments, an effective amount of a compound of the invention is from about 10 pg to about 100 mg, for example, from about 10 pg to about 50 pg, from about 50 pg to about 150 pg, from about 150 pg to about 250 pg, from about 250 pg to about 500 pg, from about 500 pg to about 750 pg, from about 750 pg to about 1 ng, from about 1 ng to about 10 ng, from about 10 ng to about 50 ng, from about 50 ng to about 150 ng, from about 150 ng to about 250 ng, from about 250 The amount of the present invention can be from about 1 μg to about 500 ng, from about 500 ng to about 750 ng, from about 750 ng to about 1 μg, from about 1 μg to about 10 μg, from about 10 μg to about 50 μg, from about 50 μg to about 150 μg, from about 150 μg to about 250 μg, from about 250 μg to about 500 μg, from about 500 μg to about 750 μg, from about 750 μg to about 1 mg, from about 1 mg to about 50 mg, from about 1 mg to about 100 mg, or from about 50 mg to about 100 mg. The amount can be the amount of a single dose or can be the total daily amount. The total daily amount can be in the range of 10 pg to 100 mg, or can be in the range of 100 mg to about 500 mg, or can be in the range of 500 mg to about 1000 mg.

[0456] In some embodiments, a single dose of a compound of the invention is administered. In other embodiments, multiple doses of a compound of the invention are administered. Where multiple doses are administered over a period of time, the D-peptide compound is administered twice a day (qid), once a day (qd), once every other day (qod), once every two days, three times a week (tiw), or twice a week (biw) over a period of time. For example, the compound is administered qid, qd, qod, tiw, or biw over a period of time from one day to about two years or longer. For example, the compound is administered at any of the aforementioned frequencies for one week, two weeks, one month, two months, six months, one year, or two years or longer, depending on various factors.

[0457] Any of a variety of methods can be used to determine whether a treatment method is effective. For example, a biological sample obtained from an individual who has been treated using the methods of the present invention can be analyzed for the presence and / or extent of angiogenesis. Assessing the effectiveness of a treatment method in a subject can include assessing the subject before, during, and / or after treatment using any convenient method. Aspects of the methods of the present invention further include the step of assessing the subject's therapeutic response to the treatment.

[0458] In some embodiments, the method includes assessing the condition of the subject, including diagnosing or assessing one or more symptoms of the subject associated with the disease or condition of interest being treated (e.g., as described herein). In some embodiments, the method includes obtaining a biological sample from the subject, and analyzing the sample, for example, for the presence of angiogenesis associated with the disease or condition of interest (e.g., as described herein). The sample may be a cell sample. In some cases, the sample is a biopsy. The assessment step of the method of the present invention may be performed one or more times before, during, and / or after administration of the compounds of the present invention using any suitable method.

[0459] In some cases, for example, the compounds of the present invention or salts thereof as defined herein can be used in medicine, in particular for in vivo diagnosis or imaging of diseases or conditions associated with angiogenesis, such as by PET. In certain embodiments, the compound is a modified compound including a detectable label, and the method further includes detecting a label in a subject. The choice of label depends on the detection means. Any suitable label and detection system can be used in the present method, see, for example, Baker, "The whole picture", Nature, 463, 2010, pp. 977-980. In certain embodiments, the compound includes a fluorescent label suitable for optical detection. In certain embodiments, the compound includes a radioactive label for detection using positron emission tomography (PET) or single photon emission computed tomography (SPECT). In some cases, the compound includes a paramagnetic label suitable for tomography detection. As described above, the compounds of the present invention may be labeled, but in some methods, the compound is unlabeled and imaging is performed using a secondary labeling agent. In certain embodiments, the methods of the present invention include diagnosing a disease condition in a subject by comparing the number, size, and / or intensity of the marker loci to corresponding baseline values. The baseline value can represent an average level in a population of subjects without the disease, or a previous level measured in the same subject.

[0460] In some cases, the radiolabeled compound can be administered to the subject in an amount sufficient to produce the desired signal for PET imaging. In some cases, a sufficient radionuclide dose is 0.01 to 100 mCi, such as 0.1 to 50 mCi or 1 to 20 mCi, based on 70 kg of body weight. Therefore, any suitable physiologically acceptable carrier or excipient can be used to formulate the radiolabeled compound for administration. For example, the compound can be optionally suspended or dissolved in an aqueous medium with the addition of a pharmaceutically acceptable excipient, and the resulting solution or suspension is subsequently sterilized. Also provided is the use of a radiolabeled compound or a salt thereof as described herein for the manufacture of a radiopharmaceutical for use in the following method: in vivo imaging, such as PET imaging, such as imaging of a disease or condition associated with angiogenesis; involving administering a radiopharmaceutical to a human or animal body and generating an image of at least a portion of the body.

[0461] In some embodiments, the method is a method for in vivo diagnosis or imaging of a disease or condition associated with angiogenesis, which involves administering a radiopharmaceutical to the body, for example, into the vascular system, and using PET to produce an image of at least a portion of the body to which the radiopharmaceutical has distributed, wherein the radiopharmaceutical comprises a radiolabeled compound or a salt thereof.

[0462] In some embodiments, the method is a method of monitoring the effect of treating the human or animal body with a drug, e.g., a cytotoxic agent, to combat a condition associated with angiogenesis, e.g., cancer, comprising administering to the body a radiolabeled compound or a salt thereof and detecting uptake of the compound by a cellular receptor, e.g., an endothelial cell receptor, e.g., an α.v.β.3 receptor, the administration and detection being optionally repeated, e.g., before, during, and after treatment with the drug.

[0463] In some embodiments, the method is a method for in vivo diagnosis or imaging of a disease or condition associated with angiogenesis, comprising administering a D-peptide compound to a subject and imaging at least a portion of the subject. In certain embodiments, the imaging comprises PET imaging, and the administration comprises administering the compound to the subject's vascular system. In some cases, the method further comprises detecting the uptake of the compound by a cell receptor. In some cases, the target is VEGF-A, and the subject is human. In certain embodiments, the method comprises administering a therapeutic antibody, such as avastin, to the subject, wherein the disease or condition is a condition associated with cancer.

[0464] The inventive method can be a diagnostic method for detecting the expression of a target protein in a specific cell, tissue or serum in vitro or in vivo. In some cases, the inventive method is a method for imaging the target protein in a subject in vivo. The method may include administering a compound to a subject who exhibits symptoms of a disease condition associated with the target protein. In some cases, the subject is asymptomatic. The inventive method may further include monitoring the disease progression and / or therapeutic response of a subject who has previously been diagnosed with the disease.

[0465] The VEGF-A binding compounds of the present invention can be used as affinity purification agents. In this process, the compound can be immobilized on a solid phase, such as a Sephadex resin or filter paper, using any convenient method. The VEGF-A binding compound of the present invention is contacted with a sample containing the VEGF-A protein (or fragment thereof) to be purified, and the support is then washed with a suitable solvent that will remove substantially all material in the sample except the VEGF protein bound to the immobilized compound. Finally, the support is washed with another suitable solvent, such as a glycine buffer, pH 5.0, which releases the VEGF-A protein from the immobilized compound.

[0466] The VEGF-A binding compounds of the present invention may also be used in diagnostic assays for VEGF-A protein, for example, to detect its expression in specific cells, tissues, or serum. Such diagnostic methods may be applicable to cancer diagnosis. For diagnostic applications, the compounds of the present invention may be modified as described above.

[0467] Combination therapy

[0468] In some embodiments, the compounds of the present invention may be administered in combination with one or more additional active agents or therapies. Any suitable agent may be used, including compounds suitable for treating the diseases targeted by the methods of the present invention. The terms "agent," "compound," and "drug" are used interchangeably herein. Additional active agents or therapies include, but are not limited to, small molecules; antibodies; antibody fragments; aptamers; L-proteins; second target binding molecules, such as a second D-peptide compound; chemotherapeutic agents; surgery; catheter devices; and radiation. Combination therapy includes administering a single pharmaceutical dosage formulation containing a compound of the present invention and one or more additional agents; and administering a compound of the present invention and one or more additional agents in their own separate pharmaceutical dosage formulations. For example, a compound of the present invention and a cytotoxic agent, a chemotherapeutic agent, or a growth inhibitory agent may be administered to a patient together in a single dose composition, such as a combination formulation, or each agent may be administered in a separate dose formulation. In the case of using a separate dose formulation, the compound of the present invention and one or more additional agents may be administered concurrently, or at separate staggered times, such as sequentially.

[0469] The terms "co-administered" and "in combination with" include the simultaneous, concurrent, or sequential administration of two or more therapeutic agents (e.g., a D-peptide compound and a second agent) without specific time limits. In one embodiment, the agents are present in a cell or in a subject at the same time, or exert their biological or therapeutic effects at the same time. In one embodiment, the therapeutic agents are in the same composition or unit dosage form. In other embodiments, the therapeutic agents are in separate compositions or unit dosage forms. In certain embodiments, the first agent (e.g., a D-peptide compound) can be administered prior to (e.g., minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concurrently with, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of the second therapeutic agent.

[0470] "Simultaneous administration" of a known therapeutic drug and a pharmaceutical composition of the present disclosure means administering the D-peptide compound and the second agent at a time when both the known drug and the composition of the present disclosure will have a therapeutic effect. Such simultaneous administration may involve administering the drug concurrently (i.e., simultaneously), before, or after administration of the D-peptide compound of the present invention. The routes of administration of the two agents may vary, with representative routes of administration being described in more detail below. One of ordinary skill in the art will readily determine the appropriate timing, sequence, and dosage for administering a particular drug and compound of the present disclosure.

[0471] In some embodiments, the compounds (e.g., a D-peptide compound of the invention and a second agent) are administered to a subject within twenty-four hours of each other, e.g., within 12 hours of each other, within 6 hours of each other, within 3 hours of each other, or within 1 hour of each other. In certain embodiments, the compounds are administered within 1 hour of each other. In certain embodiments, the compounds are administered substantially simultaneously. Substantially simultaneous administration means that the compounds are administered to a subject within about 10 minutes or less of each other, e.g., within 5 minutes or less or 1 minute or less of each other.

[0472] Also provided are pharmaceutical formulations of the compounds of the invention and a second active agent. In pharmaceutical dosage forms, the compounds can be administered in the form of their pharmaceutically acceptable salts, or they can be used alone or in suitable association and combination with other pharmaceutically active compounds.

[0473] In representative embodiments, dosage levels of about 0.01 mg to about 140 mg per kilogram of body weight per day are suitable, or, dosage levels of about 0.5 mg to about 7 g per patient per day are suitable. Those skilled in the art will readily appreciate that dosage levels can vary depending on the specific compound, the severity of the symptoms, and the subject's sensitivity to side effects. The dosage of a given compound can be readily determined by those skilled in the art by a variety of means.

[0474] The amount of active ingredient that can be combined with the carrier material to produce a single dosage form will vary depending on the subject being treated and the particular mode of administration. For example, a formulation intended for oral administration to humans may contain 0.5 mg to 5 g of active agent, mixed with an appropriate and convenient amount of carrier material, which may vary from about 5% to about 95% of the total composition. A unit dosage form will generally contain between about 1 mg and about 500 mg of active ingredient, for example, 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 800 mg, or 1000 mg.

[0475] However, it will be understood that the specific dosage level for any particular patient will depend upon a variety of factors including age, weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the severity of the particular disease undergoing therapy.

[0476] Any suitable second agent is used in the methods of the present invention. In some cases, the second active agent specifically binds to a target protein selected from the group consisting of platelet-derived growth factor (PDGF), VEGF-B, VEGF-C, VEGF-D, EGF, EGFR, Her2, PD-1, PD-L1, OX-40, LAG3, Ang2, IL-1, IL-6, and IL-17. The second active agent of interest includes, but is not limited to, pegpleranib (Fovista), ranibizumab (Lucentis), trastuzumab (Herceptin), bevacizumab (Cancer Stop), aflibercept (Eylea), nivolumab, atezolizumab, durvalumab, gefitinib, erlotinib, and pembrolizumab.

[0477] For the treatment of cancer, the compounds of the present invention can be administered in combination with a chemotherapeutic agent selected from the group consisting of taxanes, nucleoside analogs, steroids, anthracyclines, thyroid hormone replacement drugs, thymidylate-targeted drugs, chimeric antigen receptor / T cell therapy, chimeric antigen receptor / NK cell therapy, apoptosis regulator inhibitors (e.g., B cell CLL / lymphoma 2 (BCL-2) BCL-2 like protein 1 (BCL-XL) inhibitors), CARP-1 / CCAR1 (cell division cycle and apoptosis regulator 1) inhibitors, colony stimulating factor-1 receptor (CSF1R) inhibitors, CD47 inhibitors, cancer vaccines (e.g., dendritic cell vaccines that induce Th17), and other cell therapies. Specific chemotherapeutic agents include, for example, gemcitabine, docetaxel, bleomycin, erlotinib, gefitinib, lapatinib, imatinib, dasatinib, nilotinib, bosutinib, crizotinib, ceritinib, trametinib, bevacizumab, sunitinib, sorafenib, trastuzumab, trastuzumab-emtacin conjugate, rituximab, ipilimumab, rapamycin, temsirolimus, everolimus, methotrexate, doxorubicin, nab-paclitaxel, fluphenazine, cisplatin, carboplatin, 5-fluorouracil, tesumab, paclitaxel, prednisone, levothyroxine, pemetrexed, navitoclax, ABT-199.

[0478] In order to treat cancer (e.g., melanoma, non-small cell lung cancer or lymphoma, such as Hodgkin's lymphoma), the compounds of this invention can be administered in combination with immune checkpoint inhibitors. Any suitable checkpoint inhibitor can be utilized, including but not limited to cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) inhibitors, programmed death 1 (PD-1) inhibitors, and PD-L1 inhibitors. Exemplary checkpoint inhibitors of interest include but are not limited to ipilimumab, pembrolizumab, and nivolumab. In certain embodiments, in order to treat cancer and / or inflammatory diseases, the compounds of this invention can be administered in combination with colony stimulating factor-1 receptor (CSF1R) inhibitors. CSF1R inhibitors of interest include but are not limited to emactuzumab.

[0479] Any suitable cancer vaccine therapy and pharmaceutical agent can be used in combination with the immunomodulatory polypeptide compositions and methods of the present invention. To treat cancers, such as ovarian cancer, the compounds of the present invention can be administered in combination with vaccination therapies, such as dendritic cell (DC) vaccines that promote Th1 / Th17 immunity. Th17 cell infiltration is associated with a significant increase in overall survival in ovarian cancer patients. In some cases, immunomodulatory polypeptides can be used as adjuvant therapy in combination with vaccinations that induce Th17.

[0480] Also of interest are agents that are: CARP-1 / CCAR1 (cell division cycle and apoptosis regulator 1) inhibitors, including but not limited to those described in Rishi et al., Journal of Biomedical Nanotechnology, Vol. 11, No. 9, September 2015, pp. 1608-1627 (20); and CD47 inhibitors, including but not limited to anti-CD47 antibody agents, such as Hu5F9-G4.

[0481] utility

[0482] For example, the compounds of the present invention as described above are used in a variety of applications. Applications of interest include, but are not limited to, therapeutic applications, research applications, and screening applications. Each of these different applications will now be reviewed in more detail below.

[0483] Therapeutic applications

[0484] The compounds of the present invention are useful in a variety of therapeutic applications. Therapeutic applications of interest include those in which the activity of the target is a cause or a contributing factor to the progression of a disease. Thus, the compounds of the present invention are useful in treating a variety of different conditions in which modulation of the activity of the target in a subject is desired.

[0485] The compounds of the invention are useful in treating disorders associated with their target VEGF-A.Examples of disease conditions that can be treated with the compounds of the invention are described above.

[0486] In certain embodiments, disease conditions include, but are not limited to, cancer, inhibition of angiogenesis and metastasis, osteoarthritis pain, chronic low back pain, cancer-related pain, age-related macular degeneration (AMD), diabetic macular edema (DME), idiopathic pulmonary fibrosis (IPF), and graft survival of transplanted corneas.

[0487] In one embodiment, the present disclosure provides a method for treating a VEGF-A-associated condition in a subject. The method generally involves administering a compound of the present invention to a subject having a VEGF-A-associated condition in an amount effective to treat at least one symptom of the VEGF-A-associated condition. VEGF-A-associated conditions are generally characterized by excessive vascular endothelial cell proliferation, vascular permeability, edema, or inflammation, such as cerebral edema associated with injury, stroke, or tumor; edema associated with inflammatory conditions, such as psoriasis or arthritis, including rheumatoid arthritis; asthma; generalized edema associated with burns; ascites and pleural effusions associated with tumors, inflammation, or trauma; chronic airway inflammation; capillary leak syndrome; sepsis; kidney disease associated with increased protein leakage; and ocular conditions, such as age-related macular degeneration and diabetic retinopathy. Such conditions include breast cancer, lung cancer, colorectal cancer, and kidney cancer.

[0488] Research Applications

[0489] The compounds and methods of the present invention are used in a variety of research applications. The compounds and methods of the present invention can be used to analyze the role of target proteins in regulating various biological processes, including but not limited to angiogenesis, inflammation, cell growth, metabolism, transcriptional regulation and phosphorylation regulation. Other target protein binding molecules, such as antibodies, are also applicable in similar areas of biological research. See, for example, Sidhu and Fellhouse, "Synthetic therapeutic antibodies", Nature: Chemical Biology, 2006, 2 (12), 682-688. Such methods can be easily modified for a variety of research applications of the compounds and methods of the present invention.

[0490] Diagnostic applications

[0491] The compounds and methods of the present invention are useful in a variety of diagnostic applications, including, but not limited to, the development of clinical diagnostics, such as in vitro diagnostics or in vivo tumor imaging agents. Such applications are useful for diagnosing a disease state or its susceptibility, or for confirming a diagnosis thereof. The methods are also useful for monitoring disease progression and / or treatment response in patients previously diagnosed with a disease.

[0492] Diagnostic applications of interest include diagnosis of disease conditions such as those described above, including but not limited to: cancer, inhibition of angiogenesis and metastasis, osteoarthritis pain, chronic low back pain, cancer-related pain, age-related macular degeneration (AMD), diabetic macular edema (DME), idiopathic pulmonary fibrosis (IPF), and graft survival of transplanted corneas. In some methods, the same compound can serve as both a therapeutic agent and a diagnostic agent.

[0493] Other target protein binding molecules, such as aptamers and antibodies, can also be used in the development of clinical diagnostics. Such methods can be easily modified for a variety of diagnostic applications of the compounds and methods of the present invention, see, for example, Jayasena, "Aptamers: An Emerging Class of Molecules That Rival Antibodies in Diagnostics", Clinical Chemistry, 1999, 45, 1628-1650.

[0494] pharmaceutical preparations

[0495] Pharmaceutical preparations are also provided. Pharmaceutical preparations are compositions comprising compounds (alone or in the presence of one or more additional active agents) present in a pharmaceutically acceptable vehicle. The term "pharmaceutically acceptable" means approved by the regulatory agencies of the federal government or state governments or listed in the U.S. Pharmacopeia (US Pharmacopeia) or other generally recognized pharmacopeias for use in mammals, such as humans. The term "vehicle" refers to a diluent, adjuvant, excipient or carrier, with which the compounds of the present invention are formulated for use in mammals. Such pharmaceutical vehicles can be liquids, such as water and oil, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Pharmaceutical vehicles can be saline, gum arabic, gelatin, starch paste, talc, keratin, colloidal silicon dioxide, urea, etc. In addition, adjuvants, stabilizers, thickeners, lubricants and colorants can be used. When applied to mammals, the compounds and compositions of the present invention and pharmaceutically acceptable vehicles, excipients or diluents can be sterile. In some cases, when the compounds of the invention are administered intravenously, aqueous media are used as vehicles, such as water, saline solutions, and aqueous dextrose and glycerol solutions.

[0496] The pharmaceutical composition can be in the form of capsule, tablet, pill, granule, buccal tablet, powder, granule, syrup, elixir, solution, suspension, emulsion, suppository or its sustained release formulation, or any other form suitable for use in mammals. In some cases, the pharmaceutical composition is formulated into a pharmaceutical composition suitable for oral or intravenous use to humans for use according to conventional procedures. Examples of suitable pharmaceutical vehicles and their formulation methods are described in Remington: Pharmaceutical Science and Practice (Remington:The Science and Practice of Pharmacy), Alfonso R.Gennaro, Mack Publishing Co., Easton, Pennsylvania (Easton, Pa.), 19th edition, 1995, the 86th, 87th, 88th, 91st and 92nd chapters, which are incorporated herein by reference.

[0497] The choice of excipient will be determined in part by the particular compound, as well as the particular method used to administer the composition.Thus, there is a wide variety of suitable formulations of the pharmaceutical compositions of the present invention.

[0498] The administration of the compounds of the present disclosure may be systemic or local. In certain embodiments, administration to a mammal will cause systemic release of the compounds of the present invention (e.g., into the bloodstream). Methods of administration may include enteral routes, such as oral, buccal, sublingual, and rectal; topical administration, such as transdermal and intradermal; and parenteral administration. Suitable parenteral routes include injection via a subcutaneous needle or catheter, such as intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal, intraarterial, intraventricular, intrathecal, and intracameral injections, and non-injection routes, such as intravaginal, rectal, or nasal administration. In certain embodiments, the compounds and compositions of the present invention are administered orally. In certain embodiments, it may be necessary to administer one or more compounds of the present invention locally to the area in need of treatment. For example, this can be achieved by: local infusion during surgery; topical administration, such as in combination with a postoperative wound dressing; by injection; by means of a catheter; by means of a suppository; or by means of an implant, the implant being a porous, non-porous, or colloidal material, including a membrane, such as a silicone rubber membrane, or a fiber.

[0499] The compounds of the present invention can be formulated into injection preparations by dissolving, suspending or emulsifying the compounds of the present invention in an aqueous or non-aqueous solvent, such as vegetable oil or other similar oils, synthetic aliphatic acid glycerides, esters of high-carbon aliphatic acids or propylene glycol; and containing conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives as necessary.

[0500] In some embodiments, formulations suitable for oral administration may include: (a) liquid solutions, such as an effective amount of the compound dissolved in a diluent such as water or saline; (b) capsules, sachets, or tablets, each containing a predetermined amount of the active ingredient in solid or particulate form; (c) suspensions in appropriate liquids; and (d) suitable emulsions. Tablet forms may include one or more of the following: lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, as well as other excipients, colorants, diluents, buffers, wetting agents, preservatives, flavorings, and pharmacologically compatible excipients. Buccal tablet forms may include flavorings, the active ingredient typically in sucrose and acacia or tragacanth, and lozenges, which include the active ingredient in an inert matrix such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, etc., which contain, in addition to the active ingredient, excipients as described herein.

[0501] The formulations of the present invention can be made into aerosol formulations for administration via inhalation. These aerosol formulations can be placed in acceptable pressurized propellants such as dichlorodifluoromethane, propane, nitrogen, etc. They can also be formulated as pharmaceuticals for non-pressurized preparations, such as for use in a nebulizer or atomizer.

[0502] In some embodiments, formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions that may contain antioxidants, buffers, bacteriostats, and solutes that make the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions that may include suspending agents, solubilizers, thickeners, stabilizers, and preservatives. The formulations may be presented in unit dose or multi-dose sealed containers, such as ampoules and vials, and may be stored under freeze-dried (lyophilized) conditions, requiring only the addition of sterile liquid excipients for injection, such as water, just before use. Ready-to-use injection solutions and suspensions can be prepared from the aforementioned sterile powders, granules, and tablets.

[0503] Preparations suitable for topical application can be presented as creams, gels, pastes or foams, which, in addition to containing the active ingredient, also contain a suitable carrier. In some embodiments, the topical formulation contains one or more components selected from a formatting agent, a thickening agent or gelling agent and an emollient or lubricant. Long-term formatting agents include long-chain alcohols, such as stearyl alcohol, and glycerol ethers or esters and oligo(ethylene oxide) ethers or their esters. Thickening agents and gelling agents include, for example, polymers of acrylic acid or methacrylic acid and its esters, polyacrylamide and naturally occurring thickening agents, such as agar, carrageenan, gelatin and guar gum. Examples of emollients include triglycerides, fatty acid esters and amides; waxes, such as beeswax, spermaceti or carnauba wax; phospholipids, such as lecithin; and sterols and their fatty acid esters. Topical formulations may further include other components, such as astringents, fragrances, pigments, skin penetration enhancers, sunscreens (e.g., sunblocking agents), etc.

[0504] The compounds of the present disclosure can also be formulated for oral administration. For oral pharmaceutical formulations, suitable excipients include pharmaceutical grade carriers, such as mannitol, lactose, glucose, sucrose, starch, cellulose, gelatin, magnesium stearate, saccharin sodium and / or magnesium carbonate. In order to be used for oral liquid formulations, the composition can be prepared as a solution, suspension, emulsion or syrup, supplied in a solid or liquid form suitable for hydration in an aqueous carrier, such as a saline solution, a dextrose aqueous solution, glycerol or ethanol, preferably water or normal saline. If necessary, the composition may also contain a small amount of non-toxic auxiliary substances, such as a wetting agent, an emulsifier or a buffer. The compound of the present invention may also be incorporated into, for example, conventionally available existing nutritional drug formulations, which may also include herbal extracts.

[0505] Unit dosage forms for oral or rectal administration, such as syrups, elixirs, and suspensions, can be provided, wherein each dosage unit, such as a teaspoonful, a tablespoonful, a tablet, or a suppository, contains a predetermined amount of a composition comprising one or more inhibitors. Similarly, unit dosage forms for injection or intravenous administration can include the inhibitor in the composition as a solution in sterile water, normal saline, or another pharmaceutically acceptable carrier.

[0506] As used herein, the term "unit dosage form" refers to physically discrete units suitable as unit dosages for human and animal subjects, each unit containing a predetermined quantity of a compound of the present invention calculated to produce the desired effect in combination with a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications for the novel unit dosage forms of the present invention depend on the specific compound used and the effect to be achieved, as well as the pharmacodynamics associated with each compound in the subject.

[0507] Dosage levels may vary depending on the specific compound, the nature of the delivery vehicle, etc. The required dosage for a given compound can be readily determined by a variety of means.

[0508] In the context of the present invention, the dose administered to an animal, particularly a human, should be sufficient to achieve a prophylactic or therapeutic response in the animal within a reasonable timeframe, as described in more detail below. The dose will depend on various factors, including the potency of the specific compound used, the condition of the animal, and the weight of the animal, as well as the severity of the disease and the stage of the disease. The size of the dose will also be determined by the presence, nature, and extent of any adverse side effects that may accompany the administration of a specific compound.

[0509] In pharmaceutical dosage forms, the compounds may be administered in the form of a free base, a pharmaceutically acceptable salt thereof, or they may be used alone or in suitable association and combination with other pharmaceutically active compounds.

[0510] In some embodiments, the pharmaceutical composition comprises a compound of the present invention that specifically binds to a target protein with high affinity and a pharmaceutically acceptable vehicle. In certain embodiments, the target protein is a VEGF protein, and the compound of the present invention is a VEGF antagonist.

[0511] Reagent test kit

[0512] Also provided are kits comprising compounds of the present disclosure. Kits of the present disclosure may include one or more doses of compounds and optionally one or more additional active agents of one or more doses. Conveniently, the formulation may be provided in unit dosage form. In such kits, in addition to the container containing, for example, a unit dose of the formulation, there is an informational drug instruction sheet describing the use of the formulation of the present invention in the method of the present invention, such as instructions for treating cell conditions associated with pathogenic angiogenesis using the unit dose of the present invention. The term kit refers to a packaged active agent or agent. In some embodiments, the system or kit of the present invention includes a compound of the present invention (e.g., as described herein) dosage and a second active agent (e.g., as described herein) dosage in an amount that can effectively treat a disease or condition associated with angiogenesis of a subject (e.g., as described herein).

[0513] In addition to the components mentioned above, the kit of the present invention may further include components for use of the kit, such as instructions for practicing the inventive method. Instructions are generally recorded on a suitable recording medium. For example, instructions can be printed on a substrate such as paper or plastic. Therefore, instructions can be present in the kit in the form of a drug insert, in the label of the container of the kit or its components (i.e., related to packaging or sub-packaging), etc. In other embodiments, instructions exist in the form of an electronic storage data file on a suitable computer-readable storage medium such as a CD-ROM, a disk, a hard disk drive (HDD), a portable flash drive, etc. In other embodiments, there are no actual instructions in the kit, but a method for obtaining instructions from a remote source, such as via the Internet, is provided. An example of this embodiment is a kit comprising a website, on which instructions can be viewed and / or instructions can be downloaded from the website. Like instructions, this means for obtaining instructions is recorded on a suitable substrate.

[0514] In some embodiments, the kit comprises a first dose of a pharmaceutical composition of the invention and a second dose of a pharmaceutical composition of the invention. In certain embodiments, the kit further comprises a second angiogenesis modulator.

[0515] It should be understood that the present invention is not limited to the particular embodiments described, as these embodiments may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

[0516] Where a range of values ​​is provided, it is to be understood that the present invention encompasses every intervening value between the upper and lower limits of the range, to the tenth of the unit of the lower limit, and any other stated or intermediate value within the stated range, unless the context clearly indicates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the present invention, subject to the constraints of any specifically excluded limits within the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed within the present invention.

[0517] Certain ranges are presented herein that are preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that follows it, as well as for numbers that are close to or near the number that follows the term. In determining whether a number is close to or near a specifically recited number, the unrecited number that is close or near may be a number that, in the context in which it appears, provides a substantial equivalent to the specifically recited number.

[0518] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials will now be described.

[0519] All publications and patents cited in this specification are incorporated herein by reference to the same extent as if each individual publication or patent was specifically and individually indicated as incorporated by reference and are incorporated by reference to disclose and describe the methods and / or materials in connection with the cited publication. Citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0520] It should be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that the claims can be drafted to exclude any optional elements. Thus, this statement is intended to serve as antecedent basis for the use of exclusive terminology such as "solely," "only," and the like in connection with the recitation of claim elements, or for the use of a "negative" limitation.

[0521] As will be apparent to those skilled in the art after reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features that can be readily separated or combined with the features of any other several embodiments without departing from the scope or spirit of the invention. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0522] Although the apparatus and methods have been or will be described in terms of functional interpretation for the sake of grammatical fluidity, it is to be expressly understood that the claims shall not be construed as necessarily being limited in any manner to the construction of “means” or “steps” unless expressly formulated under 35 U.S.C. §112, but shall be given the meaning and full scope of equivalents as provided by the definition in the claims, and to the extent expressly specified under 35 U.S.C. §112, the full range of statutory equivalents under 35 U.S.C. §112 shall apply.

[0523] definition

[0524] The term "peptide" refers to a portion consisting primarily of amino acid residues linked together as a polypeptide. The term "peptide" is meant to include compounds in which one, two, or more residues of a conventional polypeptide sequence have been replaced by a peptide mimetic. A peptide mimetic is a small organic group designed to mimic a peptide or amino acid residue. The peptide mimetic group of the peptide portion may include a non-natural or synthetic backbone group linked to a conventional polypeptide backbone, and optional side chain groups that mimic the side chain groups of any suitable amino acid residue of interest. In some embodiments, in a peptide compound consisting primarily of amino acid residues, 2 or fewer residues are partially replaced by a peptide mimetic for every 10 amino acid residues of the parent polypeptide sequence. Any suitable peptide mimetic group and chemical substance can be used in the peptide compounds of the present invention. The term peptide also means to include multimeric peptide compounds in which two or more peptide compounds of interest are covalently linked. The term peptide also means to include modified peptide compounds in which a non-protein moiety has been covalently linked to the compound.

[0525] The terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to polymeric forms of amino acids of any length. Unless expressly indicated otherwise, "polypeptide," "peptide," and "protein" may include genetically encoded and non-encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The terms include polypeptides in which one or more conventional amino acids have been replaced by non-naturally occurring or synthetic amino acids. A polypeptide may be of any length, e.g., 2 or more amino acids, 4 or more amino acids, 10 or more amino acids, 20 or more amino acids, 30 or more amino acids, 40 or more amino acids, 50 or more amino acids, 60 or more amino acids, 100 or more amino acids, 300 or more amino acids, 500 or more amino acids, or 1000 or more amino acids.

[0526] For the polypeptide sequences and motifs depicted herein, unless otherwise indicated, capital letter codes refer to L-amino acid residues, and lowercase letter codes refer to D-amino acid residues. The amino acid residue glycine is represented as G or Gly. "a" is alanine. "c" is cysteine. "d" is aspartic acid. "e" is glutamic acid. "f" is phenylalanine. "h" is histidine. "i" is isoleucine. "k" is lysine. "l" is leucine. "m" is methionine. "n" is asparagine. "o" is ornithine. "p" is proline. "q" is glutamine. "r" is arginine. "s" is serine. "t" is threonine. "v" is valine. "w" is tryptophan. "y" is tyrosine. It should be understood that for any of the sequences and motifs described herein, e.g., sequences defining a peptide compound that specifically binds to VEGF-A, also encompasses mirror-image compounds that specifically bind to VEGF-A. The present disclosure is intended to encompass both forms of the compounds of the present invention, e.g., an L-peptide compound that specifically binds to D-VEGF-A and a D-peptide compound that specifically binds to L-VEGF-A. It will be understood that the D-VEGF-A protein can be targeted primarily in various in vitro applications, whereas the L-VEGF-A protein can be targeted for various in vitro and / or in vivo applications.

[0527] The term "analog" of an amino acid residue refers to a residue having a side chain group that is a structural and / or functional analog of the side chain group of a reference amino acid residue. In some cases, amino acid analogs share the main chain structure and / or side chain structure of one or more natural amino acids, with the difference being one or more modified groups in the molecule. Such modifications may include, but are not limited to, substitution of atoms (e.g., N) for related atoms (e.g., S), addition of groups (e.g., methyl or hydroxyl, etc.) or atoms (e.g., F, Cl, or Br, etc.), deletion of groups, substitution of covalent bonds (single bonds substituted for double bonds, etc.), or combinations thereof. For example, amino acid analogs may include α-hydroxy acids and α-amino acids, etc. In some cases, amino acid residue analogs are substituted forms of amino acids. The term "substituted form" of an amino acid residue refers to a residue having a side chain group that includes one or more additional substituents that are not present in the side chain of the reference amino acid residue.

[0528] The terms "aromatic amino acid" and "aromatic residue" are used interchangeably to refer to an amino acid residue in which the side chain group includes an aryl, substituted aryl, heteroaryl, or substituted heteroaryl group. In some cases, the side chain group is an aryl-alkyl, substituted aryl-alkyl, heteroaryl-alkyl, or substituted heteroaryl-alkyl group. The term is intended to include naturally occurring and non-naturally occurring α-amino acids. Naturally occurring aromatic residues of interest include phenylalanine, tyrosine, tryptophan, and histidine.

[0529] The terms "carbocyclic amino acid" and "carbocyclic residue" are used interchangeably to refer to amino acid residues in which the side chain groups include aryl or saturated carbocyclic groups. In some cases, the side chain groups are cycloalkyl-alkyl or substituted cycloalkyl-alkyl. Non-naturally occurring side chain groups of interest include, but are not limited to, cyclohexyl-CH2-, cyclopentyl-CH2, cyclohexyl-(CH2)2-, and cyclopentyl-(CH2)2-.

[0530] The terms "heterocyclic amino acid" and "heterocyclic residue" are used interchangeably to refer to an amino acid residue in which the side chain group includes a heterocyclic group, such as a heteroaryl group or a saturated heterocyclic group. In some cases, the side chain group is a heterocycle-alkyl group or a substituted heterocycle-alkyl group. The term is intended to include both naturally occurring and non-naturally occurring α-amino acids. Naturally occurring heterocyclic residues of interest include tryptophan and histidine.

[0531] The terms "non-polar amino acid residue" and "non-polar residue" refer to an amino acid residue that includes a side chain that is hydrogen (i.e., G) or a non-polar group. In some cases, the non-polar amino acid side chain is a hydrophobic group. The term is intended to include both naturally occurring and non-naturally occurring α-amino acids. Naturally occurring non-polar amino acid residues of interest include naturally occurring hydrophobic residues.

[0532] The terms "hydrophobic amino acid" and "hydrophobic residue" are used interchangeably to refer to an amino acid residue in which the side chain group is a hydrophobic group. The term is intended to include both naturally occurring and non-naturally occurring α-amino acids. Naturally occurring hydrophobic residues of interest include alanine, isoleucine, leucine, phenylalanine, proline, and valine.

[0533] The terms "polar amino acid" and "polar residue" are used interchangeably to refer to an amino acid residue in which a side chain group includes a polar group or a charged group. In some cases, the polar group can be a hydrogen bond donor or acceptor. The term is intended to include both naturally occurring and non-naturally occurring α-amino acids. Naturally occurring polar residues of interest include arginine, asparagine, aspartic acid, histidine, lysine, serine, threonine, tyrosine, cysteine, methionine, glutamic acid, glutamine, and tryptophan.

[0534] The terms "scaffold" and "scaffold domain" are used interchangeably and refer to a reference peptide framework motif from which a peptide compound of the invention is derived or against which a peptide compound of the invention can be compared, for example, via sequence or structure alignment methods. The structural motif of a scaffold domain can be based on naturally occurring protein domain structures. For a particular protein domain structural motif, there may be several related base sequences, any one of which can provide a specific three-dimensional structure of the scaffold domain. Scaffold domains can be defined based on characteristic consensus sequence motifs. Figure 14Shown is a possible consensus sequence for the GA scaffold domain based on alignment and comparison of 16 related naturally occurring protein domain sequences that provide the three-helix bundle structural motif of the GA scaffold domain.

[0535] The terms "parent amino acid sequence," "parent sequence," and "parent polypeptide" refer to a polypeptide comprising an amino acid sequence from which a variant peptide compound is generated and against which the variant peptide compound is compared. The parent polypeptide lacks one or more of the modifications or variant amino acids disclosed herein and may be functionally different than the variant peptide compounds as disclosed herein. The parent polypeptide may be a native domain sequence (e.g., SEQ ID NOs: 2-21), a native domain scaffold sequence with pre-existing amino acid sequence modifications (e.g., any convenient point mutations or truncations known to confer desired physical properties to the domain, such as increased stability or solubility), or a non-naturally occurring consensus sequence (e.g., a sequence based on a consensus motif of several native domains of interest, see e.g., Figure 14 ).

[0536] The terms "corresponding residue" and "residue corresponding to..." are used to refer to amino acid residues at equivalent positions in the variant and parent sequences, e.g. Figure 13 It is understood that Figure 13 The numbering scheme of the present invention is not intended to define the minimum or maximum number of residues that must be included in the sequence of the compounds of the invention. The compounds of the invention based on the 53-residue numbering scheme may include any convenient number of residues sufficient to retain the three-helix bundle structural motif. In some cases, the compounds of the invention include less than 53 residues, including N-terminal and / or C-terminal truncated sequences (e.g., as described herein).

[0537] The terms "variant amino acid" and "variant residue" are used interchangeably to refer to a specific residue of a compound of the invention that has been modified or mutated compared to the basic scaffold domain. Variant residues encompass those residues that have been selected (e.g., through mirror screening, affinity maturation and / or point mutations) to provide a desired domain motif structure that specifically binds to the target. When a compound includes an amino acid mutation or modification at a specific position compared to the scaffold domain, the amino acid residue of the peptide compound located at those specific positions is referred to as a "variant amino acid." Such variant amino acids can impart different functions to the resulting peptide compound, such as specific binding to the target protein, improved water solubility, ease of chemical synthesis, metabolic stability, and the like. Aspects of the present disclosure include peptide compounds that are selected from a phage display library based on a GA scaffold domain and further developed (e.g., through additional affinity maturation and / or point mutations), and thus include several variant amino acids integrated with the GA scaffold domain.

[0538] The terms "variant domain" and "variant motif" refer to the arrangement of variant amino acids incorporated at specific positions of a scaffold domain. A variant motif may encompass continuous and / or discontinuous sequences of residues. A variant motif may encompass variant amino acids located at one face of the compound structure. A variant domain may be considered to be incorporated into or integrated with the basic scaffold domain structure or sequence. In the compounds of the present invention, the scaffold domain may provide a stable three-dimensional protein structural motif, such as a naturally occurring protein domain, while the variant domain may be defined by an arrangement of a characteristic minimum number of variant residues at a modified surface of the structure that is capable of specific binding to the target protein.

[0539] The term "framework residues" refers to the remaining amino acid residues of the scaffold domain of a peptide compound that are not variant amino acids. Thus, the structure or sequence motif composed of framework residues is defined by the corresponding arrangement of residues of the underlying scaffold domain structure or sequence. The sequence and structure of the compounds of the present invention can be defined by a combination of variant and framework residues.

[0540] The term "mutation" refers to a deletion, insertion or substitution of an amino acid residue or a nucleotide residue relative to a reference sequence, such as a scaffold sequence.

[0541] The term "domain" refers to a continuous or discontinuous sequence of amino acid residues. A domain can include one or more regions or segments. The terms "region" and "segment" are used interchangeably to refer to a continuous sequence of amino acid residues that, in some cases, can define specific secondary structural features.

[0542] The term "non-core mutation" refers to an amino acid mutation of a peptide compound that is located at a position in the structure that is not part of the hydrophobic core of the structure. The amino acid residues in the hydrophobic core of the peptide compound are not significantly solvent exposed, but tend to form intramolecular hydrophobic contacts. The method for specifying hydrophobic core residues is described by Dahiyat et al. ("Probing the role of packing specificity in protein design", Proceedings of the National Academy of Sciences of the United States of America, 1997, 94, 10172-10177), wherein the PDB structure is used to calculate which side chains expose less than 10% of their surface area to the solvent. In some cases, the De Grado heptapeptide repeat model (De Grado et al. "Analysis and design of three-chain coiled coils and three-helix bundles", Folding and Design 1998, 3: R29-R40) can be used to define the "a" and "d" residues of the hydrophobic core, as depicted in Figure 6. Such methods can be modified for use with GA domain scaffolds.

[0543] The term "surface mutation" refers to an amino acid mutation in a scaffold domain located at a solvent-exposed position in the structure. Such variant amino acid residues at surface positions of a D-peptide compound may be able to directly interact with the target molecule, regardless of whether such interaction occurs. In some cases, the DeLaduo heptad repeat model can be used to define highly solvent-exposed "c" and "g" residues, as depicted in Figure 6.

[0544] The term "boundary mutation" refers to an amino acid mutation in a scaffold that is located at a boundary position between the hydrophobic core and the solvent-exposed surface in the structure. Such variant amino acid residues at the boundary position of a peptide compound may partially contact the hydrophobic core residues and / or be partially solvent-exposed and may be capable of certain interactions with the target molecule, regardless of whether such interactions occur. One criterion for describing the core, surface, and boundary residues of a structure is described by Mayo et al. Nature: Structural Biology, 5(6), 1998, 470-475. In some cases, the Delado heptapeptide repeat model can be used to define at least partially solvent-exposed "c" and "g" residues, as depicted in Figures 6 and 7B. Such methods and criteria can be modified for use with the compounds of the present invention.

[0545] The term "linker sequence" refers to a continuous sequence of amino acid residues or their analogs that connects two peptide motifs or regions. In some cases, the linker sequence is a loop or turn region connecting two antiparallel helical regions (e.g., as described herein).

[0546] The term "stable" refers to a compound that is able to maintain a folded state under physiological conditions at a certain temperature so that it retains at least one of its normal functional activities, such as binding to a target protein. The stability of a compound can be determined using standard methods. For example, the "thermal stability" of a compound can be determined by measuring the thermal melting ("Tm") temperature. Tm is the temperature at which half of the compound becomes unfolded, measured in ° C. In some cases, the higher the Tm, the more stable the compound.

[0547] The terms "similar," "conservative," and "highly conservative" amino acid substitutions are defined as shown below in Table 6. The determination of whether an amino acid residue substitution is similar, conservative, or highly conservative can be based on the side chains of the amino acid residues rather than the polypeptide backbone.

[0548] Table 6: Classification of amino acid substitutions

[0549]

[0550]

[0551] "Specificity determining motif" refers to the arrangement of variant amino acids incorporated at specific positions in a variant scaffold domain that provides specific binding of the variant domain to the target protein. The motif may encompass continuous and / or non-continuous sequences of residues. The motif may encompass variant amino acids that are located at one face of the compound structure and that are capable of contacting the target protein, or may encompass variant residues that do not provide contact with the target but rather provide modifications to the native domain structure that enhance binding to the target. The motif may be considered to be incorporated into a basic scaffold domain structure or sequence, such as the three-helix bundle of a naturally occurring GA or Z domain, or integrated with the three-helix bundle.

[0552] Compounds that "specifically bind" to an epitope or binding site of a target protein are well known terms in the art, and methods for determining such specific or preferential binding are also well known in the art. A compound exhibits "specific binding" if it associates more frequently, more rapidly, for a longer duration, and / or with greater affinity with a specific cell or substance (target protein) than with an alternative cell or substance. A D-peptide compound "specifically binds" to a target if it binds with greater affinity, avidity, more readily, and / or for a longer duration than it binds to other substances. For example, a compound that specifically or preferentially binds to a VEGF epitope or site is an antibody that binds to this epitope or site with greater affinity, avidity, more readily, and / or for a longer duration than it binds to other VEGF epitopes or non-VEGF epitopes. By reading this definition, it should also be understood that, for example, a compound that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Therefore, "specific binding" does not necessarily require (but may include) exclusive binding. Typically, but not necessarily, reference to binding means specific binding.

[0553] Compounds may contain one or more asymmetric centers and may therefore produce enantiomers, diastereomers, and other stereoisomeric forms, which can be defined as (R)- or (S)- in terms of absolute stereochemistry or as (D)- or (L)- for amino acids and polypeptides. The present disclosure is intended to include all such possible isomers and their racemic, diastereomeric, and optically pure forms. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless otherwise specified, it is contemplated that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included.

[0554] The term "target protein" refers to all members of the target family, as well as fragments and enantiomers thereof, and protein mimics thereof. Unless otherwise expressly described, the target protein of interest described herein is intended to include all members of the target family, as well as fragments and enantiomers thereof, and protein mimics thereof. The target protein can be any protein of interest, such as a therapeutic or diagnostic target. The term "target protein" is intended to include recombinant and synthetic molecules, which can be prepared or purchased commercially using any convenient recombinant expression method or using any convenient synthetic method; as well as fusion proteins containing the target molecule, and synthetic L-proteins or D-proteins.

[0555] As used herein, the term "VEGF" or its non-abbreviated form "vascular endothelial growth factor" refers to the protein product encoded by the VEGF gene. The term VEGF includes all members of the VEGF family, such as VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and fragments and enantiomers thereof. The term VEGF is intended to include recombinant and synthetic VEGF molecules, which can be prepared using any convenient recombinant expression method or using any convenient synthetic method or purchased commercially (e.g., R&D Systems, catalog number 210-TA, Minneapolis, Minn.); as well as fusion proteins containing VEGF molecules and synthetic L-proteins or D-proteins. VEGF is involved in angiogenesis (the de novo formation of the embryonic circulatory system) and angiogenesis (the growth of blood vessels from existing vessels), and can also participate in the growth of lymphatic vessels in a process called lymphangiogenesis. Members of the VEGF family stimulate cellular responses by binding to tyrosine kinase receptors (VEGFRs) on the cell surface, causing them to dimerize and become activated via transphosphorylation. The VEGF receptor has an extracellular portion containing seven immunoglobulin-like domains, a single transmembrane spanning region, and an intracellular portion containing a split tyrosine kinase domain. VEGF-A binds to VEGFR-1 (Flt-1) and VEGFR-2 (KDR / Flk-1). VEGFR-2 appears to mediate several cellular responses to VEGF. VEGF, its biological activity, and its receptors are well studied and described in Matsumoto et al. (VEGF receptor signal transduction Sci STKE. 2001: RE21) and Marti et al. (Angiogenesis in ischemic disease, Thrombosis and Hemostasis, 1999 Suppl 1: 44-52). The amino acid sequence of an exemplary VEGF is found in NCBI's Genbank database, and a complete description of VEGF proteins and their roles in various diseases and conditions is found in NCBI's Online Mendelian Inheritance in Man database.

[0556] Exemplary embodiments

[0557] Aspects of the present disclosure are embodied in the clauses and exemplary embodiments set forth below.

[0558] Item 1. A D-peptide compound that specifically binds to VEGF-A, provided that the compound does not comprise a GB1 domain scaffold.

[0559] Item 2. A D-peptide compound according to Item 1, comprising: a VEGF-A binding two-helical complex comprising at least two antiparallel helical regions [helix A] and [helix B] that together define a VEGF-A binding surface, wherein the VEGF-A binding surface comprises six or more VEGF-A contact residues independently selected from non-polar, aromatic, heterocyclic and carbocyclic residues.

[0560] Item 3. The D-peptide compound according to Item 2, wherein [Helix A] and [Helix B] each comprise a heptad repeat sequence (abcdefg) n , and wherein the six or more VEGF-A contact residues are located at the c and g positions of the heptad repeat sequence.

[0561] Item 4. The D-peptide compound according to Item 1, comprising:

[0562] VEGF-A binds to a three-helix bundle comprising helical regions [helix 1], [helix 2], and [helix 3], each of which contains a heptad repeat sequence (abcdefg) n , and are configured to define a hydrophobic core comprising substantially a and d residues;

[0563] wherein [helix 2] and [helix 3] are arranged antiparallel to each other and together define the VEGF-A binding gg face of the three-helix bundle, wherein the VEGF-A binding gg face comprises six or more VEGF-A contact residues independently selected from non-polar, aromatic, heterocyclic and carbocyclic residues.

[0564] Item 5. The D-peptide compound of Item 4, wherein the three-helix bundle is a GA domain motif of formula (I):

[0565] [Helix 1]-[Linker 1]-[Helix 2]-[Linker 2]-[Helix 3]

[0566] (I)

[0567] wherein [Linker 1] and [Linker 2] are independently peptide linker sequences of between 1 and 10 residues.

[0568] Item 6. A D-peptide compound according to any one of items 4 to 5, wherein the six or more VEGF-A contacting amino acid residues include four or more aromatic amino acid residues, which are configured to contact VEGF-A and are located at the c and g solvent-exposed positions of the gg face.

[0569] Item 7. The D-peptide compound according to any one of items 4 to 6, wherein [helix 2] comprises a heptad repeat sequence [c 1 d 1 e 1 f 1 g 1 a 2 b 2 c 2 d 2 ], and [helix 3] contains a heptad repeat sequence [e 1 f 1 g 1 a 2 b 2 c 2 d 2 e 2 f 2 g 2 a 3 b 3 c 3 d 3 e 3 ],in:

[0570] Residue d of [helix 2] 2 、a 2 and d 1 Residue a of [helix 3] 2 d 2 and a 3 interact with each other; and

[0571] Residue c of [helix 2] 2 、g 1 and c 1 and residue g of [helix 3] 1 Each is independently an aromatic, heterocyclic or carbocyclic residue.

[0572] Clause 8. The D-peptide compound of any one of clauses 2 to 7, wherein the VEGF-A binding surface comprises the following configuration of VEGF-A contact residues at the c and g positions of the heptad repeats of helix A and helix B:

[0573]

[0574] in:

[0575] Each h* is independently histidine or an analog thereof;

[0576] f* is phenylalanine or an analog thereof; and

[0577] Each u is independently a non-polar amino acid residue.

[0578] Clause 9. The D-peptide compound according to any one of clauses 4 to 5, wherein:

[0579] [Helix 2] contains a sequence of the formula:

[0580] h*jxxf*jxh*j (SEQ ID NO: 151)

[0581] [Helix 3] contains a sequence of the formula:

[0582] h*jxujxxuj (SEQ ID NO: 152)

[0583] in:

[0584] Each h* is independently histidine or an analog thereof;

[0585] f* is phenylalanine or its analogue;

[0586] Each u is independently a non-polar amino acid residue.

[0587] Each j is independently a hydrophobic residue; and

[0588] Each x is independently an amino acid residue.

[0589] Item 10. The D-peptide compound according to Item 9, wherein [Helix 2] is defined by a sequence of the formula:

[0590] zh*jxxf*jxh*jz (SEQ ID NO: 153)

[0591] wherein each z is independently a helix-terminating residue.

[0592] Item 11. The D-peptide compound according to Item 10, wherein each helix-terminating residue (z) is independently selected from d, p and G.

[0593] Item 12. The D-peptide compound of any one of items 5 to 11, wherein [Linker 2] is 2 amino acid residues or less in length and comprises a tyrosine residue or an analog thereof.

[0594] Clause 13. The D-peptide compound of any one of clauses 5 to 12, wherein [Helix 2]-[Linker 2]-[Helix 3] comprises a sequence of the formula:

[0595] zh*jxxf*jxh*jzy*xxh*jxujxxujx(SEQ ID NO:154)

[0596] in:

[0597] y* is tyrosine or its analogue;

[0598] Each h* is independently histidine or an analog thereof;

[0599] f* is phenylalanine or its analogue;

[0600] Each u is independently a non-polar amino acid residue.

[0601] Each j is independently a hydrophobic residue; and

[0602] Each x is independently an amino acid residue.

[0603] Item 14. A D-peptide compound according to any one of items 5 to 13, wherein [Linker 1] has a sequence of the formula

[0604] z(x) n e*z (SEQ ID NO: 148)

[0605] in:

[0606] each x is an amino acid and n is 1, 2 or 3;

[0607] each z is independently a helix-terminating residue (e.g., G or p); and

[0608] e* is glutamic acid or an analog thereof.

[0609] Clause 15. The D-peptide compound of any one of clauses 5 to 14, wherein [Linker 1]-[Helix 2]-[Linker 2]-[Helix 3] comprises a sequence of the formula:

[0610] zxxe*zh*jxxf*jxh*jzy*xxh*jxujxxujx(SEQ ID NO:155)

[0611] in:

[0612] e* is glutamic acid or its analogue;

[0613] Each z is independently a helix-terminating residue;

[0614] y* is tyrosine or its analogue;

[0615] Each j is independently a hydrophobic residue;

[0616] Each u is independently a non-polar amino acid residue; and

[0617] Each x is independently an amino acid residue.

[0618] Clause 16. The D-peptide compound of any one of clauses 4 to 15, wherein [helix 2] is defined by a sequence of the formula:

[0619] z 26 hj28 xxfj 32 xhj 35 z 36 (SEQ ID NO: 101).

[0620] in:

[0621] z 26 selected from d, p and G;

[0622] z 36 Selected from p and G;

[0623] j 28 、j 32 and j 35 are each independently a hydrophobic residue; and

[0624] Each x is independently an amino acid residue.

[0625] Item 17. The D-peptide compound according to Item 16, wherein j 28 、j 32 and j 35 Independently selected from a, i, l and v.

[0626] Item 18. The D-peptide compound according to Item 17, wherein j 28 、j 32 and j 35 is the corresponding residue of the GA scaffold domain selected from any one of SEQ ID NOs: 1-21 of US62 / 865,469 filed on June 24, 2019.

[0627] Clause 19. The D-peptide compound of any one of clauses 4 to 18, wherein [helix 2] is defined by a sequence selected from the group consisting of:

[0628] a)phvx 29 x 30 fix 33 hap (SEQ ID NO: 102)

[0629] in:

[0630] x 29 Selected from f and i;

[0631] x 30 and x 33 are independently selected from polar amino acid residues; and

[0632] b) an amino acid sequence that is 80% or more identical (eg, 2 residue changes) to the sequence defined in a).

[0633] Item 20. The D-peptide compound according to Item 19, wherein:

[0634] x 29 for i;

[0635] x 30 is s or n; and

[0636] x 33 is n.

[0637] Clause 21. The D-peptide compound of any one of clauses 4 to 20, wherein [helix 3] is defined by a sequence of the formula:

[0638] xxhj 41 xuj 44 xxuj 48 xxx (SEQ ID NO: 103)

[0639] in:

[0640] j 41 、j 44 and j 48 Each is independently a hydrophobic residue;

[0641] Each u is independently a non-polar amino acid residue; and

[0642] Each x is independently an amino acid residue.

[0643] Item 22. The D-peptide compound according to Item 21, wherein j 41 、j 44 and j 48 Independently selected from a, i, l and v.

[0644] Item 23. The D-peptide compound according to Item 21, wherein j 41 、j 44 and j 48 are the corresponding residues of the GA scaffold domain selected from SEQ ID NOs: 1-21 of US62 / 865,469 filed on June 24, 2019.

[0645] Item 24. The D-peptide compound of Item 21, wherein [Helix 3] is defined by a sequence selected from the group consisting of:

[0646] a)x 38 x 39 hvx 42 Glx 45 x 46 aix 49 x 50 a (SEQ ID NO: 98)

[0647] in:

[0648] x 38 Select from v, e, k, r;

[0649] x 39 、x 42 、x 46 and x 50 are independently selected from hydrophilic amino acid residues (e.g., n, s, d, e, and k); and

[0650] x 45 and x 49 are independently selected from l, k, r, and e; and

[0651] b) an amino acid sequence that is 80% or more identical (eg, 2 residue changes) to the sequence defined in a).

[0652] Item 25. The D-peptide compound according to Item 24, wherein: x 38 is v; x 39 is s; x 42 is n; x 45 k, x 46 is n; x 49 is l; and x 50 is k.

[0653] Item 26. A D-peptide compound according to any one of items 4 to 25, wherein the VEGF-A binding domain of the compound comprises 6 or more variant amino acid residues relative to the reference GA scaffold sequence, wherein the 6 or more variant amino acids are selected from: e at position 25; p at position 26; h at position 27; v at position 28; i at position 29; s at position 30; f at position 31; h at position 34; p at position 36; y at position 37; s at position 39; h at position 40; G at position 43; and a at position 47.

[0654] Item 27. The D-peptide compound of Item 26, wherein the compound comprises p at position 26, f at position 31, and p at position 36.

[0655] Item 28. The D-peptide compound of Item 26, wherein the compound comprises the following variant amino acids: p at position 26, i at position 29, and s at position 30.

[0656] Clause 29. The D-peptide compound of any one of clauses 26 to 28, wherein the compound comprises h at positions 27, 34, and 40.

[0657] Clause 30. The D-peptide compound of any one of clauses 26 to 29, wherein the compound comprises G at position 43; and a at position 47.

[0658] Clause 31. The D-peptide compound of any one of clauses 26 to 30, wherein the compound comprises v at position 28.

[0659] Clause 32. The D-peptide compound of any one of clauses 1 to 31, wherein the compound comprises an amino acid sequence selected from the group consisting of:

[0660] a)llknakedaiaelkkcGitephvisfinhapyvshvnGlknailka; and

[0661] b) Amino acid sequences that are 85% or more identical to the sequences defined in a).

[0662] Item 33. The D-peptide compound according to any one of items 4 to 32, wherein [Helix 1] comprises a sequence selected from the group consisting of: a) l 6 lknakedaiaelkka 21 (SEQ ID NO: 74); and b) an amino acid sequence that is 75% or more identical to the sequence defined in a).

[0663] Clause 34. The D-peptide compound of any one of clauses 1 to 33, wherein the compound comprises a sequence selected from the group consisting of: a) G 22 itephvisfinhapyvshvnGlknailka 51 (SEQ ID NO: 84); and b) an amino acid sequence that is 75% or more identical to the sequence defined in a).

[0664] Clause 35. A D-peptide compound according to any one of clauses 1 to 34, wherein the compound comprises a peptide framework sequence selected from the group consisting of: a) l 6 lknakedaiaelkkaGit…in.a..v..vn..kn.ilka 51 (SEQ ID NO: 156); and b) an amino acid sequence that is 88% or more identical to the sequence defined in a).

[0665] Clause 36. A D-peptide compound according to any one of clauses 1 to 35, wherein the compound comprises a peptide framework sequence selected from the group consisting of: a) t 1 idqwllknakedaiaelkkaGit…in.a..v..vn..kn.ilkaha 53(SEQ ID NO: 157); and b) an amino acid sequence that is 90% or more identical to the sequence defined in a).

[0666] Item 37. A D-peptide compound according to any one of items 1 to 36, wherein the compound comprises a sequence selected from SEQ ID NOs: 22-71 of US62 / 865,469 filed on June 24, 2019.

[0667] Clause 38. The D-peptide compound of any one of clauses 1 to 37, further comprising an attached non-proteinaceous polymer moiety.

[0668] Clause 39. The D-peptide compound of any one of clauses 1 to 37, further comprising a linked specific binding moiety.

[0669] Item 40. The D-peptide compound of Item 39, wherein the linked specific binding moiety is a second D-peptide binding domain.

[0670] Clause 41. The D-peptide compound of any one of clauses 39-40, wherein the compound comprises a multimeric configuration of the VEGF binding GA domain.

[0671] Clause 42. The D-peptide compound of any one of clauses 40 to 41, wherein the compound is a homodimer and comprises two linked VEGF-A binding GA domains.

[0672] Item 43. The D-peptide compound of Item 42, wherein the VEGF-A binding GA domains are linked via a polymeric linker via the N-terminal residue.

[0673] Item 44. The D-peptide compound of Item 42, wherein the VEGF-A binding GA domain motif is linked via a peptide linker through the N-terminal residue.

[0674] Clause 45. The D-peptide compound of any one of clauses 40 to 41, wherein the compound is a heterodimer.

[0675] Item 46. A D-peptide compound according to Item 45, wherein the second D-peptide binding domain specifically binds to a target protein selected from the group consisting of: PDGF, VEGF-B, VEGF-C, VEGF-D, EGF, EGFR, Her2, Her3, PD-1, PD-L1, CTLA4, OX-40, DR3, Ang-2, LAG3, HSA and Ig.

[0676] Item 47. The D-peptide compound of any one of Items 1 to 46, wherein the compound has a K of 100 nM or less (e.g., 30 nM or less, 10 nM or less, 3 nM or less, 1 nM or less, etc.). D The value specifically binds to VEGF-A protein.

[0677] Item 48. A D-peptide compound according to any one of items 1 to 47, wherein the VEGF binding GA domain comprises between 45 and 60 residues (e.g., between 46 and 55 residues, between 50 and 54 residues, etc.).

[0678] Item 49. A pharmaceutical composition comprising the D-peptide compound according to any one of Items 1 to 48, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0679] Clause 50. The pharmaceutical composition of clause 49, wherein the composition is formulated for treating an ocular disease or condition.

[0680] Clause 51. A method of treating or preventing a disease or condition associated with angiogenesis in a subject, the method comprising administering to a subject in need thereof an effective amount of a compound according to any one of clauses 1 to 48, or an effective amount of a pharmaceutical composition according to any one of clauses 49 to 50.

[0681] Item 52. The method of Item 51, wherein the disease or condition associated with angiogenesis is cancer (e.g., breast cancer, skin cancer, colorectal cancer, pancreatic cancer, prostate cancer, lung cancer, or ovarian cancer), inflammatory diseases, atherosclerosis, rheumatoid arthritis, macular degeneration, retinopathy, and skin diseases (e.g., rosacea).

[0682] Item 53. The method of Item 51, wherein the disease or condition associated with angiogenesis is diabetic macular edema (DME).

[0683] Item 54. The method of Item 51, wherein the disease or condition associated with angiogenesis is wet age-related macular degeneration (AMD).

[0684] Clause 55. The method of any one of clauses 51 to 54, further comprising administering to the subject an effective amount of a second active agent.

[0685] Item 56. The method of Item 55, wherein the second active agent is a D-peptide compound.

[0686] Item 57. The method of Item 55, wherein the second active agent is a small molecule, a chemotherapeutic agent, an antibody, an antibody fragment, an aptamer, or an L-protein.

[0687] Item 58. A method according to any one of items 55 to 57, wherein the second active agent specifically binds to a target protein selected from the group consisting of platelet-derived growth factor (PDGF), VEGF-B, VEGF-C, VEGF-D, EGF, EGFR, Her2, Her3, PD-1, PD-L1, CTLA4, OX-40, DR3, LAG3, Ang2, IL-1, IL-6 and IL-17.

[0688] Item 59. The method of Item 55, wherein the second active agent specifically binds to PDGF-B.

[0689] Item 60. The method of Item 55, wherein the second active agent is selected from: pegylated (Fovastatin), ranibizumab (Lentuzumab), trastuzumab (Hecanpin), bevacizumab (Cancer Stop), aflibercept (Cerevisia), nivolumab, atezolizumab, durvalumab, gefitinib, erlotinib, and pembrolizumab.

[0690] Clause 61. A method for in vivo diagnosis or imaging of a disease or condition associated with angiogenesis, comprising administering to a subject a D-peptide compound according to any one of clauses 1 to 49, and imaging at least a portion of the subject.

[0691] Item 62. The method of Item 61, wherein the imaging comprises PET imaging and the administering comprises administering the compound to the vascular system of the subject.

[0692] Item 63. The method of Item 61, further comprising detecting uptake of the compound by a cellular receptor.

[0693] Clause 64. The method of clause 61, further comprising administering to the subject acancerstatin, wherein the disease or condition is a condition associated with cancer.

[0694] The following examples are offered by way of illustration and not limitation.

[0695] Examples

[0696] The following examples are presented to provide a complete disclosure and description of how to make and use the present invention to one of ordinary skill in the art and are not intended to limit the scope of what the inventors consider to be their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be considered. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

[0697] General methods in molecular and cellular biochemistry can be found, for example, in standard textbooks: Molecular Cloning: A Laboratory Manual, 3rd edition (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th edition (Ausubel et al., eds., John Wiley 1999); Protein Methods (Bollag et al., John Wiley 1996); Nonviral Vectors for Gene Therapy (Wagner et al., eds., Academic Press 1999); Viral Vectors (Kaplift and Loewy, eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits, ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology. Biotechnology) (Doyle and Griffiths, John Wiley 1998), the disclosures of which are incorporated herein by reference. Reagents, cloning vectors, cells, and kits for use in or related to the methods described herein are available from commercial suppliers, such as BioRad, Agilent Technologies, Thermo Fisher Scientific, Sigma-Aldrich, New England Biolabs (NEB), Takara Bio USA, Inc., and the like, as well as repositories, such as Addgene, Inc., American Type Culture Collection (ATCC), and the like.

[0698] Example 1: Selection of D-peptide compounds

[0699] Compounds of the invention were identified via mirror-image screening of a scaffolded GA domain phage display library for binding to a synthetic D-VEGF-A target protein using methods as described by Uppalapati et al. in WO 2014 / 140882. Figure 13A depiction of the GA domain library is shown, including the basic 53 residue scaffold sequence (SEQ ID NO: 2) and the mutation positions shown in bold at positions 25, 27, 28, 31, 34, 36, 37, 39, 40, 43, 44, and 47 of the scaffold, which define the variation in the phage display library.

[0700] Briefly, 5 μg / ml D-VEGFA was plated onto NUNC Maxisorp plates. After blocking, a pool of eight scaffolds, including the GA domain library, was added to the plates after depletion on empty wells. Bound phages were eluted and amplified overnight in OmniMax2 T1R cells. For rounds 3 and 4, approximately 1 × 10 13 Compared with the standard concentration of cfu / ml, a lower concentration of the amplified phage pool (approximately 5×10 11 cfu / ml) because the elution concentration in round 2 was too high. Several hits were obtained from various libraries, including 17 different sequences from the GA domain library. Based on the sequence identity between clones, three representative clones including compound 1 were selected (see Figure 15 After cloning into the p3 fusion vector for affinity maturation, compound 1 maintained its binding to D-VEGFA.

[0701] For the first round of affinity maturation, a soft randomization strategy (Fairbrother et al., 1998) was used, wherein the polynucleotides encoding each of the randomized positions 25, 27, 28, 31, 34, 36, 37, 39, 40, 43, 44, and 47 were doped with hand-mixed bases to bias the natural nucleotide at 70%, with the other three nucleotides appearing at 10% frequency. This resulted in a 40% chance of retaining the amino acid found in the parental sequence of compound 1 at each of these positions. Affinity maturation libraries were constructed by a site-directed mutagenesis protocol (Fellouse et al.) using the following oligonucleotides and ssDNA from the original sequence of the GA domain as templates.

[0702] AAGGCTGGTATCACC(N4)(N2)(N4)GAC(N2)(N1)(N4)(N3)(N4)(N4)TTCAAC(N4)(N4)(N4)ATCAAT(N4)(N1)(N4)GCG(N2)(N2)(N4) (N4)(N1)(N4)GTG(N4)(N2)(N4)(N3)(N1)(N4)GTTAAC(N3)(N2)(N1)(N2)(N4)(N3)AAGAAC(N3)(N1)(N3)ATCCTGAAAGCTCAC(SEQ ID NO:130)

[0703] Where N1 is a mixture of 70% A, 10% C, 10% G and 10% T

[0704] N2 is a mixture of 10% A, 70% C, 10% G and 10% T

[0705] N3 is a mixture of 10% A, 10% C, 70% G and 10% T

[0706] N4 is a mixture of 10% A, 10% C, 10% G and 70% T

[0707] Affinity maturation libraries were panned against D-VEGFA using standard procedures (Fellouse et al.). 24 clones from round 3 were analyzed and ranked by affinity using competitive ELISA. Compound 1.1 was selected from this list as the clone of interest. Sequence identifiers for selected positions of all clones are shown in Figure 26 The results were compared with compound 1 and the native GA domain (GA-wt). In this study, positions 27, 28, 31, 36, and 44 were highly conserved or retained as His27, Val28, Phe31, Pro36, and Leu44 in all clones. Aromatic residues His, Tyr, and Phe predominated at position 34. His or Asp residues predominated at position 40. Glu or Ala predominated at position 47.

[0708] A second round of affinity maturation was performed to improve the affinity and stability of compound 1.1. Given that the Pro residue is largely conserved in position 36, changes in the main chain conformation may alter the orientation of helix 2 relative to the core residues and may affect the stability of the selected compound 1.1. In addition, surface-exposed residues near the C-terminus may form additional contacts. Therefore, the following positions, including core and surface-exposed positions, were selected for further optimization: positions 15, 18, 19, 21, 23, 25, 26, 28, 29, 30, 47, 48, 49, 50, 51 and 52. The soft randomization strategy was again used for site-directed mutagenesis with the following oligonucleotides

[0709] GCGAAAGAAGATGCT(N1)(N4)(N4)GCAGAA(N2)(N4)(N2)(N1)(N1)(N1)AAG(N2)(N2)(N4)GGT(N1)(N4)(N2 )ACC(N2)(N1)(N1)(N2)(N1)(N2)CAT(N2)(N4)(N4)(N4)(N4)(N2)(N1)(N1)(N2)TTTATCAATCACGCGC(SEQ ID NO:131)

[0710] GTTAACGGGCTGAAGAAC(N2)(N2)(N2)(N1)(N4)(N2)(N2)(N4)(N2)(N1)(N1)(N1)(N2)(N2)(N4)(N2)(N1)(N2)GCCGGGAGCTCTGGAG(SEQ ID NO:132)

[0711] The library was constructed and panned for D-VEGFA using a modified protocol. Given that D-VEGF-A is highly stable and remains folded even in the presence of 3M guanidine hydrochloride (GuHCl), it was hypothesized that selecting binders in the presence of low to medium concentrations of denaturants would select clones with both increased affinity and stability. In this procedure, the library or amplified phage pool was resuspended in PBT buffer (PBS, 0.2% BSA, 0.05% Tween 20), and each round of selection used a denaturant guanidine hydrochloride (GuHCl) at varying concentrations. Phage were incubated at 37°C for 2 hours for equilibrium. Selection was also performed at 37°C.

[0712] The following conditions apply for each round.

[0713]

[0714] After four rounds of affinity maturation, several clones were sequenced and evaluated by competitive ELISA analysis, and compound 1.1.1 was selected as the clone of interest. Cys21 was identified as a bystander mutation and reverted to Ala (e.g., to eliminate the possibility of disulfide dimerization), resulting in the lead compound of interest, compound 1.1.1 (C21A).

[0715] Additionally, various scaffolded phage display libraries described by Uppalapati et al. in WO2014 / 140882 were screened for binding to a synthetic D-VEGF-A target protein. Several scaffolded domain libraries generated hit clones during phage display screening studies, indicating that D-peptide compounds of the invention that specifically bind to VEGF-A may possess one of a variety of basic scaffold domains. Initially, hit clones were selected from the GA domain scaffolded library for further investigation.

[0716] Table 7: List of scaffolds that generated hits against D-VEGFA

[0717] SCF2-DGCR8 dimerization domain-56aa

[0718] SCF3-Get5 C-terminal domain-41aa

[0719] SCF7-KorB C-terminal domain-58aa

[0720] SCF8-Lsr2 dimerization domain-55aa

[0721] SCF15-Symfoil 4P (designed beta-trefoil)-42aa

[0722] SCF24-Golgin245 GRIP domain-51aa

[0723] C-terminal domain of SCF28-Ku-51aa

[0724] SCF 32-GA domain of protein G-53aa

[0725] SCF29-Cue2 Cue domain-49aa

[0726] SCF37-PEM1-like protein-44aa

[0727] SCF40-nucleotide exchange factor C-terminal domain-60aa

[0728] SCF42-transcription factor anti-termination protein-59aa

[0729] SCF44-This protein-65aa

[0730] SCF53-Rhodnin kazal inhibitor-51aa

[0731] SCF55-anti-TRAP-48aa

[0732] SCF56-TNF receptor 17 (BCMA)-39aa

[0733] SCF63-Fyn SH3-61aa

[0734] SCF64-E3 ubiquitin-protein ligase UBR5-65aa

[0735] SCF65-DNA repair endonuclease XPF-63aa

[0736] SCF66-rad23 hom.B, xpcB domain-61aa

[0737] SCF70-LEM domain of Emerin-47aa

[0738] SCF75-GspC-68aa

[0739] SCF95-protein Z-58aa

[0740] SCF96-B1 domain of protein G (GB1)-55aa

[0741] Example 2: Synthesis and folding of D-peptide compounds

[0742] Selected compounds were synthesized and purified using conventional Fmoc solid phase peptide synthesis methods. In some cases, additional point mutations were included, for example, as described herein. Compounds were folded in buffer and assessed for VEGF-A inhibitory activity as described herein.

[0743] Example 3: X-ray crystal structure of VEGF-A complex

[0744] The X-ray crystal structure of compound 1.1.1 (CA) in complex with L-VEGF-A was obtained. Figure 1 A view of the X-ray crystal structure of exemplary compound 1.1.1 (c21a) (white sticks) in complex with VEGF-A (space filling diagram) is shown. The complex is dimeric. Figure 1 and 2 In the figure, the binding site residues of VEGF-A that contact the compound are depicted in pink. The VEGF-A (8-109) binding site residues are indicated in bold: GQNHHEVVKFMDVYQRSYCH PIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITM QIMRIKPHQGQHIGEMSFLQHNKCECRPKKD (SEQ ID NO: 88);

[0745] The individual binding sites in the dimer are defined by the following residues:

[0746] Chain A: K...

Claims

1. A multivalent D-peptide compound that specifically binds to VEGF, comprising: A D-peptide Z domain that is capable of specifically binding to the first binding site of VEGF; and D-peptide GA domain, which is able to specifically bind to the second binding site of VEGF.

2. The D-peptide compound according to claim 1, wherein: The first binding site comprises the amino acid side chains E90, F62, D67, I69, E70, K110, P111, H112 and Q113 of VEGF; The second binding site comprises amino acid side chains F43, M44, Y47, Y51, N88, D89, L92, I72, K74, M107, I109, Q115 and I117 of VEGF; and The first binding site and the second binding site each at least partially overlap with a VEGFR2 binding site on a VEGF target protein.

3. A D-peptide compound according to claim 1 or 2, wherein the D-peptide Z domain comprises a VEGF-specific determining motif (SDM), wherein the VEGF-specific determining motif comprises 5 or more variant amino acid residues (e.g., 6 or more, such as 6, 7, 8, 9 or 10) at positions selected from 9, 10, 13, 14, 17, 24, 27, 28, 32 and 35.

4. The D-peptide compound according to claim 2, wherein the D-peptide Z domain is according to any one of claims 30 to 41.

5. The D-peptide compound according to any one of claims 1 to 3, wherein the D-peptide GA domain comprises a VEGF-specific determining motif (SDM), wherein the VEGF-specific determining motif comprises 5 or more (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16) variant amino acid residues at positions selected from 25, 27, 30, 31, 34, 36, 37, 39, 40, and 42-48.

6. The D-peptide compound according to claim 4, wherein the D-peptide GA domain is according to any one of claims 42 to 60. 7 . The D-peptide compound according to claim 1 , further comprising a linking component that covalently links the D-peptide Z domain and the D-peptide GA domain.

8. The D-peptide compound according to claim 7, wherein the linking component is a linker (e.g., an N-terminal to N-terminal linker or a C-terminal to C-terminal linker) that connects the terminal amino acid residue of the D-peptide Z domain to the terminal amino acid residue of the D-peptide GA domain.

9. The D-peptide compound according to claim 8, wherein the linking component is an N-terminal to N-terminal linker.

10. The D-peptide compound according to claim 9, wherein the N-terminal to N-terminal linker is (PEG) linking the N-terminal amino acid residues of the D-peptide Z domain and the D-peptide GA domain. n A bifunctional linker wherein n is 2-20 (eg, n is 5, 6, 7, 8, 9, 10, 11, or 12).

11. The D-peptide compound according to claim 7, wherein the linking component is: A linker connecting the amino acid side chain of the D-peptide Z domain and the terminal amino acid residue of the D-peptide GA domain, wherein when the D-peptide Z domain and the D-peptide GA domain simultaneously bind to the target protein, the amino acid side chain and the terminal amino acid residue are close to each other; or A linker connecting the terminal amino acid residue of the D-peptide Z domain and the amino acid side chain of the D-peptide GA domain, wherein when the D-peptide Z domain and the D-peptide GA domain simultaneously bind to the target protein, the terminal amino acid residue and the amino acid side chain are close to each other.

12. The D-peptide compound according to claim 11, wherein the connecting component is a linker connecting the amino acid side chains of the D-peptide Z domain and the amino acid side chains of the D-peptide GA domain, and when the D-peptide Z domain and the D-peptide GA domain simultaneously bind to VEGF, the amino acid side chains are close to each other. 13 . The D-peptide compound according to claim 12 , wherein the linker connects the side chain of the amino acid residue at position 7 of the D-peptide Z domain and the side chain of the amino acid residue at position 19 of the D-peptide GA domain.

14. The D-peptide compound according to claim 13, wherein the D-peptide GA domain and the D-peptide Z domain are connected by the k 7 The residues are related to the k of the D-peptide GA domain 19 Linkers connect residues.

15. The D-peptide compound according to any one of claims 6 to 14, wherein the linking component connects the N-terminus of the D-peptide Z domain to the proximal residue (e.g., k 19 , k 20 or k 50 residue).

16. The D-peptide compound according to any one of claims 1 to 15, wherein the D-peptide GA domain comprises an inter-helical linker between the proximal amino acid residues at positions 7 and 38 of the domain.

17. The D-peptide compound of claim 16, wherein the inter-helical linker has a backbone length of 3 to 7 atoms as measured between the α-carbons of the proximal amino acid residues.

18. The D-peptide compound according to claim 16 or 17, wherein the inter-helical linker comprises one or more groups selected from the group consisting of: C (1-6) Alkyl, substituted C (1-6) Alkyl, -(CHR) n -CONH-(CHR) m -and-(CHR) n -SS-(CHR) m -, wherein each R is independently H, C (1-6) Alkyl or substituted C (1-6) Alkyl, and n+m=0-5 (eg, n+m=2, 3, 4 or 5).

19. The D-peptide compound according to any one of claims 7 to 18, wherein the linking component is configured to link the D-peptide GA domain and the D-peptide Z domain, whereby the domains are capable of simultaneously binding to VEGF-A.

20. The D-peptide compound according to any one of claims 7 to 19, wherein the linking component comprises one or more groups selected from the group consisting of amino acid residues, polypeptides, (PEG) n A linker (e.g., n is 2-50, 3-50, 4-50, 6-50, or 6-20), a modified PEG moiety, a C (1-6) Alkyl linker, substituted C (1-6) Alkyl linker, -CO(CH2) m CO-、-NR(CH2) p NR-, -CO(CH2) m NR-, -CO(CH2) m O-, -CO(CH2) m S- and a linked chemoselective functional group (e.g., -CONH-, -OCONH-, click chemistry conjugates such as 1,2,3-triazole, maleimide-thiol conjugated thiosuccinimide, haloacetyl-thiol conjugated thioether, etc.), wherein m is 1 to 6, p is 2-6, and each R is independently H, C (1-6) Alkyl or substituted C (1-6) alkyl.

21. The D-peptide compound according to any one of claims 1 to 20, wherein the compound is divalent.

22. The D-peptide compound according to any one of claims 1 to 20, wherein the compound is trivalent.

23. The D-peptide compound according to any one of claims 1 to 20, wherein the compound is tetravalent.

24. The D-peptide compound according to any one of claims 1 to 23, wherein the compound further comprises a second D-peptide Z domain homologous to the first D-peptide Z domain.

25. The D-peptide compound according to any one of claims 1 to 24, wherein the compound further comprises a second D-peptide GA domain homologous to the first D-peptide GA domain.

26. The D-peptide compound according to any one of claims 23 to 25, wherein the compound comprises four D-peptide domains configured as a dimer of two divalent D-peptide compounds, each of which comprises a D-peptide Z domain and a D-peptide GA domain.

27. The D-peptide compound according to claim 26, wherein the D-peptide Z domain and the D-peptide GA domain of each divalent D-peptide compound are linked by the k 7 Residues and k of D-peptide GA domain 19 Linkers connect residues.

28. The D-peptide compound according to claim 17, wherein the linker is: wherein n and m are independently 1-6 (eg, 1, 2 or 3). 29 . The D-peptide compound according to claim 28 , wherein the two divalent D-peptide compounds are covalently linked via a linker between the C-terminal amino acid residues of the D-peptide Z domains of the divalent D-peptide compounds.

30. The D-peptide compound of claim 29, wherein the C-terminal to C-terminal linker comprises: *-Alanine-Lysine(-*) wherein each -* is a bond to the D-peptide domain.

31. A D-peptide compound that specifically binds to VEGF, comprising: A D-peptide Z domain comprising: a) A VEGF specificity determining motif (SDM) defined by the following amino acid residues: w 9 d 10 --w 13 x 14 --r 17 ------x 24 --k 27 x 28 ---x 32 --y 35 (SEQ ID NO:160) in: x 14 Selected from l, r and t; x 24 Selected from h, i, l, r and v; x 28 Selected from G, r and v; x 32 is selected from a, r, h, s, and t; and x 35 Selected from k or y; b) a VEGF SDM having 80% or more (e.g., 90% or more) identity with the SDM residues defined in (a); or c) a VEGF SDM having 1 to 3 amino acid residue substitutions relative to the SDM residues defined in (a), wherein the 1 to 3 amino acid residue substitutions are selected from: i) substitution of analogous amino acid residues according to Table 6; ii) conservative amino acid residue substitution according to Table 6; iii) substitution of highly conserved amino acid residues according to Table 6; and iv) Amino acid residue substitution according to the motif defined in Figure 33A.

32. The D-peptide compound according to claim 31, wherein the SDM residue defined in (a) is: w 9 d 10 --w 13 r 14 --r 17 ------l 24 --k 27 r 28 ---s 32 --y 35 (SEQ ID NO:161) or w 9 d 10 --w 13 r 14 --r 17 ------v 24 --k 27 r 28 ---r 32 --y 35 (SEQ ID NO:162)。 33. The D-peptide compound according to claim 32, wherein the VEGF SDM is defined by the following residues: w 9 d 10 --w 13 r 14 --r 17 ------l 24 --k 27 r 28 ---s 32 --y 35 (SEQ ID NO:161) or w 9 d 10 --w 13 r 14 --r 17 ------v 24 --k 27 r 28 ---r 32 --y 35 (SEQ ID NO:162)。 34. The D-peptide compound according to any one of claims 31 to 33, wherein the SDM residue is contained in a peptide framework sequence comprising: a) The peptide framework residues are defined by the following amino acid residues: --n 11 a--e 15 oh 18 lpnln-e 25 q--a 29 you 33 l-; b) a peptide framework residue that is 80% or more (eg, 90% or more) identical to the residue defined in (a); or c) peptide framework residues having 1 to 3 amino acid residue substitutions relative to the residues defined in (a), wherein the 1 to 3 amino acid residue substitutions are selected from: i) substitution of analogous amino acid residues according to Table 6; ii) conservative amino acid residue substitutions according to Table 6; and iii) Substitution of highly conserved amino acid residues according to Table 6.

35. The D-peptide compound according to any one of claims 31 to 34, comprising an SDM-containing sequence having 80% or more (e.g., 85% or more, 90% or more, or 95% or more) identity with the following amino acid sequence: w 9 d 10 naw 13 x 14 eir 17 hlpnlnx 24 eqk 27 x 28 afix 32 sly 35 (SEQ ID NO:133) in: x 14 Selected from l, r and t; x 24 Selected from h, i, l, r and v; x 28 Selected from G, r and v; x 32 is selected from a, r, h, s, and t; and x 35 Selected from k or y.

36. The D-peptide compound according to any one of claims 31 to 35, wherein the D-peptide Z domain is a three-helix bundle of the following structural formula: [Spiral 1 (#8-18) ]-[Linker 1 (#19-24) ]-[Spiral 2 (#25-36) ]-[Linker 2 (#37-40) ]-[Spiral 3 (#41-54) ] in: # represents the reference position of the amino acid residues contained in the D-peptide GA domain; and Helix 3 (#41-54) Comprising a peptide framework sequence selected from the group consisting of: <h2 style=";text-align:left;direction:ltr">a)s<h2 style=";text-align:left;direction:ltr"> 41 <h2 style=";text-align:left;direction:ltr"> agree<h2 style=";text-align:left;direction:ltr"> 54 <h2 style=";text-align:left;direction:ltr"> (SEQ ID NO:134); b) a sequence that is 70% or more (e.g., 75% or more, 80% or more, 85% or more, or 90% or more) identical to the sequence set forth in (a); or c) a sequence having 1 to 5 amino acid residue substitutions relative to the sequence set forth in (a), wherein the 1 to 5 amino acid residue substitutions are selected from: i) substitution of analogous amino acid residues according to Table 6; ii) conservative amino acid residue substitutions according to Table 6; and iii) Substitution of highly conserved amino acid residues according to Table 6.

37. The D-peptide compound according to any one of claims 31 to 36, wherein the D-peptide Z domain further comprises a C-terminal peptide framework sequence selected from: a)d 36 dpsqsanllaeakklndaqapk 58 (SEQ ID NO:135); and b) a sequence having 70% or more (eg, 75% or more, 80% or more, 85% or more, or 90% or more) identity to the sequence set forth in (a).

38. The D-peptide compound according to any one of claims 31 to 37, wherein the D-peptide Z domain further comprises an N-terminal peptide framework sequence selected from the group consisting of: a) v 1 dnkfnke 8 (SEQ ID NO:136); and b) a sequence having 60% or more (eg, 75% or more, 85% or more) identity to the sequence set forth in (a).

39. The D-peptide compound according to any one of claims 31 to 38, comprising: (a) a sequence selected from one of compounds 978333 to 978337 (SEQ ID NOs: 114-118), 980181 (SEQ ID NO: 119), 980174 to 980180 (SEQ ID NOs: 120-126), and 981188 to 981190 (SEQ ID NOs: 127-129); (b) a sequence having 80% or greater sequence identity to the sequence defined in (a); or (c) a sequence having 1 to 10 amino acid substitutions relative to the sequence defined in (a), wherein the 1 to 10 amino acid substitutions are: i) analogous amino acid substitutions according to Table 6; ii) a conservative amino acid substitution according to Table 6; or iii) highly conservative amino acid substitutions according to Table 6.

40. The D-peptide compound according to claim 39, comprising the amino acid sequence of one of compounds 978333 to 978337 and 980181 (SEQ ID NOs: 114-119).

41. The D-peptide compound according to any one of claims 31 to 40, wherein the compound is dimeric.

42. The D-peptide compound according to any one of claims 31 to 40, wherein the compound further comprises a second D-peptide Z domain homologous to the first D-peptide Z domain.

43. A D-peptide compound that specifically binds to VEGF, comprising: A D-peptide GA domain comprising: a) A VEGF specificity determining motif (SDM) defined by the following amino acid residues: e 25 phvisf--h 34 -p 36 x 37 -s 39 h--G 43 ---a 47 (SEQ ID NO:149) where x 37 Selected from s, n and y; b) a VEGF SDM having 80% or more (e.g., 90% or more) identity with the SDM residues defined in (a); or c) a VEGF SDM having 1 to 3 amino acid residue substitutions relative to the SDM residues defined in (a), wherein the 1 to 3 amino acid residue substitutions are selected from: i) substitution of analogous amino acid residues according to Table 6; ii) conservative amino acid residue substitution according to Table 6; iii) substitution of highly conserved amino acid residues according to Table 6; and iv) Amino acid residue substitution according to the motif defined in Figure 26.

44. The D-peptide compound according to claim 43, wherein the VEGF SDM defined in (a) is further defined by the following residues: c 7 -----------------e 25 phvisf--h 34 -p 36 x 37 c 38 sh--G 43 ---a 47 (SEQ ID NO:150) where x 37 Selected from s and n.

45. The D-peptide compound according to claim 43 or 44, further comprising the following segments (I)-(II): x 1 x 2 x 3 qwx 6 x 7 (I) x 37 x 38 (II) in: x 1 to x 3 independently selected from any D-amino acid residues; x 6 Selected from i and v; x 37 is selected from s and n; and x 7 and x 38 are amino acid residues connected via an intradomain linker having a backbone of 3 to 7 atoms in length, such as in amino acid residue x 7 and x 38 Measured between the α-carbons.

46. ​​The D-peptide compound according to claim 45, wherein x 1 to x 3 Independently selected from f, h, i, p, r, y, n, s and v.

47. The D-peptide compound according to claim 45 or 46, wherein x 6 is v.

48. The D-peptide compound according to any one of claims 44 to 47, wherein x 37 is n.

49. The D-peptide compound according to any one of claims 44 to 48, wherein said x 7 The amino acid residue and the x 38 The amino acid residues are linked via an intra-disulfide domain linkage and are selected from: Cysteine 7 -Cysteine 38 disulfide; Homocysteine 7 -Cysteine 38 disulfide; Cysteine 7 -Homocysteine 38 disulfides; and Homocysteine 7 -Homocysteine 38 Disulfide.

50. The D-peptide compound according to claim 49, wherein x 7 and x 38 Each is cysteine, and the intradomain linker comprises c 7 Amino acid residues and c 38 Disulfide linkages between amino acid residues.

51. The D-peptide compound according to any one of claims 45 to 50, wherein the intradomain linker comprises the x 7 The side chain of the amino acid residue is 38 The side chains of the amino acid residues are linked by amide bonds.

52. The D-peptide compound according to claim 51, wherein the amide bond connection is at the following x 7 and x 38 Between one of the D-amino acid residue pairs: Aspartate7 and Dap38; Aspartate 7 and Dab38; Aspartate-7 and ornithine-38; Glu7 and Dap38; Glutathione 7 and Dap38; and glutamate 7 and ornithine 38; Wherein Dap is α,β-diaminopropionic acid, and Dab is α,γ-diaminobutyric acid.

53. The D-peptide compound according to any one of claims 45 to 52, having a binding affinity (K D ) is 3-fold or more potent than a control compound lacking the intradomain linker (i.e., K D 3 times lower).

54. The D-peptide compound according to any one of claims 43 to 53, wherein the D-peptide GA domain comprises a three-helix bundle of the following formula: [Spiral 1 (#6-21) ]-[Linker 1 (#22-26) ]-[Spiral 2 (#27-35) ]-[Linker 2 (#36-37) ]-[Spiral 3 (#38-51) ] in: # represents the reference position of the amino acid residues contained in the D-peptide GA domain; and Spiral 1 (#6-21) Comprising a peptide framework sequence selected from the group consisting of: a)x 6 x 7 knakedaiaelkka 21 (SEQ ID NO:138) in: x 6 is selected from l, v and i; and x 7 selected from l and c; and b) a sequence that is 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more) identical to the sequence defined in (a).

55. The D-peptide compound according to claim 54, wherein the D-peptide GA domain comprises an N-terminal peptide framework sequence selected from the group consisting of: a)x 1 x 2 x 3 qwx 6 x 7 knakedaiaelkkaGit 24 (SEQ ID NO:139) in: x 1 Selected from t, y, f, i, p and r; x 2 Selected from i, h, n, p and s; x 3 Selected from d, i and v; x 6 is selected from l, v and i; and x 7 selected from l and c; and b) a sequence that is 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more) identical to the sequence defined in (a).

56. The D-peptide compound according to any one of claims 43 to 55, wherein the D-peptide GA domain further comprises a C-terminal peptide framework sequence selected from: a) ilkaha (SEQ ID NO: 140); and b) a sequence that is 50% or more (eg, 65% or more, or 80% or more) identical to the sequence defined in (a).

57. The D-peptide compound according to claim 56, wherein the D-peptide GA domain comprises the following sequence: x 1 x 2 x 3 qwx 6 x 7 knakedaiaelkkagitephvisfinhapx 37 x 38 shvnGlknailkaha 53 (SEQ ID NO:141) in: x 1 Selected from t, y, f, i, p and r; x 2 Selected from i, h, n, p and s; x 3 Selected from d, i and v; x 6 Selected from l, v and i; x 7 Selected from l and c; x 37 Selected from t, y, n and s; x 38 Selected from v and c; x 39 Selected from e and s; x 40 Selected from h and e; x 43 is selected from g and a; and x 47 Selected from a and e.

58. The D-peptide compound according to any one of claims 43 to 57, comprising: (a) a sequence selected from one of compounds 11055, 979102, and 979107-979110 (SEQ ID NOs: 108-113); b) a sequence that is 80% or more (eg, 90% or more) identical to the sequence defined in (a); or c) a sequence having 1 to 10 amino acid residue substitutions relative to the sequence defined in (a), wherein the 1 to 10 amino acid residue substitutions are selected from: i) substitution of analogous amino acid residues according to Table 6; ii) conservative amino acid residue substitutions according to Table 6; and iii) Substitution of highly conserved amino acid residues according to Table 6.

59. The D-peptide compound according to claim 58, comprising one of compounds 11055, 979102, and 979107-979110 (SEQ ID NOs: 108-113).

60. The D-peptide compound according to any one of claims 43 to 59, further comprising a second D-peptide GA domain homologous to the first D-peptide GA domain.

61. The D-peptide compound according to any one of claims 43 to 59, wherein the compound is dimeric.

62. A pharmaceutical composition comprising: A D-peptide compound according to any one of clauses 1 to 61, or a pharmaceutically acceptable salt thereof; and Pharmaceutically acceptable excipients.

63. The pharmaceutical composition of claim 62, wherein the composition is formulated for treating an ocular disease or condition.

64. A method of treating or preventing a disease or condition associated with angiogenesis in a subject, the method comprising administering to a subject in need thereof an effective amount of a D-peptide compound that specifically binds to VEGF according to any one of clauses 1 to 60, or a pharmaceutically acceptable salt thereof.

65. The method of claim 64, wherein the disease or condition associated with angiogenesis is cancer (e.g., breast cancer, skin cancer, colorectal cancer, pancreatic cancer, prostate cancer, lung cancer, or ovarian cancer), inflammatory disease, atherosclerosis, rheumatoid arthritis, macular degeneration, retinopathy, and skin disease (e.g., rosacea).

66. The method of claim 64, wherein the disease or condition associated with angiogenesis is diabetic macular edema (DME).

67. The method of claim 64, wherein the disease or condition associated with angiogenesis is wet age-related macular degeneration (AMD).

68. The method of any one of claims 64 to 67, further comprising administering to the subject an effective amount of a second active agent.

69. The method of claim 68, wherein the second active agent is a D-peptide compound.

70. The method of claim 68, wherein the second active agent is selected from the group consisting of a small molecule, a chemotherapeutic agent, an antibody, an antibody fragment, an aptamer, and an L-protein.

71. The method of any one of claims 68 to 70, wherein the second active agent specifically binds to a target protein selected from the group consisting of platelet-derived growth factor (PDGF), VEGF-B, VEGF-C, VEGF-D, EGF, EGFR, Her2, Her3, PD-1, PD-L1, CTLA4, OX-40, DR3, LAG3, Ang2, IL-1, IL-6, FcRn, CD3, BCMA, and IL-17.

72. The method of claim 71, wherein the second active agent is selected from the group consisting of pegpleranib (Fovista), ranibizumab (Lucentis), trastuzumab (Herceptin), bevacizumab (Avastin), aflibercept (Eylea), nivolumab (Opdivo), atezolizumab, durvalumab, gefitinib, erlotinib, and pembrolizumab (Keytruda).

73. The method of claim 68, wherein the second active agent is a D-peptide compound that is an antagonist of PD-1.

74. A method for in vivo diagnosis or imaging of a disease or condition associated with angiogenesis, comprising: administering to a subject a D-peptide compound that specifically binds to VEGF according to any one of claims 1 to 60; and At least a portion of the subject is imaged.

75. The method of claim 74, wherein the imaging comprises PET imaging and the administering comprises administering the compound to the vasculature of the subject.

76. The method of claim 74, further comprising detecting uptake of the compound by a cellular receptor.

77. The method of claim 74, further comprising administering bevacizumab to the subject, wherein the disease or condition associated with angiogenesis is cancer.

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