Patent Application
20210061861
Vascular endothelial cell growth factor (VEGF-A), is a key regulator of both normal and abnormal or pathological angiogenesis. In addition to being an angiogenic factor in angiogenesis and vasculogenesis, VEGF is a pleiotropic growth factor that exhibits multiple biological effects in other physiological processes, such as endothelial cell survival, vessel permeability and vasodilation, monocyte chemotaxis and calcium influx. Angiogenesis is an important cellular event in which vascular endothelial cells proliferate to form new vessels from an existing vascular network. Angiogenesis is implicated in the pathogenesis of a variety of disorders, such as tumors, proliferative retinopathies, age-related macular degeneration (AMD), rheumatoid arthritis (RA), and psoriasis. Angiogenesis is essential for the growth of most primary tumors and their subsequent metastasis in a variety of cancers.
The concentration of VEGF-A in eye fluids is correlated to the presence of active proliferation of blood vessels in patients with diabetic and other ischemia-related retinopathies. Furthermore, VEGF is localized in choroidal neovascular membranes in patients affected by AMD. Wet AMD is preceded by dry AMD, a condition characterized by the development of yellow-white deposits under the retina, along with variable thinning and dysfunction of the retinal tissue, although lacking any abnormal new blood vessel growth. Dry AMD converts to wet AMD when new and abnormal blood vessels invade the retina. This abnormal new blood vessel growth is called choroidal neovascularization (CNV). Anti-VEGF-A drugs find use in the treatment of wet AMD.
VEGF-A targeted therapies find use in the treatment of a variety of cancers. However, in some cases, patients eventually develop resistance to such therapy. Combination therapies that target VEGF-A and one more additional cancer targets are currently of interest, e.g., Programmed cell death protein 1 (PD-1) or Programmed death-ligand 1 (PD-L1). For example, a combination therapy targeting VEGF-A and PD-L1 using bevacizumab and atezolizumab showed a reduced risk of disease progression or death in patients with PD-L1 positive metastatic renal cell carcinoma.
The ability to manipulate the interactions of proteins such as VEGF-A is of interest for both basic biological research and for the development of therapeutics and diagnostics. Protein ligands can form large binding surfaces with multiple contacts to a target molecule that leads to binding events with high specificity and affinity. For example, antibodies are a class of protein that has yielded specific and tight binding ligands for various target proteins. In addition, 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 a D-protein antagonist of VEGF-A. Because of the diversity of target molecules of interest and the binding properties of protein ligands, the preparation of binding proteins with useful functions is of interest.
D-peptidic compounds that specifically bind to vascular endothelial cell growth factor (VEGF) are provided. The subject compounds can include a VEGF-A binding GA domain. The subject compounds can include a VEGF-A binding Z domain motif. Also provided are multivalent compounds that include two or more of the subject D-peptidic domains connected via linking components. The multivalent (e.g., bivalent, trivalent, tetravalent, etc.) D-peptidic compounds can include multiple distinct domains that specifically bind to different binding sites on a target protein to provide for high affinity binding to, and potent activity against, the VEGF target protein. D-peptidic GA and Z domains that find use in the multivalent compounds are also provided, which polypeptides have specificity-determining motifs (SDM) for specific binding to VEGF (e.g., VEGF-A). Since the target protein is homodimeric (e.g., VEGF-A), the D-peptidic compounds may be similarly dimeric, and include a dimer of multivalent (e.g., bivalent) D-peptidic compounds. The subject D-peptidic compounds find use in a variety of applications in which specific binding to VEGF-A target is desired. Methods for 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 a subject for age-related macular degeneration (AMD) or cancer.
As summarized above, aspects of this disclosure include multivalent D-peptidic compounds that specifically bind with high affinity to VEGF. This disclosure provides a class of multivalent compounds that is 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 interactions between a compound and a target protein that can occur at two or more separate and distinct sites of a target protein molecule. The multivalent D-peptidic compounds are capable of multiple binding interactions that can occur cooperatively to provide for high affinity binders to target proteins and potent biological effects on the function of the target protein. The term “multimeric” refers to a compound that includes two (i.e., dimeric), three (i.e., trimeric) or more monomeric peptidic units (e.g., domains) When the multimeric compound is homologous each peptidic unit can have the same binding property, i.e. each monomeric unit is capable of binding to the same binding site(s) on a VEGF target protein molecule. Such multimeric compounds can find use in binding target proteins that occur naturally as homodimers or are capable of multimerization. A dimeric compound can bind simultaneously to the two identical binding sites on the two molecules of the VEGF target protein homodimer. In some instances, depending on the target protein, the multivalent D-peptidic compounds of this disclosure can be multimerized, e.g., a dimeric bivalent D-peptidic compound can include a dimer of two bivalent D-peptidic compounds. In certain cases, the multimeric compound is heterologous and each peptidic unit (e.g., domain or bivalent unit) specifically binds a different target site or protein.
The multivalent peptidic compound includes at least two peptidic domains where each domain has a specificity determining motif composed of variant amino acids configured to provide a interface of specific protein-protein interactions at a binding site. When multiple peptidic domains are linked together they can simultaneously contact the target protein and provide multiple interfaces at multiple binding sites. The multiple protein-protein binding interactions can occur cooperatively via an avidity effect to provide for significantly higher effective affinities than is possible to achieve for any one D-peptidic domain alone. The present disclosure discloses use of mirror image phage display screening using scaffolded small protein domain libraries to produce multiple peptidic domains binding multiple target binding sites, and that such domains can be successfully linked to produce high affinity binders exhibiting a strong avidity effect. The multimeric compounds demonstrated by the inventors have affinity comparable to or better than corresponding antibody agents and provide for effective biological activity against VEGF target protein in vivo.
In general, the VEGF target protein is a naturally occurring L-protein and the compound is a D-peptidic compound. It is understood that for any of the D-peptidic compounds described herein, a L-peptidic version of the compound is also included in the present disclosure, which specifically binds to a D-VEGF target protein. The subject peptidic compounds were identified in part by using methods of mirror image screening of a variety of scaffolded domain phage display libraries for binding to a synthetic D-VEGF target protein.
D-peptidic compounds can provide a number of desirable properties for therapeutic applications in comparison to a corresponding L-polypeptide, such as proteolytic stability, substantially reduced immunogenicity and long in vivo half life. The D-peptidic compounds of this disclosure are generally significantly smaller in size by comparison to an antibody agent for VEGF. In some cases, the smaller size and properties of the subject compounds provide for routes of administration, tissue distribution and tissue penetration, and dosage regimens that are superior to antibody-based therapeutics.
This disclosure provides a multivalent D-peptidic compound including at least first and second D-peptidic domains. The first and second D-peptidic domains can specifically bind to distinct non-overlapping binding sites of the target protein and can be linked to each other via a linking component (e.g., as described herein). The linking component can be configured to allow for simultaneous or sequential binding to the target protein. By “sequential binding” it is meant that binding of the first D-peptidic domain to the target can increases the likelihood binding by the second D-peptidic domain will occur, even if binding does not occur simultaneously.
The first and second D-peptidic domains can be heterologous to each other, i.e., the domains are of different domain types. For example, the first D-peptidic domain may be a variant GA domain and the second D-peptidic domain may 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 that are directed towards two different binding sites on the VEGF.
When the multivalent D-peptidic compound includes only two such domains it can be termed bivalent. Trivalent, tetravalent and higher multivalencies are also possible. Trivalent D-peptidic compounds can include three D-peptidic domains connected via two linking components in a linear fashion, or via a single trivalent linking component. Trivalent D-peptidic compounds can include two of the same D-peptidic compounds connected via a disulfide linkage between two cysteine residues on each D-peptidic compound and a linking component between one of the disulfide linked D-peptidic compounds and a third D-peptidic compound. Tetravalent and higher multivalent compounds can similarly be linked, either in a linear fashion via bivalent linking components, or in a branched configuration via one or more multivalent or branched linking components.
The term “linking component” is meant to cover multivalent moieties capable of establishing covalent links between two or more D-peptidic domains of the subject compounds. Sometimes, the linking component is bivalent. Alternatively, the linking component is trivalent or dendritic. A linking component may be installed during synthesis of D-peptidic domain polypeptides, or post-synthesis, e.g., via conjugation of two or more D-peptidic domains that are already folded. A linking component may be installed in a subject compound via conjugation of two D-peptidic domains using a bifunctional linker. A linking component may also be designed such that it may be incorporated during synthesis of the D-peptidic domain polypeptides, e.g., where the linking component is itself peptidic and is prepared via solid phase peptide synthesis (SPPS) of a sequence of amino acid residues. In addition, chemoselective functional groups and/or linkers may be installed during polypeptide synthesis to provide for facile conjugation of a D-peptidic domain after SPPS.
Any convenient linking groups or linkers can be adapted for use as a linking component in the subject multivalent compounds. Linking groups and linker units of interest include, but are not limited to, amino acid residue(s), polypeptide, PEG units, (PEG)n linker (e.g., where n is 2-50, such as 2-40, 2-30, 2-20 or 2-10), terminal-modified PEG (e.g., —NH(CH2)mO[(CH2)2O](CH2)pCO—, or —NH(CH2)mO.[(CH2)2O]n(CH2)mNH—, or —CO(CH2)pO[(CH2)2O]n(CH2)pCO— linking groups where m is 2-6, p is 1-6 and n is 1-50, such as 1-20, 1-12 or 1-6), C1-C6alkyl or substituted C1-C6alkyl linkers, C2-C12alkyl or substituted C2-C12alkyl linkers, succinyl (e.g., —COCH2CH2CO—) units, diaminoethylene units (e.g., —NRCH2CH2NR— wherein R is H, alkyl or substituted alkyl), —CO(CH2)mCO—, —NR(CH2)pNR—, —CO(CH2)mNR—, —CO(CH2)mO—, —CO(CH2)mS— (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, e.g., connected via linking functional groups such as amide (e.g., —CONH— or —CONR— where R is C1-C6alkyl), sulfonamide, carbamate, carbonyl (—CO—), ether, thioether, ester, thioester, amino (—NH—) and the like. The linking component can be peptidic, e.g., a linker including a sequence of amino acid residues. The linking component can be a linker of formula -(L1)a-(L2)b-(L3)c-(L4)d-(L5)e-, where L1 to L5 are each 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. Other linkers are also possible, as shown in the multimeric compounds described herein.
The linking component can include a terminal-modified PEG linker that is connected to the D-peptidic compounds using any convenient linking chemistry. PEG is polyethylene glycol. The term “terminal-modified PEG” refers to polyethylene glycol of any convenient length where one or both of the terminals are modified to include a chemoselective functional group suitable for conjugation, e.g., to another linking group moiety or to the terminal or sidechain of a peptidic compound. The Examples section describes use of several exemplary terminal-modified PEG bifunctional linkers having terminal maleimide functional groups for conjugating chemoselectively to a thiol group, such as a cysteine residue installed in the sequence of a D-peptidic domain. The D-peptidic compounds can be modified at the N- and/or C-terminals of the GA domain motifs to include one or more additional amino acid residues that can provide for a particular linkage or linking chemistry to connect to a multivalent linking group group, such as a cysteine or a lysine.
Chemoselective reactive functional groups that may be utilized in linking the subject peptidic compounds via a linking group, include, but are not limited to: an amino group (e.g., a N-terminal amino or a lysine sidechain group), an azido group, an alkynyl group, a phosphine group, a thiol (e.g., a cysteine residue), a C-terminal thioester, aryl azides, maleimides, carbodiimides, N-hydroxysuccinimide (NHS)-esters, hydrazides, PFP-esters, hydroxymethyl phosphines, psoralens, imidoesters, pyridyl disulfides, isocyanates, aminooxy-, aldehyde, keto, chloroacetyl, bromoacetyl, and vinyl sulfones.
Any convenient multivalent linker may be utilized in the subject multimers. By multivalent is meant that the linker includes two or more terminal or sidechain groups suitable for attachment to components of the subject compounds, e.g., peptidic domains, as described herein. In some cases, the multivalent linker is bivalent or trivalent. In some instances, the multivalent linker is a dendrimer scaffold. Any convenient dendrimer scaffold may be adapted for use in the subject multimers. The dendrimer scaffold is a branched molecule that includes at least one branching point and two or more terminals suitable for connecting to the N-terminal or C-terminal of a domain via optional linkers. The dendrimer scaffold may be selected to provide a desired spatial arrangement of two or more domains. In some cases, the spatial arrangement of the two or more domains is selected to provide for a desired binding affinity and avidity for the VEGF target protein.
In some cases, the multivalent linker group is derived from/includes a chemoselective reactive functional group that is capable of conjugating to a compatible function group on a second peptidic domain. In certain cases, the multivalent linker group is a specific binding moiety (e.g., biotin or a peptide tag) that is capable of specifically binding to a multivalent binding moiety (e.g., a streptavidin or an antibody). In certain cases, the multivalent linker group is a specific binding moiety that is capable of forming a homodimer or a heterodimer directly with a second specific binding moiety of a second compound. As such, in some cases, where the compound includes a molecule of interest that includes a multivalent linker group, the compound may be part of a multimer. Alternatively, the compound may be a monomer that is capable of being multimerized either directly with one or more other compounds, or indirectly via binding to a multivalent binding moiety.
This disclosure provides multivalent compounds that bind VEGF-A. The multivalent VEGF-A binding compound can be bivalent and include two distinct variant domains connected via a linking component (e.g., as described herein). Exemplary single D-peptidic domains that specifically bind VEGF-A are disclosed herein that bind to one of two different binding sites on the target protein.
D-peptidic variant GA domain polypeptides which can be linked to a D-peptidic variant Z domain polypeptide in order 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).
D-peptidic variant Z domain polypeptides which can be linked to a D-peptidic variant GA domain polypeptide in order 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).
In
In some cases, the N-terminal to N-terminal linker can be installed by extending the polypeptide sequence of the domains to incorporate a cysteine residues that provide for conjugation to a maleimide comprising homobifunctional PEG linker. For example, both compounds 11055 and 978336 were chemically synthesized with additional N-terminal cysteine residues, which were conjugated with a bis-maleimide PEG8 linker using conventional methods to provide for an N-terminal to N-terminal linkage (
where 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. In some cases of L1, n+m is 2-6, such as 3, 4 or 5. In some cases of L1, n and m are each 2. In some cases of L1, n and m are each 3. In some cases of L1, p, q and r are each 1. In some cases of L1, p is 0. In some cases of L1, q is 0. In some cases of L1, r is 0. In some cases of L1, s is 2. In some cases of L1, s is 3.
Aspects of this disclosure include multimeric (e.g., dimeric, trimeric or tetrameric, etc) D-peptidic compounds that include any two or more of the subject variant domain polypeptides and/or bivalent compounds described herein. A multimer of the present disclosure can refer to a compound having two or more homologous domains or two or more homologous bivalent compounds. As such, a dimer of a bivalent compound can include two molecules of any one of the bivalent compounds described herein, connected via a linking component. The target molecule VEGF-A can be a homodimer, and a homologous dimeric compound can provide for binding to analogous sites on each VEGF-A target monomer. For example,
Any convenient peptidic domains can be utilized in the subject compounds. A variety of small protein domains are utilized in phage display screening that can be adapted for use in methods of mirror image screening against target proteins as described herein. A small peptidic domain of interest can consist of a single chain polypeptide sequence 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. The peptidic domain can have a molecular weight (MW) of 1 to 20 kilodaltons (kDa), such as 2 to 15 kDa, 2 to 10 kDa, 2 to 8 kDa, 3 to 8 kDa or 4 to 6 kDa.
The peptidic domain can be a three helix bundle domain. A three helix bundle domain has a structure consisting of two parallel helices and one anti-parallel helix joined by loop regions. Three helix bundle domains of interest include, but are not limited to, GA domains, Z domains and albumin-binding domains (ABD) domains.
Based on the present disclosure, it is understood that several of the amino acid residues of the peptidic domain motif which are not located at the target binding surface of the structure can be modified without having a significant detrimental effect on three dimensional structure or the target binding activity of the resulting modified compound. As such, several amino acids modifications/mutations can be incorporated into the subject compounds as needed in order to impart a desirable property on the compound, including but not limited to, increased water solubility, ease of chemical synthesis, cost of synthesis, conjugation site, interhelix linkage site, stability, isoelectric point (pI), aggregation resistance and/or reduced non-specific binding. The positions of the mutations may be selected so as to avoid or minimize any disruption to the specificity determining motif (SDM) or the underlying three dimensional structure of the target binding domain motif that provides for specific binding to the target protein. For example, mutation of solvent exposed positions on the opposite side of the domain structure from the binding surface can be made to introduce desirable variant amino acid residues, e.g., to increase solubility or provide a desirable protein pI, or incorporate a conjugation or linkage site. In some cases, based on the three dimensional structure of the target binding domain motif, the positions of mutations can be selected to provide for increased stability (e.g., via introduction of variant amino acid(s) into the core packing residues of the structure) or increased binding affinity (e.g., via introduction of variant amino acid(s) in the SDM). In some instances, the compound includes two or more, such as 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.
This disclosure provides D-peptidic Z domains that specifically bind 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 a Z domain. It is understood that a variety of underlying Z domain scaffolds or peptidic framework sequences can be utilized to provide the characteristic three dimensional structure of the Z domain.
The term “Z domain” refers to a peptidic domain having a three-helix bundle tertiary structure that is related to the immunoglobulin G binding domain of protein A. In the Protein Data Bank (PDB), structure 2spz provides an exemplary Z domain structure. See also,
[Helix 3]-[Linker 1]-[Helix 2]-[Linker 2]-[Helix 1]
wherein [Linker 1] and [Linker 2] are independently peptidic linking sequences of between 1 and 10 residues and [Helix 1], [Helix 2] and [Helix 3] are as described above for the GA domain.
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, U.S. Pat. No. 9,469,670 and a 33-residue minimized Z-domain of protein A described by Tjhung et al. (Front. Microbiol., 28 Apr. 2015), the disclosures of which are herein incorporated by reference in their entirety.
For purposes of describing some exemplary VEGF-A specific Z domains of this disclosure, a numbered 57 residue scaffold sequence of
D-peptidic Z domain compounds can specifically bind VEGF-A at a binding site defined by the amino acid sidechains E90, F62, D67, I69, E70, K110, P111, H112 and Q113 of VEGF.
Exemplary VEGF-A binding D-peptidic Z domains include those described in Table 4 and by the sequences of 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 structures and sequence variants described in the present disclosure, it is understood that a number of amino acid substitutions may be made to the sequences of the exemplary compounds while retaining specific binding to VEGF-A. By selecting positions of the variant Z domain where variability is tolerated without adversely affecting the three dimensional architecture of the Z domain, a number of amino acid substitutions may be incorporated.
As such, this disclosure includes a sequence 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 substitutions for amino acids based on physical properties of the amino acid sidechains, e.g., according to Table 6. Sometimes, an amino acid of a sequence 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 for a conservative amino acid substitution or a highly conservative amino acid substitution according to Table 6.
This disclosure includes VEGF-A binding D-peptidic Z domains described by a sequence having 80% or more sequence identity with a sequence of 978333 to 978337 and 980181 (SEQ ID NOs: 114-119), 980174-980180, and 981188-981190 (SEQ ID NOs: 120-129) such as 85% or more, 87% or more, 89% or more, 91% or more, 93% or more, 94% or more, 96% or more, 98% or more sequence identity.
The VEGF-A binding D-peptidic Z domains can have amino acid residues at positions 9, 10, 13, 14, 17, 24, 27, 28, 32 and 35 of a Z domain scaffold that are defined by the specificity-determining motif (SDM) depicted in
wherein: x14, x24, x28 and x32 are each independently any amino acid residue. In certain cases of the SDM: x14 is selected from l, r and t; x24 is selected from h, i. l, r and v; x28 is selected from G, r and v; and x32 is selected from a, r, h, s and t. In certain cases, the specificity-determining motifs (SDM) is:
In some embodiments, the D-peptidic compound that specifically binds VEGF comprises a D-peptidic Z domain comprising a VEGF specificity-determining motif (SDM) defined by the following amino acid residues:
wherein:
In some embodiments of the VEGF SDM, x14 is 1. In some embodiments of the VEGF SDM, x14 is r. In some embodiments of the VEGF SDM, x14 is t.
In some embodiments of the VEGF SDM, x24 is h. In some embodiments of the VEGF SDM, x24 is i. In some embodiments of the VEGF SDM, x24 is 1. In some embodiments of the VEGF SDM, x24 is r. In some embodiments of the VEGF SDM, x24 is v.
In some embodiments of the VEGF SDM, x28 is G. In some embodiments of the VEGF SDM, x28 is r. In some embodiments of the VEGF SDM, x28 is v.
In some embodiments of the VEGF SDM, x32 is a. In some embodiments of the VEGF SDM, x32 is r. In some embodiments of the VEGF SDM, x32 is h. In some embodiments of the VEGF SDM, x32 is s. In some embodiments of the VEGF SDM, x32 is t.
In some embodiments of the VEGF SDM, x35 is k. In some embodiments of the VEGF SDM, x35 is y.
In some embodiments, the VEGF SDM is defined by the following residues:
In some embodiments of the GA domain, the SDM residues are comprised in a peptidic framework sequence comprising peptidic framework residues defined by the following amino acid residues: --n11a--e15i-h18lpnln-e25q--a29fi-s33l-.
In some embodiments, the GA domain comprises a SDM-containing sequence having 80% or more (e.g., 85% or more, 90% or more, or 95% or more) identity to the amino acid sequence:
wherein:
x14 is selected from l, r and t;
x24 is selected from h, i, l, r and v;
x28 is selected from G, r and v;
x32 is selected from a, r, h, s and t; and
x35 is selected from k or y.
In some embodiments of the SDM-containing sequence, x14 is 1. In some embodiments of the SDM-containing sequence, x14 is r. In some embodiments of the SDM-containing sequence, x14 is t.
In some embodiments of the SDM-containing sequence, x24 is h. In some embodiments of the SDM-containing sequence, x24 is i. In some embodiments of the SDM-containing sequence, x24 is 1. In some embodiments of the SDM-containing sequence, x24 is r. In some embodiments of the SDM-containing sequence, x24 is v.
In some embodiments of the SDM-containing sequence, x28 is G. In some embodiments of the SDM-containing sequence, x28 is r. In some embodiments of the SDM-containing sequence, x28 is v.
In some embodiments of the SDM-containing sequence, x32 is a. In some embodiments of the SDM-containing sequence, x32 is r. In some embodiments of the SDM-containing sequence, x32 is h. In some embodiments of the SDM-containing sequence, x32 is s. In some embodiments of the SDM-containing sequence, x32 is t. p In some embodiments of the SDM-containing sequence, x35 is k. In some embodiments of the SDM-containing sequence, x35 is y.
In some embodiments of the compound, Helix 3(#41-54) of the Z domain comprises a peptidic framework sequence s41 anllaeakklnda54 (SEQ ID NO: 134).
In some embodiments the D-peptidic Z domain comprises a C-terminal peptidic framework sequence: d36dpsqsanllaeakklndaqapl58 (SEQ ID NO: 135).
In some embodiments the D-peptidic Z domain comprises a N-terminal peptidic framework sequence: v1dnkfnke8 (SEQ ID NO: 136).
VEGF-binding GA domain
The term “GA domain” and “GA domain motif” refer to a peptidic domain having a three-helix bundle tertiary structure that is related to the albumin binding domain of protein G. In the Protein Data Bank (PDB) structure 1tf0 provides an exemplary GA domain structure.
A GA domain can be described by the structural formula:
[Helix 1]-[Linker 1]-[Helix 2]-[Linker 2]-[Helix 3]
where [Helix 1], [Helix 2] and [Helix 3] are helical regions of a characteristic three-helix bundle linked via peptidic linkers [Linker 1] and [Linker 2]. In the three-helix bundle, [Helix 1], [Helix 2] and [Helix 3] are linked peptidic regions wherein [Helix 2] is configured substantially anti-parallel to a two-helix complex of parallel alpha helices [Helix 1] and [Helix 3]. [Linker 1] and [Linker 3] can each independently include a sequence of 1 to 10 amino acid residues. In some cases, [Linker 1] is longer in length than [Linker 3]. The GA domain can be a peptidic sequence of between 30 and 90 residues, such as 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 certain instances, the GA domain motif is a peptidic sequence of between 35 and 55 residues, such as between 40 and 55 residues, or between 45 and 55 residues. In certain embodiments, the GA domain motif is a peptidic sequence of 45, 46, 47, 48, 49, 50, 51, 52 or 53 residues.
In some embodiments, the D-peptidic GA domain is a three-helix bundle of the structural formula:
[Helix 1(#6-21)]-[Linker 1(#22-26)]-[Helix 2(#27-35)]-[Linker 2(#36-37)]-[Helix 3(#38-51)]
wherein: # denotes reference positions of amino acid residues comprised in the D-peptidic GA domain, e.g., according to the numbering scheme shown in
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 herein incorporated by reference in its entirety, and which includes a GA domain and phage display library having a scaffold sequence (G148-GA3) with library mutations at positions 25, 27, 31, 34, 36, 37, 39, 40, 43, 44 and 47 of the scaffold. Other GA domains of interest include but are not limited to those described in U.S. Pat. Nos. 6,534,628 and 6,740,734, the disclosures of which are herein incorporated by reference in their entirety.
The variant GA domains of this disclosure can have a specificity-determining motif (SDM) that includes 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 instances, the specificity-determining motif (SDM) further includes a variant amino acid at position 28 of a GA domain.
This disclosure includes variant GA domain compounds having an interhelix linker or bridge between adjacent residues of helix 1 and helix 3. The term “locked variant GA domain” and “locked GA domain” refers to a variant GA domain that includes a structure stabilizing linker between any two helices of GA domain. Sometimes, the linked adjacent residues are located at the ends of the helices 1 and 3.
A variety of compatible natural and non-naturally occurring amino acid residues can be incorporated at positions 7 and 38 of a GA domain and which are able to be conjugated to each other to provide for the interhelix linker. Compatible residues include, but are not limited to, aspartate or glutamate linked to serine or cysteine via ester or thioester linkage, aspartate or glutamate linked to ornithine or lysine via an amide linkage. As such, the interhelix linker can include one or more groups selected from C(1-6)alkyl, substituted C(1-6)alkyl, —(CHR)n—CONH—(CHR)m—, and —(CHR)n—S—S—(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 residues can be utilized to incorporate compatible chemoselective tags at the amino acid residue sidechains of positions 7 and 38, e.g., click chemistry tags such as azide and alkyne tags, which can be conjugated to each other post polypeptide synthesis.
Incorporation of an intradomain linker can provide an increase in stability and/or binding affinity for VEGF target protein. In some cases, the binding affinity (KD) of the D-peptidic compound for VEGF is 3-fold or more stronger (i.e., a 3-fold lower KD) than a control polypeptide lacking the intradomain linker, such as 5-fold or more stronger, 10-fold or more stronger, 30 fold or more stronger, or even stronger. Exemplary locked variant GA domain compounds that specifically bind VEGF-A are described below in greater detail.
A variant GA domain polypeptide can include a N-terminal region from position 1 to about position 6 that can be considered non-overlapping with Helix 2 and Helix 3 because this region is not directly involved in contacts with the adjacent helix 2-loop-helix 3 region of the folded three helix bundle structure (see e.g.,
In some embodiments, a D-peptidic GA domain includes one or more (e.g., both) of the following segments (I)-(II):
wherein:
x1 to x3 are independently selected from any D-amino acid residue;
x6 is selected from i and v;
x37 is selected from s and n; and
x7 and x38 are amino acid residues connected via an intradomain/interhelix linker having a backbone of 3 to 7 atoms in length as measured between the alpha-carbons of amino acid residues x7 and x38. In some embodiments of formula (I), x1 to x3 are independently selected from f, h, i, p, r, y, n, s and v. In some embodiments of formula (I), x6 is v. In some embodiments of formula (II), X37 is n.
The intradomain/interhelix linker can be composed of a disulfide bridge or linkage between sidechains of the x7 and x38 amino acid residues. Any convenient natural or non-naturally occurring thiol containing amino acids can be utilized to provide the intradomain linker Amino acid residues x7 and x38 that can be connected via a disulfide linkage include: cysteine7-cysteine38 disulfide; homocysteine7-cysteine38 disulfide; cysteine7-homocysteine38 disulfide; and homocysteine7-homocysteine38 disulfide. Alternatively, the intradomain/interhelix linker can include an amide bond linkage between sidechains of the x7 and x38 amino acid residues. Any convenient natural or non-naturally occurring amine and carboxylic acid containing amino acids can be utilized to provide the intradomain linker Amino acid residues x7 and x38 that can be connected via an amide linkage include: Asp7-Dap38, Asp7-Dab38, Asp7-0rn38, Glu7-Dap38, Glu7-Dap38 and Glu7-0rn38, where Dap is α,β-diaminopropionic acid, Dab is α,γ-diaminobutyric acid and Orn is ornithine. The pairs of x7 and x38 residues can be D-amino acid residues. Any convenient chemoselective functional groups and conjugates thereof may be utilized to achieve an intradomain/interhelix linkage, including but not limited to, azide-alkyne, thiol-maleimide, thiol-haloacetyl, thiol-vinyl sulfone, ester, thioester, amide, ether and thioether.
The D-peptidic variant GA domain compound can specifically bind to VEGF-A at a binding site defined by the amino acid sidechains F43, M44, Y47, Y51, N88, D89, L92, I72, K74, M107, 1109, Q115 and I117 of VEGF-A (see
In some cases, a VEGF-A binding motif includes at least two antiparallel helical regions [Helix A] and [Helix B] that are in contact with each other and together define a VEGF-A binding face. That portion of a VEGF-A binding motif that includes the antiparallel complex of [Helix A] and [Helix B] can be referred to as a “two-helix complex” structure.
The two-helix complex can be further stabilized by any convenient methods, including but not limited to, incorporation of residues that provide for desirable helix-helix packing interactions or hydrophilicity at solvent exposed positions, incorporation of interhelix linkages, incorporation of intrahelix linkages, incorporation of a constrained turns or linker that connects the helices, and linkage to a third peptidic region capable of stabilizing contacts with both [Helix A] and [Helix B].
In some embodiments, the two-helix complex can be stabilized by a third helix (Helix C) which contacts both [Helix A] and [Helix B] at the opposite side of the VEGF-A binding face of the compound and which 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 that is a small protein tertiary structure including three substantially parallel or antiparallel alpha helices. The three helices are based on a linear sequence of linked helical regions arranged in a parallel-antiparallel-parallel configuration in the three-helix bundle structure.
DeGrado et al. (Analysis and design of three-stranded coiled coils and three-helix bundles”, Folding & Design 1998, 3: R29-R40) provides a model for the assembly of three-stranded coiled coils and three-helix bundles, the disclosure of which is herein incorporated by reference in its entirety. Three-helix bundles can be single stranded structures with loops connecting helices that have regular contacts with each other in a non-polar core. The three helices of the structure can show an approximate seven-residue repeat motif, designated by the letters in italics a-g, i.e., (abcdefg)n. The heptad designations a, c, d, e, f and g do not correspond to the single letter codes for particular amino acids, but rather to positions in the heptad sequence. Non-polar residues can occur at positions a and d of the heptad including sidechain groups packing into the center of the structure to provide hydrophobic stabilization. The non-polar a and d residues can pack into layers. In some cases, charged sidechains can occur at the interfacial e and g positions, where the non-polar portions of their sidechains can shield the hydrophobic core and the polar portions can engage in electrostatic or hydrogen bonding interactions. In some cases, solvent exposed positions b and c can be occupied by polar residues. In some instances, position f is highly solvent exposed and can be occupied by polar or charged residues.
In certain embodiments of the compounds described herein, a numbering scheme is utilized for convenience and simplicity to refer to particular positions in the structure and/or sequence of the compounds, e.g., positions at which particular variant amino acid residues of interest are incorporated into a GA scaffold domain. This numbering scheme is based on that utilized for the 53 residue GA scaffold domain depicted in
In some cases, the subject compounds include one or more variations relative to a numbered parent sequence, such as, a N-terminal truncation (e.g., from position 1), a C-terminal truncation (e.g., from position 53), a deletion (e.g., of a single residue position at any convenient location of the parent sequence), an insertion (e.g., of 1, 2, 3 or more contiguous residues between two particular numbered positions of a parent sequence). In certain cases, such variations which are incorporated into the subject compounds substantially preserve the three dimensional structure of the three-helix bundle that provides for specific binding to the target. The subject compounds can 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, e.g., as described in the following embodiments.
As described herein, the subject compounds can have a three-helix bundle structure where particular solvent exposed variant amino acids located at particular positions of [Helix 2] and [Helix 3] can form contacts with the VEGF-A. In some cases, additional contacts can occur at particular residues of [Linker 2] and/or [Linker 1].
Selected amino acid residues can be located at the VEGF-A binding surface of the subject compounds and configured to interact with VEGF-A (e.g., located at the solvent exposed c and/or g positions of the g-g face defined by Helix 2 and Helix 3).
The hydrophobic core of the subject compounds can include a and d residues of [Helix 2] which contact corresponding d and a residues of [Helix 3].
As depicted in
In certain instances, the subject compound includes a helix terminating residue that provides for an increase in the angle between Helix 2 and 3, e.g., an increase of about 5 degrees or more, such as about 10 degrees or more, or about 15 degrees or more. See e.g.,
In some embodiments, [Helix 2] comprises the heptad repeat sequence [c1d1e1g1a2b2c2d2] and [Helix 3] comprises the heptad repeat sequence [e1f1g1a2b2c2d2e2f2a3b3c3d3e3], where the individual heptad repeat residues can be numbered. In certain cases of this arrangement of [Helix 2] and [Helix 3], residues d2, a2 and d′ of [Helix 2] interact with residues a2, d2 and a3 of [Helix 3] to form a network of structure stabilizing interactions. In certain cases, residues c2, g1 and c1 of [Helix 2] and residue g1 of [Helix 3] are each independently an aromatic, heterocyclic or carbocyclic residue which are configured to contact VEGF-A.
The VEGF-A binding surface of the subject compounds can be defined by a configuration of aromatic amino acid residues located at the c and g positions of the heptad repeat model which residues are configured on the surface to interact with VEGF-A. In some cases, the VEGF-A binding surface includes 2 or more, 3 or more aromatic amino acid residues, such as 4 or more, or 5 or more aromatic amino acid residues located at the c and g positions of the heptad repeat sequences.
In some embodiments of formula (I), [Helix 2] comprises a sequence of the formula:
wherein: each “A” 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 that find use in formula (II) include, but are not limited to, h, f, y and w, and substituted versions thereof. In some instances of formula (II), the first Λ is h, for y. The second A residue can be an aromatic residue comprising an aryl, heteroaryl, substituted aryl or substituted heteroaryl ring (e.g., a reside having a sidechain of formula —CH2—Ar where Ar is aryl or substituted aryl). In some instances of formula (II), the second Λ is for y, or a substituted version thereof. The second A residue can be configured on the binding surface of the GA domain motif structure to interact with a VEGF-A protein, e.g., to project into the deep pocket on the surface of VEGF-A depicted in
In some embodiments of formula (I) and (II), [Helix 2] comprises a sequence of formula (III):
wherein:
each h* is independently histidine or an analog thereof;
f* is phenylalanine or an analog thereof;
each j is independently a hydrophobic residue; and
each x is independently an amino acid residue.
In some embodiments of formula (III), the [Helix 2] comprises a sequence of the formula: hvxxf*jxh*j. The residue f* of formula (III) can be configured on the binding surface of the GA domain motif structure to interact with a VEGF-A protein, e.g., to project into the deep pocket on the surface of VEGF-A depicted in
In certain embodiments of formulae (II) and (III), h*27, P31 and h*34 are each variant residues. In certain embodiments of formulae (II) and (III), j28 and x29 are each variant residues. In certain embodiments of formulae (II) and (III), j28, x29 and x30 are each variant residues. In some instances of formulae (II) and (III), each j is independently selected from a, i, l and v. In some instances of formula (II) and (III), the first j residue is valine. In some cases, the heptad repeat register of formulae (II) and (III) is b′a′gfedcba.
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:
wherein: each h*, f*, each j and each x are as defined above; and z26 and z36 are each independently a helix-terminating residue. It is understood that, in some cases, the helix-terminating residues are not considered to be helical residues of the structure but merely define the termination of the [Helix 2] region and the beginning of a turn or loop structure. The residue and each h* residue can be configured on the binding surface of the GA domain motif structure to make specific contact with a target VEGF-A protein, e.g., as described herein. In some embodiments of formula (IV), the [Helix 2] comprises a sequence of the formula:
The term “helix-terminating residue” refers to an amino acid residue that has a high free energy penalty for forming a helix structure relative to an analogous alanine residue. In some cases, a high free energy helix penalty is referred to as a helix propensity value and is 0.5 kcal/mol or greater as defined by the method of Pace and Scholtz where higher values indicate increased penalty (“A Helix Propensity Scale Based on Experimental Studies of Peptides and Proteins”, Biophysical Journal Volume 75 July 1998 422-427). In some cases, a helix-terminating residue is a naturally occurring residue that has a helix propensity value of 0.5 or more (kcal/mol), such as 0.55 or more, 0.60 or more, 0.65 or more or 0.70 or more. For example, proline has a helix propensity value of 3.16 kcal/mol and glycine has a helix propensity value of 1.00 kcal/mol, as shown in Table 1. The helix propensity values of non-naturally occurring helix-terminating residues may be estimated by using the value of the closest naturally occurring residue having a sidechain group that is a structural analog. In some instances of formula (IV), the helix-terminating residues z26 and z36 are independently selected from from d, n, G and p. In some instances of formula (IV), the helix-terminating residues are independently selected from d, G and p. In some instances of formula (IV), the helix-terminating residues are independently selected from G and p. In some instances of formula (IV), the helix-terminating residues z26 and z36 are each p. In some instances of formula (IV), z36 is p
In certain embodiments of formula (IV), z26 is a framework residue, e.g., a residue corresponding to a residue of a scaffold domain motif. In certain cases of formula (IV), z26 is a variant residue, e.g., a residue that differs from the corresponding residue of a scaffold domain motif such as one or more of SEQ ID NOs: 1-21. In certain instances of formula (IV), z36 is a variant residue. In certain embodiments of formula (IV), h*27, f*31 and h*34 are each variant residues. In some embodiments of formula (IV), j28 and x29 are each variant residues. In some instances of formula (IV), j28, x29 and x30 are each variant residues. In certain embodiments of formula (IV), h*27 is selected from h, y and f. In certain embodiments of formula (IV), h*34 is selected from h, y and f.
In some embodiments of the compound, [Helix 2] is defined by a sequence of the formula:
wherein: each j is independently a hydrophobic residue; and each x is an amino acid residue. In certain instances, each j is a residue independently selected from a, i, f, l and v. In certain cases, each j is a residue independently selected from a, i, l and v. In certain cases, each j is a residue independently selected from a, i and v. In certain cases of formula (V), j28 is V. In certain instances of formula (V), j29 is a, l or v. In some embodiments of formula (V), j29 is i. In some instances of formula (V), j32 is i. In certain cases of formula (V), j36 is a. In certain instances of formula (V), x30 is a polar residue. In some cases of formula (V), x33 is a polar residue. In certain embodiments of formula (V), x30 and x33 are independently selected from d, e, k, n, r, s, t and q. In certain instances of formula (V), x30 and x33 are independently selected from s and n. In certain cases of formula (V), x30 is s. In some cases of formula (V), x33 is n. In some embodiments of formula (V), the [Helix 2] comprises a sequence of the formula: p26hvjxfjxhjp37 (SEQ ID NO: 137).
In some embodiments of the compound, [Helix 2] in defined by a sequence of the formula (VI):
wherein:
Z26 is selected from d, p and G;
j29 is selected from f and i;
x30 is selected from n and s;
x33 is selected from n and s; and
z37 is selected from p and G.
In some cases of formula (VI), z26 is p. In some instances of formula (VI), j29 is i. In certain cases of formula (VI), x30 is s. In some embodiments of formula (VI), x33 is n. In some instances of formula (VI), z37 is p.
In some instances of the compound, [Helix 2] is defined by a sequence selected from:
a) phvj29x30fix33hap (VII) (SEQ ID NO: 95) wherein: j29 is selected from f and i; and x30 and x33 are independently a polar amino acid residue; and
b) an amino acid sequence which has 80% or greater identity to the sequence of formula (VII) defined in a), such as 90% or greater identity to the sequence defined in a).
In some instances of the sequence of formula (VII) defined in a), x30 and x33 are independently selected from n, s, d, e and k. In some instances of the sequence of formula (VII) defined in a), j29 is i. In some instances the sequence of formula (VII) defined in a), x30 is s or n. In some instances the sequence of formula (VII) defined in a), x33 is n. In some instances the sequence of formula (VII) defined in a), j29 is i; x30 is s or n; and x33 is n.
In some embodiments of the compound, [Helix 2] has 66% identity or greater to the sequence of SEQ ID NO: 74, such as 77% identity or greater or 88% identity or greater to the sequence of SEQ ID NO: 74.
In some embodiments of formula (I), [Helix 3] comprises a sequence of the formula:
wherein: each “A” is independently an D-aromatic amino acid; each j is independently a hydrophobic residue; each u is independently a non-polar amino acid residue; and each x is independently an amino acid residue. In some cases, the heptad repeat register of formula (VIII) is edcbag′f′e′d′. In some instances of formula (VIII), the Λ is an aromatic residue comprising a heteroaryl or substituted heteroaryl ring (e.g., an aromatic residue comprising a sidechain group capable of hydrogen bonding to the VEGF-A). In certain instances, Λ is histidine or a substituted version thereof.
In some embodiments of formulae (I) or (VIII), [Helix 3] comprises a sequence of the formula (IX):
wherein j, x, u are as defined above and h* is histidine or an analog thereof. In some cases, the heptad repeat register of formula (IX) is gfedcbag′f′e′d′c′. In some instances of formula (IX), h* is histidine. In some instances of formula (IX), h* is a histidine analog (e.g., a residue having a sidechain including an alkyl-cycloalkyl group, such as a -alkyl-cyclopentyl or alkyl-cyclohexyl, or substituted version thereof). In some instances of formula (IX), h* is a substituted histidine. In some instances of formula (XI), u43 is G. In some instances of formula (IX), u47 is a. In some instances of formula (IX), x38 is v. In some instances of formula (IX), x39 is s. 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), in is v. In some instances of formula (IX), j44 is l. In some instances of formula (IX), j48 is i. In some instances of formula (IX), x51 is a hydrophobic residue. In some instances of formula (IX), x51 is a. In some instances of formula (IX), x42 is n. In some instances of formula (IX), x45 is k or r. In some instances of formula (IX), x45 is k. In some instances of formula (IX), x46 is n. In some instances of formula (IX), x49 is l. In some instances of formula (IX), Helix 3 is capped with a C-terminal sequence of residues. In certain instances, Helix 3 of formula (IX) includes additional residues x50x51, where x is an amino acid residue. In some cases, x50 is k or r. In some instances of formula (IX), x50 is k and x51 is a. In some instances of formula (IX), x50 is e and x51 is d. In some instances of formula (IX), x50 is G and x51 is r. In certain instances, Helix 3 of formula (IX) includes a C-terminal region selected from one of SEQ ID NO: 85-87. In some cases, [Helix 3] includes the heptad repeat register of gfedcbag′f′e′d′c′b′a′. It is understood that a variety of truncations (e.g., truncations of 1, 2 or 3 residues) and extensions (e.g., extensions of 1, 2, 3 or more residues) can be utilized at the C-terminal of [Helix 3] without significantly disrupting the three helix bundle structure or the variant domain, e.g., as depicted in
In some instances of formulae (IX), [Helix 3] is defined by a sequence selected from:
a) x38x39hvx42Glx45x46aix (X) (SEQ ID NO: 97) wherein: x38 is selected from v, e, k, r; x39, x42 and x46 are independently selected from a polar amino acid residue; and x45 and x49 are independently selected from l, k, r and e; and
b) an amino acid sequence which has 75% or greater identity to the sequence of formula (X) defined in a), such as 83% identity or greater or 91% identity or greater to the sequence defined in a).
In some instances of formulae (IX), [Helix 3] is defined by a sequence selected from:
a) x38x39hvx42Glx45x46aix49x50a (XI) (SEQ ID NO: 98) wherein: x38 is selected from v, e, k, r; x39, x42, x46 and x50 are independently selected from a polar amino acid residue; and x45 and x49 are independently selected from l, k, r and e; and
b) an amino acid sequence which has 78% or greater identity to the sequence of formula (XI) defined in a), such as 85% identity or greater or 92% identity or greater to the sequence defined in a).
In some instances of formulae (X)-(XI), x39, x42, x46 and x50 are independently selected from n, s, d, e and k. In some instances of formulae (X)-(XI), x38 is V. In some instances of formulae (X)-(XI), x45 is k. In some instances of formulae (X)-(XI), x49 is l. In some instances of formulae (X)-(XI), x39 is s. In some instances of formulae (X)-(XI), x42 is n. In some instances of formulae (X)-(XI), x46 is n. In some instances of formula (XI), x50 is k.
In some embodiments of the compound, [Helix 3] has 65% identity or greater to the sequence of SEQ ID NO: 79, such as 75% identity or greater, 83% identity or greater or 91% identity or greater to the sequence of SEQ ID NO: 79. In some embodiments of the compound, [Helix 3] has 70% identity or greater to the sequence of SEQ ID NO: 82, such as 78% identity or greater, 85% identity or greater or 92% identity or greater to the sequence of SEQ ID NO: 82.
In formula (I), [Linker 2] is a peptidic linker that connects [Helix 2] and [Helix 3] and which can make optional additional contacts with the surface of VEGF-A. [Linker 2] can be any convenient length. In some cases, [Linker 2] is a shorter linker than [Linker 1]. The N-terminal residue of [Linker 2] that is adjacent to [Helix 2] can be considered to be a helix-terminating residue, e.g., as described herein. In some cases, the C-terminal residue of [Linker 2] that is adjacent to [Helix 3] can be considered to be a helix-terminating residue, e.g., as described herein. In some cases, [Linker 2] can include 4 amino acid residues or less, such as 3 or less or 2 or less. In some instances, [Linker 2] has the same number of residues as the corresponding helices-connecting loop region of a native GA scaffold domain. In certain embodiments of formula (I), [Linker 2] is zx where z is a 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 contacting 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 version thereof. In some instances of [Linker 2], x is tyrosine or an analog thereof. In certain instances, [Linker 2] includes a helix terminating proline residue that provides for a modified Helix 2 to Helix 3 interhelix angle (i.e., angle between axes of the helices), e.g., as described herein. See
A tyrosine analog can be incorporated at position 37 in Linker 2, e.g., an analog including an substituted or unsubstituted, alkyl-aryl or alkyl-heteroaryl extended sidechain group that can make closer contacts (e.g., hydrophobic contacts and/or a hydrogen bond) with adjacent residues of VEGF-A.
In some embodiments of formula (I), [Helix 2]-[Linker 2]-[Helix 3] comprises a sequence of the formula (XII) that defines a VEGF-A binding surface:
wherein:
each z is a helix-terminating residue;
y* is tyrosine or an analog thereof;
each h* is independently histidine or an analog thereof;
f* is phenylalanine or an analog thereof;
each u is independently a non-polar residue.
each j is independently a hydrophobic residue; and
each x is independently an amino acid residue.
In certain instances, Helix 3 of formula (XII) includes additional residues x50x51, where x is an amino acid residue. In some cases, x50 is k or r. In some instances of extended formula (XII), x50 is k and x51 is a. In some cases of extended formula (XII), x50 is e and x51 is d. In certain instances of formula (XII), x50 is G and x51 is r. In certain instances, Helix 3 of formula (XII) includes a C-terminal region selected from one of SEQ ID NO: 85-87. In some embodiments of extended formula (XII), x51 is framework residue. In some embodiments of extended formula (XII), x51 is a non-polar residue (u). In some embodiments of extended formula (XII), x51 is a hydrophobic residue.
In some embodiments of the compound, [Helix 2]-[Linker 2]-[Helix 3] has 70% identity or greater to the sequence of SEQ ID NO: 80, such as 75% identity or greater, 83% identity or greater, 87% identity or greater, 91% identity or greater or 95% identity or greater to the sequence of SEQ ID NO: 80. In some embodiments of the compound, [Helix 2]-[Linker 2]-[Helix 3] has 70% identity or greater to the sequence of SEQ ID NO: 83, such as 80% identity or greater, 84% identity or greater, 88% identity or greater, 92% identity or greater or 96% identity or greater to the sequence of SEQ ID NO: 83.
In certain instances of formula (I), [Linker 1] has a sequence of the formula:
wherein: x′ is a polar residue; each x is an amino acid and n is an integer from 1-6; and each z is independently a helix-terminating residue, e.g., the first z is a Helix 1-terminating resdiue and the second z is a Helix 2-terminating residue. In certain instances, 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 certain cases, n is 1, 2 or 3. In certain instances of formula (XIII), [Linker 1] has a sequence of the formula (XIV):
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 formulae (XIII) and (XIV), each z is selected from G and p. In some instances of formulae (XIII) and (XIV), n is 2.
In certain instances of formula (I), [Linker 1]-[Helix 2]-[Linker 2]-[Helix 3] comprises a sequence of the formula:
wherein:
e* is glutamic acid or an analog thereof;
each z is independently a helix-terminating residue;
y* is tyrosine or an analog thereof;
each j is independently a hydrophobic residue;
each u is independently a non-polar amino acid residue; and
each x is independently an amino acid residue.
In some instances of formulae (I), (XII) and (XV), [Helix 2] is defined by a sequence of the formula (XVI):
wherein:
z26 is selected from d, p and G;
In certain instances, j28, j32 and j35 are corresponding residues of a GA scaffold domain selected from SEQ ID NO: 1-21. In some cases, j28, j32 and j35 are independently selected from a, i, l and v.
In some instances of formulae (I), (XII), (XV) and (XVI), [Helix 2] is defined by a sequence selected from: a) phvx29x30fix33hap (XVII) (SEQ ID NO: 102) wherein: x29 is selected from f and i; and x30 and x33 are independently selected from a polar amino acid residue; and
b) an amino acid sequence which has 80% or greater identity to the sequence of formula (XVII) defined in a) (e.g., 90% or greater identity).
In some instances of formulae (XVI)-(XVII), x30 and x33 are independently selected from n, s, d, e and k. In some instances of formulae (XVI)-(XVII), x29 is i. In some instances of formulae (XVI)-(XVII), x30 is s or n. In some instances of formulae (XVI)-(XVII), x33 is n. In some instances of formulae (XVI)-(XVII), x29 is i; x30 is s or n; and x33 is n.
In some instances of formulae (I), (XII) and (XV), [Helix 3] is defined by a sequence of the formula (XVIII):
wherein:
j41, j44 and j48 and are each independently a hydrophobic residue;
each u is independently a non-polar amino acid residue; and
each x is independently an amino acid residue.
In some cases, x50 is k or r. In some instances of formula (XVIII), x50 is k and x51 is a. In some instances of formula (XVIII), x50 is e and x51 is d. In some instances of formula (XVIII), x50 is G and x51 is r. In certain instances, Helix 3 of formula (XVIII) includes a C-terminal region selected from one of SEQ ID NO: 85-87. In some embodiments of formula (XVIII), x51 is framework residue. In some embodiments of formula (XVIII), x51 is a non-polar residue (u). In some embodiments of formula (XVIII), x51 is a hydrophobic residue. In some embodiments of formula (XVIII), j41, j44 and j48 are independently selected from a, i, l and v. In some embodiments of formula (XVIII), j41, j44 and j48 are corresponding residues of a GA scaffold domain selected from SEQ ID NO: 1-21.
In some instances of formulae (I), (XII) and (XV), [Helix 3] is defined by a sequence selected from : a) x38x39hvx42Glx45x46aix49x50a (XIX) (SEQ ID NO: 104) wherein:
b) an amino acid sequence which has 80% or greater identity to the sequence of formula (XIX) defined in a) (e.g., 90% or greater identity).
In some instances of formula (XIX), x39, x42, x46 and x50 are independently selected from n, s, d, e and k. In some instances of formula (XIX), x38 is V. In some instances of formula (XIX), x45 is k. In some instances of formula (XIX), x49 is 1. In some instances of formula (XIX), x39 is s. In some instances of formula (XIX), x42 is n. In some instances of formula (XIX), x46 is n. In some instances of formula (XIX), x50 is k.
In certain cases, [Helix 1] comprises the following consensus sequence: l7..a10ke.ai.elk..21, where the residues at positions 8, 9, 13, 16, 20 and 21 are defined by any one of the corresponding residues of the sequences of the GA domains of Table 3. In certain cases, [Helix 1] comprises a sequence of 15 residues having 66% or more % identity, such as 73% or more, 80% or more, 86% or more, or 93% or more % identity, to the following sequence. l6lknakedaiaelkk20.
In some embodiments of the compound, [Linker 1]-[Helix 2]-[Linker 2]-[Helix 3] has 70% identity or greater to the sequence of SEQ ID NO: 81, such as 78% identity or greater, 82% identity or greater, 85% identity or greater, 89% identity or greater, 92% identity or greater or 96% identity or greater to 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 greater to the sequence of SEQ ID NO: 84, such as 80% identity or greater, 83% identity or greater, 86% identity or greater, 90% identity or greater, 93% identity or greater or 96% identity or greater to the sequence of SEQ ID NO: 84.
Any convenient N-terminal alpha-helical segments of GA domains of interest can be adapted for use in the subject compounds. In some cases, [Helix 1] includes a sequence of N-terminal residues from about position 6 up to about position 20.
In certain cases, [Helix 1] comprises the following consensus sequence: l7..a10ke.ai.elk..21 (SEQ ID NO: 105), where the residues at positions 8, 9, 13, 16, 20 and 21 are defined by any one of the corresponding residues of the sequences of SEQ ID NO: 2-21. In certain cases, [Helix 1] comprises a sequence of 15 residues having 66% or more % identity, such as 73% or more, 80% or more, 86% or more, or 93% or more % identity, to the following sequence l6lknakedaiaelkk20 (SEQ ID NO: 74).
Described herein are D-peptidic GA domains having VEGF specificity-determining motifs (SDM) defined by a configuration of variant amino acid residues comprised in an underlying sequence of peptidic framework residues. Based on the present disclosure, it is understood that variations of any of the SDMs and peptidic framework residues/sequences are also encompassed by the present disclosure. In some embodiments, the GA domain includes a VEGF SDM having 50% or more, 60% or more, 65% or more, 70% or more, such as 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more identity with any one of embodiments of SDM residues and/or peptidic framework residues defined herein. In some embodiments, the GA domain includes a VEGF SDM having 1 to 5, e.g., 1 to 4, or 1 to 3 amino acid residue substitutions (e.g., 1, 2, 3, 4 or 5 substitutions) relative to any one of embodiments of SDM residues and/or peptidic framework residues defined herein. In certain embodiments, the 1 to 3 amino acid residue substitutions are selected from similar, conservative or highly conserved amino acid residue substitutions according to Table 6.
In some embodiments of the D-peptidic compound that specifically binds VEGF, the D-peptidic GA domain comprises a VEGF specificity-determining motif (SDM) defined by the following amino acid residues:
wherein x37 is selected from s, n, and y. In some embodiments of the VEGF SDM, x37 is s. In some embodiments of the VEGF SDM, x37 is n. In some embodiments of the VEGF SDM, x37 is y.
In some embodiments, the VEGF SDM is further defined by the following residues:
wherein x37 is selected from s and n. In some embodiments of the VEGF SDM, x37 is s. In some embodiments of the VEGF SDM, x37 is n.
In some embodiments of the GA domain, Helix 1(#6-21) comprises a peptidic framework sequence: x6x7knakedaiaelkka20 (SEQ ID NO: 138)
wherein: x6 is selected from l, v, and i; and x7 is selected from l and c.
In some embodiments of Helix 1, x6 is l. In some embodiments of Helix 1, x6 is v. In some embodiments of Helix 1, x6 is i.
In some embodiments, the GA domain comprises an N-terminal peptidic framework sequence:
wherein:
x1 is selected from t, y, f, i, p and r;
x2 is selected from i, h, n, p, and s;
x3 is selected from d, i, and v;
x6 is selected from l, v, and i; and
x7 is selected from l and c.
In some embodiments of the peptidic framework sequence, x1 is t. In some embodiments of the peptidic framework sequence, x1 is y. In some embodiments of the peptidic framework sequence, x1 is f. In some embodiments of the peptidic framework sequence, x1 is i. In some embodiments of the peptidic framework sequence, x1 is p. In some embodiments of the peptidic framework sequence, x1 is r.
In some embodiments of the peptidic framework sequence, x2 is i. In some embodiments of the peptidic framework sequence, x2 is h. In some embodiments of the peptidic framework sequence, x2 is n. In some embodiments of the peptidic framework sequence, x2 is p. In some embodiments of the peptidic framework sequence, x2 is s.
In some embodiments of the peptidic framework sequence, x3 is d. In some embodiments of the peptidic framework sequence, x3 is i. In some embodiments of the peptidic framework sequence, x3 is v.
In some embodiments of the peptidic framework sequence, x6 is 1. In some embodiments of the peptidic framework sequence, x6 is v. In some embodiments of the peptidic framework sequence, x6 is i.
In some embodiments of the peptidic framework sequence, x7 is 1. In some embodiments of the peptidic framework sequence, x7 is c.
In some embodiments, the D-peptidic GA domain comprises a C-terminal peptidic framework sequence: ilkaha (SEQ ID NO: 140).
In some embodiments, the D-peptidic GA domain comprises a sequence:
wherein:
x1is selected from t, y, f, i, p and r;
x2 is selected from i, h, n, p, and s;
x3 is selected from d, i, and v;
x6 is selected from l, v, and i;
x7 is selected from l and c;
x37 is selected from t, y, n, and s;
x38 is selected from v and c;
x39 is selected from e and s;
x40 is selected from h and e;
x43 is selected from g and a; and
x47 selected from is a and e.
In some embodiments, x1 is t. In some embodiments, x1 is y. In some embodiments, x1 is f. In some embodiments, x1 is i. In some embodiments, x1 is p. In some embodiments, x1 is r. In some embodiments, x2 is i. In some embodiments, x2 is h. In some embodiments, x2 is n. In some embodiments, x2 is p. In some embodiments, x2 is s. In some embodiments, x3 is d. In some embodiments, x3 is i. In some embodiments, x3 is v. In some embodiments, x6 is l. In some embodiments, x6 is v. In some embodiments, x6 is i. In some embodiments, x7 is l. In some embodiments, x7 is c. In some embodiments, x37 is t. In some embodiments, x37 is y. In some embodiments, x37 is n. In some embodiments, x37 is s. In some embodiments, x38 is v. In some embodiments, x38 is c. In some embodiments, x39 is e. In some embodiments, x39 is s. In some embodiments, x40 is h. In some embodiments, x40 is e. In some embodiments, x43 is g. In some embodiments, x43 is a. In some embodiments, x47 is a. In some embodiments, x47 is e.
In some embodiments, D-peptidic compound comprises a sequence selected from one of compounds 11055, 979102 and 979107-979110 (SEQ ID NOs: 108-113).
In some embodiments, D-peptidic compound comprises a sequence having 80% or more (e.g., 90% or more) identity with one of compounds 11055, 979102 and 979107-979110 (SEQ ID NOs: 108-113).
In some embodiments, D-peptidic 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, such as 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 similar, conservative and highly conserved amino acid residue substitutions, e.g., according to Table 6.
Based on the present disclosure, it is understood that several of the amino acid residues of the GA domain motif which are not located at the VEGF-A binding surface of the structure can be modified without having a detrimental effect on the VEGF-A binding activity of the resulting modified compound. As such, any convenient amino acids can be incorporated into the subject compounds to impart a desirable property, including but not limited to, increased water solubility, ease of chemical synthesis, cost, bioconjugation site, stability, pI, aggregation, reduced non-specific binding and/or specific binding to a second target protein. The positions of the mutations may selected so as to minimize any disruption to the structure of the VEGF-A binding GA domain motif or specific binding to the target VEGF-A protein, e.g., by selecting positions on opposite sides of the structure from the VEGF-A binding surface. In some instances, the compound includes two or more, such as 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 target VEGF-A protein.
For example, in some cases, one or more of the c, f and b residues of Helix 1 and the c andfresidues of Helices 2 and 3 can be modified since those residues are not directly involved in VEGF-A binding and solvent exposed (see heptad model of
indicates data missing or illegible when filed
In some cases, the subject peptidic compounds were selected from a phage display library based on a GA scaffold domain and further developed (e.g., via additional affinity maturation and/or point mutations), to include several variant amino acids integrated with a GA scaffold domain. The variant motif comprises the variant amino acids and can define a VEGF-A binding surface of the subject compounds. SEQ ID NO: 25 shows a variant motif of exemplary compound 1.1.1(c21a). Aspects of the VEGF-A binding surface of the subject compounds are described above. It is understood that a variety of underlying GA scaffold domain sequences can be utilized in the subject compounds to provide a three-helix bundle scaffold structure in which the variant domain is incorporated. The structure of a subject compound can be defined by a combination of variant and framework domains. The sequence of a subject compound can be defined by a combination of variant and framework residues. As such, in some instances, the framework residues of a structural or sequence motif can be defined by the corresponding residues of a scaffold domain structure or sequence.
For example, a comparison of scaffold SCF32 (SEQ ID NO:2) and compound 1.1.1(c21a) (SEQ ID NO:24) gives a variant motif (SEQ ID NO:25) and a framework domain (SEQ ID NO:26). Aspects of the variant motif are described herein. It is understood that a variety of modifications can be incorporated into the framework domain without having a significant adverse effect on the three helix bundle structure or VEGF-A binding surface.
In some cases, modifications to residues that make contact with the hydrophobic core of a GA scaffold domain (e.g., a and d residues of the heptad repeat model as depicted in
In certain instances, each a and d residue of [Helix 2] is a residue capable of imparting stability on the modified three-helix bundle structure of the subject compound. In certain cases, one or more of the a and d residues of the subject compound, e.g., at positions 28, 32 and 35 of [Helix 2] provide intramolecular contacts, that define in part the hydrophobic core of the compound. In certain embodiments of [Helix 2], each a and d residue is independently a hydrophobic residue. In certain cases of [Helix 2], each a and d residue is selected from a, i, f, m, l and v. In some embodiments of [Helix 2], each a and d residue is selected from a, i, f, l and v. In certain instances of [Helix 2], each a and d residue is selected from a, i, 1 and v. In some instances of [Helix 2], the a and d residues at positions 32 and 35 are part of a scaffold domain (e.g., framework residues that have the same identity as corresponding residues of a scaffold domain motif).
In certain instances, the “d” residues of [Helix 2] and [Helix 3] that are closest to the g-g face of the structure which contacts the VEGF-A can make contact with the protein. In such cases, the VEGF-A contacting “d” residues can be revered to as boundary residues. It is understood that the
Table 3 sets forth a list of sequences of exemplary scaffold domains, exemplary compounds, and exemplary compound regions of interest. In some embodiments of formula (I)-(XIX), the residues correspond to the residues located at the same positions of one of SEQ ID NOs: 22-71 set forth in Table 3. In certain embodiments of formula (I), the compound comprises a sequence of residues having 85% or more % identity, such as 88% or more, 90% or more, 92% or more, 94% or more, 96% or more, or 98% or more % identity, to one of SEQ ID NOs: 22-71. In some cases, the sequence identity comparison is based on sequence regions having the same length, e.g., 48 residue, 49 residues, 50 residues, 51 residues, 52 residues or 53 residues in length. These subject compounds can be further mutated to incorporate residues at surface positions of the GA domain motif not involved in contacting the target VEGF-A protein. The residues can be selected to confer on the resulting modified compound a desirable property (e.g., as described herein).
Aspects of the present disclosure include compounds (e.g., as described herein), salts thereof (e.g., pharmaceutically acceptable salts), and/or solvate or hydrate forms thereof. It will be appreciated that all permutations of salts, solvates and hydrates are meant to be encompassed by the present disclosure. In some embodiments, the subject compounds are provided in the form of pharmaceutically acceptable salts. Compounds containing amine and/or nitrogen containing heteraryl groups may be basic in nature and accordingly may react with any number of inorganic and organic acids to form pharmaceutically acceptable acid addition salts. Acids commonly employed to form such salts include inorganic acids such as hydrochloric, hydrobromic, hydriodic, sulfuric and phosphoric acid, as well as organic acids such as para-toluenesulfonic, methanesulfonic, oxalic, para-bromophenylsulfonic, carbonic, succinic, citric, benzoic and acetic acid, and related inorganic and organic acids. Such pharmaceutically acceptable salts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephathalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycollate, maleate, tartrate, methanesulfonate, propanesulfonates, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, hippurate, gluconate, lactobionate, and the like 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.
The variant D-peptidic domains of the subject multivalent compounds may define a binding surface area of a suitable size for forming protein-protein interactions of high functional affinity (e.g., equilibrium dissociation constant (KD)) and 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). The variant D-peptidic domains may each include a surface area of between 600 and 1800 Å2, such as between 800 and 1600 Å2, between 1000 and 1400 Å2, between 1100 and 1300 Å2, or about 1200 Å2.
In some cases, the multivalent D-peptidic compound specifically binds a target protein with a binding affinity (KD) 10-fold or more stronger, such as 30-fold or more, 100-fold or more, 300-fold or more, 1000-fold or more, or even more, than each of the binding affinities of the first and second D-peptidic domains alone for the target protein. A peptidic compound's affinity of a target protein can be determined by any convenient methods, such as using an SPR binding assay or an ELISA binding assay (e.g., as described herein). In certain cases, the multivalent D-peptidic compound has a binding affinity (KD) for the target protein of 3 nM or less, such as 1 nM or less, 300 pM or less, 100 pM or less, and the binding affinities of the first and second D-peptidic domains alone for the target protein are each independently 100 nM or more, such as 200 nM or more, 300 nM or more, 400 nM or more, 500 nM or more, or 1 uM or more. The effective binding affinity of the multivalent D-peptidic compound as a whole may be optimized to provide for a desirable biological potency and/or other property such as in vivo half-life. By selecting individual D-peptidic domains having a particular individual affinities for their target binding site, the overall functional affinity of the multivalent D-peptidic compound can be optimized, as needed.
Potency of the compounds can be assessed using any convenient assays, such as via an ELISA assay measuring IC50 as described in the experimental section herein. In some instances, the subject multivalent compound has in vitro antagonist activity against the target protein that is at least 10-fold more potent, such as at least 30-fold, at least 100-fold, at least 300-fold, at least 1000-fold more potent, than the potency of each of the first and second D-peptidic domains alone.
In certain embodiments, the subject peptidic compounds specifically bind to VEGF-A target protein with high affinity, e.g., as determined by an SPR binding assay or an ELISA assay. The subject compounds may exhibit an affinity for VEGF-A of 1 uM or less, such as 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.
The subject D-peptidic compounds may exhibit a specificity for VEGF-A, e.g., as determined by comparing the affinity of the compound for VEGF-A protein with that for a reference protein (e.g., an albumin protein), that is 5:1 or more 10:1 or more, such as 30:1 or more, 100: 1 or more, 300:1 or more, 1000:1 or more, or even more. In some cases, specificity can be a difference in binding affinities by a factor of 103 or more, such as 104 or more, 105 or more, 106 or more, or even more. In some cases, the peptidic compounds may be optimized for any desirable property, such as protein folding, protease stability, thermostability, compatibility with a pharmaceutical formulation, etc. Any convenient methods may be used to select the D-peptidic compounds, e.g., structure-activity relationship (SAR) analysis, affinity maturation methods, or phage display methods.
Also provided are D-peptidic compounds that have high thermal stability. In some cases, the compounds having high thermal stability have a melting temperature of 50° C. or more, such as 60° C. or more, 70° C. or more, 80° C. or more, or even 90° C. or more. Also provided are D-peptidic compounds that have high protease stability. The subject D-peptidic compounds are resistant to proteases and can have long serum and/or saliva half-lives. Also provided are D-peptidic compounds that have a long in vivo half-life. As used herein, “half-life” refers to the time required for a measured parameter, such the potency, activity and effective concentration of a compound to fall to half of its original level, such as half of its original potency, activity, or effective concentration at time zero. Thus, the parameter, such as potency, activity, or effective concentration of a polypeptide molecule is generally measured over time. For purposes herein, half-life can be measured in vitro or in vivo. In some cases, the peptidic compound has a half-life of 1 hour or longer, such as 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 may be measured by any convenient method, e.g., by incubating the compound in human EDTA blood or serum for a designated 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.
Also provided are D-peptidic compounds that have low immunogenicity, e.g., are non-immunogenic. In certain embodiments, the D-peptidic compounds have low immunogenicity compared to an L-peptidic compound. In certain embodiments, the D-peptidic compounds are 10% or less, 20% or less, 30% or less, 40% or less, 50% or less, 70% or less, or 90% or less immunogenic compared to an L-peptidic compound, in an immunogenicity assay such as that described by Dintzis et al., “A Comparison of the Immunogenicity of a Pair of Enantiomeric Proteins” Proteins: Structure, Function, and Genetics 16:306-308 (1993).
Also provided are D-peptidic compounds that have been optimized for binding affinity and specificity to VEGF-A by affinity maturation, e.g., second generation D-peptidic compounds based on a parent compound that binds to VEGF-A. In some embodiments, the affinity maturation of a subject compound may include holding a fraction of the variant amino acid positions as fixed positions while the remaining variant amino acid positions are varied to select optimal amino acids at each position. A parent D-peptidic compound may be selected as a scaffold for an affinity maturation compound. In some cases, a number of affinity maturation compounds are prepared that include mutations at limited subsets of the variant amino acid positions of the parent, while the rest of the variant positions are held as fixed positions. The positions of the mutations may be tiled through the scaffold sequence to produce a series of compounds such that mutations at every variant position are represented and a diverse range of amino acids are substituted at every position (e.g., all 20 naturally occurring amino acids). Mutations that include deletion or insertion of one or more amino acids may also be included at variant positions of the affinity maturation compounds. An affinity maturation compound may be prepared and screened using any convenient method, e.g., phage display library screening, to identify second generation compounds having an improved property, e.g., increased binding affinity for a target molecule, protein folding, protease stability, thermostability, compatibility with a pharmaceutical formulation, etc.
In some embodiments, the affinity maturation of a subject compound may include holding most or all of the variant amino acid positions in the variable regions of the parent compound as fixed positions, and introducing contiguous 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 may be used to optimize the compound variants for any desirable property, such as protein folding, protease stability, thermostability, compatibility with a pharmaceutical formulation, etc.
Aspects of the present disclosure include compounds (e.g., as described herein), salts thereof (e.g., pharmaceutically acceptable salts), and/or solvate, hydrate and/or prodrug forms thereof. It will be appreciated that all permutations of salts, solvates, hydrates, and prodrugs are meant to be encompassed by the present disclosure.
In some embodiments, the subject compounds, or a prodrug form thereof, are provided in the form of pharmaceutically acceptable salts. Compounds containing an amine or nitrogen containing heteraryl group may be basic in nature and accordingly may react with any number of inorganic and organic acids to form pharmaceutically acceptable acid addition salts. Acids commonly employed to form such salts include inorganic acids such as hydrochloric, hydrobromic, hydriodic, sulfuric and phosphoric acid, as well as organic acids such as para-toluenesulfonic, methanesulfonic, oxalic, para-bromophenylsulfonic, carbonic, succinic, citric, benzoic and acetic acid, and related inorganic and organic acids. Such pharmaceutically acceptable salts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephathalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycollate, maleate, tartrate, methanesulfonate, propanesulfonates, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, hippurate, gluconate, lactobionate, and the like 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.
Any convenient D-peptidic compound (e.g., as described herein) may be multimerized, to provide a multimer of D-peptidic compounds. In certain embodiments, the multimer includes two or more D-peptidic compounds, such as 2 (e.g., a dimer), 3 (e.g., a trimer) or 4 or more compounds (e.g., a tetramer or a dendrimer, etc). In some cases, the multimer is described by the formula:
Y-(GA)n
where: Y is a multivalent linking group; n is an integer greater than one; and GA is a D-peptidic compound comprising a GA domain motif (e.g., as described herein). In certain cases, n is 2. In certain cases, n is 3.
In certain cases, the multimer is a dimer of one of the formulae:
where each GA is independently a D-peptidic compound (e.g., as described herein); and Y is a linker connected to the N-terminal (N-GA) or the C-terminal (GA-C) of the compounds. In certain cases, the dimer is a homodimer of two identical GA domain motifs that each specifically bind VEGF-A. In certain instances, the dimer is a heterodimer. The heterodimer can be a dimer of two distinct GA domain motifs that each specifically bind VEGF-A, or a dimer of a subject D-peptidic compound and a second D-peptidic binding domain.
Any convenient linking groups can be utilized in the subject multimers. The terms “linker”, “linkage” and “linking group” are used interchangeably and refer to a linking moiety that covalently connects two or more compounds. In some cases, the linker is divalent. In certain cases, the linker is a branched or trivalent linking group. In some cases, the linker has a linear or branched backbone of 200 atoms or less (such as 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) in length. A linking moiety may be a covalent bond that connects two groups or a linear or branched chain of between 1 and 200 atoms in length, for example 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, where the linker may be linear, branched, cyclic or a single atom. In certain cases, one, two, three, four or five or more carbon atoms of a linker backbone may be optionally substituted with a sulfur, nitrogen or oxygen heteroatom. In certain instances, when the linker includes a PEG group, every third atom of that segment of the linker backbone is substituted with an oxygen. The bonds between backbone atoms may be saturated or unsaturated, usually not more than one, two, or three unsaturated bonds will be present in a linker backbone. The linker may include one or more substituent groups, for example an alkyl, aryl or alkenyl group. A linker may include, without limitations, oligo(ethylene glycol), ethers, thioethers, disulfide, amides, carbonates, carbamates, tertiary amines, alkyls, which may be straight or branched, e.g., methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), and the like. The linker backbone may include a cyclic group, for example, an aryl, a heterocycle or a cycloalkyl group, where 2 or more atoms, e.g., 2, 3 or 4 atoms, of the cyclic group are included in the backbone. A linker may be cleavable or non-cleavable. A linker may be peptidic, e.g., a linking sequence of residues.
Y can include any convenient group(s) or linker units, including but not limited to, amino acid residue(s), PEG, modified PEG (e.g., —NH(CH2)mO[(CH2)2O]n(CH2)pCO— linking groups where m is 2-6, p is 1-6 and n is 1-50, such as 1-12 or 1-6), C2-C12 alkyl linkers, —CO—CH2CH2CO— units, and combinations thereof (e.g., linked via functional groups such as amide bonds, sulfonamide bonds, carbamates, ether bonds, ester bonds, or —NH—). In some instances, Y is peptidic. 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 also possible, as shown in the multimeric compounds described herein.
In some instances, Y comprises a modified PEG linker that is connected to the D-peptidic compounds using any convenient linking chemistry. PEG is a polyethylene glycol or a modified polyethylene glycol. By modified PEG is meant that a polyethylene glycol or any convenient length where one or both of the terminals are modified to include a chemoselective functional group suitable for conjugation, e.g., to another linking group moiety or to the terminal or sidechain of a peptidic compound. Table 9 and and Examples section describe several exemplary homodimers of compound 1.1.1 (c21a) connected via either the N-terminals or C-terminals of the compounds. The D-peptidic compounds can be modified at the N- and/or C-terminals of the GA domain motifs to include one or more additional amino acid residues that can provide for a particular linkage or linking chemistry to connect to the Y group, such as a cysteine or a lysine.
Chemoselective reactive functional groups that may be utilized in linking the subject peptidic compounds via a linking group, include, but are not limited to: an amino group (e.g., a N-terminal amino or a lysine sidechain group), an azido group, an alkynyl group, a phosphine group, a thiol (e.g., a cysteine residue), a C-terminal thioester, aryl azides, maleimides, carbodiimides, N-hydroxysuccinimide (NHS)-esters, hydrazides, PFP-esters, hydroxymethyl phosphines, psoralens, imidoesters, pyridyl disulfides, isocyanates, aminooxy-, aldehyde, keto, chloroacetyl, bromoacetyl, and vinyl sulfones.
Any convenient multivalent linker may be utilized in the subject multimers. By multivalent is meant that the linker includes two or more terminal groups suitable for attachment to a subject compound, e.g., as described herein. In some cases, the multivalent linker is divalent or trivalent. In some instances, the multivalent linker Y is a dendrimer scaffold. Any convenient dendrimer scaffold may be adapted for use in the subject multimers. The dendrimer scaffold is a branched molecule that includes at least one branching point and two or more terminals suitable for connecting to the N-terminal or C-terminal of a GA domain motif via optional linkers. The dendrimer scaffold may be selected to provide a desired spatial arrangement of two or more GA domain motifs. In some cases, the spatial arrangement of the two or more GA domain motifs is selected to provide for a desired binding affinity and avidity for the target protein.
In some cases, the D-peptidic compounds each independently include a specific binding moiety (e.g., a biotin or a peptide tag) where the D-peptidic compounds can be bound to each other via a multivalent binding moiety (e.g., a streptavidin, an avidin or an antibody) that specifically binds the specific binding moiety. In some embodiments, the two or more D-peptidic compounds, e.g., 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, connected via an optional linker, to either the N-terminal or C-terminal of the compound. In certain cases, the terminal biotin moiety is Biotin-(Gly)n- where n is 1 to 6 or Biotin-Ahx- (Ahx=6-aminohexanoic acid residue).
Any convenient molecules or moieties of interest may be attached to the subject D-peptidic compounds. The molecule of interest may be peptidic or non-peptidic, naturally occurring or synthetic. Molecules of interest suitable for use in conjunction with the subject compounds include, but are not limited to, an additional protein domain, a polypeptide or amino acid residue, a peptide tag, a specific binding moiety, a polymeric moiety such as a polyethylene glycol (PEG), a carbohydrate, a dextran or a polyacrylate, a linker, a half-life extending moiety, a drug, a toxin, a detectable label and a solid support. In some cases, the molecule of interest may confer on the resulting peptidic compounds enhanced and/or modified properties and functions including, but not limited to, increased water solubility, ease of chemical synthesis, cost, bioconjugation site, stability, isoelectric point (pI), aggregation, reduced non-specific binding and/or specific binding to a second target protein, e.g., as described herein.
In some embodiments of any one of the VEGF-A binding GA domain motif sequences described herein, the motif may be extended to include one or more additional residues at the N-terminal and/or C-terminal of the sequence, such as two or more, three or more, four or more, five or more, 6 or more, or even more additional residues. Such additional residues may be considered part of the GA domain motif even though they do not provide a VEGF-A binding interaction. Any convenient residues may be included at the N-terminal and/or C-terminal of the VEGF-A binding GA domain motif to provide for a desirable property or group, such as increased solubility via a water soluble group, a linkage for dimerization or multimerization, a linkage for connecting to a label or a specific binding moiety.
In some cases, the subject modified compound is described by formula:
X-L-Z
where 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, where L is attached to X at any convenient location (e.g., the N-terminal, C-terminal or via the sidechain of a surface residue not involved in binding to the target).
The D-peptidic compounds may include one or more molecules of interest, e.g., a N-terminal moiety and/or a C-terminal moiety. In some instances, the molecule of interest is covalently attached via the alpha-amino group of the N-terminal residue, or is covalently attached to the alpha-carboxyl acid group of the C-terminal residue. In other instances, an molecules of interest is attached to the motif via a sidechain group of a residue (e.g., via a c, k, d ore residue).
The molecules of interest may include a polypeptide or a protein domain. Polypeptides and protein domains of interest include, but are not limited to: gD tags, c-Myc epitopes, FLAG tags, His tags, fluorescence proteins (e.g., GFP), beta-galactosidase protein, GST, albumins, immunoglobulins, Fc domains, or similar antibody-like fragments, leucine zipper motifs, a coiled coil domain, a hydrophobic region, a hydrophilic region, a polypeptide comprising a free thiol which forms an intermolecular disulfide bond between two or more multimerization domains, a “protuberance-into-cavity” domain, beta-lactoglobulin, or fragments thereof.
The molecules of interest may include a half-life extending moiety. The term “half-life extending moiety” refers to a pharmaceutically acceptable moiety, domain, or “vehicle” covalently linked or conjugated to the subject compound, that prevents or mitigates in vivo proteolytic degradation or other activity-diminishing chemical modification of the subject compound, increases half-life or other pharmacokinetic properties (e.g., rate of absorption), reduces toxicity, improves solubility, increases biological activity and/or target selectivity of the subject compound with respect to a target of interest, increases manufacturability, and/or reduces immunogenicity of the subject compound, compared to an unconjugated form of the subject compound.
In certain embodiments, the half-life extending moiety is a polypeptide that binds a serum protein, such as an immunoglobulin (e.g., IgG) or a serum albumin (e.g., human serum albumin (HSA)). Polyethylene glycol is an example of a useful half-life extending moiety. Exemplary half-life extending moieties include a polyalkylene glycol moiety (e.g., PEG), a serum albumin or a fragment thereof, a transferrin receptor or a transferrin-binding portion thereof, and a moiety comprising a binding site for a polypeptide that enhances half-life in vivo, a copolymer of ethylene glycol, a copolymer of propylene glycol, a carboxymethylcellulose, a polyvinyl pyrrolidone, a poly-1,3-dioxolane, a poly-1,3,6-trioxane, an ethylene/maleic anhydride copolymer, a polyaminoacid (e.g., polylysine), a dextran n-vinyl pyrrolidone, a poly n-vinyl pyrrolidone, a propylene glycol homopolymer, a propylene oxide polymer, an ethylene oxide polymer, a polyoxyethylated polyol, a polyvinyl alcohol, a linear or branched glycosylated chain, a polysialic acid, a polyacetal, a long chain fatty acid, a long chain hydrophobic aliphatic group, an immunoglobulin Fc domain (see, e.g., U.S. Pat. No. 6,660,843), an albumin (e.g., human serum albumin; see, e.g., U.S. Pat. No. 6,926,898 and US 2005/0054051; U.S. Pat. No. 6,887,470), a transthyretin (TTR; see, e.g., US 2003/0195154; 2003/0191056), or a thyroxine-binding globulin (TBG).
An extended half-life can also be achieved via a controlled or sustained release dosage form of the subject compounds, e.g., as described by Gilbert S. Banker and Christopher T. Rhodes, Sustained and controlled release drug delivery system. In Modern Pharmaceutics, Fourth Edition, Revised and Expanded, Marcel Dekker, New York, 2002, 11. This can be achieved through a variety of formulations, including liposomes and drug-polymer conjugates.
In certain embodiments, the half-life extending moiety is a fatty acid. Any convenient fatty acids may be used in the subject modified compounds. See e.g., 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.
In certain embodiments, the compound is modified to include a specific binding moiety. The 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 binds to the complementary second moiety with an affinity of at least 10−7M (e.g., as measured by a KD 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). Complementary binding moiety pairs of specific binding moieties include, but are not limited to, a ligand and a receptor, an antibody and an antigen, complementary polynucleotides, complementary protein homo- or heterodimers, an aptamer and a small molecule, a polyhistidine tag and nickel, and a chemoselective reactive group (e.g., a thiol) and an electrophilic group (e.g., with which the reactive thiol group can undergo a Michael addition). The specific binding pairs may include analogs, derivatives and fragments of the original specific binding member. For example, an antibody directed to a protein antigen may also recognize peptide fragments, chemically synthesized, labeled protein, derivatized protein, etc. so long as an epitope is present. Protein domains of interest that find use as specific binding moieties include, but are not limited to, Fc domains, or similar antibody-like fragments, leucine zipper motifs, a coiled coil domain, a hydrophobic region, a hydrophilic region, a polypeptide comprising a free thiol which forms an intermolecular disulfide bond between two or more multimerization domains, or a “protuberance-into-cavity” domain (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.
In certain embodiments, the molecule of interest is a linked specific binding moiety that specifically binds a target protein. The linked specific binding moiety can be an antibody, an antibody fragment, an aptamer or a second D-peptidic binding domain. The linked specific binding moiety can specifically bind any convenient target protein, e.g., a target protein that is desirable to target in conjunction with VEGF-A in the subject methods of treatment. 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 certain instances, the linked specific binding moiety is a second D-peptidic binding domain that targets PDGF-B.
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, biocytin, etc. In some cases, the biotin moiety is capable of specifically binding with high affinity to a chromatography support that contains immobilized avidin, neutravidin or streptavidin. Biotin moieties can bind to streptavidin with an affinity of at least 10−8M. In some cases, a monomeric avidin support may be used to specifically bind a biotin-containing compound with moderate affinity thereby allowing bound compounds to be later eluted competitively from the support (e.g., with a 2 mM biotin solution) after non-biotinylated polypeptides have been washed away. In certain instances, the biotin moiety is capable of binding to an avidin, neutravidin or streptavidin in solution to form a multimeric compound, e.g., a dimeric, or tetrameric complex of D-peptidic compounds with the avidin, neutravidin or streptavidin. A biotin moiety may also include a linker, e.g., -LC-biotin, -LC-LC-Biotin, -SLC-Biotin or -PEGn-Biotin where n is 3-12 (commercially available from Pierce Biotechnology). In certain embodiments, the compound is modified to include a detectable label. Examples of detectable labels include labels that permit both the direct and indirect measurement of the presence of the subject peptidic compound. Examples of labels that permit direct measurement of the compound include radiolabels, fluorophores, dyes, beads, nanoparticles (e.g., quantum dots), chemiluminescers, colloidal particles, paramagnetic labels and the like. Radiolabels may include radioisotopes, such as 35S, 14C, 125I, 3H, 64Cu and 131I. The subject compounds can be labeled with the radioisotope using any convenient techniques, such as those described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al., Ed. Wiley-Interscience, New York, N.Y., Pubs. (1991), and radioactivity can be measured using scintillation counting or positron emission. Examples of detectable labels which permit indirect measurement of the presence of the modified compound include enzymes where a substrate may provide for a colored or fluorescent product. For example, the compound may include a covalently bound enzyme capable of providing a detectable product signal after addition of suitable substrate. Instead of covalently binding the enzyme to the compound, the compound may include a first member of specific binding pair which specifically binds with a second member of the specific binding pair that is conjugated to the enzyme, e.g. the compound may be covalently bound to biotin and the enzyme conjugate to streptavidin. Examples of suitable enzymes for use in conjugates include horseradish peroxidase, alkaline phosphatase, malate dehydrogenase and the like. Where not commercially available, such enzyme conjugates may be readily produced by any convenient techniques.
In certain embodiments, the detectable label is a fluorophore. The term “fluorophore” refers to a molecule that, when excited with light having a selected wavelength, emits light of a different wavelength, which may emit light immediately or with a delay after excitation. Fluorophores, include, without limitation, fluorescein dyes, e.g., 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, e.g. Cy3, CY5, Cy5.5, QUASAR™ dyes etc.; dansyl derivatives; rhodamine dyes e. g. 6-carboxytetramethylrhodamine (TAMRA), CAL FLUOR dyes, tetrapropano-6-carboxyrhodamine (ROX). BODIPY fluorophores, ALEXA dyes, Oregon Green, pyrene, perylene, benzopyrene, squarine dyes, coumarin dyes, luminescent transition metal and lanthanide complexes and the like. The term fluorophore includes excimers and exciplexes of such dyes.
In some embodiments, the compound includes a detectable label, such as a radiolabel. In certain embodiments, the radiolabel suitable for use in PET, SPECT and/or MR imaging. In certain embodiments, the radiolabel is a PET imaging label. In certain cases, the compound is radiolabeled with 18F, 64Cu, 68Ga, 99mTc or 86Y.
The detectable label may be attached to the peptidic compound at any convenient position and via any convenient chemistry. Methods and materials of interest include, but are not limited to those described by U.S. Pat. No. 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 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, labeling with succinimidyl [18F]fluorobenzoate (SFB) (e.g., Vaidyanathan et al., 1992, Int. J. Rad. Appl. Instrum. B 19:275), other acyl compounds (Tada et al., 1989, Labeled Compd. Radiopharm. 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, Bioconj Chem. 18:1987).
Any convenient synthetic methods or bioconjugation methods may be utilized in preparing the subject modified D-peptidic compounds. In certain cases, the detectable label is connected to the compound via an optional linker. In certain embodiments, the detectable label is connected to the N-terminal of the compound. In certain embodiments, the detectable label is connected to the C-terminal of the compound. In certain embodiments, the detectable label is connected to a non-terminal residue of the compound, e.g., via a side chain moiety. In certain embodiments, the detectable label is connected to the N-terminal peptidic extension moiety of the compound via an optional linker. In some cases, the N-terminal peptidic extension moiety is modified to include a reactive functional group which is capable of reacting with a compatible functional group of a radiolabel containing moiety. Any convenient reactive functional groups, chemistries and radiolabel containing moieties may be utilized to attach a detectable label to the compound, including but not limited to, click chemistry, an azide, an alkyne, a cyclooctyne, copper-free click chemistry, a nitrone, a chelating group (e.g., selected from DOTA, TETA, NOTA, NODA, (tert-Butyl)2NODA, NETA, C-NETA, L-NETA, S-NETA, NODA-MPAA, and NODA-MPAEM), a propargyl-glycine residue, etc.
In certain instances, the molecule of interest is a second active agent, e.g., an active agent or drug that finds use in conjunction with targeting VEGF-A in the subject methods of treatment. In certain instances, the molecule of interest is a small molecule, a chemotherapeutic, an antibody, an antibody fragment, an aptamer, or a L-protein. In some embodiments, the compound is modified to include a moiety that is useful as a pharmaceutical (e.g., a protein, nucleic acid, organic small molecule, etc.). Exemplary pharmaceutical proteins include, e.g., cytokines, antibodies, chemokines, growth factors, interleukins, cell-surface proteins, extracellular domains, cell surface receptors, cytotoxins, etc. Exemplary small molecule pharmaceuticals include small molecule toxins or therapeutic agents.
Any convenient therapeutic or diagnostic agent (e.g., as described herein) can be conjugated to a D-peptidic compound. A variety of therapeutic agents including, but not limited to, anti-cancer agents, antiproliferative agents, cytotoxic agents and chemotherapeutic agents are described below in the section entitled Combination Therapies, any one of which can be adapted for use in the subject modified compounds. 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, Ado-trastuzumab emtansine, Rituximab, Ipilimumab, Rapamycin, Temsirolimus, Everolimus, Methotrexate, Doxorubicin, Abraxane, Folfirinox, Cisplatin, Carboplatin, 5-fluorouracil, Teysumo, Paclitaxel, Prednisone, Levothyroxine, Pemetrexed, navitoclax, ABT-199. Any exemplary cytotoxic agents that find use in ADC can be adapted for use in the subject modified D-peptidic compounds. Cytotoic agents of interest include, but are not limited to, auristatins (e.g., MMAE, MMAF), maytansines, dolastatins, calicheamicins, duocarmycins, pyrrolobenzodiazepines (PBDs), 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). The cell penetrating peptide may facilitate cellular uptake of the molecule. Any convenient tag polypeptides and their respective antibodies may be used. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu 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., Molecular and Cellular Biology, 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 the Flag-peptide [Hopp et al., BioTechnology 6:1204-1210 (1988)]; the 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 the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. U.S.A. 87:6393-6397 (1990)].
In certain embodiments, the compound may include a cell penetrating peptide (e.g., tat). The cell penetrating peptide may facilitate cellular uptake of the molecule. Any convenient tag polypeptides and their respective antibodies may be used. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu 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., Molecular and Cellular Biology, 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 the Flag-peptide [Hopp et al., BioTechnology 6:1204-1210 (1988)]; the 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 the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. U.S.A. 87:6393-6397 (1990)].
The molecules of interest may be attached to the subject modified compounds via any convenient method. In some cases, a molecules of interest is attached via covalent conjugation to a terminal amino acid residue, e.g., at the amino terminal or at the carboxylic acid terminal. The molecule of interest may be attached to the peptidic GA domain motif via a single bond or a suitable linker, e.g., a PEG linker, a peptidic linker including one or more amino acids, or a saturated hydrocarbon linker. A variety of linkers (e.g., as described herein) find use in the subject modified compounds. Any convenient reagents and methods may be used to include a molecule of interest in a subject GA domain motif, for example, conjugation methods as described in G. T. Hermanson, “Bioconjugate Techniques” Academic Press, 2nd Ed., 2008, solid phase peptide synthesis methods, or fusion protein expression methods. Functional groups that may be used in covalently bonding the domain, via an optional linker, to produce the modified compound include: hydroxyl, sulfhydryl, amino, and the like. Certain moieties on the molecules of interest and/or GA domain motif may be protected using convenient blocking groups, see, e.g. Green & Wuts, Protective Groups in Organic Synthesis (John Wiley & Sons) 3rd Ed. (1999). The particular molecule of interest and site of attachment to the GA domain motif may be chosen so as not to substantially adversely interfere with the desired binding activity, e.g. for the target VEGF-A protein.
The molecule of interest may be peptidic. It is understood that a molecule of interest may further include one or more non-peptidic groups including, but not limited to, a biotin moiety and/or a linker. Any convenient protein domains may be adapted and utilized as molecules of interest in the subject modified peptidic compounds. Protein domains of interest include, but are not limited to, any convenient serum protein, serum albumin (e.g., human serum albumin; see, e.g., U.S. Pat. No. 6,926,898 and US 2005/0054051; U.S. Pat. No. 6,887,470), a transferrin receptor or a transferrin-binding portion thereof, immunoglobulin (e.g., IgG), an immunoglobulin Fc domain (see, e.g., U.S. Pat. No. 6,660,843), a transthyretin (TTR; see, e.g., US 2003/0195154; 2003/0191056), a thyroxine-binding globulin (TBG), or a fragment thereof.
A multimerizing group is any convenient group that is capable of forming a multimer (e.g., a dimer, a trimer, or a dendrimer), e.g., by mediating binding between two or more compounds (e.g., directly or indirectly via a multivalent binding moiety), or by connecting two or more compounds via a covalent linkage. In some cases, the multimerizing group Z is a chemoselective reactive functional group that conjugates to a compatible function group on a second D-peptidic 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., a streptavidin or an antibody). In some cases, the compound includes a multimerizing group and is a monomer that has not yet been multimerized.
Chemoselective reactive functional groups for inclusion in the subject peptidic compounds, include, but are not limited to: an azido group, an alkynyl group, a phosphine group, a cysteine residue, a C-terminal thioester, aryl azides, maleimides, carbodiimides, N-hydroxysuccinimide (NHS)-esters, hydrazides, PFP-esters, hydroxymethyl phosphines, psoralens, imidoesters, pyridyl disulfides, isocyanates, aminooxy-, aldehyde, keto, chloroacetyl, bromoacetyl, and vinyl sulfones.
Also provided are polynucleotides that encode a sequence corresponding to the subject peptidic compounds as described herein. The polynucleotide can encode a L-peptidic compound that specifically binds to a D-VEGF-A target protein.
In some embodiments, the polynucleotide encodes a peptidic compound that includes 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 certain instances, the polynucleotide encodes a peptidic compound sequence of between 35 and 55 residues, such as between 40 and 55 residues, or between 45 and 55 residues. In certain embodiments, the polynucleotide encodes a peptidic compound sequence of 45, 46, 47, 48, 49, 50, 51, 52 or 53 residues.
In certain embodiments, the polynucleotide is a replicable expression vector that includes a nucleic acid sequence encoding a L-peptidic compound that may be expressed in a protein expression system. In certain embodiments, the polynucleotide is a replicable expression vector that includes a nucleic acid sequence encoding a gene fusion, where the gene fusion encodes a fusion protein including the L-peptidic compound fused to all or a portion of a viral coat protein.
In certain embodiments, the subject polynucleotides are capable of being expressed and displayed in a cell-based or cell-free display system. Any convenient display methods may be used to display L-peptidic compounds encoded by the subject polynucleotides, such as cell-based display techniques and cell-free display techniques. In certain embodiments, cell-based display techniques include phage display, bacterial display, yeast display and mammalian cell display. In certain embodiments, cell-free display techniques include mRNA display and ribosome display.
The herein-described compounds may be employed in a variety of methods. One such method includes contacting a subject compound with a VEGF-A target protein under conditions suitable for binding of VEGF-A to produce a complex. In some embodiments, the method includes administering a D-peptidic compound to a subject, where the compound binds to VEGF-A in the subject.
A subject compound may inhibit at least one activity of its VEGF-A target in the range of 10% to 100%, e.g., 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, a subject compound may inhibit its VEGF-A target with an IC50 of 1×10−5 M or less (e.g., 1×10−6 M or less, 1×10−7M or less, 1×10−8M or less, 1×10−9 M or less, 1×10−10 M or less, or 1×10−11 M or less). In certain assays, a subject compound may inhibit its VEGF-A target with an IC20 of 1×10−6 M 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). In certain assays, a subject compound may inhibit its VEGF-A target with an IC10 of 1×10−6 M 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). In assays in which a mouse is employed, a subject compound may have an ED50 of less than 1 μg/mouse (e.g., 1 ng/mouse to about 1 μg/mouse).
In some embodiments, the subject method is an in vitro method that includes contacting a sample with a subject compound that specifically binds with high affinity to a target molecule. In certain embodiments, the sample is suspected of containing the target molecule and the subject method further comprises evaluating 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 D-peptidic. In certain embodiments, the subject compound is a modified compound that includes a label, e.g., a fluorescent label, and the subject method further includes detecting the label, if present, in the sample, e.g., using optical detection. In certain embodiments, the compound is modified with a support, such that any sample that does not bind to the compound may be removed (e.g., by washing). The specifically bound target protein, if present, may then be detected using any convenient means, such as, using the binding of a labeled target specific probe or using a fluorescent protein reactive reagent. In another embodiment of the subject method, 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 L-peptidic. In certain embodiments, the target VEGF-A protein is a L-protein and the compound is D-peptidic.
In certain embodiments, a subject compound may be contacted with a cell in the presence of VEGF-A, and a VEGF-A response phenotype of the cell monitored. Exemplary VEGF-A assays include assays using isolated protein in cell free systems, in vitro using cultured cells or in vivo assays. Exemplary VEGF-A assays include, but are not limited to a receptor tyrosine kinase inhibition assay (see, e.g., Cancer Research Jun. 15, 2006; 66:6025-6032), an in vitro HUVEC proliferation assay (FASEB Journal 2006; 20: 2027-2035; Wells et al., Biochemistry 1998, 37, 17754-17764), an in vivo solid tumor disease assay (U.S. Pat. No. 6,811,779) and an in vivo angiogenesis assay (FASEB Journal 2006; 20: 2027-2035). The descriptions of these assays are hereby incorporated by reference. The protocols that may be employed in these methods are numerous and include, but are not limited to cell-free assays, e.g., binding assays; cellular assays in which a cellular phenotype is measured, e.g., gene expression assays; and in vivo assays that involve a particular animal (which, in certain embodiments may be an animal model for a condition related to the target). In certain cases, the assay may be a vascularization assay. In certain embodiments, the target protein is VEGF-A and the subject compound inhibits VEGF-A dependent angiogenesis. In certain embodiments, the target protein is VEGF-A and the subject compound inhibits VEGF-A dependent cellular proliferation. In certain instances, the target protein is VEGF-A and the compound inhibits VEGFR2 phosphorylation.
In some embodiments, the subject method is in vivo and includes administering to a subject a D-peptidic compound that specifically binds with high affinity to a target molecule. In certain embodiments, the compound is administered as a pharmaceutical preparation. A variety of subjects are treatable according to the subject methods. Generally such subjects are “mammals” or “mammalian,” where these terms are used broadly to describe organisms which are within the class mammalia, including the orders carnivore (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs and rats), and primates (e.g., humans, chimpanzees and monkeys). In some embodiments, the subject is human. The subject can be a subject in need of prevention of treatment of a disease or condition associated with angiogenesis in a subject (e.g., as described herein).
The subject compounds can bind to and inhibit VEGF-A and may thus be useful for 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 referred to herein. Reference is also made in this regard to WO 98/47541. Diseases and conditions associated with angiogenesis include different forms of cancer and metastasis, for example, breast, skin, colorectal, pancreatic, prostate, lung or ovarian cancer. Other diseases and conditions associated with angiogenesis are inflammation (for example, chronic inflammation), atherosclerosis, rheumatoid arthritis and gingivitis. Further diseases and conditions associated with angiogenesis are arteriovenous alformations, astrocytomas, choriocarcinomas, glioblastomas, gliomas, hemangiomas (childhood, capillary), hepatomas, hyperplastic endometrium, ischemic myocardium, endometriosis, Kaposi sarcoma, macular degeneration, melanoma, neuroblastomas, occluding peripheral artery disease, osteoarthritis, psoriasis, retinopathy (diabetic, proliferative), scleroderma, seminomas and ulcerative colitis. In some cases, the disease or condition associated with angiogenesis is cancer (e.g., breast, skin, colorectal, pancreatic, prostate, lung or ovarian cancer), an inflammatory disease, atherosclerosis, rheumatoid arthritis, macular degeneration and retinopathy. Of particular interest is treatment of diabetic macular edema (DME) or age-related macular degeneration (AMD).
The VEGF-A binding subject compounds are useful in the treatment of various neoplastic and non-neoplastic diseases and disorders. Neoplasms and related conditions that are amenable to treatment include breast carcinomas, lung carcinomas, gastric carcinomas, esophageal carcinomas, colorectal carcinomas, liver carcinomas, ovarian carcinomas, thecomas, arrhenoblastomas, cervical carcinomas, endometrial carcinoma, endometrial hyperplasia, endometriosis, fibrosarcomas, choriocarcinoma, head and neck cancer, nasopharyngeal carcinoma, laryngeal carcinomas, hepatoblastoma, Kaposi's sarcoma, melanoma, skin carcinomas, hemangioma, cavernous hemangioma, hemangioblastoma, pancreas carcinomas, retinoblastoma, astrocytoma, glioblastoma, Schwannoma, oligodendroglioma, medulloblastoma, neuroblastomas, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcomas, urinary tract carcinomas, thyroid carcinomas, Wilm's tumor, renal cell carcinoma, prostate carcinoma, abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome.
Non-neoplastic conditions that are amenable to treatment include rheumatoid arthritis, psoriasis, atherosclerosis, diabetic and other proliferative retinopathies including retinopathy of prematurity, retrolental fibroplasia, neovascular glaucoma, age-related macular degeneration, thyroid hyperplasias (including Grave's disease), corneal and other tissue transplantation, chronic inflammation, lung inflammation, nephrotic syndrome, preeclampsia, ascites, pericardial effusion (such as that associated with pericarditis), and pleural effusion.
The term “treating” or “treatment” as used herein means the treating or treatment of a disease or medical condition in a patient, such as a mammal (such as a human) that includes: (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 or causing regression of the disease or medical condition in a patient; (c) suppressing the disease or medical condition, for example by, slowing or arresting the development of the disease or medical condition in a patient; or (d) alleviating a symptom of the disease or medical condition in a patient. As such, treatment also includes situations where the pathological condition, or at least symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or stopped, e.g., terminated, such that the subject no longer suffers from the pathological condition, or at least the symptoms that characterize the pathological condition. Treatment may also manifest in the form of a modulation of a surrogate marker of the disease condition, e.g., as described above.
Aspects of the present disclosure include methods of preventing or treating AMD, such as wet age-related macular degeneration (AMD). Age-related macular degeneration (AMD) is a leading cause of severe visual loss in the elderly population. The exudative form of AMD is characterized by choroidal neovascularization and retinal pigment epithelial cell detachment. Because choroidal neovascularization is associated with a dramatic worsening in prognosis, the subject VEGF-binding compounds find use in reducing the severity of AMD. In certain instances, the subject is a patient suffering from dry AMD and administration of a compound according to the subject methods prevents the occurrence, or reduces the severity, of wet AMD in the subject.
In certain embodiments, the subject methods include administering a compound, such as a VEGF-A binding compound, and then detecting the compound after it has bound to its target protein. In some methods, the same compound can serve as both a therapeutic and a diagnostic compound.
The VEGF-A binding compounds of the present disclosure are therapeutically useful for treating any disease or condition which is improved, ameliorated, inhibited or prevented by removal, inhibition, or reduction of a VEGF-A protein, or a fragment thereof.
In some embodiments, the subject method is a method of modulating angiogenesis in a subject, the method comprising administering to the subject an effective amount of a subject compound that specifically binds with high affinity to a VEGF-A protein. In certain embodiments, the method further comprises diagnosing the presence of a disease condition in the subject. In certain embodiments, the disease condition is a condition that may be treated by enhancing angiogenesis. In certain embodiments, the disease condition is a condition that may be treated by decreasing angiogenesis. In certain embodiments, the subject method is a method of inhibiting angiogenesis and the compound is a VEGF-A antagonist.
In some embodiments, the subject method is a method of treating a subject suffering from a cellular proliferative disease condition, the method including administering to the subject an effective amount of a subject compound that specifically binds with high affinity to a VEGF-A protein so that the subject is treated for the cellular proliferative disease condition.
In some embodiments, the subject method is a method of inhibiting tumor growth in a subject, the method comprising administering to a subject an effective amount of a subject compound that specifically binds with high affinity to the VEGF-A protein. In certain embodiments, the tumor is a solid tumor. In certain embodiments, the tumor is a non-solid tumor.
The amount of compound administered can be determined using any convenient methods to be an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the unit dosage forms of the present disclosure will depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the subject.
In some embodiments, an effective amount of a subject compound is an amount that ranges 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, from about 70 ng/ml to about 300 ng/ml, from about 60 ng/ml to about 100 ng/ml, from about 65 ng/ml to about 85 ng/ml, from about 70 ng/ml to about 90 ng/ml, from about 200 ng/ml to about 900 ng/ml, from about 200 ng/ml to about 800 ng/ml, from about 200 ng/ml to about 700 ng/ml, from about 200 ng/ml to about 600 ng/ml, from about 200 ng/ml to about 500 ng/ml, from about 200 ng/ml to about 400 ng/ml, or from about 200 ng/ml to about 300 ng/ml).
In some embodiments, an effective amount of a subject compound is an amount that ranges from about 10 pg to about 100 mg, e.g., 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 ng 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 a single dose amount or can be a total daily amount. The total daily amount can range from 10 pg to 100 mg, or can range from 100 mg to about 500 mg, or can range from 500 mg to about 1000 mg.
In some embodiments, a single dose of the subject compound is administered. In other embodiments, multiple doses of the subject compound are administered. Where multiple doses are administered over a period of time, the D-peptidic compound is administered twice daily (qid), daily (qd), every other day (qod), every third day, three times per week (tiw), or twice per week (biw) over a period of time. For example, a compound is administered qid, qd, qod, tiw, or biw over a period of from one day to about 2 years or more. For example, a 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 more, depending on various factors.
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 with a subject method can be assayed for the presence and/or extent of angiogenesis. Assessment of the effectiveness of the methods of treatment on the subject can include assessment of the subject before, during and/or after treatment, using any convenient methods. Aspects of the subject methods further include a step of assessing the therapeutic response of the subject to the treatment.
In some embodiments, the method includes assessing the condition of the subject, including diagnosing or assessing one or more symptoms of the subject which are 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 assaying the sample, e.g., for the presence of angiogenesis that is associated with the disease or condition of interest (e.g., as described herein). The sample can be a cellular sample. In some cases, the sample is a biopsy. The assessment step(s) of the subject method can be performed at one or more times before, during and/or after administration of the subject compounds, using any convenient methods.
In some cases, a subject compound or a salt thereof, e.g., as defined herein, finds use in medicine, particularly in the in vivo diagnosis or imaging, for example by PET, of a disease or condition associated with angiogenesis. In certain embodiments, the compound is a modified compound that includes a detectable label, and the method further includes detecting the label in the subject. The selection of the label depends on the means of detection. Any convenient labeling and detection systems may be used in the subject methods, see e.g., Baker, “The whole picture,” Nature, 463, 2010, p977-980. In certain embodiments, the compound includes a fluorescent label suitable for optical detection. In certain embodiments, the compound includes a radiolabel 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 tomographic detection. The subject compound may be labeled, as described above, although in some methods, the compound is unlabelled and a secondary labeling agent is used for imaging. In certain embodiments, the subject methods include diagnosis of a disease condition in a subject by comparing the number, size, and/or intensity of labeled loci, to corresponding baseline values. The base line values can represent the mean levels in a population of undiseased subjects, or previous levels determined in the same subject.
In some cases, radiolabelled compounds may be administered to subjects for PET imaging in amounts sufficient to yield the desired signal. In certain instances, the radionuclide dosage is of 0.01 to 100 mCi, such as 0.1 to 50 mCi, or 1 to 20 mCi, which is sufficient per 70 kg bodyweight. The radiolabelled compounds may therefore be formulated for administration using any convenient physiologically acceptable carriers or excipients. For example, the compounds, optionally with the addition of pharmaceutically acceptable excipients, may be suspended or dissolved in an aqueous medium, with the resulting solution or suspension then being sterilized. Also provided is the use of a radiolabelled compound or a salt thereof as described herein for the manufacture of a radiopharmaceutical for use in a method of in vivo imaging, e.g., PET imaging, such as imaging of a disease or condition associated with angiogenesis; involving administration of the radiopharmaceutical to a human or animal body and generation of an image of at least part of said body.
In some embodiments, the method is a method for in vivo diagnosis or imaging of a disease or condition associated with angiogenesis involving administering a radiopharmaceutical to said body, e.g. into the vascular system and generating an image of at least a part of said body to which said radiopharmaceutical has distributed using PET, wherein said radiopharmaceutical comprises a radiolabelled compound or a salt thereof.
In some embodiments, the method is a method of monitoring the effect of treatment of a human or animal body with a drug, e.g., a cytotoxic agent, to combat a condition associated with angiogenesis e.g., cancer, said method comprising administering to said body a radiolabelled compound or a salt thereof and detecting the uptake of the compound by cell receptors, such as endothelial cell receptors, e.g., alpha.v.beta.3 receptors, the administration and detection optionally being effected repeatedly, e.g. before, during and after treatment with said drug.
In some embodiments, the method is a method for in vivo diagnosis or imaging of a disease or condition associated with angiogenesis comprising administering to a subject a D-peptidic compound and imaging at least a part of the subject. In certain embodiments, the imaging comprises PET imaging and the administering comprises administering the compound to the vascular system of the subject. In some instances, the method further comprising detecting uptake of the compound by cell receptors. In certain instances, the target is VEGF-A and the subject is human. In certain embodiments, the method includes administering a therapeutic antibody, e.g., avastin, to the subject, wherein the disease or condition is a condition associated with cancer.
The subject methods may be diagnostic methods for detecting the expression of a target protein in specific cells, tissues, or serum, in vitro or in vivo. In some cases, the subject method is a method for in vivo imaging of a target protein in a subject. The methods may include administering the compound to a subject presenting with symptoms of a disease condition related to a target protein. In some cases, the subject is asymptomatic. The subject methods may further include monitoring disease progression and/or response to treatment in subjects who have been previously diagnosed with the disease.
The subject VEGF-A binding compounds may be used as affinity purification agents. In this process, the compounds are immobilized on a solid phase such a Sephadex resin or filter paper, using any convenient methods. The subject VEGF-A binding compound is contacted with a sample containing the VEGF-A protein (or fragment thereof) to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the VEGF protein, which is bound to the immobilized compound. Finally, the support is washed with another suitable solvent, such as glycine buffer, pH 5.0 that will release the VEGF-A protein from the immobilized compound.
The subject VEGF-A binding compounds may also be useful in diagnostic assays for VEGF-A protein, e.g., detecting its expression in specific cells, tissues, or serum. Such diagnostic methods may be useful in cancer diagnosis. For diagnostic applications, the subject compound may be modified as described above.
In some embodiments, the subject compounds may be administered in combination with one or more additional active agents or therapies. Any convenient agents may be utilized, including compounds useful for treating diseases that are targeted by the subject methods. The terms “agent,” “compound,” and “drug” are used interchangeably herein. Additional active agents or therapies include, but are not limited to, a small molecule, an antibody, an antibody fragment, an aptamer, a L-protein, a second target-binding molecule such as a second D-peptidic compound, a chemotherapeutic agent, surgery, catheter devices, and radiation. Combination therapy includes administration of a single pharmaceutical dosage formulation which contains the subject compound and one or more additional agents; as well as administration of the subject compound and one or more additional agent(s) in its own separate pharmaceutical dosage formulation. For example, a subject compound and a cytotoxic agent, a chemotherapeutic agent or a growth inhibitory agent can be administered to the patient together in a single dosage composition such as a combined formulation, or each agent can be administered in a separate dosage formulation. Where separate dosage formulations are used, the subject compound and one or more additional agents can be administered concurrently, or at separately staggered times, e.g., sequentially.
The terms “co-administration” and “in combination with” include the administration of two or more therapeutic agents (e.g., a D-peptidic compound and a second agent) either simultaneously, concurrently or sequentially within no specific time limits. In one embodiment, the agents are present in the cell or in the subject's body at the same time or exert their biological or therapeutic effect 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, a first agent (e.g., a D-peptidic 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), concomitantly with, or subsequent to (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 a second therapeutic agent.
“Concomitant administration” of a known therapeutic drug with a pharmaceutical composition of the present disclosure means administration of the D-peptidic compound and second agent at such time that both the known drug and the composition of the present disclosure will have a therapeutic effect. Such concomitant administration may involve concurrent (i.e. at the same time), prior, or subsequent administration of the drug with respect to the administration of a subject D-peptidic compound. Routes of administration of the two agents may vary, where representative routes of administration are described in greater detail below. A person of ordinary skill in the art would have no difficulty determining the appropriate timing, sequence and dosages of administration for particular drugs and compounds of the present disclosure.
In some embodiments, the compounds (e.g., a subject D-peptidic compound and a second agent) are administered to the subject within twenty-four hours of each other, such as 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. By administered substantially simultaneously is meant that the compounds are administered to the subject within about 10 minutes or less of each other, such as 5 minutes or less, or 1 minute or less of each other.
Also provided are pharmaceutical preparations of the subject compounds and the second active agent. In pharmaceutical dosage forms, the compounds may be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds.
Dosage levels of the order of from about 0.01 mg to about 140 mg/kg of body weight per day are useful in representative embodiments, or alternatively about 0.5 mg to about 7 g per patient per day. Those of skill will readily appreciate that dose levels can vary as a function of the specific compound, the severity of the symptoms and the susceptibility of the subject to side effects. Dosages for a given compound are readily determinable by those of skill in the art by a variety of means.
The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, a formulation intended for the oral administration of humans may contain from 0.5 mg to 5 g of active agent compounded with an appropriate and convenient amount of carrier material which may vary from about 5 to about 95 percent of the total composition. Dosage unit forms will generally contain between from about 1 mg to about 500 mg of an active ingredient, such as 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 800 mg, or 1000 mg.
It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the age, body 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.
Any convenient second agents can find use in the subject methods. In some cases, the second active agent specifically binds a target protein selected from 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. Second active agents of interest include, but are not limited to, pegpleranib (Fovista), ranibizumab (Lucentis), trastuzumab (Herceptin), Bevacizumab (Avastin), aflibercept (Eylea), nivolumab, atezolizumab, Durvalumab, gefitinib, erlotinib and Pembrolizumab.
For the treatment of cancer, the subject compounds 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 therapies, Chimeric Antigen Receptor/NK cell therapies, apoptosis regulator inhibitors (e.g., B cell CLL/lymphoma 2 (BCL-2) BCL-2-like 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 vaccine (e.g., a Th17-inducing dendritic cell vaccine) 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, Ado-trastuzumab emtansine, Rituximab, Ipilimumab, Rapamycin, Temsirolimus, Everolimus, Methotrexate, Doxorubicin, Abraxane, Folfirinox, Cisplatin, Carboplatin, 5-fluorouracil, Teysumo, Paclitaxel, Prednisone, Levothyroxine, Pemetrexed, navitoclax, ABT-199.
For the treatment of cancer (e.g., melanoma, non-small cell lung cancer or a lymphoma such as Hodgkin's lymphoma), the subject compounds can be administered in combination with an immune checkpoint inhibitor. Any convenient checkpoint inhibitors 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, for treatment of cancer and/or inflammatory disease, the subject compounds can be administered in combination with a colony-stimulating factor-1 receptor (CSF1R) inhibitors. CSF1R inhibitors of interest include, but are not limited to, emactuzumab.
Any convenient cancer vaccine therapies and agents can be used in combination with the subject immunomodulatory polypeptide compositions and methods. For treatment of cancer, e.g., ovarian cancer, the subject compounds can be administered in combination with a vaccination therapy, e.g., a dendritic cell (DC) vaccination agent that promotes Th1/Th17 immunity. Th17 cell infiltration correlates with markedly prolonged overall survival among ovarian cancer patients. In some cases, the immunomodulatory polypeptide finds use as adjuvant treatment in combination with Th17-inducing vaccination.
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 by Rishi et al., Journal of Biomedical Nanotechnology, Volume 11, Number 9, September 2015, pp. 1608-1627(20), and CD47 inhibitors, including, but not limited to, anti-CD47 antibody agents such as Hu5F9-G4.
The compounds of the invention, e.g., as described above, find use 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 are now reviewed in greater details below.
The subject compounds find use in a variety of therapeutic applications. Therapeutic applications of interest include those applications in which the activity of the target is the cause or a compounding factor in disease progression. As such, the subject compounds find use in the treatment of a variety of different conditions in which the modulation of target activity in the host is desired.
The subject compounds are useful for treating a disorder relating to its target, VEGF-A. Examples of disease conditions which may be treated with compounds of the invention are described above.
In certain embodiments, the disease conditions include, but are not limited to: cancer, inhibition of angiogenesis and metastasis, osteoarthritis pain, chronic lower 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 one embodiment, the present disclosure provides a method of treating a subject for a VEGF-A-related condition. The method generally involves administering a subject compound to a subject having a VEGF-A related disorder in an amount effective to treat at least one symptom of the VEGF-A related disorder. VEGF-A related conditions are generally characterized by excessive vascular endothelial cell proliferation, vascular permeability, edema or inflammation such as brain edema associated with injury, stroke or tumor; edema associated with inflammatory disorders such as psoriasis or arthritis, including rheumatoid arthritis; asthma; generalized edema associated with burns; ascites and pleural effusion associated with tumors, inflammation or trauma; chronic airway inflammation; capillary leak syndrome; sepsis; kidney disease associated with increased leakage of protein; and eye disorders such as age related macular degeneration and diabetic retinopathy. Such conditions include breast, lung, colorectal and renal cancer.
The subject compounds and methods find use in a variety of research applications. The subject compounds and methods may be used to analyze the roles of target proteins in modulating various biological processes, including but not limited to, angiogenesis, inflammation, cellular growth, metabolism, regulation of transcription and regulation of phosphorylation. Other target protein binding molecules such as antibodies have been similarly useful in similar areas of biological research. See e.g., Sidhu and Fellhouse, “Synthetic therapeutic antibodies,” Nature Chemical Biology, 2006, 2(12), 682-688. Such methods can be readily modified for use in a variety of research applications of the subject compounds and methods.
The subject compounds and methods find use in a variety of diagnostic applications, including but not limited to, the development of clinical diagnostics, e.g., in vitro diagnostics or in vivo tumor imaging agents. Such applications are useful in diagnosing or confirming diagnosis of a disease condition, or susceptibility thereto. The methods are also useful for monitoring disease progression and/or response to treatment in patients who have been previously diagnosed with the disease.
Diagnostic applications of interest include diagnosis of disease conditions, such as those conditions described above, including but not limited to: cancer, inhibition of angiogenesis and metastasis, osteoarthritis pain, chronic lower back pain, cancer-related pain, age-related macular degeneration (AMD), diabetic macular edema (DME), ideopathic pulmonary fibrosis (IPF) and graft survival of transplanted corneas. In some methods, the same compound can serve as both a treatment and diagnostic reagent.
Other target protein binding molecules, such as aptamers and antibodies, have also found use in the development of clinical diagnostics. Such methods can be readily modified for use in a variety of diagnostics applications of the subject compounds and methods, see for example, Jayasena, “Aptamers: An Emerging Class of Molecules That Rival Antibodies in Diagnostics,” Clinical Chemistry, 1999, 45, 1628-1650.
Also provided are pharmaceutical preparations. Pharmaceutical preparations are compositions that include a compound (either 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 a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, such as humans. The term “vehicle” refers to a diluent, adjuvant, excipient, or carrier with which a compound of the invention is formulated for administration to a mammal. Such pharmaceutical vehicles can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical vehicles can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents may be used. When administered to a mammal, the compounds and compositions of the invention and pharmaceutically acceptable vehicles, excipients, or diluents may be sterile. In some instances, an aqueous medium is employed as a vehicle when the compound of the invention is administered intravenously, such as water, saline solutions, and aqueous dextrose and glycerol solutions.
Pharmaceutical compositions can take the form of capsules, tablets, pills, pellets, lozenges, powders, granules, syrups, elixirs, solutions, suspensions, emulsions, suppositories, or sustained-release formulations thereof, or any other form suitable for administration to a mammal. In some instances, the pharmaceutical compositions are formulated for administration in accordance with routine procedures as a pharmaceutical composition adapted for oral or intravenous administration to humans. Examples of suitable pharmaceutical vehicles and methods for formulation thereof are described in Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro ed., Mack Publishing Co. Easton, Pa., 19th ed., 1995, Chapters 86, 87, 88, 91, and 92, incorporated herein by reference.
The choice of excipient will be determined in part by the particular compound, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical composition of the present invention. Administration of compounds of the present disclosure may be systemic or local. In certain embodiments administration to a mammal will result in systemic release of a compound of the invention (for example, 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 hypodermic needle or catheter, for example, intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal, intraarterial, intraventricular, intrathecal, and intracameral injection and non-injection routes, such as intravaginal rectal, or nasal administration. In certain embodiments, the compounds and compositions of the invention are administered orally. In certain embodiments, it may be desirable to administer one or more compounds of the invention locally to the area in need of treatment. This may be achieved, for example, by local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
The subject compounds can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
In some embodiments, formulations suitable for oral administration can include (a) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, or saline; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as solids or granules; (c) suspensions in an appropriate liquid; and (d) suitable emulsions. Tablet forms can include one or more of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients. Lozenge forms can include the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles including the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are described herein.
The subject formulations can be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They may also be formulated as pharmaceuticals for non-pressured preparations such as for use in a nebulizer or an atomizer.
In some embodiments, formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
Formulations suitable for topical administration may be presented as creams, gels, pastes, or foams, containing, in addition to the active ingredient, such carriers as are appropriate. In some embodiments the topical formulation contains one or more components selected from a structuring agent, a thickener or gelling agent, and an emollient or lubricant. Frequently employed structuring agents include long chain alcohols, such as stearyl alcohol, and glyceryl ethers or esters and oligo(ethylene oxide) ethers or esters thereof. Thickeners and gelling agents include, for example, polymers of acrylic or methacrylic acid and esters thereof, polyacrylamides, and naturally occurring thickeners such as agar, carrageenan, gelatin, and guar gum. Examples of emollients include triglyceride esters, fatty acid esters and amides, waxes such as beeswax, spermaceti, or carnauba wax, phospholipids such as lecithin, and sterols and fatty acid esters thereof. The topical formulations may further include other components, e.g., astringents, fragrances, pigments, skin penetration enhancing agents, sunscreens (e.g., sunblocking agents), etc.
A compound of the present disclosure may also be formulated for oral administration. For an oral pharmaceutical formulation, suitable excipients include pharmaceutical grades of carriers such as mannitol, lactose, glucose, sucrose, starch, cellulose, gelatin, magnesium stearate, sodium saccharine, and/or magnesium carbonate. For use in oral liquid formulations, the composition may be prepared as a solution, suspension, emulsion, or syrup, being supplied either in solid or liquid form suitable for hydration in an aqueous carrier, such as, for example, aqueous saline, aqueous dextrose, glycerol, or ethanol, preferably water or normal saline. If desired, the composition may also contain minor amounts of non-toxic auxiliary substances such as wetting agents, emulsifying agents, or buffers. A compound of the invention may also be incorporated into existing nutraceutical formulations, such as are available conventionally, which may also include an herbal extract.
Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more inhibitors Similarly, unit dosage forms for injection or intravenous administration may include the inhibitor(s) in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.
The term “unit dosage form,” as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the novel unit dosage forms of the present invention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.
Dose levels can vary as a function of the specific compound, the nature of the delivery vehicle, and the like. Desired dosages for a given compound are readily determinable by a variety of means.
The dose administered to an animal, particularly a human, in the context of the present invention should be sufficient to effect a prophylactic or therapeutic response in the animal over a reasonable time frame, e.g., as described in greater detail below. Dosage will depend on a variety of factors including the strength of the particular compound employed, the condition of the animal, and the body weight of the animal, as well as the severity of the illness and the stage of the disease. The size of the dose will also be determined by the existence, nature, and extent of any adverse side-effects that might accompany the administration of a particular compound.
In pharmaceutical dosage forms, the compounds may be administered in the form of a free base, their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds.
In some embodiments, a pharmaceutical composition includes a subject compound that specifically binds with high affinity to a target protein, and a pharmaceutically acceptable vehicle. In certain embodiments, the target protein is a VEGF protein and the subject compound is a VEGF antagonist.
Also provided are kits that include compounds of the present disclosure. Kits of the present disclosure may include one or more dosages of the compound, and optionally one or more dosages of one or more additional active agents. Conveniently, the formulations may be provided in a unit dosage format. In such kits, in addition to the containers containing the formulation(s), e.g. unit doses, is an informational package insert describing the use of the subject formulations in the methods of the invention, e.g., instructions for using the subject unit doses to treat cellular conditions associated with pathogenic angiogenesis. The term kit refers to a packaged active agent or agents. In some embodiments, the subject system or kit includes a dose of a subject compound (e.g., as described herein) and a dose of a second active agent (e.g., as described herein) in amounts effective to treat a subject for a disease or condition associated with angiogenesis (e.g., as described herein).
In addition to the above-mentioned components, a subject kits may further include instructions for using the components of the kit, e.g., to practice the subject method. The instructions are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or sub-packaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, Hard Disk Drive (HDD), portable flash drive, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.
In some embodiments, a kit includes a first dosage of a subject pharmaceutical composition and a second dosage of a subject pharmaceutical composition. In certain embodiments, the kit further includes a second angiogenesis modulatory agent.
It is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this 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 are now described.
All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The 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.
It is 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 is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.
While the apparatus and method has or will be described for the sake of grammatical fluidity with functional explanations, it is to be expressly understood that the claims, unless expressly formulated under 35 U.S.C. § 112, are not to be construed as necessarily limited in any way by the construction of “means” or “steps” limitations, but are to be accorded the full scope of the meaning and equivalents of the definition provided by the claims under the judicial doctrine of equivalents, and in the case where the claims are expressly formulated under 35 U.S.C. §112 are to be accorded full statutory equivalents under 35 U.S.C. § 112.
The term “peptidic” refers to a moiety that is composed primarily of amino acid residues linked together as a polypeptide. The term “peptidic” is meant to include compounds in which one, two or more residues of a conventional polypeptide sequence have been replaced with a peptidomimetic. A peptidomimetic is a small organic group designed to mimic a peptide or amino acid residue. A peptidomimetic group of a peptidic moiety can include a non-naturally occurring or synthetic backbone group linked to the conventional polypeptide backbone and an optional sidechain group that mimics the sidechain group of any convenient amino acid residue of interest. In some embodiments, a peptidic compound that is composed primarily of amino acid residues has 2 residues or less per 10 amino acid residues of a parent polypeptide sequence replaced with a peptidomimetic moiety. Any convenient peptidomimetic groups and chemistries can be utilized in the subject peptidic compounds. The term peptidic is also meant to include multimeric peptidic compounds where two or more peptidic compounds of interest are covalently linked. The term peptidic is also meant to include modified peptidic compounds where a non-proteinaceous moiety has been covalently linked to the compound.
The terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to a polymeric form of amino acids of any length. Unless specifically indicated otherwise, “polypeptide,” “peptide,” and “protein” can include genetically coded and non-coded 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 with 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 or 1000 or more amino acids.
For the polypeptide sequences and motifs depicted herein, unless noted otherwise, capital letter codes refer to L-amino acid residues and small 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. “1” 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 is understood that for any of the sequences and motifs described herein, e.g., sequences defining a peptidic compound that specifically binds VEGF-A, a mirror image compound is also encompassed which specifically binds to the mirror image of VEGF-A. The present disclosure is meant to encompass both versions of the subject compounds, e.g., L-peptidic compounds that specifically bind D-VEGF-A and D-peptidic compounds that specifically bind L-VEGF-A. It is understood that D-VEGF-A protein may be targeted primarily in a variety of in vitro applications, while L-VEGF-A protein may be targeted for a variety of in vitro and/or in vivo applications.
The term “analog” of an amino acid residue refers to a residue having a sidechain group that is a structural and/or functional analog of the sidechain group of the reference amino acid residue. In some instances, the amino acid analogs share backbone structures, and/or the side chain structures of one or more natural amino acids, with difference(s) being one or more modified groups in the molecule. Such modification may include, but is not limited to, substitution of an atom (such as N) for a related atom (such as S), addition of a group (such as methyl, or hydroxyl, etc.) or an atom (such as F, Cl or Br, etc.), deletion of a group, substitution of a covalent bond (single bond for double bond, etc.), or combinations thereof. For example, amino acid analogs may include α-hydroxy acids, and α-amino acids, and the like. In some cases, an analog of an amino acid residue is a substituted version of the amino acid. The term “substituted version” of an amino acid residue refers to a residue having a sidechain group that includes one or more additional substituents on the sidechain group that are not present in the sidechain of the reference amino acid residue.
The terms “aromatic amino acid” and “aromatic residue” are used interchangeably to refer to an amino acid residue where the sidechain group includes an aryl, a substituted aryl, a heteroaryl or a substituted heteroaryl group. In some cases, the sidechain group is an aryl-alkyl, a substituted aryl-alkyl, a heteroaryl-alkyl or a substituted heteroaryl-alkyl group. The terms are meant to include naturally occurring and non-naturally occurring alpha-amino acids. Naturally occurring aromatic residues of interest include phenylalanine, tyrosine, tryptophan and histidine.
The terms “carbocyclic amino acid” and “carbocyclic residue” are used interchangeably to refer to an amino acid residue where the sidechain group includes an aryl or a saturated carbocyclic group. In some cases, the sidechain group is an cycloalkyl-alkyl or a substituted cycloalkyl-alkyl group. Non-naturally occurring sidechain groups of interest include, but are not limited to, cyclohexyl-CH2—, cyclopentyl-CH2, cyclohexyl-(CH2)2— and cyclopentyl-(CH2)2—.
The terms “heterocyclic amino acid” and “heterocyclic residue” are used interchangeably to refer to an amino acid residue where the sidechain group includes a heterocyclic group, such as a heteroaryl group or a saturated heterocyclic group. In some cases, the sidechain group is an heterocycle-alkyl or a substituted heterocycle-alkyl group. The terms are meant to include naturally occurring and non-naturally occurring alpha-amino acids. Naturally occurring heterocyclic residues of interest include tryptophan and histidine.
The terms “non-polar amino acid residue” and “non-polar residue” refer to an amino acid residue that includes a sidechain that is hydrogen (i.e., G) or a non-polar group. In some cases, a non-polar amino acid sidechain is a hydrophobic group. The terms are meant to include naturally occurring and non-naturally occurring alpha-amino acids. Naturally occurring non-polar amino acid residues of interest include naturally occurring hydrophobic residues.
The terms “hydrophobic amino acid” and “hydrophobic residue” are used interchangeably to refer to an amino acid residue where the sidechain group is a hydrophobic group. The terms are meant to include naturally occurring and non-naturally occurring alpha-amino acids. Naturally occurring hydrophobic residues of interest include alanine, isoleucine, leucine, phenylalanine, proline and valine.
The terms “polar amino acid” and “polar residue” are used interchangeably to refer to an amino acid residue where the sidechain group includes a polar group or charged group. In certain cases, the polar group is capable of being a hydrogen bond donor or acceptor. The terms are meant to include naturally occurring and non-naturally occurring alpha-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.
The terms “scaffold” and “scaffold domain” are used interchangeably and refer to a reference peptidic framework motif from which a subject peptidic compound arose, or against which the subject peptidic compound is able to be compared, e.g., via a sequence or structural alignment method. The structural motif of a scaffold domain can be based on a naturally occurring protein domain structure. For a particular protein domain structural motif, several related underlying sequences may be available, any one of which can provide for the particular three-dimensional structure of the scaffold domain. A scaffold domain can be defined in terms of a characteristic consensus sequence motif.
The terms “parent amino acid sequence”, “parent sequence” and “parent polypeptide” refer to a polypeptide comprising an amino acid sequence from which a variant peptidic compound arose and against which the variant peptidic compound is being compared. The parent polypeptide lacks one or more of the modifications or variant amino acids disclosed herein and can differ in function compared to a variant peptidic compound as disclosed herein. The parent polypeptide may be a native domain sequence (e.g., SEQ ID NO: 2-21), a native domain scaffold sequence having pre-existing amino acid sequence modifications (such as any convenient point mutations or truncations known to confer a desirable physical property upon the domain, e.g., increased stability or solubility), or a non-naturally occurring consensus sequence (e.g., a sequence of a consensus motif based on several native domains of interest, see e.g.,
The terms “corresponding residue” and “residue corresponding to” are used to refer to an amino acid residue located at equivalent positions of variant and parent sequences, e.g., as defined by the GA domain numbering scheme shown in
The terms “variant amino acid” and “variant residue” are used interchangeably to refer to the particular residues of a subject compound which are modified or mutated by comparison to an underlying scaffold domain. The variant residues encompass those residues that were selected (e.g., via mirror image screening, affinity maturation and/or point mutation(s)) to provide for a desirable domain motif structure that specific binds to the target. When a compound includes amino acid mutations or modifications at particular positions by comparison to a scaffold domain, the amino acid residues of the peptidic compound located at those particular positions are referred to as “variant amino acids.” Such variant amino acids may confer on the resulting peptidic compounds different functions, such as specific binding to a target protein, increased water solubility, ease of chemical synthesis, metabolic stability, etc. Aspects of the present disclosure include peptidic compounds that were selected from a phage display library based on a GA scaffold domain and further developed (e.g., via additional affinity maturation and/or point mutations), and as such include several variant amino acids integrated with a GA scaffold domain.
The terms “variant domain” and “variant motif” refers to an arrangement of variant amino acids incorporated at particular locations of a scaffold domain. The variant motif can encompass a continuous and/or a discontinuous sequence of residues. The variant motif can encompass variant amino acids located at one face of the compound structure. The variant domain may be considered to be incorporated into, or integrated with, an underlying scaffold domain structure or sequence. In the subject compounds, the scaffold domain can provide a stable three-dimensional protein structural motif, e.g., of a naturally occurring protein domain, while the variant domain can be defined by an arrangement of characteristic minimum number of variant residues at a modified surface of the structure that is capable of specifically binding a target protein.
The term “framework residues” refers to residual amino acid residues of a scaffold domain of a peptidic compound that are not variant amino acids. As such, a structural or sequence motif composed of framework residues is defined by the corresponding arrangement of residues of an underlying scaffold domain structure or sequence. The sequence and structure of a subject compound can be defined by a combination of variant and framework residues.
The term “mutation” refers to a deletion, insertion, or substitution of an amino acid(s) residue or nucleotide(s) residue relative to a reference sequence, such as a scaffold sequence. 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 a particular secondary structural feature.
The term “non-core mutation” refers to an amino acid mutation of a peptidic compound that is located at a position in the structure that is not part of the hydrophobic core of the structure. Amino acid residues in the hydrophobic core of a peptidic compound are not significantly solvent exposed but rather tend to form intramolecular hydrophobic contacts. A methodology used to specify hydrophobic core residues is described by Dahiyat et al., (“Probing the role of packing specificity in protein design,” Proc. Natl. Acad. Sci. USA, 1997, 94, 10172-10177) where a PDB structure was used to calculate which side chains expose less than 10% of their surface area to solvent. In some cases, Degrado's heptad repeat model (DeGrado et al. “Analysis and design of three-stranded coiled coils and three-helix bundles”, Folding & Design 1998, 3: R29-R40) can be utilized to define “a” and “d” residues of a hydrophobic core, as depicted in
The term “surface mutation” refers to an amino acid mutation in a scaffold domain that is located at a position in the structure that is solvent exposed. Such variant amino acid residues at surface positions of a D-peptidic compound can be capable of interacting directly with a target molecule, whether or not such an interaction occurs. In some cases, Degrado's heptad repeat model can be utilized to define “c” and “g” residues that are highly solvent exposed, as depicted in
The term “boundary mutation” refers to an amino acid mutation in a scaffold that is located at a position in the structure that is at the boundary between the hydrophobic core and the solvent exposed surface. Such variant amino acid residues at boundary positions of a peptidic compound may be in part contacting hydrophobic core residues and/or in part solvent exposed and capable of some interaction with a target molecule, whether or not such an interaction occurs. One criteria for describing 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, Degrado's heptad repeat model can be utilized to define “c” and “g” residues that are at least partially solvent exposed, as depicted in
The term “linking sequence” refers to a continuous sequence of amino acid residues, or analogs thereof, that connect two peptidic motifs or regions. In certain cases, a linking sequence is a loop or turn region (e.g., as described herein) connecting two antiparallel helical regions.
The term “stable” refers to a compound that is able to maintain a folded state under physiological conditions at a certain temperature, such that it retains at least one of its normal functional activities, for example binding to a target protein. The stability of the compound can be determined using standard methods. For example, the “thermostability” of a compound can be determined by measuring the thermal melt (“Tm”) temperature. The Tm is the temperature in degrees Celsius at which half of the compound becomes unfolded. In some instances, the higher the Tm, the more stable the compound.
The terms “similar,” “conservative,” and “highly conservative” amino acid substitutions are defined as shown in Table 6, below. The determination of whether an amino acid residue substitution is similar, conservative, or highly conservative can be based on the side chain of the amino acid residue and not the polypeptide backbone.
A “specificity determining motif” refers to an arrangement of variant amino acids incorporated at particular locations of a variant scaffold domain that provides for specific binding of the variant domain to a target protein. The motif can encompass continuous and/or a discontinuous sequences of residues. The motif can encompass variant amino acids located at one face of the compound structure and which are capable of contacting the target protein, or can encompass variant residues which do not provide contacts with the target but rather provide for a modification to the natural domain structure that enhances binding to the target. The motif may be considered to be incorporated into, or integrated with, an underlying scaffold domain structure or sequence, e.g., a three helix bundle of a naturally occurring GA or Z domain.
A compound that “specifically binds” to an epitope or binding site of a target protein is a term well understood in the art, and methods to determine such specific or preferential binding are also well known in the art. A compound exhibits “specific binding” if it associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular cell or substance (target protein) than it does with alternative cells or substances. A D-peptidic compound “specifically binds” to a target if it binds with greater affinity, avidity, more readily, and/or with greater 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 this epitope or site with greater affinity, avidity, more readily, and/or with greater duration than it binds to other VEGF epitopes or non-VEGF epitopes. It is also understood by reading this definition 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. As such, “specific binding” does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to binding means specific binding.
The compounds may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)- or, as (D)- or (L)- for amino acids and polypeptides. The present disclosure is meant to include all such possible isomers, as well as, their racemic, diastereomeric, and optically pure forms. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included.
The term “a target protein” refers to all members of the target family, and fragments and enantiomers thereof, and protein mimics thereof. The target proteins of interest that are described herein are intended to include all members of the target family, and fragments and enantiomers thereof, and protein mimics thereof, unless explicitly described otherwise. The target protein may 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 using any convenient recombinant expression methods or using any convenient synthetic methods, or purchased commercially, as well as fusion proteins containing a target molecule, as well as synthetic L- or D-proteins.
The term “VEGF” or its non-abbreviated form “vascular endothelial growth factor”, as used herein, refers to the protein products 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 methods or using any convenient synthetic methods, or purchased commercially (e.g. R & D Systems, Catalog No. 210-TA, Minneapolis, Minn.), as well as fusion proteins containing a VEGF molecule, as well as synthetic L- or D-proteins. VEGF is involved in both vasculogenesis (the de novo formation of the embryonic circulatory system) and angiogenesis (the growth of blood vessels from pre-existing vasculature) and can also be involved in the growth of lymphatic vessels in a process known as lymphangiogenesis. Members of the VEGF family stimulate cellular responses by binding to tyrosine kinase receptors (the VEGFRs) on the cell surface, causing them to dimerize and become activated through transphosphorylation. The VEGF receptors have an extracellular portion containing 7 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 of the cellular responses to VEGF. VEGF, its biological activities, and its receptors are well studied and are described in Matsumoto et al. (VEGF receptor signal transduction Sci STKE. 2001:RE21 and Marti et al (Angiogenesis in ischemic disease. Thromb Haemost. 1999 Suppl 1:44-52). Amino acid sequences of exemplary VEGFs are found in the NCBI's Genbank database and a full description of VEGF proteins and their roles in various diseases and conditions is found in NCBI's Online Mendelian Inheritance in Man database.
Aspects of the present disclosure are embodied in the clauses and exemplary embodiments set forth below.
a VEGF-A binding three-helix bundle comprising helical regions [Helix 1], [Helix 2] and [Helix 3] each comprising a heptad repeat sequence (abcdefg)n and configured to define a hydrophobic core substantially comprising a and d residues;
wherein [Helix 2] and [Helix 3] are configured antiparallel to each other and together define a VEGF-A binding g-g face of the three-helix bundle comprising six or more VEGF-A contacting residues independently selected from non-polar, aromatic, heterocyclic and carbocyclic residues.
[Helix 1]-[Linker 1]-[Helix 2]-[Linker 2]-[Helix 3] (I)
wherein [Linker 1] and [Linker 2] are independently peptidic linking sequences of between 1 and 10 residues.
residues d2, a2 and d1 of [Helix 2] interact with residues a2, d2 and a3 of [Helix 3]; and
residues c2, g1 and c1 of [Helix 2] and residue g1 of [Helix 3] are each independently an aromatic, heterocyclic or carbocyclic residue.
wherein:
each h* is independently histidine or an analog thereof;
f* is phenylalanine or an analog thereof; and
each u is independently a non-polar amino acid residue.
[Helix 2] comprises a sequence of the formula:
[Helix 3] comprises a sequence of the formula:
wherein:
wherein each z is independently a helix-terminating residue.
wherein:
wherein:
each xis an amino acid and n is 1, 2 or 3;
each z is independently a helix-terminating residue (e.g., G or p); and
e* is glutamic acid or an analog thereof.
wherein:
wherein:
a) phvx29x30fix33hap (SEQ ID NO: 102)
wherein:
b) an amino acid sequence which has 80% or greater identity (e.g., 2 residue changes) to the sequence defined in a).
wherein:
a) x38x39hvx42Glu45x46aix49x50a (SEQ ID NO: 98)
wherein:
b) an amino acid sequence which has 80% or greater identity (e.g., 2 residue changes) to the sequence defined in a).
a) llknakedaiaelkkcGitephvisfinhapyvshvnGlknailka; and
b) an amino acid sequence which has 85% or greater identity to the sequence defined in a).
l6lknakedaiaelkkaGit.......in.a..v..vn..kn.ilka51 (SEQ ID NO: 156); and b) an amino acid sequence which has 88% or greater identity to the sequence defined in a).
tlidqwllknakedaiaelkkaGit.......in.a..v..vn..kn.ilkaha53 (SEQ ID NO: 157); and b)
an amino acid sequence which has 90% or greater identity to the sequence defined in a).
The following examples are offered by way of illustration and not by way of limitation.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HaRBor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference. Reagents, cloning vectors, cells, and kits for methods referred to in, or related to, this disclosure are available from commercial vendors 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 e.g., Addgene, Inc., American Type Culture Collection (ATCC), and the like.
The subject compounds 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 WO2014/140882.
Briefly, 5 ug/ml of D-VEGFA was coated in NUNC Maxisorp plates. After blocking, a pool of 8 scaffold libraries including the GA domain library, was added to the plate after depletion on an empty well. The bound phage was eluted and amplified overnight in OmniMax2 T1R cells. For Rounds 3 and 4, a lower concentration of amplified phage pools (˜5×1011 cfu/ml) was used compared to the standard concentration of 1 x 1013 cfu/ml, as the elute concentration was too high in Round 2. Several hits were obtained from various libraries including 17 different sequences from GA domain library. Based on sequence identity between the clones, 3 representative clones including Compound 1 (See
For the first round of affinity maturation, a soft-randomization strategy was utilized (Fairbrother et.al, 1998) where the polynucleotides encoding each of the randomized positions 25, 27, 28, 31, 34, 36, 37, 39, 40, 43, 44 and 47 were doped with handmixed bases so that the native nucleotide is baised at 70% and the other three nucleotides occur at 10% frequency. This allows for a 40% chance of retention of amino acids found in the parent sequence of Compound 1 in each of these positions. The affinity maturation library was constructed by site-directed mutagenesis protocols (Fellouse et.al.) using the following oligonucleotide and ssDNA from GA domain original sequence as template.
N2 is a mix of 10% A, 70% C, 10% G and 10% T
N3 is a mix of 10% A, 10% C, 70% G and 10% T
N4 is a mix of 10% A, 10% C, 10% G and 70% T
The affinity maturation library was panned against D-VEGFA using standard procedures (Fellouse et.al.) 24 clones from Round 3 were analyzed and a competitive ELISA was performed to rank them by affinity. Compound 1.1 was selected as a clone of interest from this list. A sequence logo of selected positions of all the clones is shown in
A second round of affinity maturation was performed to improve the affinity and stability of Compound 1.1. Given that a Pro residue was heavily conserved in Position 36, the change in backbone conformation may alter the orientation of Helix2 with respective to core residues and possibly affect the stability of selected Compound 1.1. Moreover, surface exposed residues near the C-terminus can 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. Again a soft randomization strategy was used with the following oligonucleotides for site directed mutagenesis
The library was constructed and panned against D-VEGFA with a modified protocol. Given that D-VEGF-A is highly stable and retains fold even at 3M guanidine hydrochloride (GuHCl), it was hypothesized that selection of binders in the presence of a low-medium concentration of denaturant can select for clones with improved affinity and stability at the same time. In this procedure, the library or the amplified phage pool was resuspended in PBT buffer (PBS, 0.2% BSA, 0.05% Tween20) with varying concentrations of denaturant guanidine hydrochloride (GuHCl) for each round of selection. The phage was incubated at 37° C. for 2 hours for equilibration. The selections were also carries out at 37° C. The following conditions were used for each round.
After four rounds of affinity maturation several clones were sequenced and Compound 1.1.1 was selected as a clone of interest via assessment with a competitive ELISA assay. Cys21 was identified as a bystander mutation and reverted back to Ala (e.g., to eliminate possibility of disulfide dimerization) to give a lead compound of interest, compound 1.1.1(C21A).
In addition, a variety of the scaffolded phage display libraries that are described by Uppalapati et al. in WO2014/140882 were screened for binding to synthetic D-VEGF-A target protein. Several of the scaffolded domain libraries produced hit clones during phage display screening studies indicating that the subject D-peptidic compounds that specifically bind VEGF-A can have one of a variety of underlying scaffold domains. Initially, the hit clones from the GA domain scaffolded library were selected for further investigation.
Selected compounds were synthesized and purified using conventional Fmoc solid phase peptide synthesis methods. In some cases, additional point mutations were included, e.g., as described herein. Compounds were folded in buffer and assessed for VEGF-A inhibition activity as described herein.
An X-ray crystal structure of Compound 1.1.1(C A) in complex with L-VEGF-A was obtained.
where a single binding site in the dimer is defined by the following residues of:
The binding affinity of compounds of interest for VEGF-A was measured using a surface plasmon resonance (SPR) assay.
Compounds of interest were assessed for VEGF-A binding in a competitive phage ELISA assay.
1-1.1
5-5.4
Compounds of interest were assessed for inhibition of VEGF-A:VEGFR1 in an Octet assay. Exemplary conditions: VEGF-A at 10 nM, inhibitor at nM conc.; VEGF-A:VEGFR1 Kd=25 pM.
A series of dimers of modified compound 1.1.1 (c21a) were prepared having linkers of various lengths by conjugation of a variety of PEG-based linkers to either the N- or C-terminals of the compounds using cysteine maleimide or disulfide conjugation chemistry. A cysteine residue was incorporated either at the C-terminal or N-terminal of the compound and dimerization was achieved via cysteine-maleimide conjugation chemistry with a bifunctional modified PEG linker. Structures of exemplary dimeric compounds are shown below:
The resulting dimeric compounds were assayed for VEGF-A inhibition activity in an octet assay.
Conditions: VEGF-A at 10 nM, inhibitor at 20 nM (or 25 nM*); VEGF-A:VEGFR1 Kd=25 pM. 100%=100 nM of (1.1.1 (c21a)) dimer C-C-linked with PEG11 linker
Based on an analysis of the X-ray crystal structure as shown in
A D-peptidic VEGF-A antagonist was identified using mirror image phage display screening of a GA-domain library. See U.S. 62/688,272, filed Jun. 21, 2018, by Uppalapati et al. and entitled “D-Peptidic VEGF-A Binding Compounds and Methods for Using the Same”. Exemplary compound 11055 (
In order to engineer a high affinity variant of 11055, a pIII-fused phage display library was designed based on an analysis of the X-ray crystal structure of compound 11055 bound to VEGF-A. A 2.3 angstrom resolution structure was solved of 11055 in complex with VEGF-A using hanging drop method. Diffraction quality crystals were grown in 0.1 M Tris pH 8.5, 0.2 M calcium chloride, 18% w/v PEG 4000. The structure was solved by molecular replacement. The crystal structure shows two molecules of 11055 bound to a VEGF-A homodimer where they occupy identical binding sites on the VEGF-A monomers, these sites overlap with the VEGFR2 receptor binding site of VEGF-A (
Based on this structure a library was designed to further stabilize the variant GA domain three helix structure. A total of 7 amino acid residues were selected for randomization at the packing interface between helix 1 (H1) and the loop connecting helix 2 (H2) and helix 3 (H3) (
Based on the X-ray crystal structure described above (
The affinity matured D-peptidic compounds were characterized in a VEGF-A blocking ELISA in order to measure their antagonistic activity. Here, a VEGFR1-Fc fusion was coated overnight on Maxisorp plates at 1 μg/mL in PBS. 1 nM biotinylated-VEGF-A was mixed with antagonist titrations and binding of biotinylated-VEGF-A to VEGFR1-Fc was detected with streptavidin-HRP. Variant compound 979110 blocks VEGF-A binding to VEGFR1 and exhibited an inhibition constant (IC50) in this assay of 3.5 nM, 14.8-fold better than 11055 (52 nM), consistent with the improved binding affinity (
A HUVEC cell proliferation assay was used to assess the ability of the D-peptidic compounds to block VEGF-A signaling. Here, HUVEC cell proliferation is increased in the presence of recombinant VEGF-A and antagonist compounds that block VEGF-A signaling reduce HUVEC cell proliferation. The apparent IC50 of compound 979110 in the HUVEC assay was 131 nM, which is 4-fold more potent than parent compound 11055, but remains 185-fold weaker than bevacizumab (Avastin), (
The structures of VEGF-A in complex with VEGF receptors, VEGFR1 and VEGFR2 are available and reveal multivalent interactions between Ig-like domains of VEGFR1 or VEGFR2 and two identical binding sites on the VEGF-A homodimer (Markovic-Mueller et al., Structure (2017), 25, 341-352)(Brozzo et al., Blood (2012), 119(7), 1781-1788.). An overlay of the compound 11055/VEGF-A complex structure with VEGFR2 highlights significant overlap between the 11055 binding epitope and one of the Ig-like domains of VEGFR2 (Domain 2, D2) (See
A new phage display library based on the Z-domain scaffold was generated as a pVIII-fusion to M13 phage. Ten positions were selected within the Z-domain for randomization using kunkel mutagenesis with trinucleotide codons representing all amino acids except cysteine (
To further characterize the VEGF-A binding site for compound 978336 a 2.9 angstrom resolution crystal structure was solved of L-VEGF-A in complex with 978336. Diffraction quality crystals were grown in 0.1M Bis-Tris, pH 5.5, 0.15 M magnesium chloride, 25% w/v PEG 3350 using the hanging drop method. The structure was solved by molecular replacement. Two molecules of 978336 were bound to identical binding sites on a single VEGF-A homodimer (
Structure-based affinity maturation methods were used to improve upon the VEGF-A binding affinity of compound 978336. Based on the consensus sequence of VEGF-A binding polypeptides defined in
Given the D-peptidic antagonist compounds 11055 and 978336 bind to non-overlapping epitopes on VEGF-A and directly block both the D2 and D3 binding sites, we engineered a chemically linked conjugate of compounds 11055 and 978336 in order to assess the overall effect on binding to target and antagonistic activity. Both compounds 11055 and 978336 were chemically synthesized with additional N-terminal cysteine residues, which were conjugated with a bis-maleimide PEG8 linker using conventional methods to provide for an N-terminal to N-terminal linkage (
The new heterodimer, compound 979111, exhibited a VEGF-A binding affinity of 1.7 nM as measured by SPR (
To further improve both the affinity and potency of the D-peptidic compounds, a scheme was devised for the chemical linkage of the monomeric D-protein antagonists into a dimeric bivalent antagonist. Conceptually, two 980181 polypeptides are tethered to each other through their carbon-termini and then a polypeptide 979110 is site-specifically conjugated to each of the 980181 polypeptides in the dimer to provide a tetradomain D-protein that would mimic VEGF receptor engagement.
A synthesis strategy was developed whereby two components would be synthesized in parallel using solid-phase peptide synthesis methods and a single click conjugation step would assemble the full tetradomain compound for final purification (
Since the D-protein tetrdomain compound is capable of sub-nanomolar binding to VEGF-A, a more accurate characterization of its antagonistic activity could be obtained in the VEGF-A/VEGFR1 blocking ELISA using a sub-nanomolar concentration of VEGF-A and long-incubation equilibrium binding conditions. Specifically, 150 pM of VEGF-A was incubated overnight with the antagonist titrations, then incubated on plate-coated VEGFR1-Fc for 5 hr to allow any free VEGF-A to bind the receptor. Under these conditions, the affinity matured monomer 979110 had an IC50 of 7 nM while the D-protein tetadomain compounds exhibited potent IC50s of 128 pM (980870) and 163 pM (980871), in agreement with their sub-nanomolar binding affinities (
To translate these findings to VEGF signaling blockade, we used a cell-based assay for VEGFR2 signaling in a 293 luciferase reporter cell line. Here VEGF-A activates VEGFR2 signaling in 293 cells resulting in luciferase expression as a functional readout. Inhibition of VEGF-A signaling in this system results in a loss of luciferase signal. In an effort to mimic the ELISA conditions, 150 pM of VEGF-A was used to elicit a measurable luciferase signal and the antagonist were titrated to block this activity. Here, 979110 showed an IC50 of 6.1 nM while the tetradomain D-proteins showed sub-nanomolar IC50s of 180 pM (980870) and 90 pM (980871), in very good agreement with the in vitro ELISA results (
A chemically synthesized D-protein blocks VEGF signaling with antibody-like potency, exhibits efficacy in ophthalmic and oncology disease models, and evades the humoral anti-drug antibody response.
Mirror-image phage display and structure-guided optimization were used to engineer a fully synthetic D-protein that antagonizes VEGF-A using a receptor mimicry mechanism. Phage panning against mirror-image D-VEGF-A yielded independent proteins that bound canonical receptor interaction sites. Crystal structures guided affinity maturation and the design of a chemical linkage to create a heterodimeric D-protein that tightly bound natural VEGF-A, inhibiting signaling activity at picomolar concentrations. The D-protein VEGF antagonist described herein, prepared by total chemical synthesis, prevented vascular leakage in a rabbit eye model of wet age-related macular degeneration, slowed tumor growth in the MC38 syngeneic mouse tumor model and was non-immunogenic during treatment or following subcutaneous immunization.
D-Proteins are mirror-image molecules composed entirely of D-amino acids and the achiral amino acid glycine. D-proteins resist digestion by endogenous proteases, avoiding fragmentation into peptides required for immunologic presentation (1, 4, 8), and are reported to not stimulate an immune response, even when emulsified in a strong adjuvant and repeatedly administered by subcutaneous injection (1, 2).
The antagonist of VEGF of as described herein was able to completely block vascular leakage induced by VEGF-A in a rabbit eye model of wet AMD. Furthermore, cross-species activity against human and murine VEGF-A enabled demonstration of tumor growth inhibition in the MC38 syngeneic mouse model and lack of immunogenicity following treatment. In addition, there was complete absence of a humoral antibody response following repeated subcutaneous immunization with our D-protein antagonist emulsified in an adjuvant.
Mirror-Image Protein Phage Display
To develop a multivalent D-protein antagonist, protein binders to non-overlapping epitopes on VEGF-A were identified. The 53-residue GA domain and 58-residue Z domain proteins derived from bacterial protein G and protein A respectively (22, 23), were selected as two different 3-helix bundle scaffolds for phage display because of their high stability, small size, and ease of chemical synthesis. M13 phage display libraries were generated for the Z and GA-domain scaffolds containing 10 and 12 hard-randomized library positions, respectively (
Both GA and Z-domain hits were synthesized as D-proteins (RFX-11055 and RFX-978336, respectively) for further characterization as binders to the natural L-protein form of VEGF-A. Titrations of the D-protein binders performed against L-VEGF-A using surface plasmon resonance (SPR) revealed binding affinities of 43 nM for the GA-domain binder RFX-11055 and 168 nM for the Z-domain binder RFX-978336 (
Antagonists of VEGF-A signaling need to block the VEGF receptors from interacting with two binding sites formed at the interface of the symmetrical VEGF-A homodimer (16, 24). To assess VEGF antagonism, a non-equilibrium VEGF-A121 blocking ELISA was employed that measures binding of biotinylated VEGF-A isoform 121 (VEGF-A121-biot) to VEGFR1-Fc coated on a plate (Sup methods). Both RFX-11055 and RFX-978336 exhibited inhibition of VEGF-A121 binding to VEGFR1 with apparent IC50 values of 52 nM and 935 nM, respectively (
Structure-Guided Affinity Maturation of D-Protein VEGF-A Antagonists
In order to guide further optimization of the D-protein antagonists, two independent crystal structures of VEGF-A in complex with RFX-11055 and RFX-978336 at 2.3 Å and 2.9 Å, respectively were solved (
Based on the 3-helix bundle structure of RFX-11055, a seven-residue soft randomization library was designed to stabilize the packing between the N-terminal helix 1 and the helix 2-3 loop (
Affinity maturation of RFX-978336 involved selecting VEGF-A contact residues showing minimal conservation from the initial panning for further interrogation using soft-randomization. A total of 4 residues were selected and Kunkel mutagenesis was used to soft-randomize each residue (
The affinity-matured D-proteins were evaluated in a non-equilibrium VEGF-A121 blocking ELISA to measure their antagonistic activity. RFX-979110 blocked VEGF-A121 binding to VEGFR1-Fc with an IC50 of 3.5 nM, a 15-fold improvement over RFX-11055 and approaching the potency of bevacizumab, which had an IC50 of 1.8 nM in this assay (
Total Chemical Synthesis of a Heterodimeric D-Protein Antagonist of VEGF-A Signaling\
The affinity and potency of the monomeric D-proteins were enhanced by chemically linking them together, recapitulating the interactions between the VEGF receptor D2 and D3 domains and VEGF-A. Based on the structures of RFX-11055 and RFX-978336 bound to VEGF-A, and their similarity to RFX-979110 and RFX-980181, site-specifically linkage was carried out between them through chemically modified lysine side chains K19 and K7, respectively, using a Click reaction to create a heterodimeric D-protein construct designed to mimic natural receptor engagement (
SPR titrations of the heterodimeric D-protein RFX-980869 demonstrated ultra-high binding affinities with KD measurement of 0.07 nM, similar to that of bevacizumab at 0.16 nM (
To further characterize its antagonistic activity a VEGF-A121/VEGFR1 blocking ELISA was employed using a sub-nanomolar concentration of VEGF-A121 under long-incubation equilibrium binding conditions (Supplementary methods). Under these conditions, the affinity-matured monomer RFX-979110 showed an IC50 of 7.6 nM, while the D-protein heterodimer exhibited an IC50 value of 0.31 nM, in reasonable agreement with the affinity measured by SPR (
To demonstrate the effects of these D-protein antagonists on VEGF signaling a cell-based luciferase reporter assay was used driven by VEGFR2 receptor activation. In this assay, 150 pM of VEGF-A activates VEGFR2 signaling causing an increase in luciferase expression, while inhibition of VEGF-A results in a decrease in luciferase expression. The monomeric D-protein RFX-979110 had an IC50 of 6.1 nM while the heterodimeric D-protein RFX-980869 exhibited a sub-nanomolar IC50 value of 0.49 nM, equivalent to bevacizumab (IC50 of 0.53 nM) in blocking VEGFR2 signaling (
RFX-980869 Exhibits Potent Activity In Vivo and is Non-Immunogenic
The activity of RFX-980869 was explored in a rabbit eye model for wet AMD and a syngeneic mouse tumor model in order to demonstrate applications in both ophthalmic and oncology diseases, respectively. In the rabbit eye model for wet AMD, intravitreal challenge with exogenous VEGF-A165 induces vascular leakage of the retina that can be monitored using fluorescein angiography (FA). VEGF-A blockade can prevent the diffuse leakage of fluorescein into the eye, which serves as a measure of efficacy. Here, we tested RFX-980869 for dose-dependent efficacy and durability in comparison to aflibercept. After a single intravitreal administration of RFX-980869 at 0.25 mg or 1.0 mg per eye, rabbits were challenged with exogenous VEGF-A twice over a 1-month period (Day 2 and Day 23) and their eyes were examined three days later (Day 5 and Day 26). Notably, a single dose of RFX-980869 at either 0.25 mg or 1 mg was able to significantly block the vascular leakage observed in control eyes following both VEGF challenges (
To assess the tumor growth inhibition potential of RFX-980869, the cross-reactivity of RFX-980869 with mouse VEGF-A (data not shown) was studied and used the syngeneic MC38 mouse tumor model. MC38 colon cancer tumors were established in C57BL6 mice transgenic for human PD-1, and reached 82 mm3 prior to initiation of treatment. Nivolumab was used as a positive control since we could not find published precedence for the efficacy of VEGF-A antagonists in a syngeneic MC38 tumor model. RFX-980869 dosed daily at 6 mg/kg for 2 weeks exhibited inhibition of tumor growth similar to nivolumab dosed biweekly at 3 mg/kg (
To highlight the non-immunogenic potential of our heterodimeric D-protein antagonist, mouse serum was analyzed for anti-drug-antibodies (ADAs) at the termination of the tumor study. In this fully immuno-competent mouse tumor model, plasma from both the low and high dose RFX-980869 treatment groups exhibited a complete lack of an IgG titer response against RFX-980869, while the nivolumab treatment groups had saturating levels of IgG titer (
Discussion
Mirror-image protein phage display and structure-guided optimization was used to independently mature two different 3-helix bundles into D-protein antagonists that occupied the D2 and D3 binding sites on VEGF-A (
VEGF-A surface area, achieving picomolar affinity while replicating a mechanism that closely resembles VEGF receptor binding. By blocking all four receptor interaction sites on VEGF-A, the resulting neutralization of VEGF-A is likely to be irreversible on the timescale of turnover and clearance in vivo. Like aflibercept, which utilizes a receptor decoy mechanism to block VEGF-A (25, 26), the heterodimeric D-protein VEGF antagonist described herein also uses receptor mimicry, blocking all of the VEGF receptor binding sites on VEGF-A albeit in a much smaller, chemically synthesized D-protein format.
The heterodimeric D-protein VEGF antagonist described herein is half the size of brolucizumab, is readily soluble in PBS (pH 7.4), and is amenable to high-dose formulations. Its small size enables better retinal penetration and rapid systemic clearance after leaving the eye. Moreover, its properties including increased proteolytic stability and lack of immunogenicity provide further advantages in the durability of a therapeutic response, lower inflammation, and an absence of ADA from long-term chronic treatment.
Protein Synthesis Reagents
Fmoc-D-amino acids were purchased from Chengdu Zhengyuan Company, Ltd and Chengdu Chengnuo New-Tech Company, Ltd. Fmoc-D-Ile-OH was purchased from Chemimpex International, Inc. Fmoc-D-propargylglycine (Fmoc-D-Pra-OH) was purchased from Haiyu Biochem. MBHA Resin was purchased from Sunresin New Materials Co. Ltd., Xian Rink Amide linker was purchased from Chengdu Tachem Company, Ltd. Chloro-(2-Cl)-trityl-resin was purchased from Tianjin Nankai Hecheng Science and Technology Company, Ltd. Fmoc-NH2(PEG)n-COOH and other PEG linkers were purchased from Biomatrik Inc. 2-Azidoacetic acid was purchased from Amatek Scientific Company Ltd. Sodium ascorbate was purchased from TCI (Shanghai) Ltd. Copper sulfate pentahydrate (CuSO4.5H2O) was purchased from Energy Chemical.
D-VEGF-A Synthesis and Refolding
The D-VEGF-A polypeptide chain (COOH acid, residues 8-109 (33)) was chemically synthesized using solid phase peptide synthesis (SPPS) and native chemical ligation, and folded to form the protein covalent homodimer, using methods adapted from our previous work (21). Individual peptide fragments corresponding to 1: Gly1-to-D-Tyr18, 2: D-Cys19-to-D-Arg49, 3: D-Cys50-to-D-Asp102, were synthesized using standard Fmoc chemistry protocols for stepwise SPPS. Fragments 1 and 2 were synthesized on NH2NH-(2-Cl)trityl-resin and fragment 3 was synthesized from pre-loaded Wang Resin. Briefly, preloaded Fmoc-aminoacyl-Wang Resin was initially swelled with DMF (10 mL/g) for 1 hour, then treated with 20% piperidine/DMF (30 min) to remove the Fmoc group and washed again with DMF (5 times). Fmoc-D-amino acid residues were coupled by addition of a pre-activated solution of 3 equivalents each of protected amino acid (0.4 M in DMF), diisopropylcarbodiimide (DIC), and hydroxybenzotriazole (HOBt) to the resin. After 1-2 h, the ninhydrin test showed the reaction was completed and the resin was washed with DMF (3 times). To remove the Fmoc group, piperidine (20% in DMF) was added to the resin for 30 min. After removal of the final Fmoc group, the resin was rinsed with DMF (3 times) and MeOH (2 times), dried under vacuum, then taken up in 85% TFA, 5% thioanisole, 5% EDT, 2.5% phenol and 2.5% water for cleavage. After 2 h, the resin was washed with TFA and the eluted peptide was concentrated by bubbling nitrogen gas. The crude peptides were precipitated with cold ether, pelleted by centrifugation, and washed with cold ether 2 times before drying under vacuum. Peptide residue was dissolved in water, purified by preparative reverse phase HPLC and analyzed by HPLC and MS.
Ligations between D-peptide-hydrazide fragments and D-Cys-peptide fragments were performed as follows: D-Peptide-hydrazide was dissolved in Buffer A (0.2M sodium phosphate containing 6 M GnHCl, pH 3.0), cooled to −15° C. in an ice-salt bath, and gently stirred by magnetic stirrer. NaNO2 (7 equivalents) was added and the solution stirred for 20 min to oxidize the D-peptide-hydrazide to the peptide-azide. A solution of 4-mercaptophenyl acetic acid (MPAA) (50 eq) dissolved in Buffer B (0.2M sodium phosphate containing 6 M GnHCl, pH 7.0) was quickly added to the solution containing the newly-formed D-Peptide-azide (equal volume) to eliminate excess NaNO2 and to convert the peptide-azide to the peptide-MPAA thioester. Then a solution of D-Cys-peptide in Buffer B (equal volume) was added to the solution containing the newly formed peptide-MPAA thioester. And the reaction mixture was adjusted to pH 7 with NaOH to initiate overnight native chemical ligation. Reaction progress was monitored by analytic RP-HPLC until completion, then treated by TCEP before HPLC purification.
Purification of the ligated peptide product was performed on a CXTHLC6000/Hanbon NU3000 prep system on Phenomenex C18/YMC C4 silica with columns of dimension 21.2×250 mm/20.0×250 mm. Crude peptides were loaded onto the prep column and eluted at a flow rate of 5 mL per minute with a shallow gradient of increasing concentrations of solvent B (0.1% TFA in 80% acetonitrile) in solvent A (0.1% TFA in water). Fractions containing the purified target peptide were identified by analytical LC-MS, combined, and lyophilized.
Final linear D-VEGF-A peptide was folded at pH 8.4 in aqueous Gu.HCl (0.15 M) containing a glutathione-reduced (2 mM)/glutathione-oxidized (0.4 mM) redox couple and stirred for 5 days to reach completion (21). Folded D-VEGF-A was purified by RP-HPLC.
Phage Display Libraries and Panning
Naïve GA- and Z-domain scaffold libraries were constructed as fusions to the N-terminal gene 8 major coat protein by previously described methods (34). Randomization of desired library positions (
All phage selections were executed according to previously established protocols (34). Briefly, selections with the peptide libraries were performed using biotinylated D-VEGF captured with streptavidin-coated magnetic beads (Promega). Initially, three rounds of selection were completed with decreasing amounts of D-VEGF (2.0 mM, 1.0 mM, and 0.5 mM). The phage pools were then transferred to a N-terminal gene 3 minor coat protein display vector and subjected to an additional three rounds of panning with decreasing amounts of D-VEGF (200 nM, 100 nM, and 50 nM) and increased wash times. Individual phage clones were then sent in for sequencing analysis.
Synthesis of D-Protein Binders
The polypeptide chains of the affinity matured D-proteins RFX-979110 and RFX-98018 (
Synthesis of the D-Protein Heterodimer
Step 1: Preparation of Azido-PEG3-D-979110 Resin.
Fmoc-aminoacyl-Rink Amide MBHA Resin was swelled in DMF (10-15 mL/g resin) for 1 hour. The suspension was filtered, exchanged into DMF containing 20% piperidine, and kept at room temperature for 0.5 hr under continuous nitrogen gas perfusion. The resin was then washed 5 times with DMF. Fmoc-D-amino acid-OH, DIC, HOBt and DMF were added to the resin. The suspension was kept at room temperature for 1 hr while a stream of nitrogen was bubbled through it. The ninhydrin test was used to monitor the coupling reaction until completion. The remaining D-amino acids corresponding to the affinity matured D-protein RFX-979110 were coupled to the peptidyl resin sequentially. Azido-PEG3-COOH was coupled to the primary amine of Lys19. After assembly of the amino acid sequence of the protected RFX-979110 polypeptide chain was complete, the final Fmoc group was removed by treatment with DMF containing 20% piperidine. The peptidyl-resin was washed with DMF (5 times), MeOH (2 times), DCM (2 times) and MeOH (2 times), then dried under vacuum overnight.
Step 2: Cleavage and Deprotection of Azido-PEG3-D-979110-Resin.
Cleavage solution (TFA/Thioanisole/EDT/Phenol/H2O=87.5/5/2.5/2.5/2.5 v/v, 10 mL/g peptide Resin) was added to the dried Azido-PEGS-D-979110-resin. The suspension was shaken for 3 h and was filtered and the filtrate collected. Cold ether was added to the filtrate to precipitate the peptide which was recovered by centrifugation. The white precipitate was washed with ether twice, then dried under vacuum overnight to give crude Azido-PEG3-D-979110 as a white solid.
Step 3: Oxidation and Purification. Crude Azido-PEG3-D-979110 was oxidized using I2.
Briefly, peptide (23.5 mg) was dissolved in 11 mL of 30% ACN and mixed with 330 μL of CH3COOH. An I2/MeOH solution was added dropwise until the mixture was pale yellow then aqueous sodium ascorbate was added dropwise to quench excess I2. Purification of oxidized Azido-PEG3-D-979110 was performed on a CXTH LC6000/Hanbon NU3000 prep system on Phenomenex C18 silica with columns of dimension 21.2×250 mm. Crude peptides were loaded onto the prep column and eluted at a flow rate of 5 mL/min with a shallow gradient of increasing concentrations of solvent B (0.1% TFA in 80% acetonitrile in water) in solvent A (0.1% TFA in water). Fractions containing the pure target peptide were identified by analytical LC-MS, and were combined and lyophilized to give purified Azido-PEG3-D-979110 for subsequent click reaction with (Alkynyl-PEG2)-D-980181.
Step 4: Preparation ofAlkynyl-PEG2-D-980181 Resin.
Fmoc-aminoacyl-Rink Amide MBHA Resin was swelled in DMF (10-15 mL/g resin) for 1 hour. The suspension was filtered, exchanged into DMF containing 20% piperidine, and kept at room temperature for 0.5 hr under continuous nitrogen gas perfusion. The resin was then washed 5 times with DMF. Fmoc-D-amino acid-OH, DIC, HOBt and DMF were added to the resin. The suspension was kept at room temperature for 1 hr while a stream of nitrogen was bubbled through it. The ninhydrin test was used to monitor the coupling reaction until completion. The remaining D-amino acids corresponding to the affinity matured D-protein 980181 polypeptide chain were added to the sequentially, in order. Alkynyl-PEG2-COOH was coupled to the primary amine of Lys7. After assembly of the amino acid sequence of the protected RFX-979181 polypeptide chain was complete, the final Fmoc group was removed by treatment with DMF containing 20% piperidine.
The peptidyl-resin was washed with DMF (5 times), MeOH (2 times), DCM (2 times) and MeOH (2 times), then dried under vacuum overnight.
Step 5: Cleavage and Deprotection of Alkynyl-PEG2-D-980181.
Cleavage solution (TFA/TIS/H2O 95/2.5/2.5v/v, 10 mL/g peptide Resin) was added into the alkynyl-PEG2-D-980181 homodimer resin. The mixture was shaken for 3 h and the filtrate was collected. Cold ether was added to the filtrate to precipitate the peptide which was collected by centrifugation. The white precipitate was washed with ether twice and dried under vacuum overnight to give crude alkynyl-PEG2-D-980181 homodimer as a white solid.
Step 6: Purification.
Purification of crude alkynyl-PEG2-D-980181 homodimer was performed on a CXTH LC6000/Hanbon NU3000 prep system on YMC C4 silica with columns of dimension 21.2×250 mm. Crude peptides were loaded onto the prep column and eluted at a flow rate of 10 mL per minute with a shallow gradient of increasing concentrations of solvent B (0.1% TFA in 80% acetonitrile in water) in solvent A (0.1% TFA in water). Fractions containing the pure target peptide were identified by analytical LCMS, combined, and lyophilized to give purified alkynyl-PEG2-D-980181 homodimer used for the click reaction with azido-PEGn-D-979110.
Step 7: Click Reaction and Purification.
Azido-PEG3-D-979110 and the alkynyl-PEG2-D-980181 were dissolved in ethanol:H2O (v/v, 1:1), then 0.2 M CuSO4 was added to the reaction mixture, followed by the addition of 0.2M of sodium ascorbate, and the reaction mixture was stirred at 30° C. for 2 h. The reaction mixture was loaded onto RP-HPLC without further workup and purified by gradient elution as described above. Fractions containing the desired product were identified by LCMS, combined, and lyophilized. Observed mass (LC-MS): 13174.0 Da; Calculated masses (average isotope composition): 13176.8 Da.
LC-MS Analysis of D-Proteins
Analytical RP-HPLC was performed on a HP 1090 system with Waters C4/Phenomenex C18 silica columns (4.6×150 mm, 3.5 μm/4.6×150 mm, 5.0 μm particle size) at a flow rate of 1.0 mL/min (50° C. column temperature). Peptides were eluted from the column using a 1.0% B/min gradient of water/0.1% TFA (solvent A) versus 80% acetonitrile in water/0.1% TFA (solvent B). Peptide masses were obtained by in-line electrospray MS detection using an Agilent 6120 LC/MSD ion trap.
Surface Plasmon Resonance Affinity Measurements
Surface plasmon resonance (SPR) binding measurements were carried out on a Biacore S200 (GE). Biotinylated VEGF-A(8-109) was immobilized on a biotin CAPture chip (GE) and serial dilutions of D-proteins were flowed over the chip at 30 μL/min in running buffer (10 mM Hepes, pH 7.4, 150 mM NaCl, 0.05% P20). Association reactions were 60 seconds for RFX-11055, -978336, -979110 and -980181 and 120 seconds for RFX-980869. Dissociation reactions were carried out in running buffer for either 120 seconds (RFX-11055, -978336, -979110, -980181) or 360 seconds (RFX-980869). All measurements were carried out at 25° C. SPR data are representative of multiple independent titrations. Kinetic fits were performed using Biacore software using a global single site binding model.
Expression and Purification of VEGF-A for Crystallography
The gene sequence for the VEGF-A (8-109) polypeptide chain was cloned into the expression vector pET21b with a His6-tag and TEV cleavage site sequence added at the N-terminus. The recombinant plasmid was transformed into E. coli BL21-Gold, grown in LB medium supplemented with Ampicillin (100 μg/ml) and expression of the His-tagged protein was induced by 0.3 mM isopropyl-b-D-thiogalactoside (IPTG) at 16° C. overnight. Cells were harvested by centrifugation and then stored at −80° C.
Pelleted cells from 30 L of culture were resuspended in 1 L buffer A (20 mM Tris, pH 8.0, 400 mM NaCl) and then passed through high-pressure homogenization (3 cycles). His-tagged protein from supernatant was captured on a Ni-NTA resin column (30 ml). The column was washed with 20 CV of Buffer A containing 20 mM imidazole, 5 CV of Buffer C (20 mM Tris, pH 8.0, 1M NaCl) and 10 CV of buffer A containing 50 mM imidazole. The His6-tagged-TEV site-VEGF-A protein was eluted with a high concentration of imidazole (0.25 M) in buffer A (5 CV). The eluted protein was digested with TEV protease at a 1:20 ratio (TEV: Protein) and dialyzed against 5 L buffer (20 mM Tris, pH 8.0, 50 mM NaCl.) at 4° C. overnight. Cleaved sample was loaded onto a 2nd Ni-NTA column to remove free His-tag. Eluted VEGF-A protein was further purified by ion exchange chromatography on a Resource Q column (6 ml). A final SEC polishing step was performed using HiLoad 16/60 Superdex 75 pg column equilibrated with buffer A. Monodisperse VEGF-A peak fractions were identified by absorbance at 280 nm and were combined and concentrated to 10-15 mg/mL in the buffer A. Final purified VEGF-A(8-109) protein was 95% pure as assessed by SDS-PAGE analysis and the molecular weight was confirmed by direct injection MS.
Crystallography of VEGF-A/D-Protein Complexes
VEGF-A/RFX-11055 complex. Crystals for VEGF-A/RFX-11055 were grown by hanging drop vapor diffusion at 18° C. The drop was composed of 0.8 μL of VEGF-A/D-protein complex (2.72 mg/ml VEGF-A and 0.5 mM RFX-11055) mixed 1:1 with 0.8 μl of the crystallization solution containing 0.2 M Calcium Chloride, 0.1 M Tris pH 8.5, 18% w/v PEG 4000. Crystals were soaked in a cryo-protectant solution containing crystallization solution plus 20% (v/v) glycerol and were flash-frozen in liquid nitrogen. The diffraction data were collected at the Shanghai Synchrotron Radiation Facility beam line BL19U1 to 2.31 Angstroms resolution and processed in space group P212121 using XDS. The structure was solved by molecular replacement using Phaser with VEGF structure (PDB ID: 3QTK) as the search model. Structure refinement and model building on the initial model were iteratively performed between Refmac5 and Coot. There are two copies of the {VEGF-A plus RFX-11055} complexes in an asymmetric unit. The detailed data processing and structure refinement statistics are listed in
VEGF-A/RFX-978336 Complex.
Crystals for VEGF-A/RFX-978336 were grown by hanging drop vapor diffusion at 18° C. The drop was composed of 0.8 μL of VEGF-A/D-protein complex (5.44 mg/ml VEGF-A and 0.46 mM RFX-978336) mixed 1:1 with 0.80 of the crystallization solution containing 0.15 M Magnesium Chloride, 0.1 M Bis-Tris pH 5.5, 25% w/v PEG 3350. Crystals were soaked in a cryo-protectant solution containing crystallization solution plus 10% (v/v) glycerol and were flash-frozen in liquid nitrogen. The diffraction data were collected at ALS beam line 8.3.1 to 2.9 Angstroms resolution and indexed in space group P212121 using XDS. The structure was solved by molecular replacement using Phaser with VEGF structure (PDB ID: 3QTK) as the search model. Structure refinement and model building on the initial model were iteratively performed between Refmac5 and Coot. There are four copies of the {VEGF_A plus RFX-978336} complexes in an asymmetric unit. The detailed data processing and structure refinement statistics are listed in
VEGF-A121/VEGFR1-Fc Binding ELISAs
Biotinylated human VEGF-A121 (isoform 121) was purchased from Acro Biosystems (cat #VE1-H82E7). VEGFR-1-Fc was purchased from R&D Systems (cat #3516-FL-050).
Bevacizumab was manufactured by Genentech Inc. (Lot #3067997). In all cases, 1 μg/mL of VEGFR1-Fc was coated on MaxiSorp plates overnight at 4° C. The following day, coated wells were blocked with Super Block (Rockland) for 2 hr with shaking at room temp. For non-equilibrium ELISAs, titrations of D-proteins and Bevacizumab were incubated with 1.0 nM of biotinylated VEGF-A121 for 30 min before addition to blocked VEGFR1-Fc coated wells.
Antagonist/VEGF-A121 mixture was incubated on VEGFR1-Fc wells for 1 hr with shaking at room temp, washed 3 times with wash buffer (PBS, 0.05% Tween 20), and bound biotinylated VEGF-A121 was detected with streptavidin-HRP (ThermoFisher). For equilibrium binding ELISAs, titrations of D-proteins, bevacizumab, and soluble VEGFR1-Fc were incubated with 0.15 nM of biotinylated VEGF-A121 overnight at 4° C. before addition to blocked VEGFR1-Fc coated wells. Antagonist/VEGF-A121 mixture was incubated on VEGFR1-Fc wells for 5 hr with shaking at room temp and developed as above. Data plotted are mean±standard deviation of triplicate measurements. IC50 values were derived from 3-parameter fits using Prism (GraphPad) and the error reported is derived from fits.
VEGF Cell Signaling Assay
Measurement of VEGF cellular signaling was performed using the VEGF Bioassay (Promega). Briefly, HEK293 cells are engineered to express VEGFR-2 coupled to a luciferase response element (KDR/NFAT-RE HEK293). VEGF signaling through VEGFR-2 mediates expression of luciferase which can be quantified using bioluminescence. Plated cells are incubated in the presence of 0.15 nM VEGF-A165 plus D-protein or Bevacizumab titrations and incubated at 37° C., 5% CO2 for 6 hours. Following incubation Bio-Glo is added to wells according to the manufacturer's protocol and relative luminescence units (RLUs) were measured on a PerkinElmer 2300 Enspire Multimode plate reader. Data plotted are mean±standard deviation of triplicate measurements. IC50 values were derived from 3-parameter fits using Prism (GraphPad) and error reported are derived from fits.
Rabbit Wet AMD Model
Dutch Belted rabbits (1.5-2.5 kg) were purchased from Western Oregon Rabbit Company. aflibercept was purchased from Regeneron Pharmaceuticals. On Day 0 Rabbits were randomized into treatment groups (N=5 per group) and baseline ophthalmic exams were done prior to a single intravitreal injection (25 μL per eye) of RFX-980869 (0.25 mg or 1.0 mg) or Eylea (1.0 mg). Rabbits were challenged with in 1 μg VEGF-A165 in both eyes on Days 2 and 23. On Days 5 and 26 fluorescein angiography was performed on both eyes and images were taken to assess vascular leakage. Scoring of vascular leakage based on FA images was carried out at Day 5 and 26 (
MC38 Syngeneic Tumor Model in C57BL6 Mice
Female C57BL6 mice transgenic for human PD-1 (12-13 weeks) were purchased from Beijing Biocytogen Co.). Nivolumab was purchased from Bristol Myers Squibb, lot #AAY1999. MC38 tumor cells (1×106) were implanted subcutaneously in the right front flank and tumors were allowed to establish until the mean volume was 82 mm3. Mice were randomized into treatment groups (N=6 per group) on Day 0 when treatment initiation began. RFX-980869 at 2 mg/kg or 6 mg/kg was injected i.p. daily for 2 weeks (14 doses) and nivolumab at 1 mg/kg or 3 mg/kg was injected i.p. biweekly for 6 doses. All data is plotted as mean ±SEM.
Subcutaneous Immunization in BALB/c Mice
Adjuvant was purchased from TiterMax. Bevacizumab was purchased from Genentech/Roche. Female BALB/c mice (6-8 weeks) were randomized into immunization groups on Day 0 (n=5 per group) Immunizations were performed on Days 0, 21, 35 by subcutaneous injection of 25 μg of antigen. Antigens were emulsified in adjuvant (TiterMax) for injection on Day 0 and administered in PBS for Days 21 and 35. Serum pre-bleeds were performed on Days 0, 21, 35 prior to immunizations. Final bleeds for max titer response were taken on Day 42.
Although the particular embodiments have been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.
Accordingly, the preceding merely illustrates the principles of the invention. Various arrangements may be devised which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims.
This application claims the benefit of U.S. Provisional Patent Application No. 62/822,241, filed Mar. 22, 2019, and U.S. Provisional Patent Application No. 62/865,469, filed Jun. 24, 2019, which applications are incorporated herein by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62822241 | Mar 2019 | US | |
| 62865469 | Jun 2019 | US |
