WO2013138795A1 - Cell penetrating compositions for delivery of intracellular antibodies and antibody-like moieties and methods of use - Google Patents
Cell penetrating compositions for delivery of intracellular antibodies and antibody-like moieties and methods of use Download PDFInfo
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- WO2013138795A1 WO2013138795A1 PCT/US2013/032686 US2013032686W WO2013138795A1 WO 2013138795 A1 WO2013138795 A1 WO 2013138795A1 US 2013032686 W US2013032686 W US 2013032686W WO 2013138795 A1 WO2013138795 A1 WO 2013138795A1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/50—Fibroblast growth factor [FGF]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/36—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Actinomyces; from Streptomyces (G)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/22—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a Strep-tag
Definitions
- an agent intended for use as a therapeutic, diagnostic, or in other applications is often highly dependent on its ability to penetrate cellular membranes or tissues to access a target and/or induce a desired change in biological activity.
- many therapeutic drugs, diagnostic or other product candidates whether protein, nucleic acid, small organic molecule, or small inorganic molecule, show promising biological activity in vitro, many fail to reach or penetrate target cells to achieve the desired effect, often due to physiochemical properties that result in inadequate biodistribution in vivo.
- Adequate delivery into a cell or cellular compartment of interest is a particularly acute problem for larger molecules, such as antibodies and antibody-like moieties.
- proteins such as antibodies
- proteins do not penetrate cells well. It is of great interest for protein-based therapeutics, diagnostics and biological assays to identify methods and compositions that facilitate delivery of polypeptides into a cell.
- compositions and methods for delivering antibodies and antibody-mimic moieties (referred to herein as "AAM moieties” or "an AAM moiety") into a cell.
- AAM moieties referred to herein as "AAM moieties” or "an AAM moiety"
- the present disclosure is based, at least in part, on the discovery that an AAM moiety can be delivered into a cell by complexing the AAM moiety with a cell penetrating polypeptide having surface positive charge (referred to herein as a "Surf+ Penetrating Polypeptide”) .
- the present disclosure is exemplary of the important applications of Intraphilin technology.
- complexes, as well as methods for making and using such complexes comprising a Surf+ Penetrating Polypeptide portion and an AAM moiety portion.
- the disclosure provides a complex comprising a Surf+
- the AAM moiety binds to an intracellular target distinct from the Surf+ Penetrating Polypeptide.
- the target of the AAM moiety is not the Surf+ Penetrating Polypeptide to which that AAM moiety is complexed to.
- the disclosure provides a complex comprising (or consisting of) a first portion comprising a Surf+ Penetrating Polypeptide and a second portion comprising an AAM moiety that binds an intracellular target.
- the AAM moiety binds to an intracellular target distinct from the Surf+ Penetrating Polypeptide.
- the target of the AAM moiety is not the the Surf+ Penetrating Polypeptide to which that AAM moiety is complexed to.
- the disclosure provides a fusion protein comprising a Surf+
- the disclosure provides a fusion protein comprising a first polypeptide portion comprising a Surf+ Penetrating Polypeptide and a second polypeptide portion comprising an AAM moiety that binds to an intracellular target.
- the fusion protein is a single polypeptide chain.
- the disclosure provides a complex comprising (a) a polypeptide selected from the group consisting of: agouti-signaling protein precursor, band 3 anion transport protein, B-cell lymphoma 6 protein isoform 1 ,
- BCL2/adeno virus E1B 19 kDa protein-interacting protein 3, beta-defensin 1 preproprotein, cathepsin E isoform a preproprotein, charged multivesicular body protein 6, cpG-binding protein isoform 2, C-X-C motif chemokine 10 precursor, epidermal growth factor receptor isoform a precursor, histone acetyltransferase MYST3, histone acetyltransferase p300, homeobox protein Nkx-3.1,
- lethal(3)malignant brain tumor-like protein 2 male-specific lethal 3 homolog isoform a, Na(+)/H(+) exchange regulatory cofactor NHE-RF1, peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 , peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 , POU domain class 2-associating factor 1 , prostatic acid phosphatase isoform PAP precursor, receptor tyrosine-protein kinase erbB-2 isoform b, receptor tyrosine-protein kinase erbB-3 isoform 1 precursor, receptor tyrosine-protein kinase erbB-4 isoform JM-a/CVT-2 precursor, RING1 and YY1 -binding protein, sterol regulatory element- binding protein 2, stromal cell-derived factor 1 isoform gamma, talin-1, T-cell surface glycoprotein CD4 isoform 1 precursor,
- the AAM moiety binds to an intracellular target distinct from the polypeptide associated with the AAM moiety in said complex and/or the complex is a fusion protein.
- the target of the AAM moiety is not the the Surf+ Penetrating Polypeptide to which that AAM moiety is complexed to.
- Complexes and fusion proteins include, in certain embodiments, a single polypeptide chain.
- the disclosure provides a complex comprising (a) a polypeptide selected from the group consisting of: agouti-signaling protein precursor, band 3 anion transport protein, B-cell lymphoma 6 protein isoform 1 ,
- BCL2/adeno virus E1B 19 kDa protein-interacting protein 3, beta-defensin 1 preproprotein, cathepsin E isoform a preproprotein, charged multivesicular body protein 6, cpG-binding protein isoform 2, C-X-C motif chemokine 10 precursor, epidermal growth factor receptor isoform a precursor, histone acetyltransferase MYST3, histone acetyltransferase p300, homeobox protein Nkx-3.1,
- lethal(3)malignant brain tumor-like protein 2 male-specific lethal 3 homolog isoform a, Na(+)/H(+) exchange regulatory cofactor NHE-RF1, peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 , peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 , POU domain class 2-associating factor 1 , prostatic acid phosphatase isoform PAP precursor, receptor tyrosine-protein kinase erbB-2 isoform b, receptor tyrosine-protein kinase erbB-3 isoform 1 precursor, receptor tyrosine-protein kinase erbB-4 isoform JM-a/CVT-2 precursor, RING1 and YY1 -binding protein, sterol regulatory element- binding protein 2, stromal cell-derived factor 1 isoform gamma, talin-1, T-cell surface glycoprotein CD4 isoform 1 precursor,
- the AAM moiety binds to an intracellular target distinct from the polypeptide associated with the AAM moiety in said complex and/or the complex is a fusion protein.
- the target of the AAM moiety is not the the Surf+ Penetrating Polypeptide to which that AAM moiety is complexed to.
- Complexes and fusion proteins include, in certain embodiments, a single polypeptide chain.
- the disclosure provides a complex comprising (a) a polypeptide comprising an amino acid sequence at least 85%, 90%>, 95%, 96%, 97%, 98%), 99%), or 100& identical to any of the amino acid sequences set forth in Section 2 of the sequence listing and identified in such sequence listing by PDB identifier, or a domain thereof having surface positive charge, a mass of at least 4 kDa, and a charge/molecular weight ratio of at least 0.75 and (b) an AAM moiety.
- the AAM moiety binds to an intracellular target distinct from the polypeptide associated with the AAM moiety in said complex and/or the complex is a fusion protein.
- the target of the AAM moiety is not the the Surf+ Penetrating Polypeptide to which that AAM moiety is complexed to.
- the polypeptide of (a) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions relative to the sequence of any of the amino acid sequences set forth in Section 2 of the sequence listing and identified in such sequence listing by PDB identifier, or a domain thereof having surface positive charge, a mass of at least 4 kDa, and a charge/molecular weight ratio of at least 0.75.
- the amino acid substitutions are conservative substitutions. In other embodiments, at least half of the substitutions are conservative substitutions.
- the substitutions do not alter the net charge and/or charge/molecular weight of the polypeptide. In certain embodiments, the substitutions are intended to supercharge the polypeptide.
- Complexes and fusion proteins include, in certain embodiments, a single polypeptide chain.
- the disclosure provides a complex comprising (a) a polypeptide comprising an amino acid sequence at least 85%, 90%, 92%, 95%, 96%, 97%), 98%o, 99%), or 100% identical to any of the amino acid sequences set forth in Section 1 of the sequence listing and identified in such sequence listing by GenBank accession number, or a domain thereof having surface positive charge, a mass of at least 4 kDa, and a charge/molecular weight ratio of at least 0.75 and (b) an AAM moiety.
- the AAM moiety binds to an intracellular target distinct from the polypeptide associated with the AAM moiety in said complex and/or the complex is a fusion protein.
- the target of the AAM moiety is not the the Surf+ Penetrating Polypeptide to which that AAM moiety is complexed to.
- the polypeptide of (a) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions relative to the sequence any of the amino acid sequences set forth in Section 1 of the sequence listing and identified in such sequence listing by GenBank accession number, or a domain thereof having surface positive charge, a mass of at least 4 kDa, and a charge/molecular weight ratio of at least 0.75.
- the amino acid substitutions are conservative substitutions.
- at least half of the substitutions are conservative substitutions.
- the substitutions do not alter the net charge and/or
- substitutions are intended to supercharge the polypeptide.
- the complex comprises a linker (e.g., 1, 2, 3, 4, more than 4 linkers).
- a linker may interconnect the first and second portions of the complex.
- a linker may interconnect portions of the AAM moiety, such as a VH and VL domains of an scFv.
- the Surf+ Penetrating Polypeptide is a human polypeptide.
- the Surf+ Penetrating Polypeptide is a non- human polypeptide (e.g., mouse, rat, non-human primate) or is a non-naturally occurring protein or is a prokaryotic protein.
- the Surf+ Penetrating Polypeptide is a full-length, naturally occurring human polypeptide.
- the Surf+ Penetrating Polypeptide is a domain of a full length, naturally occurring human polypeptide.
- the domain of a full length, naturally occurring human polypeptide has a charge/molecular weight ratio greater than that of the full length, naturally occurring human polypeptide. In other embodiments, the domain has a charge/molecular weight ratio of at least 0.75 but the full length, naturally occurring human polypeptide has a charge/molecular weight ratio of less than 0.75. In still other embodiments, the domain has a charge/molecular weight of at least 0.75 but the full length, naturally occurring polypeptide has a net negative charge.
- domains e.g., fragments have some level of structure
- domains of full length polypeptide may be compared to their full length polypeptide based on differences in net charge (e.g., the domain has a greater or lesser net charge; the domain has a net positive charge where the full length polypeptide has a net negative charge).
- the Surf+ Penetrating Polypeptide is a domain of a full length, naturally occurring human protein, and the complex does not include the full length, naturally occurring human protein.
- the Surf+ Penetrating Polypeptide is a domain of a full length, naturally occurring human protein, and the complex does not include the full length, naturally occurring human protein.
- Penetrating Polypeptide is a domain of a full length, naturally occurring human protein, and wherein the complex does not include sufficient additional amino acid sequence from said full length, naturally occurring human protein contiguous with said domain such that the charge/molecular weight of the first portion would be less than 0.75.
- the Surf+ Penetrating Polypeptide is a domain of a full length polypeptide, and the domain is less than or about 300, 250, 200, 175, 150, 140, 130, 125, 120, 110, or less than 100 amino acid residues. In other embodiments, the Surf+ Penetrating Polypeptide is a domain of a full length polypeptide, and the domain is less than or about 90, 80, 75, 70, 65, 60, 55, 50, or 45 amino acid residues.
- Surf+ Penetrating Polypeptides have a minimal mass of 4 kDa, and thus a suitable domain for use as a Surf+ Penetrating Polypeptide has a mass of at least 4 kDa.
- Surf Penetrating Polypeptides have surface positive charge and charge/molecular weight ratio of at least 0.75.
- suitable domains for use as a Surf+ Penetrating Polypeptide also meet this criteria. Numerous exemplary domains are identified herein.
- the size of the first portion of a complex of the disclosure can be described.
- the first portion may be less than or about 500, 450, 400, 350, 300, 250, 200, 175, 150, 140, 130, 125, 120, 110, or less than 100 amino acid residues.
- the first portion may be less than or about 90, 80, 75, 70, 65, 60, 55, 50, or 45 amino acid residues.
- the first portion of the complex comprises a Surf+ Penetrating Polypeptide.
- a region of the first portion will have the characteristics of a Surf+ Penetrating Polypeptide - even if those characteristics are not applicable when considered over the entire first portion (e.g., the Surf+ Penetrating Polypeptide region of the first portion has a charge/molecular weight ratio of at least 0.75, but the entire first portion does not). It should be noted that the foregoing sizes are emeplary, and Surf+ Penetrating Polypeptides or first portions that are larger are also contemplated.
- the Surf+ Penetrating Polypeptide has an endogenous function.
- the Surf+ Penetrating Polypeptide is a polypeptide having endogenous function as a DNA binding protein or is a domain of a full length polypeptide that has endogenous function as a DNA binding protein.
- the Surf+ Penetrating Polypeptide is a polypeptide having endogenous function as an RNA binding protein or is a domain of a full length polypeptide, which full length polypeptide has endogenous function as an RNA binding protein.
- Surf+ Penetrating Polypeptide is a polypeptide having endogenous function as a heparin binding protein or is a domain of a full length polypeptide, which full length polypeptide has endogenous function as a heparin binding protein.
- the Surf+ Penetrating Polypeptide is a polypeptide having endogenous function as a C-C or C-X-C class of chemokine or is a domain of a full length polypeptide, which full length polypeptide has
- complexes do not include Surf+ Penetrating
- the Surf+ Penetrating Polypeptide is not an antibody or an antigen binding fragment of an antibody.
- the AAM for use in a complex is a full length antibody molecule or an antigen binding fragment thereof, or a bispecific antibody or antibody fragment.
- the AAM moiety is a camelid antibody, an IgNAR, or an antibody like molecule comprising a target binding domain engineered into an Fc domain of the antibody like molecule.
- the AAM moiety comprises an antibody-mimic comprising a protein scaffold, such as a fibronectin-based scaffold.
- the AAM moiety comprises a DARPin polypeptide, an Adnectin ® polypeptide or an Anticalin ® polypeptide.
- the AAM moiety comprises: a target binding scaffold from Src homology domains (e.g. SH2 or SH3 domains), PDZ domains, beta-lactamase, high affinity protease inhibitors, an EGF-like domain, a Kringle-domain, a PAN domain, a Gla domain, a SRCR domain, a Kunitz/Bovine pancreatic trypsin Inhibitor domain, a Kazal-type serine protease inhibitor domain, a Trefoil (P-type) domain, a von Willebrand factor type C domain, an Anaphylatoxin-like domain, a CUB domain, a thyroglobulin type I repeat, LDL-receptor class A domain, a Sushi domain, a Link domain, a Thrombospondin type I domain, a C-type lectin domain, a MAM domain, a von Willebrand factor type A domain, a Somatomedin B domain, a
- the two portions or components of the complex are associated non-covalently. In other embodiments, they are associated covalently. Associations may be direct or via a linker, including via a cleavable linker. The two portions of the complex may be associated via both covalent and non-covalent interactions.
- the complex is a fusion protein (e.g., the Surf+ Penetrating Polypeptide or portion comprising the Surf+ Penetrating Polypeptide is fused, directly or via a linker, to the AAM moiety or portion comprising the AAM moiety). Suitable fusion proteins include, for example, fusion as a single polypeptide chain.
- the Surf+ Penetrating Polypeptide has an overall net positive charge of +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, or greater than +20. In other embodiments, the Surf+ Penetrating Polypeptide has an overall net charge of +5 to +17, +4-+10, +3-+8, +5-+14, +7-+15, and the like. Similarly, Surf+ Penetrating Polypeptides with a range of
- the Surf+ Penetrating Polypeptide has a mass of about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or about 15 kDa.
- larger Surf+ Penetrating Polypeptides are also contemplated and described herein.
- the Surf+ Penetrating Polypeptide is a domain of naturally occurring ataxin-7 isoform a, C-C motif chemokine 24 precursor or cytochrome c, which domain has surface positive charge and a charge/molecular weight ratio greater than that of its corresponding naturally occurring, full length polypeptide.
- An exemplary domain is provided in Figures 1 and 2.
- other suitable domains include a small domain of any of those described in Figure 1 or 2 having a mass of 4 kDa, surface positive charge, and charge/molecular weight ratio of at least 0.75.
- the Surf+ Penetrating Polypeptide is a naturally occurring protein selected from C-C motif chemokine 24 precursor, beta-defensin 103 precursor, cytochrome c, fibroblast growth factor 10 precursor, signal recognition particle 14 kDa protein, C-X-C chemokine 14 precursor or fibroblast growth factor 8 isoform B precursor, or a domain of any of the foregoing, which domain has surface positive charge and a charge/molecular weight ratio of at least 0.75.
- An exemplary domain is provided in Figures 1 and 2. However, other suitable domains include a small domain of any of those described in Figure 1 or 2 having a mass of 4 kDa, surface positive charge, and charge/molecular weight ratio of at least 0.75.
- the Surf+ Penetrating Polypeptide is: a full length polypeptide or a domain of C-C motif chemokine 26 precursor; a domain of HB-EGF (proheparin-binding EGF-like growth factor precursor); a domain of protein DEK isoform 1; a domain of hepatocyte growth factor isoform 1 preprotein; a full length polypeptide or a domain of cytochrome c; a full length polypeptide or domain of C-X- C motif chemokine 24 precursor; or a domain of ataxin 7 isoform a.
- HB-EGF proheparin-binding EGF-like growth factor precursor
- a domain of protein DEK isoform 1 a domain of protein DEK isoform 1
- a domain of hepatocyte growth factor isoform 1 preprotein a full length polypeptide or a domain of cytochrome c
- a full length polypeptide or domain of C-X- C motif chemokine 24 precursor or a
- the Surf+ Penetrating Polypeptide is a domain of any of the following, which domain has a charge per molecular weight ratio of at least 0.75 but for which the corresponding full length naturally occurring polypeptide has a charge/molecular weight ratio of less than 0.75: histone-lysine N-methyltransferase MLL isoform 1 precursor; transcription factor AP-1; proheparin-binding EGF-like growth factor precursor; protein DEK isoform 1 ; hepatocyte growth factor isoform 1 preprotein; epidermal growth factor receptor isoform a precursor; forkhead box protein K2; pre-mR A-processing factor 40 homolog A; ataxin-7 isoform a, E3 SUMO-protein ligase PIAS 1 ; platelet factor 4 precursor; advanced glycosylation end product-specific receptor isoform 2 precursor; serol regulatory element-binding protein 2; histone acetyltransfer
- SAP30 heterochromatin protein 1-binding protein 3; lethal(3)malignant brain tumorlike protein 2; CCAAT/enhancer-binding protein beta; troponin T, cardiac muscle isoform 2; CREB-binding protein isoform B; cyclic AMP-dependent transcription factor ATF-2; cathepsin E isoform a preprotein; glycine receptior subunit alpha- 1 isoform 1 precursor; CREB-binding protein isoform b; pituitary adenylate cyclase- activating polypeptide precursor; mastermind-like protein 1; BCL2/adeno virus E1B 19 kDa protein-interacting protein 3; cathelicidin antimicrobial peptide; epidermal growth factor receptor isoform a precursor; transcription factor NF-E2 45 kDa subunit isoform 2; integrin beta-1 isoform ID precursor.
- the Surf+ Penetrating Polypeptide is a domain of charged multivesicular body protein 6; homeobox protein Nkx3.1 ; B-cell lymphoma 6 protein isoform 1; lethal(3)malignant brain tumor-like protein 2; cathepsin E isoform a preprotein; BCL2/adeno virus E1B 19 kDa protein-interacting protein 3; cathelicidin antimicrobial peptide.
- the Surf+ Penetrating Polypeptide is any of the foregoing or following aspects or embodiments described herein.
- HEGF heparin-binding EGF-like growth factor precursor
- Polypeptide are also domains within the scope of the disclosure and suitable for use in a complex of the disclosure.
- the Surf+ Penetrating Polypeptide is a naturally occurring human polypeptide that is modified to increase its overall net charge (e.g., it is supercharged).
- the Surf+ Penetrating Polypeptide may be a polypeptide engineered to comprise an overall charge from about +10 to about +40.
- Supercharging can also be described as the change in charge relative to what it was prior to supercharging.
- the disclosure contemplates embodiments in which a polypeptide was supercharged by increasing its net charge from negative to positive, such as by increasing by +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +20, etc.
- the disclosure contemplates embodiments in which a polypeptide is supercharged to increase the net charge on an already positively charged polypeptide.
- supercharging may increase the net charge by +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +20, etc.
- the AAM moiety binds to a target and the target is a kinase, a transcription factor, or an oncoprotein. In other embodiments, the AAM moiety binds to a target and the target is NFAT-2, calcineurin, JAK-1, JAK-2, SOCS1, SOCS3, ras or Erk. In certain embodiments, the AAM moiety binds to a target which localizes to a subcompartment of a cell (e.g., nucleus, mitochondria, cytoplasm, or cytoplasmic face of cell membrane.
- a subcompartment of a cell e.g., nucleus, mitochondria, cytoplasm, or cytoplasmic face of cell membrane.
- the complex is a fusion protein comprising the Surf+ Penetrating Polypeptide and the AAM moiety, and wherein the Surf+ Penetrating Polypeptide is N-terminal to the AAM moiety.
- the complex is a fusion protein comprising the Surf+ Penetrating Polypeptide and the AAM moiety, and wherein the Surf+ Penetrating Polypeptide is C-terminal to the AAM moiety.
- the disclosure provides a nucleic acid comprising a nucleotide sequence encoding any of the Surf+ Penetrating Polypeptides disclosed herein, or a nucleotide sequence encoding a polypeptide portion comprising a Surf+ Penetratng Polypeptide disclosed herein.
- the disclosure provides a nucleic acid comprising a nucleotide sequence encoding any of the AAM moieties disclosed herein.
- the disclosue provides a nucleic acid comprising a nucleotide sequence encoding a fusion protein comprising a complex of the disclosure.
- the disclosure provides vectors comprising any of the nucleic acids of the disclosure, as well as host cells comprising such vectors, and methods of making polypeptides and complexes.
- the disclosure provides methods of deliverying an AAM moiety into a cell.
- the method is applicable to any of the complexes discussed herein. Such a complex is provided, and cells are contacted with the complex.
- the AAM moiety is delivered into the cell.
- the disclosure provides methods of inhibiting the activity of an intracellular target in a cell and methods of binding an intracellular target in a cell. Any of the complexes described herein, including complexes formed from any combination of Surf+ Penetrating Polypeptide portions and AAM moiety portions are suitable for use in such methods.
- the disclosure provides a composition comprising a complex of the disclosure and a pharmaceutically acceptable carrier. Any of the complexes described herein, including complexes formed from any combination of Surf+ Penetrating Polypeptide portions and AAM moiety portions are suitable for use in such a composition.
- a complex of the disclosure can penetrate a cell. Similarly, in certain embodiments, a complex of the disclosure binds to the target via the AAM moiety.
- FIG. 1 is table of human polypeptides.
- FIG. 2 is a table of a subset of the human polypeptides presented in FIG. 1.
- complexes comprising (i) a cell penetrating polypeptide having surface positive charge, called a Surf+ Penetrating Polypeptide, and (ii) an antibody or antibody-mimic molecule, such as a polypeptide comprising a protein scaffold, called an AAM moiety that binds to an intracellular target.
- a cell penetrating polypeptide having surface positive charge called a Surf+ Penetrating Polypeptide
- an antibody or antibody-mimic molecule such as a polypeptide comprising a protein scaffold, called an AAM moiety that binds to an intracellular target.
- nucleic acid molecules encoding such protein complexes or encoding the Surf+ Penetrating Polypeptide or AAM moiety portion of such protein complexes, as well as methods of making and using such complexes.
- the Surf+ Penetrating Polypeptide penetrates cells and, when complexed with the AAM moiety, promotes delivery of the AAM moiety into a cell (e.g.,
- the AAM moiety can bind its intracellularly expressed or localized target molecule and impact cellular activity based on its affect on the target molecule.
- an AAM moiety may bind to an intracellular target, such as a polypeptide or peptide, and alter the activity of the target and/or the activity of the cell via one or more of the following mechanisms (i) inhibit one or more functions of the target; (ii) activate one or more functions of the target; (iii) increase or decrease the activity of the target; (iv) promote or inhibit degradation of the target; (v) change the localization of the target; and (vi) prevent binding between the target and another protein (e.g., prevent binding between the target and a binding partner).
- the proteins and complexes described herein are provided for delivery of AAM moieties, e.g., therapeutic, diagnostic and research agents, to cells in vivo, ex vivo, or in vitro.
- the portions of the complexes of the disclosure may be associated via covalent or non-covalent interactions.
- Exemplary interconnections include fusions (direct or via a linker) via a peptide bond and fusions via chemical methods (direct or via a linker).
- the association between the two portions of the molecule may persist following internalization into a cell or may be transient. For example, if the two portions of a complex are covalently linked via a cleavable linker, the association may be disrupted after the Surf+ Penetrating Polypeptide portion successfully delivers the AAM moiety into a cell (e.g., once inside the cell, the complex may optionally be disrupted).
- This disclosure provides an exemplary application of Intraphilin TM technology in which a member of a class of Surf+ Penetrating Polypeptides is delivered into a cell or is used to deliver a cargo molecule into a cell.
- certain Surf+ Penetrating Polypeptides are complexed with an AAM moiety, and these complexes are useful for delivering the AAM moiety into cells.
- Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
- a heavy chain variable domain may include a single amino acid insertion (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82.
- the Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard” Kabat numbered sequence. Maximal alignment of framework residues frequently requires the insertion of "spacer" residues in the numbering system, to be used for the Fv region.
- complex of the disclosure is used to refer to a complex comprising a Surf+ Penetrating Polypeptide portion, such as any of the Surf+ Penetrating
- the AAM moiety which may be an antibody or an antibody-mimic, binds a target expressed or otherwise present in a cell, and the Surf+ Penetrating Polypeptide functions to deliver the AAM moiety into a cell.
- antibody and “antibodies”, also known as immunoglobulins, encompass monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies formed from at least two different epitope binding fragments (e.g., bispecific antibodies), human antibodies, humanized antibodies, camelised antibodies, chimeric antibodies, murine or other non-human antibodies, single-chain Fvs (scFv), Fab fragments, F(ab')2 fragments, antibody fragments that exhibit the desired biological activity (e.g. the antigen binding portion), disulfide-linked Fvs (dsFv), and anti-idiotypic (anti-Id) antibodies (including, e.g.
- Immunoglobulins include functional fragments accepted in the art, such as Fc, Fab, scFv, Fv, or other derivatives or combinations of the immunoglobulins, domains of the heavy and light chains of the variable region (such as Fd, VI, Vk, Vh) and the constant region of an intact antibody such as CHI , CH2, CH3, CH4, CI and Ck, as well as mini-domains consisting of two beta-strands of an immunoglobulin domain connected by a structural loop.
- antibodies include immunoglobulin molecules and immunologically active or other functional fragments of immunoglobulin molecules, i.e., molecules that contain at least one antigen-binding.
- Immunoglobulin molecules can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), subisotype (e.g., IgGl , IgG2, IgG3, IgG4, IgAl and IgA2) or allotype (e.g. , Gm, e.g. , Glm(f, z, a or x),
- Antibodies may be derived from any mammal, including, but not limited to, humans, monkeys, pigs, horses, rabbits, dogs, cats, mice, etc., or other animals such as birds (e.g. chickens).
- the term "about” in the context of a given value or range refers to a value or range that is within 20%, preferably within 10%, and more preferably within 5% of the given value or range. It is convenient to point out here that "and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.
- association means that these portions are physically associated or connected with one another, either directly or via one or more additional moieties, including moieties that serve as a linking agent, to form a structure that is sufficiently stable so that the AAM moiety is delivered into a cell.
- the association may be via
- the complex may be a fusion protein in which the Surf+ Penetrating Polypeptide portion and the AAM moiety portion are connected by a peptide bond as a fusion protein, either directly or via a linker or other additional polypeptide sequence.
- the fusion protein is a single polypeptide chain.
- the AAM moiety binds to an intracellular target (e.g., a target expressed or present intracellularly) that is distinct from the Surf+ Penetrating Polypeptide present in the complex.
- an intracellular target e.g., a target expressed or present intracellularly
- Penetrating Polypeptides may be expressed endogenously inside a cell, in certain embodiments, the target molecule for the AAM moiety is not a Surf+ Penetrating Polypeptide and/or is not the same Surf+ Penetrating Polypeptide as present in that complex. In certain embodiments, the Surf+ Penetrating Polypeptide portion of a complex of the disclosure is not an antibody or antigen-binding fragment of an antibody. In certain embodiments, the Surf+ Penetrating Polypeptide portion of a complex of the disclosure is not an antibody mimic molecule.
- the term "supercharge” refers to any modification of a protein, the primary purpose of which is to increase the net charge or the surface charge of the protein to make that protein suitable for or to improve its suitability for use as a Surf+ Penetrating Polypeptide. Modifications include, but are not limited to, alterations in amino acid sequence or addition of positively charged moieties.
- a "Surf+ Penetrating Polypeptide”, as used herein, is a polypeptide capable of promoting entry into a cell and having, at least, the following characteristics: mass of at least 4 kDa, charge/molecular weight ratio of at least 0.75, and presence of surface positive charge such that the polypeptide is capable of promoting entry into a cell.
- the Surf+ Penetrating Polypeptide can itself enter into a cell and/or can be associated with an agent, such as an antibody or antibody mimic, such that it also promotes entry into the cell of the agent. In addition to having surface positive charge, the Surf+ Penetrating Polypeptide has a net positive charge.
- a Surf+ Penetrating Polypeptide may be a human polypeptide, including a full length, naturally occurring human polypeptide or a variant of a full length, naturally occurring human polypeptide having one or more amino acid additions, deletions, or substitutions. Moreover, such human polypeptides include domains of full length naturally occurring human polypeptides or a variant of such a domain having one or more amino acid additions, deletions, or substitutions. For the avoidance of doubt, the term "human polypeptide" includes domains (e.g., structural and functional fragments) unless otherwise specified.
- Surf+ Penetrating Polypeptides include human or non-human proteins engineered to have one or more regions of surface positive charge and a charge/molecular weight ratio of at least 0.75, including supercharged polypeptides.
- the present disclosure provides numerous examples of Surf+ Penetrating Polypeptides, as well as numerous examples of sub-categories of Surf+ Penetrating Polypeptides.
- the disclosure contemplates that any of the sub-categories of Surf+ Penetrating Polypeptides, as well as any of the specific polypeptides described herein may be provided as part of a complex comprising an AAM moiety. Moreover, any such complexes may be used to deliver an AAM moiety into a cell.
- a "variant of a human polypeptide” is a polypeptide that differs from a naturally occurring (full length or domain) human polypeptide by one or more amino acid substitutions, additions or deletions.
- these changes in amino acid sequence may be to increase the overall net charge of the polypeptide and/or to increase the surface charge of the polypeptide (e.g., to supercharge a polypeptide).
- changes in amino acid sequence may be for other purposes, such as to provide a suitable site for pegylation or to facilitate production.
- the variant of the human polypeptide will be sufficiently similar based on sequence and/or structure to its naturally occurring human polypeptide such that the variant is more closely related to the naturally occurring human protein than it is to a protein from a non-human organism.
- the amino acid sequence of the variant is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to a naturally occurring human protein.
- the variant of the naturally occurring human polypeptide is a Surf+ Penetrating Polypeptide having cell penetrating activity and a charge/molecular weight ratio of at least 0.75 or of greater than 0.75, but the naturally occurring human polypeptide from which the variant is derived does not have cell penetrating activity and/or has a charge/molecular weight ratio of less than 0.75.
- the variant does not result in further supercharging of the polypeptide.
- the variant results in a change in amino acid sequence but not a change in the net charge, surface charge and/or charge/molecular weight ratio of the polypeptide.
- the Surf+ Penetrating Polypeptide is a human polypeptide having surface positive charge, mass of at least 4 kDa and
- Such a human polypeptide may be a naturally occurring human polypeptide (which may also be a fragment of a naturally occurring human polypeptide), or a variant thereof having one or more amino acid additions, substitions, deletions, such as additions, substitutions or deletions that increase (or that do not change) surface positive charge,
- the Surf+ Penetrating Polypeptide is a human polypeptide that is a domain of a naturally occurring human polypeptide.
- the domain of a naturally occurring human polypeptide has a mass of at least 4 kDa and a
- the Surf+ Penetrating Polypeptide for use in the disclosure is a domain of a naturally occurring human polypeptide that has a charge/molecular weight ratio of at least 0.75 or of greater than 0.75, but the corresponding, full length, naturally occurring human protein has a charge/molecular weight ratio of less than 0.75.
- such a domain has an overall net positive charge greater than that of the corresponding, full length, naturally occurring human protein.
- a Surf+ Penetrating Polypeptide has a mass of at least 4, 5, 6, 10, 20, 50, 100, 200 kDa or 250 kDa.
- a Surf+ Penetrating Polypeptide may have a mass of about 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27 or 28 kDa.
- a Surf+ Penetrating Polypeptide may have a mass of about 4-30 kDa, about 5-25 kDa, about 4-20 kDa, about 5-18 kDa, about 5-15 kDa, about 4-12 kDa, about 5-10 kDa, and the like.
- the molecular weight of a Surf+ Penetrating Polypeptide may have a mass of at least 4, 5, 6, 10, 20, 50, 100, 200 kDa or 250 kDa.
- a Surf+ Penetrating Polypeptide may have a mass of about 4, 5, 6, 7, 8, 9, 10, 1 1 , 12,
- Polypeptide e.g., a naturally occurring or modified Surf+ Penetrating Polypeptide protein ranges from approximately 5 kDa to approximately 250 kDa, such as 10 to 250kDa, 50 to 250kDa, or 50 to lOOkDa.
- the molecular weight of the Surf+ Penetrating Polypeptide ranges from approximately 4 kDa to approximately 100 kDa.
- the molecular weight of the Surf+ Penetrating Polypeptide ranges from approximately 10 kDa to approximately 45 kDa.
- the molecular weight of the Surf+ Penetrating Polypeptide ranges from approximately 5 kDa to approximately 50 kDa.
- the molecular weight of the Surf+ Penetrating Polypeptide ranges from approximately 5 kDa to approximately 27kDa. In certain embodiments, the molecular weight of the Surf+ Penetrating Polypeptide ranges from approximately 10 kDa to approximately 60 kDa.
- the molecular weight of the Surf+ Penetrating Polypeptide is about 5 kD, about 7.5 kDa, about 10 kDa, about 12.5 kDa, about 15 kDa, about 17.5 kDa, about 20 kDa, about 22.5 kDa, about 25 kDa, about 27.5 kDa, about 30 kDa, about 32.5 kDa, or about 35 kDa.
- the mass of the Surf+ Penetrating Polypeptide including the minimal mass of 4 kDa, refers to monomer mass.
- a Surf+ Penetrating Polypeptide for use as part of a complex is a dimer, trimer, tetramer, or a higher order multimer.
- a Surf+ Penetrating Polypeptide for use in the present disclosure is selected to minimize the number of disulfide bonds.
- the Surf+ Penetrating Polypeptide may have not more than 2 or 3 or 4 disulfide bonds (e.g., the polypeptide has 0, 1 , 2, 3 or 4 disulfide bonds).
- Polypeptide for use in the present disclosure may also be selected to minimize the number of cysteines.
- the Surf+ Penetrating Polypeptide may have not more than 2 cysteines, or not more than 4 cysteines, not more than 6 cysteines or not more than 8 cysteines (e.g., 0, 1 , 2, 3, 4, 5, 6, 7, 8 cysteines).
- a Surf+ Penetrating Polypeptide for use in the present disclosure may also be selected to minimize glycosylation sites.
- the polypeptide may have not more than 1 or 2 or 3 glycosylation sites (e.g., N-linked or O-linked glycosylation; 0, 1 , 2 or 3 sites).
- a Surf+ Penetrating Polypeptide has surface positive charge.
- the Surf+ Penetrating Polypeptide also has an overall net positive charge under physiological conditions. Note that when the Surf+ Penetrating Polypeptide is a domain of a naturally occurring polypeptide, the overall net positive charge is that of the domain.
- the Surf+ Penetrating Polypeptide has an overall net positive charge of at least +4, +5, +10, +15, +20, +25, +30, +35, +40, or +50.
- a Surf+ Penetrating Polypeptide may have an overall net positive charge of about +4, +5, +6, +7, +8, +9, +10, +1 1 , +12, +13, +14, +15, +16, +17, +18, +19, +20, +21 , +22, +23, +24, +25, or greater than +25.
- the Surf+ Penetrating under physiological conditions, the Surf+ Penetrating
- Polypeptide has a pi greater than or equal to 9, such as a pi of about 9 to about 13 or a pi of between 9 and 13 (inclusive or exclusive). In other embodiments, under physiological conditions, the Surf+ Penetrating Polypeptide has a pi greater than 9 or greater than 9.5, but less than 10. In other embodiments, under physiological conditions, the Surf+ Penetrating Polypeptide has a pi of about 9-9.5, or about 9-10, or about 9.5-10, or about 10-10.5, or about 10-10.3. In other embodiments, under physiological conditions, the Surf+ Penetrating Polypeptide has a pi of about 10-1 1 , about 10.5-1 1 , about 1 1-12, about 1 1.5-12, about 12-13, or about 12.5-13.
- a Surf+ Penetrating Polypeptide may be a polypeptide that has been modified, such as to increase surface charge and/or overall net positive charge as compared to the unmodified protein, and the modified polypeptide may have increased stability and/or increased cell penetrating ability in comparison to the unmodified polypeptide. In some cases, the modified polypeptide may have cell penetrating ability where the unmodified polypeptide did not.
- Theoretical net charge serves as a convenient short hand.
- the theoretical net charge on the Surf+ Penetrating Polypeptide e.g., the naturally occurring Surf+ Penetrating Polypeptide or the modified Surf+
- Penetrating Polypeptide is at least +1 , +2, +3, +4, +5, +6, +7, +8, +9, +10, +1 1 , +12, +13, +14, +15, +16, +17, +18, +19, +20, +21 , +22, +23, +24, +25, +30, +35, +40 or +50.
- the theoretical net charge on the Surf+ Penetrating Polypeptide is about +1 , +2, +3, +4, +5, +6, +7, +8, +9, +10, +1 1 , +12, +13, +14, +15, +16, +17, +18, +19, +20, +21 , +22, +23, +24, +25, +30, +35, +40 or +50.
- the theoretical net charge on the naturally occurring Surf+ Penetrating Polypeptide can be, e.g., at least +1 , at least +2, at least +3, at least +4, at least +5, at least +10, at least +15, at least +20, at least +25, at least +30, at least +35, at least +40 or at least +50 or about +1 to +5, +1 to +10, +5 to +10, +5 to +15, +10 to +20, +15 to +20, +20 to +30, +30 to +40, or +40 to +50 and the like.
- a Surf+ Penetrating Polypeptide may be a polypeptide that has been modified, such as to increase surface charge and/or overall net positive charge as compared to the unmodified protein, and the modified polypeptide may have increased stability and/or increased cell penetrating ability in comparison to the unmodified polypeptide. In some cases, the modified polypeptide may have cell penetrating ability where the unmodified polypeptide did not.
- the Surf+ Penetrating Polypeptide has a
- charge :molecular weight ratio e.g., also referred to as charge/MW or
- charge/molecular weight of at least approximately 0.75, 0.8, 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, or 3.0. This ratio is the ratio of the theoretical net charge of the Surf+ Penetrating Polypeptide to its molecular weight in kilodaltons. In certain embodiments, the charge/molecular weight is about 0.75-2.0. In certain embodiments, the charge/molecular weight ratio of the Surf+ Penetrating Polypeptide is greater than 0.75.
- the Surf+ Penetrating Polypeptide is a domain of a naturally occurring human polypeptide where the domain has a charge/molecular weight ratio of at least 0.75 or of greater than 0.75, but the corresponding full length, naturally occurring human polypeptide has a
- the Surf+ Penetrating Polypeptide has a charge :molecular weight ratio of at least approximately 0.75 or of greater than 0.75. In certain embodiments, the Surf+ Penetrating Polypeptide has a charge: molecular weight ratio of at least approximately 0.8. In certain embodiments, the Surf+
- Penetrating Polypeptide has a charge: molecular weight ratio of at least approximately 1.0. In certain embodiments, the Surf+ Penetrating Polypeptide has a charge:
- the Surf+ Penetrating Polypeptide has a charge: molecular weight ratio of at least approximately 1.4. In certain embodiments, the Surf+ Penetrating Polypeptide has a charge: molecular weight ratio of at least approximately 1.5. In certain embodiments, the Surf+ Penetrating Polypeptide has a charge: molecular weight ratio of at least approximately 1.6. In certain embodiments, the Surf+ Penetrating Polypeptide has a charge: molecular weight ratio of at least approximately 1.7. In certain embodiments, the Surf+ Penetrating Polypeptide has a charge: molecular weight ratio of at least approximately 1.8.
- the Surf+ Penetrating Polypeptide has a charge: molecular weight ratio of at least approximately 1.9. In certain embodiments, the Surf+ Penetrating Polypeptide has a charge: molecular weight ratio of at least approximately 2.0. In certain embodiments, the Surf+ Penetrating Polypeptide has a charge: molecular weight ratio of at least approximately 2.5. In certain embodiments, the Surf+ Penetrating Polypeptide has a charge: molecular weight ratio of at least approximately 3.0.
- the Surf+ Penetrating Polypeptide is a naturally occurring human polypeptide or a domain of a naturally occurring human
- a Surf+ Penetrating Polypeptide for use in this disclosure may have an endogenous function as, for example, a DNA binding protein, an R A binding protein or a heparin binding protein.
- the disclosure provides complexes in which the Surf+ Penetrating Polypeptide Portion is (i) a domain of a naturally occurring human polypeptide having a charge/molecular weight ratio of at least 0.75 or of greater than 0.75 but for which its naturally occurring, full length human polypeptide does not have a charge/molecular weight ratio of at least 0.75 and (ii) the domain is from a naturally occurring human polypeptide having an endogenous, natural function as a DNA binding protein, an R A binding protein or a heparin binding protein.
- the Surf+ Penetrating Polypeptide does not have an endogenous function as, for example, a DNA binding protein, an RNA binding protein or a heparin binding protein.
- the Surf+ Penetrating Polypeptide does not have an endogenous function as a histone or histone - like protein. In certain embodiments, the Surf+ Penetrating Polypeptide does not have an endogenous function as a homeodomain containing protein.
- the Surf+ Penetrating Polypeptide has tertiary structure.
- the presence of such tertiary structure distinguishes Surf+ Penetrating Polypeptides from unstructured, short cell penetrating peptides (CPPs) such as poly- arginine and poly-lysine and also distinguishes Surf+ Penetrating Polypeptides from cell penetrating peptides that have some secondary structure but no tertiary structure, such as penetratin and antenapedia.
- CCPs unstructured, short cell penetrating peptides
- Surf+ Penetrating Polypeptides from cell penetrating peptides that have some secondary structure but no tertiary structure, such as penetratin and antenapedia.
- the Surf+ Penetrating Polypeptide is not an antibody or an antigen-binding fragment of an antibody. As noted above, Surf+ Penetrating
- Polypeptides are distinguishable based on numerous characteristics from various short cell penetrating peptides known in the art. For example, Surf+ Penetrating
- Polypeptides are distinguishable based on size, shape and structure, charge
- Surf+ Penetrating Polypeptides and complexes comprising a Surf+ Penetrating Polypeptide have improved cell penetration characteristics compared to short CPPs or complexes comprises short CPPs. Nevertheless, to provide further clarity, in certain
- complexes of the disclosure do not further include a short CPP.
- Penetrating Polypeptide portion of a complex of the disclosure does not include a full length sequence for HIV-Tat, or the portion thereof known in the art as imparting cell penetration activity.
- a complex of the disclosure and/or the Surf+ Penetrating Polypeptide portion of a complex of the disclosure does not contain the protein transduction domain of HIV-Tat, for example, does not contain the contiguous amino acid sequence YGRKK RQRR (SEQ ID NO: 612).
- a complex of the disclosure comprising a Surf+ Penetrating
- Polypeptide penetrates cells more efficiently than a complex comprising all or a portion of HIV-Tat fused to the same cargo.
- Penetrating Polypeptide portion of a complex of the disclosure does not include the protein transduction domain of an antennapedia protein, such as the Drosophilia antennapedia protein or a mammalian ortholog thereof.
- a complex of the disclosure and/or the Surf+ Penetrating Polypeptide portion of a complex of the disclosure does not include the protein transduction domain of the h- region of fibroblast growth factor 4 (FGF-4).
- FGF-4 fibroblast growth factor 4
- a complex of the disclosure and/or the Surf+ Penetrating Polypeptide portion of a complex of the disclosure does not include an FGF polypeptide or a 16 residue cell penetrating polypeptide fragment thereof.
- Penetrating Polypeptide portion of a complex of the disclosure does not include the 16 amino acid residue sequence referred to as penetratin: RQIKIWFQNRRMKWK (SEQ ID NO: 613).
- a complex of the disclosure and/or the Surf+ Penetrating Polypeptide portion of a complex of the disclosure does not include the 19 amino acid residue sequence referred to as SynBl :
- a complex of the disclosure and/or the Surf+ Penetrating Polypeptide portion of a complex of the disclosure does not include the following amino acid sequence referred to as transportan:
- Penetrating Polypeptide portion of a complex of the disclosure does not include the following amino acid sequence RKMLKSTRRQRR.
- a complex of the disclosure and/or the Surf+ Penetrating Polypeptide portion of a complex of the disclosure does not include the amino acid sequence selected from one or more of the following amino acid sequences: YGRKKRRQRRR (SEQ ID NO:
- WLRRIKAWLRRIKA SEQ ID NO: 615
- WLRRIKAWLRRIKAWLRRIKA SEQ ID NO: 616
- WLRRIKAWLRRIKAWLRRIKA SEQ ID NO: 616
- AGGGGYGRKKRRQRRR (SEQ ID NO: 620);
- RKKRRRESRKKRRRES (SEQ ID NO: 633); GRPRESGKKRKRKRLKP (SEQ ID NO: 634).
- VKRGLKLRHVRPRVTRMDV SEQ ID NO: 639
- VKRGLKLRHVRPRVTRDV VKRGLKLRHVRPRVTRDV
- SRRARRSPRHLGSG SEQ ID NO: 641
- KTRYYSMKKTTMKIIPFNRL (SEQ ID NO: 644)
- LGTYTQDFNKFHTFPQTAIGVGAP (SEQ ID NO: 646); TSPLNIHNGQKL (SEQ ID NO: 6)
- Penetrating Polypeptide portion of a complex of the disclosure does not include HSV- 1 structural protein Vp22 (DAATATRGRSAASRPTERPRAPARSASRPRRPVE) (SEQ ID NO: 649).
- a complex of the disclosure and/or the Surf+ Penetrating Polypeptide portion of a complex of the disclosure does not include 9 (or, optionally, does not include 7 or 8) consecutive arginine residues (e.g., poly- Arg9).
- Penetrating Polypeptide portion of a complex of the disclosure does not include 9 (or, optionally, does not include 7 or 8) consecutive lysine residues (e.g., poly-Lys9).
- a complex of the disclosure and/or the Surf+ Penetrating Polypeptide portion of a complex of the disclosure does not include the PTD of mouse transcription factor Mph-1 (YARVRRRGPRR) (SEQ ID NO: 650), Sim-2
- AKAARQAAR SEQ ID NO: 651
- HIV-1 viral protein Tat YGRKKRRQRRR
- Antennapedia protein Antp
- the Surf+ Penetrating Polypeptide is not a toxin. In certain embodiments, the Surf+ Penetrating Polypeptide is not a homeodomain. In certain embodiments, a complex of the disclosure and/or the Surf+ Penetrating
- Polypeptide portion of a complex of the disclosure does not include a homeodomain.
- the foregoing provides description for characteristics of Surf+ Penetrating Polypeptides and sub-categories of Surf+ Penetrating Polypeptides.
- the disclosure contemplates that any Surf+ Penetrating Polypeptide for use in the present disclosure may be described based on presence or absence of any one or any combination of any of the foregoing features. Additional features and specific examples of polypeptides having such features are described in greater detail below. Such features and combinations of features (including combinations with features set forth above) may also be used to describe the Surf+ Penetrating Polypeptide for use in accordance with the claimed disclosure. Any such polypeptides or categories or sub-categories may be used as part of a complex of the disclosure (e.g., the disclosure provides complexes comprising any such polypeptides).
- This section provides examples of Surf+ Penetrating Polypeptides and categories of Surf+ Penetrating Polypeptides.
- Surf+ Penetrating Polypeptides that may be used, e.g., in a complex with an AAM moiety and/or to deliver an AAM moiety into a cell as described herein, include nucleic acid binding proteins, e.g., DNA binding proteins, RNA binding proteins or heparin binding proteins.
- nucleic acid binding proteins e.g., DNA binding proteins, RNA binding proteins or heparin binding proteins.
- Naturally occurring proteins that can function as Surf+ Penetrating Polypeptides may have a natural, endogenous function, such as an endogenous function as a DNA, RNA or heparin binding protein.
- Surf+ Penetrating Polypeptides that may be used in the delivery of an AAM moiety, such as a non-antibody protein scaffold (e.g., an antibody mimic or an antibody-like molecule) or an antibody molecule, can be a DNA binding protein, such as a histone component or a histone-like protein.
- the Surf+ Penetrating Polypeptide portion comprises the histone component is histone linker HI .
- the Surf+ Penetrating Polypeptide portion comprises the histone component is core histone H2A.
- Penetrating Polypeptide portion comprises the histone component is core histone H2B.
- the Surf+ Penetrating Polypeptide portion comprises the histone component is core histone H3.
- Penetrating Polypeptide portion comprises the histone component is core histone H4.
- the the Surf+ Penetrating Polypeptide portion comprises the archael histone-like protein, HPhA.
- the the Surf+ Penetrating Polypeptide portion comprises the bacterial histone-like protein, TmHU.
- the Surf+ Penetrating Polypeptide portion does not comprise a protein select from any of the foregoing histone components or histone-like proteins. It should be noted that the foregoing proteins have endogenous, natural function as DNA binding proteins.
- the disclosure contemplates the use of human polypeptides, including full length polypeptides and domains of full length polypeptides, regardless of whether the domain with cell penetration function is also a domain that modulates DNA binding activity.
- a Surf+ Penetrating Polypeptide that is used to deliver an AAM moiety is an RNA binding protein, such as a ribosomal protein (e.g., LI 1 , S7, S9, or a small nucleolar protein (snoRNP), such as nucleolin, fibrillarin, NOP77P), an RNA polymerase (e.g., RNA polymerase I or II), an RNAse, a transcription factor (e.g., a transcriptional U protein (tUTP)), a histone acetyl transferase (hALP), an upstream binding factor (UBF), a splicing protein (e.g., a snRNP (e.g., Ul or U2) or an SR factor), a La protein, or an hnRNP (heterogeneous RNA binding protein, such as a non-antibody protein scaffold (e.g., an antibody mimic or an antibody-like molecule) or an antibody molecule, is
- the Surf+ Penetrating Polypeptide portion comprises any of the foregoing RNA binding proteins.
- the Surf+ Penetrating Polypeptide portion does not comprise a protein select from any of the foregoing RNA binding proteins. It should be noted that the foregoing proteins have endogenous, natural function as RNA binding proteins.
- the disclosure contemplates the use of human polypeptides, including full length polypeptides and domains of full length polypeptides, regardless of whether the domain with cell penetration function is also a domain that modulates RNA binding activity.
- the Surf+ Penetrating Polypeptide portion comprises a naturally occurring polypeptide, such as a naturally occurring human polypeptide.
- Naturally occurring polypeptides include, but are not limited to, DEK (ID No. : P35659), HB-EGF (ID No.: Q99075), or c-Jun (ID No.: P05412); HGF (ID No. : P14210); cyclon (ID No. :
- the complex comprises a Surf+ Penetrating Polypeptide portion comprising one of the following: U4/U6.U5 tri-snRNP-associated protein 3 (ID No.: Q8WVK2); beta-defensin (ID No.: P81534); Protein SFRS121P1 (ID No.:
- Q8N726-1 isoform 1 of prokineticin-2 (ID No.: Q9HC23-1); isoform 1 of ADP- ribosylation factor-like protein 6-interacting protein 4 (ID No.: Q66PJ3-1); isoform long of fibroblast growth factor 5 (ID No.: P12034-1); or isoform 1 of cyclin-Ll (ID No.: Q9UK58-1).
- FIG. 1 and 2 Additional exemplary Surf+ Penetrating Polypeptides are provided in Figures 1 and 2.
- the disclosure contemplates that any of the polypeptides, or fragments thereof, may be used in a complex of the disclosure. Moreover, additional suitable domains are described herein.
- the disclosure contemplates complexes comprising a Surf+ Penetrating Polypeptide-containing portion. This portion of the complex may comprise any of the Surf+ Penetrating Polypeptides provided in Figures 1 or 2, or a full length or near full length naturally occurring polypeptide provided in Figures 1 or 2, or a domain of any of the foregoing having a mass of at least 4 kDa, surface positive charge, and a charge/molecular weight ratio of at least 0.75.
- Figure 1 provides information for exemplary domains of naturally occurring human proteins that are Surf+ Penetrating Polypeptides and can be used in the instant disclosure (e.g., in a complex and/or to deliver an AAM moiety into a cell).
- Figure 2 provides similar information for a subset of the proteins provided in Figure 1. For each entry, a PDB ID number (and chain) is provided, as well as the terminal residues of the fragment, relative to the full length sequence provided in GenBank (e.g., the subsequence start and subsequence end entries). The amino acid sequence for the full length protein sequences provided in GenBank are reproduced herein below in Section 1 of the sequence listing.
- the amino acid sequence for the particular domains identified by PDB ID number and chain are reproduced below in Scetion 2 of the sequence listing.
- the five columns to the right of the protein name provide information for the exemplified fragment (e.g., for the fragment of a naturally occurring human polypeptide, which fragment is a Surf+ Penetrating Polypeptide). For example, these columns indicate the charge/molecular weight, mass, net positive charge, length (# of amino acid residues) of the fragment, and the size of the fragment relative to its corresponding full length protein (% FL).
- the next column, just to the left of the Gen Bank accession number for the full length protein, indicates the size of the full length protein.
- the four columns to the right of the Ref seq column (the accession number for the full length protein) provide information for the full length, naturally occurring protein from which the fragment is derived. This information includes the
- both the full length, naturally occurring protein and a domain have characteristics indicative of a Surf+ Penetrating Polypeptide (e.g., surface positive charge, charge/molecular weight ratio of at least 0.75, etc.).
- the full length protein does not have such characteristics, while a domain of the protein does.
- the disclosure provides complexes in which the Surf+ Penetrating Polypeptide has at least the following characteristics: surface positive charge, mass of at least 4 kDa, charge/molecular weight ratio of at least 0.75 or of greater than 0.75, and is a domain of a naturally occurring human polypeptide.
- the selected domain has a charge per molecular weight ratio greater than that of the corresponding full length, naturally occurring human polypeptide. In other embodiments, the selected domain has a charge per molecular weight ratio of at least 0.75 or greater than 0.75, but the full length, naturally occurring human polypeptide has a charge per molecular weight ratio of less than 0.75. In other embodiments, the selected domain has a net theoretical charge greater than that of the corresponding full length, naturally occurring human polypeptide. In other embodiments, the selected domain has a net positive charge and the corresponding, full length, naturally occurring human polypeptide has a net negative charge.
- the disclosure contemplates the use of any of the specified domains of full length, naturally occurring human proteins, as well as other domains having the charge and molecular weight characteristics of a Surf+ Penetrating Polypeptide. Moreover, the disclosure contemplates the use of full length, naturally occurring human polypeptides having the charge and molecular weight characteristics of a Surf+ Penetrating Polypeptide. Further, the disclosure
- complexes may comprise a full length naturally occurring human polypeptide, even though only a domain of said human polypeptide functions as a Surf+ Penetrating Polypeptide.
- the additional polypeptide sequence can optionally be used to interconnect the Surf+ Penetrating Polypeptide to the AAM moiety.
- the disclosure provides complexes comprising a first polypeptide portion that comprises a Surf+ Penetrating Polypeptide.
- Such a Surf+ Penetrating Polypeptide may optionally be provided with additional sequence endogenously present in, for example, the naturally occurring polypeptide from which the Surf+ Penetrating Polypeptide is a domain or may be present without additional sequence endogenously present in the naturally occurring polypeptide from which the Surf+ Penetrating Polypeptide is a domain.
- the presence of additional sequence from the same naturally occurring polypeptide does not result in the portion comprising the Surf+ Penetrating Polypeptide having a charge/molecular weight ratio of less than 0.75.
- the presence of additional sequence from the same naturally occurring polypeptide results in the portion comprising the Surf+ Penetrating Polypeptide having a charge/molecular weight ratio of less than 0.75.
- portion comprising a Surf+ Penetrating Polypeptide refers to the Surf+ Penetrating Polypeptide and additional sequence from the same or similar naturally or non-naturally occurring polypeptide. This portion does not include heterologous linker sequence, nuclear localization signals, or additional portions intended to have an independent and distinct biological function (e.g., a moiety to increase the half life of the complex).
- the foregoing are exemplary of sub-categories of Surf+ Penetrating
- Domains of the naturally occurring human proteins may be modified, such as by introducing one or more amino acid substitutions, deletions or additions.
- the resulting domain will still be considered a domain of a naturally occurring human polypeptide as long as the domain is readily identifiable based on sequence and/or structure as a domain of that naturally occurring human protein.
- the Surf+ Penetrating Polypeptide portion comprises (or consists of) a full length naturally occurring polypeptide or a domain of a full length polypeptide presented in Figure 2.
- the disclosure provides a complex comprising an AAM moiety associated with a human polypeptide (full length or domain) presented in Figure 2.
- the domains depicted in the figures are merely exemplary. Having identified a suitable domain, such as the domains identified by PDB in figures 1 and 2, suitable sub- domains or non-overlaping domains can be readily identified.
- the disclosure contemplates the use of any of the domains set forth in Figure 1 or 2, as well as a fragment (sub-domain; also considered a domain) thereof having a mass of at least 4kDa, surface positive charge and charge/molecular weight ratio of at least 0.75.
- Polypeptide is a full length or a domain of C-C motif chemokine 26 precursor (e.g., such as a fragment of about 71 amino acid residues beginning at position 24 of the full length protein, a net charge of + 13, and having a charge/MW of 1.55), a domain of HB-EGF (proheparin-binding EGF-like growth factor precursor, such as, a fragment of about 79 amino acid residues beginning at position 72 of the full length protein, a net positive charge of +12, and a charge/molecular weight of 1.35), a domain of protein DEK isoform 1 (e.g., such as a fragment of about 131 amino acid residues beginning at position 78 of the full length protein, a net positive charge of +19, and a charge/molecular weight of 1.26), a domain of hepatocyte growth factor isoform 1 preprotein (e.g., such as a fragment of about 131 amino acid residues beginning at position 31 of the full length protein, a net positive charge
- the disclosure provides a complex comprising an AAM moiety and any of the foregoing full length, naturally occurring human polypeptides, or a domain thereof, which domain has the charge and charge/molecular weight characteristics of a Surf+ Penetrating Polypeptide.
- the complex (a complex of the disclosure) comprises a domain of the full length, naturally occurring human polypeptide, but the complex does not comprise the full length, naturally occurring human polypeptide.
- the Surf+ Penetrating Polypeptide is a domain of any of the following, which domain has a charge per molecular weight ratio of at least 0.75 but for which the corresponding full length naturally occurring polypeptide has a charge/molecular weight ratio of less than 0.75: histone-lysine N- methyltransferase MLL isoform 1 precursor; transcription factor AP-1; proheparin- binding EGF-like growth factor precursor; protein DEK isoform 1 ; hepatocyte growth factor isoform 1 preprotein; epidermal growth factor receptor isoform a precursor; forkhead box protein K2; pre-mR A-processing factor 40 homolog A; ataxin-7 isoform a, E3 SUMO-protein ligase PIAS 1 ; platelet factor 4 precursor; advanced glycosylation end product-specific receptor isoform 2 precursor; serol regulatory element-binding protein 2; histone acetyltransferase p300; Ul small nuclear rib
- lethal(3)malignant brain tumor-like protein 2 CCAAT/enhancer-binding protein beta; troponin T, cardiac muscle isoform 2; CREB-binding protein isoform B; cyclic AMP- dependent transcription factor ATF-2; cathepsin E isoform a preprotein; glycine receptior subunit alpha- 1 isoform 1 precursor; CREB-binding protein isoform b; pituitary adenylate cyclase-activating polypeptide precursor; mastermind-like protein 1; BCL2/adeno virus E1B 19 kDa protein-interacting protein 3; cathelicidin antimicrobial peptide; epidermal growth factor receptor isoform a precursor;
- a suitable fragment is provided in Figure 1.
- other examples of this sub-category of Surf+ Penetrating Polypeptides are provided in and are immediately apparent from Figure 1.
- the disclosure provides a complex comprising an AAM moiety and any of the foregoing full length, naturally occurring human polypeptides, or a domain thereof, which domain has the charge and charge/molecular weight characteristics of a Surf+ Penetrating Polypeptide.
- the complex (a complex of the disclosure) comprises a domain of the full length, naturally occurring human polypeptide, but the complex does not comprise the full length, naturally occurring human polypeptide.
- the complex and/or the Surf+ Penetrating Polypeptide portion does not include one of the polypeptides or specific fragments provided in Figure 1.
- the complex and/or the Surf+ Penetrating Polypeptide portion does not include HRX (Uniprot number Q03164 or fragment identified at PDB 2J2S.
- the complex and/or the Surf+ Penetrating Polypeptide portion does not include c-Jun (Uniprot number P05412 or fragment identified at PDB 1 JNM. In certain embodiments, the complex and/or the Surf+ Penetrating Polypeptide portion does not include defensin 3 (Uniprot number P81534 or fragment identified at PDB 1KJ6. In certain embodiments, the complex and/or the Surf+ Penetrating Polypeptide portion does not include HBEGF (Uniprot number Q99075 or fragment identified at PDB 1XDT. In certain embodiments, the complex and/or the Surf+ Penetrating Polypeptide portion does not include N-Dek (Uniprot number P35659 or fragment identified at PDB 2JX3.
- the complex and/or the Surf+ Penetrating Polypeptide portion does not include HGF (Uniprot number PI 4210 or fragment identified at PDB 2HGF. In certain embodiments, the complex and/or the Surf+ Penetrating Polypeptide portion does not include HIST4 (Uniprot number P62805 or fragment identified at PDB 2CV5.
- the Surf+ Penetrating Polypeptide is a domain of: charged multivesicular body protein 6 (e.g., a fragment of about 39 amino acid residues having a charge/molecular weight of 1.07); homeobox protein Nkx3.1 (e.g., a fragment of about 69 amino acid residue having a charge/molecular weight of 0.96); B-cell lymphoma 6 protein isoform 1 (e.g., a fragment of about 74 amino acid residues having a charge per molecular weight of 0.93); lethal(3)malignant brain tumor-like protein 2 (e.g., a fragment of about 43 amino acid residues having a charge/molecular weight of 0.87); cathepsin E isoform a preprotein (e.g., a fragment of about 35 amino acid residues having a charge/molecular weight of 1.66);
- the disclosure provides a complex comprising an AAM moiety and any of the foregoing full length, naturally occurring human polypeptides, or a domain thereof, which domain has the charge and charge/molecular weight characteristics of a Surf+ Penetrating Polypeptide.
- the complex (a complex of the disclosure) comprises a domain of the full length, naturally occurring human polypeptide, but the complex does not comprise the full length, naturally occurring human polypeptide.
- the Surf+ Penetrating Polypeptide is selected from a domain of any of: agouti-signaling protein precursor, band 3 anion transport protein, B-cell lymphoma 6 protein isoform 1, BCL2/adeno virus E1B 19 kDa protein-interacting protein 3, beta-defensin 1 preproprotein, cathepsin E isoform a preproprotein, charged multivesicular body protein 6, cpG-binding protein isoform 2, C-X-C motif chemokine 10 precursor, epidermal growth factor receptor isoform a precursor, histone acetyltransferase MYST3, histone acetyltransferase p300, homeobox protein Nkx-3.1 , lethal(3)malignant brain tumor-like protein 2, male- specific lethal 3 homolog isoform a, Na(+)/H(+) exchange regulatory cofactor NHE- RFl, peptidyl-prolyl cis-trans is
- the selected domain is a domain presented in Figure 2, or a variant thereof.
- the complex (a complex of the disclosure) comprises a domain of the full length, naturally occurring human polypeptide, but the complex does not comprise the full length, naturally occurring human polypeptide.
- the disclosure provides a complex comprising an AAM moiety and any of the following full length (or substantially full length), naturally occurring human polypeptides: agouti-signaling protein precursor, band 3 anion transport protein, B-cell lymphoma 6 protein isoform 1, BCL2/adeno virus E1B 19 kDa protein-interacting protein 3, beta-defensin 1 preproprotein, cathepsin E isoform a preproprotein, charged multivesicular body protein 6, cpG-binding protein isoform 2, C-X-C motif chemokine 10 precursor, epidermal growth factor receptor isoform a precursor, histone acetyltransferase MYST3, histone acetyltransferase p300, homeobox protein Nkx-3.1, lethal(3)malignant brain tumor- like protein 2, male- specific lethal 3 homolog isoform a, Na(+)/H(+) exchange regulatory cofactor NHE- RF1, peptidyl-pro
- Figure 1 provides specific examples of domains that are Surf+ Penetrating Polypeptides. It should be appreciated that other fragments of the corresponding naturally occurring human proteins may also be suitable, such as an overlapping fragment that retains the surface positive charge of the recited fragment but is shorter or longer (e.g., the starting or ending residue is different but the functional core of surface positive charge is retained; the fragment retains the essential structure of the recited fragment). Fragments that retain the essential structure but differ in length may differ in mass, length, and/or charge/molecular weight. However, essential structure, surface charge and charge/molecular weight of at least 0.75 are maintained. Additionally, Figure 1 provides examples for several human polypeptides of more than one non-overlapping domain that may be used as a Surf+ Penetrating
- the Surf+ Penetrating Polypeptide portion of a complex of the disclosure is or comprises a domain of a human polypeptide, such as a domain of a naturally occurring human polypeptide.
- a complex may comprise the domain outside of its context in its full length, naturally occurring protein (e.g., the complex does not include the full length human polypeptide from which the domain is a portion).
- the domain may be provided in the context of its full length polypeptide or in the context of additional polypeptide sequence (but less than all) from the naturally occurring protein from which the Surf+ Penetrating Polypeptide is a domain (e.g., the complex does include the full length human polypeptide from which the domain is an identified portion).
- a complex of the disclosure e.g., a complex
- a complex comprises a portion comprising a Surf+ Penetrating Polypeptide and the portion comprising a Surf+ Penetrating Polypeptide is selected from a polypeptide listed in Table 1.
- the complex includes at least about 10%, 15%, 20%>, 25%, 30%>, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%o, 98%o, or 100% of the full length polypeptide, provided as contiguous amino acid residues.
- forkhead box protein 04 isoform 1 NP 005929.2 general transcription factor IIF subunit 1 NP 002087.2 glycine receptor subunit alpha- 1 isoform 1 precursor NP 001 139512.1 granulysin isoform NKG5 NP 006424.2 heparin-binding growth factor 2 NP 001997.5 hepatocyte growth factor isoform 1 preproprotein NP 000592.3 heterochromatin protein 1 -binding protein 3 NP 057371.2 histone acetyltransferase MYST3 NP 001092883.1 histone acetyltransferase p300 NP 001420.2 histone deacetylase complex subunit SAP30 NP 003855.1 histone H3-like centromeric protein A isoform a NP 001800.1 homeobox protein Hox-A9 NP 689952.1 homeobox protein Hox-B 1 NP 002135.2 homeobox protein NANOG NP 0791
- NP 001036064.1 retinoblastoma-associated protein NP 000312.2 ribonuclease HI NP 002927.2
- RNA-binding motif protein RNA-binding motif protein, Y chromosome, family 1
- TATA-box-binding protein isoform 2 NP 001 165556.1
- T-cell surface glycoprotein CD4 isoform 3 NP 001 181946.1 telomeric repeat-binding factor 1 isoform 1 NP 059523.2 telomeric repeat-binding factor 2 NP 005643.1
- THAP domain-containing protein 1 isoform 1 NP 060575.1 transcription factor AP- 1 NP 002219.1 transcription factor NF-E2 45 kDa subunit isoform 2 NP 001 129495.1 transcription factor SOX-2 NP 003097.1 transcription factor Sp 1 isoform b NP 003100.1 transcriptional activator Myb isoform 1 NP 001 123645.1 transcriptional activator Myb isoform 4 NP 001 155128.1 troponin T, cardiac muscle isoform 2 NP 001001430.1 tumor necrosis factor receptor superfamily member 13C NP 443177.1
- Refseq is the NCBI Reference Sequence ID on the web at ncbi.nlm.nih.gov/ RefSeq/
- the disclosure contemplates embodiments in which the complex comprises a domain of a full length, naturally occurring human protein, but does not include the full length, naturally occurring human protein as a contiguous amino acid sequence.
- the disclosure contemplates embodiments in which that domain is provided in the context of the full length (or substantially full length), naturally occurring protein - such that the complex comprises the full length, naturally occurring human protein, or when the Surf+ Polypeptide portion includes additional polypeptide sequence (more sequence than is necessary or sufficient to achieve cell penetration).
- a complex comprises a portion comprising a Surf+ Penetrating Polypeptide and the portion comprising a Surf+ Penetrating Polypeptide is selected from a polypeptide listed in Figure 1 or 2.
- the complex includes at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 100% of the full length polypeptide from which the Surf+ Penetrating polypeptide is a domain, provided as contiguous amino acid residues.
- the disclosure has provided numerous exemplary Surf+ Penetrating Polypeptides, including numerous human polypeptides.
- Surf+ Penetrating Polypeptides suitable for use also include polypeptides from other species, such as mouse, rat, monkey, etc.
- the disclosure contemplates use of naturally occurring polypeptides (and domains thereof having characteristics of Surf+ Penetrating Polypeptides) from these other organisms.
- the disclosure provides a complex comprising a Surf+ Penetrating Polypeptide, which is a naturally occurring mammalian polypeptide (such as mouse, rat, monkey, etc.) or domain thereof associated with an AAM moiety.
- Surf+ Penetrating Polypeptides include naturally occurring or non-human proteins that may be or have been further modified to increase positive charge (e.g., supercharged). These include polypeptides that, prior to supercharging, have a charge/molecular weight ratio of at least 0.75 or of greater than 0.75, as well as polypeptides that do not have a charge/molecular weight ratio of at least 0.75 prior to supercharging.
- An example is the +52 streptavidin described in the Examples in which streptavidin has been supercharged to have a net positive charge of +52.
- Another example is the +36 GFP described in the Examples in which GFP has been supercharged to have a net positive charge of +36.
- Surf+ Penetrating Polypeptides can be naturally-occurring, or can be produced by changing one or more conserved or non-conserved amino acids on or near the surface of a protein to more polar or charged amino acid residues.
- the amino acid residues to be modified may be hydrophobic, hydrophilic, charged, or a combination thereof.
- Surf+ Penetrating Polypeptides can also be produced by the attachment of charged moieties to the protein in order to supercharge the protein.
- Natural as well as unnatural proteins may be modified, e.g., to increase the net charge of the protein.
- proteins that may be modified include receptors, membrane bound proteins, transmembrane proteins, enzymes, transcription factors, extracellular proteins, therapeutic proteins, cytokines, messenger proteins, DNA-binding proteins, RNA-binding proteins, proteins involved in signal transduction, structural proteins, cytoplasmic proteins, nuclear proteins, hydrophobic proteins, hydrophilic proteins, etc.
- a naturally occurring Surf+ Penetrating Polypeptides, or a protein to be modified for supercharging may be derived from any species of plant, animal, and/or microorganism.
- the protein is a mammalian protein.
- the protein is a human protein.
- the naturally occurring Surf+ Penetrating Polypeptide, or the protein to be modified is derived from an organism typically used in research.
- the naturally occurring Surf+ Penetrating Polypeptide, or the protein to be modified may be from a primate ⁇ e.g., ape, monkey), rodent ⁇ e.g., rabbit, hamster, gerbil), pig, dog, cat, fish ⁇ e.g., Danio rerio), nematode ⁇ e.g., C. elegans), yeast ⁇ e.g., Saccharomyces cerevisiae), or bacteria ⁇ e.g., E. coli).
- the protein is non- immunogenic.
- the protein is non-antigenic.
- the protein does not have inherent biological activity or has been modified to have no biological activity.
- the protein is chosen based on its targeting ability.
- the term supercharging is used to refer to changes made to the Surf+ Penetrating Polypeptide or changes made to a polypeptide such that it functions as and meets the definition of a Surf+ Penetrating Polypeptide, but do not include changes in charge or charge density that result from association with the AAM moiety.
- the naturally occurring Surf+ Penetrating Polypeptides, or the protein to be modified is one whose structure has been characterized, for example, by NMR or X-ray crystallography. In some embodiments, the naturally occurring Surf+ Penetrating Polypeptides, or the protein to be modified, is one whose structure has been predicted, for example, by threading homology modeling or de novo structure prediction. In some embodiments, the naturally occurring Surf+ Penetrating Polypeptides, or the protein to be modified, is one whose structure has been correlated and/or related to biochemical activity (e.g., enzymatic activity, protein-protein interactions, etc.).
- biochemical activity e.g., enzymatic activity, protein-protein interactions, etc.
- the inherent biological activity of a modified protein is reduced or eliminated to reduce the risk of deleterious and/or undesired effects.
- the biological activity of the modified protein can be increased or potentiated, or a non-naturally occurring biological activity of the protein may be generated as a result of the charge modification concomitant with the creation of the charged-modified Surf+ Penetrating Polypeptides.
- the surface residues of a protein to be modified may be identified using any method known in the art.
- surface residues are identified by computer modeling of the protein.
- the three-dimensional structure of the protein is known and/or determined, and surface residues are identified by visualizing the structure of the protein. Homology modeling and de novo structure prediction are two methods for modeling the 3-D structure of a protein; such methods are particularly useful in the absence of an NMR or crystal structure.
- surface residues are predicted using computer software.
- an Accessible Surface Area (ASA) is used to predict surface exposure. A high ASA value indicates a surface exposed residue, whereas a low ASA value indicates the exclusion of solvent interactions with the residue.
- an Average indicates a surface exposed residue
- AvNAPSA Neighbor Atoms per Sidechain Atom
- AvNAPSA is an automated measure of surface exposure which has been implemented as a computer program.
- a low AvNAPSA value indicates a surface exposed residue, whereas a high value indicates a residue in the interior of the protein.
- the software is used to predict the secondary structure and/or tertiary structure of a protein, and surface residues or near-surface residues are identified based on this prediction.
- the prediction of surface residues is based on hydrophobicity and hydrophilicity of the residues and their clustering in the primary sequence of the protein.
- surface residues of the protein may also be identified using various biochemical techniques, for example, protease cleavage, surface modification, derivatization, labeling, hydrogen-deuterium exchange experiments, etc.
- biochemical techniques for example, protease cleavage, surface modification, derivatization, labeling, hydrogen-deuterium exchange experiments, etc.
- conserved residues are identified by aligning the primary sequence of the protein of interest with related proteins. These related proteins may be from the same family of proteins. Related proteins may also be the same protein from a different species. For example, conserved residues may be identified by aligning the sequences of the same protein from different species. For example, proteins of similar function or biological activity may be aligned.
- a residue is considered conserved if over 50%, over 60%, over 70%>, over 75%, over 80%), over 90%>, or over 95% of the sequences have the same amino acid in a particular position.
- the residue is considered conserved if over 50%, over 60%, over 70%, over 75%, over 80%, over 90%, or over 95% of the sequences have the same or a similar ⁇ e.g., valine, leucine, and isoleucine; glycine and alanine; glutamine and asparagine; or aspartate and glutamate) amino acid in a particular position.
- conserved residues may be determined first or the surface residues may be determined first. The order does not matter.
- a computer software package may determine surface residues and/or conserved residues, and may optionally do so simultaneously. Important residues in the protein may also be identified by mutagenesis of the protein. For example, alanine scanning of the protein can be used to determine the important amino acid residues in the protein. In some embodiments, site-directed mutagenesis may be used. In certain embodiments, conserving the original biological activity of the protein is not important, and therefore, the steps of identifying the conserved residues and preserving them are not performed.
- each of the surface residues is identified as hydrophobic or hydrophilic.
- residues are assigned a hydrophobicity score.
- each surface residue may be assigned an octanol/water logP value.
- hydrophobicity parameters may also be used.
- hydrophobicity parameters may be used in the inventive method to determine which residues to modify.
- hydrophilic or charged residues are identified for modification.
- Near-surface residues are residues that are either a) not surface residues but immediately adjacent in primary amino acid sequence or within a three-dimensional structure or b) not surface residues that can become surface residues upon the alteration of a polypeptide's tertiary structure. The contribution of near-surface residues in a Surf+ Penetrating Polypeptideis determined using the methods described herein.
- At least one identified surface residue or near-surface residue is chosen for modification.
- hydrophobic residue(s) are chosen for modification.
- hydrophilic and/or charged residue(s) are chosen for modification.
- more than one residue is chosen for modification.
- 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 of the identified residues are chosen for modification.
- over 10, over 15, over 20, or over 25 residues are chosen for modification.
- multiple variants of a protein are produced and tested to determine the best variant in terms of delivery of a biological moiety to a cell, pharmacokinetics, stability, biocompatibility, and/or biological activity, or a biophysical property such as expression level.
- a library of protein variants is generated in an in vivo system containing an expression host such as phage, bacteria, yeast or mammalian cells, or in an in vitro system such as mRNA display, ribosome display, or polysome display.
- Such a library may contain 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or over 10 9 , possible variants (including substitutions, deletions of one or more residues, and insertion of one or more residues).
- Surf+ Penetrating Polypeptides may be created from polypeptides for which no structural information such as crystal structure is known or available.
- residues chosen for modification are mutated into more hydrophilic residues (including positively charged residues).
- residues are mutated into more hydrophilic natural amino acids.
- residues are mutated into amino acids that are positively charged at physiological pH.
- a residue may be changed to an arginine, or lysine, or histidine.
- all the residues to be modified are changed into the same alternate residue.
- all the chosen residues are changed to an arginine residue, a lysine residue or a histidine residue.
- the chosen residues are changed into different residues; however, all the final residues are positively charged at physiological pH.
- to create a positively charged protein all the residues to be mutated are converted to arginine or lysine or histidine residues, or a combination thereof.
- all the chosen residues for modification are aspartate, glutamate, asparagine, and/or glutamine, and these residues are mutated into arginine, lysine or histidine.
- a protein may be modified to increase the overall net charge on the protein.
- the theoretical net charge is increased, relative to its unmodified protein, by at least +1, at least +2, at least +3, at least +4, at least +5, at least +10, at least +15, at least +20, at least +25, at least +30, at least +35, or at least +40.
- the chosen amino acids are changed into non-ionic, polar residues (e.g., cysteine, serine, threonine, tyrosine, glutamine, and asparagine).
- increasing the overall net charge comprises increasing the total number of positively charged residues on or near the surface.
- the amino acid residues mutated to charged amino acids residues are separated from each other by at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or at least 25 amino acid residues in the primary amino acid sequence.
- the amino acid residues mutated to positively charged amino acids residues e.g., arginine, lysine or histidine
- the amino acid residues mutated to positively charged amino acids residues are separated from each other by at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or at least 25 amino acid residues in the primary amino acid sequence.
- fewer than two or only two, three, four or five consecutive amino acids are modified to generate a charge-modified Surf+ Penetrating Polypeptide.
- a surface projection is present in the polypeptide, more than two, three, four, five, six, seven, eight, nine, or ten
- a surface exposed loop, helix, turn, or other secondary structure may contain only 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 or more than 30 charged residues. Distributing the charged residues over the surface of the protein may allow for more stable proteins. In certain embodiments, only 1 , 2, 3, 4, or 5 residues per 15-20 amino acids of the primary sequence are mutated to charged amino acids (e.g., arginine, lysine or histidine). In certain embodiments, on average only 1 , 2, 3, 4, or 5 residues per 10 amino acids of the primary sequence are mutated to charged amino acids (e.g., arginine, lysine or histidine).
- At least 50%, at least 60%, at least 70%>, at least 80%>, or at least 90% of the mutated charged amino acid residues of a charge-modified Surf+ Penetrating Polypeptide are solvent exposed. In certain embodiments, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the mutated charged amino acids residues of the charge-modified Surf+ Penetrating Polypeptide are on the surface of the protein. In certain embodiments, less than 5%, less than 10%, less than 20%), less than 30%>, less than 40%>, less than 50%> of the mutated charged amino acid residues are not solvent exposed. In certain embodiments, less than 5%, less than 10%, less than 20%, less than 30%, less than 40%, less than 50% of the mutated charged amino acid residues are internal amino acid residues.
- amino acids are selected for modification using one or more predetermined criteria.
- ASA or AvNAPSA values may be used to identify aspartic acid, glutamic acid, asparagine, and/or glutamine residues with ASA values above a certain threshold value or AvNAPSA values below a certain threshold value, and one or more (e.g., all) of these residues may be changed to arginine, lysine or histidine.
- ASA calculations are used to identify aspartic acid, glutamic acid, asparagine, and/or glutamine residues with ASA above a certain threshold value, and one or more (e.g., all) of these are changed to arginine, lysine or histidine.
- AvNAPSA is used to identify aspartic acid, glutamic acid, asparagine, and/or glutamine residues with AvNAPSA below a certain threshold value, and one or more (e.g., all) of these are changed to arginines.
- AvNAPSA is used to identify aspartic acid, glutamic acid, asparagine, and/or glutamine residues with AvNAPSA below a certain threshold value, and one or more (e.g., all) of these are changed to lysines.
- AvNAPSA is used to identify aspartic acid, glutamic acid, asparagine, and/or glutamine residues with AvNAPSA below a certain threshold value, and one or more (e.g., all) of these are changed to histidines.
- solvent-exposed residues are identified by the number of neighbors. In general, residues that have more neighbors are less solvent-exposed than residues that have fewer neighbors. In some embodiments, solvent-exposed residues are identified by half sphere exposure, which accounts for the direction of the amino acid side chain (Hamelryck, 2005, Proteins, 59:8-48; incorporated herein by reference). In some embodiments, solvent-exposed residues are identified by computing the solvent exposed surface area, accessible surface area, and/or solvent excluded surface of each residue. See, e.g., Lee et ah, J. Mol. Biol. 55(3):379-400, 1971; Richmond, J. Mol. Biol. 178:63-89, 1984; each of which is incorporated herein by reference.
- the modifications are made by site-directed mutagenesis of the polynucleotide encoding the protein.
- Other techniques for introducing mutations are discussed in Molecular Cloning: A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch, and Maniatis (Cold Spring Harbor Laboratory Press: 1989); the treatise, Methods in Enzymology (Academic Press, Inc., N.Y.); Ausubel et al. Current Protocols in Molecular Biology (John Wiley & Sons, Inc., New York, 1999); each of which is incorporated herein by reference.
- the modified protein is expressed and tested.
- a series of variants is prepared, and each variant is tested to determine its biological activity and its stability.
- the variant chosen for subsequent use may be the most stable one, the most active one, or the one with the greatest overall combination of activity and stability. After a first set of variants is prepared an additional set of variants may be prepared based on what is learned from the first set. Variants are typically created and over-expressed using recombinant techniques known in the art.
- protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of this disclosure.
- any protein fragment of a reference protein meaning a polypeptide sequence at least one amino acid residue shorter than a reference polypeptide sequence but otherwise identical
- any protein that includes a stretch of about 20, about 30, about 40, about 50, or about 100 amino acids which are about 40%, about 50%, about 60%>, about 70%>, about 80%), about 90%o, about 95%, or about 100% identical to any of the sequences described herein can be utilized in accordance with the disclosure.
- a protein sequence to be utilized in accordance with the disclosure includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations as shown in any of the sequences provided or referenced herein.
- Antibody or Antibody-Mimic Moiety (AAM Moiety)
- the disclosure provides complexes comprising a Surf+ Penetrating
- AAM moiety an antibody or antibody-mimic moiety (AAM moiety) portion that is associated with the Surf+ Penetrating Polypeptide portion.
- AAM moiety an antibody or antibody-mimic moiety
- This section of the application describes the AAM moiety portion of complexes of the disclosure and provides numerous representative examples.
- the disclosure contemplates that any such AAM moiety may be associated with any Surf+ Penetrating Polypeptide or category of Surf+ Penetrating Polypeptide to form a complex (e.g., may be associated to a portion comprising or consisting of a Surf+ Penetrating Polypeptide).
- Such a complex has cell penetrating ability (e.g., cell penetrating ability provided by the Surf+ Penetrating Polypeptide portion) and promotes delivery of the AAM moiety into a cell.
- AAM moieties for use in the context of the present disclosure bind to intraceullar targets (e.g., bind to targets expressed or otherwise present inside a cell). Accordingly, the present disclosure provides complexes and methods for delivering the AAM moiety into a cell where it can bind its target molecule.
- an "AAM moiety” is an antibody or an antibody mimic molecule that specifically binds to a target molecule expressed or otherwise present intracellularly (an intracellular target).
- An antibody-mimic molecule is also referred to as an antibody-like molecule.
- An antibody-mimic binds to a target molecule, but binding is mediated by binding units other than antigen binding portions comprising at least a variable heavy or variable light chain of an antibody.
- binding to target is mediated by a different antigen-binding unit, such as a protein scaffold or other engineered binding unit.
- a different antigen-binding unit such as a protein scaffold or other engineered binding unit.
- target refers to a molecule expressed or otherwise present inside a cell to which an AAM moiety specifically binds (e.g., binds with affinity and specificity distinct from non-specific interactions).
- the target is a peptide or polypeptide, including peptides or polypeptides that are glycosylated, phosphorylated or otherwise post-translationally modified.
- intracellular target refers to molecules expressed or otherwise present in a cell so that the target can be contacted while inside the cell by an AAM moiety. For example, a secreted polypeptide that is taken up by a cell is, for some period of time, present inside a cell.
- such a secreted polypeptide may be an intracellular target available to be contacted by an AAM moiety.
- the intracellular target is a target whose endogenous localization is inside a cell (e.g., the target is not secreted).
- the AAM moiety binds to a target expressed or otherwise present intracellularly, and that target is distinct from the Surf+ Penetrating Polypeptide to which the AAM moiety is complexed.
- the Surf+ Penetrating Polypeptide or Surf+ Penetrating Polypeptide portion to which the AAM moiety is complexed is not also the endogenous target of the AAM moiety.
- the Surf+ Penetrating Polypeptide may itself bind to or have some affinity for the same target. This, however, is permissible and is not intended to be excluded by the foregoing description.
- a complex of the disclosure comprises an AAM moiety, wherein the AAM moiety is an antibody that binds to a target molecule expressed inside a cell.
- a complex of the disclosure comprises an AAM moiety, wherein the AAM moiety is an antibody-mimic (e.g., a protein comprising a protein scaffold or other binding unit that binds to a target expressed inside a cell).
- the AAM moiety binds to its target, and that target is a polypeptide expressed in a cell.
- the AAM moiety binds its target molecule, such as a polypeptide, with high affinity (e.g., with an affinity of at least 10 "6 , 10 "7 , 10 “8 , 10 “9 , 10 “10 , or 10 "11 M, or with an affinity in the range of 10 "6 to 10 "8 , 10 "7 to 10 "10 , or 10 "9 to 10 “11 M).
- the AAM moiety binds to its target with an affinity at least 100, at least 1000, or at least 10000 times tighter than its affinity for another polypeptide. Regardless of the affinity with which an AAM moiety binds its target, binding is understood to not include nonspecific binding (e.g., binding due to background or general stickiness of polypeptides).
- the target may also be expressed extracellularly.
- the primary aim is to facilitate delivery of the AAM moiety into a cell to promote binding of the AAM moiety to target expressed inside a cell.
- the target moiety such as a polypeptide
- the target polypeptides are described in greater detail in the portion of the disclosure entitled "Applications". However, thiese serve only as examples.
- Binding of an AAM moiety to a target is generally intended to have one or more biological consequences or utilities.
- binding of an AAM moiety may be useful for inhibiting the activity of the target, such as by preventing binding to another protein, by promoting degradation of the target, or by sequestering the target away from its necessary site of action.
- Binding of an AAM moiety may also be useful for labeling a target to facilitate visualization or monitoring of cells expressing the target.
- Given a particular known target polypeptide numerous methods exist for identifying AAM moieties that bind to the target and that have a desired function, e.g., that inhibit activity of the target or that bind to the target without altering activity (so as to serve as a suitable labeling agent). Exemplary methods of making and testing AAM moieties that bind a target are described herein.
- an AAM moiety is an antibody-mimic comprising a protein scaffold.
- Scaffold-based AAM moieties have positioning or structural components and target-contacting components in which the target contacting residues are largely concentrated.
- a scaffold-based AAM moiety comprises a scaffold comprising two types of regions, structural and target contacting. The target contacting region shows more variability than does the structural region when a scaffold-based AAM moiety to a first target is compared with a scaffold-based AAM moiety of a second target (where both AAM moieties are of the same category, e.g., both are Adnectins or both are Anticalins ® ).
- the structural region tends to be more conserved across AAM moieties that bind different targets. This is analogous to the CDRs and framework regions of antibodies.
- the first class corresponds to the loops, and the second class corresponds to the anti-parallel strands.
- the AAM moiety is a subunit-based AAM moiety.
- These AAM moieties are based on an assembly of subunits which provide distributed points of contact with the target that form a domain that binds with high affinity to the target (e.g. as seen with DARPins).
- an AAM moiety for use as part of a complex of the disclosure has a molecular weight of 5-250, 10-200, 5-15, 10-30, 15-30, 20-25 kD.
- AAM moieties can comprise one or more polypeptide chains.
- AAM moieties can be antibody-based or non-antibody-based.
- AAM moieties suitable for use in the compositions and methods featured in the disclosure include antibody molecules, such as full-length antibodies and antigen- binding fragments thereof, and single domain antibodies, such as camelids.
- an antibody molecule is complexed with an Surf+ Penetrating Polypeptide for delivery of the antibody molecule into a cell.
- the antibody molecule binds an intracellular target, e.g., an intracellular polypeptide, such as to inhibit, label or activate the target, e.g., for treatment of a disorder, for labeling to monitor expression or as a diagnostic, for research or clinical purposes.
- AAM moieties include polypeptides engineered to contain a scaffold protein, such as a DARPin, an Adnectin ® , or an Anticalin ® . These are exemplary of antibody-mimic moieties that, in the context of the disclosure, may be complexed with a Surf+ Penetrating Polypeptide to promote delivery of the AAM moiety into a cell.
- the scaffold protein e.g., the AAM moiety portion of the complex
- binds an intracellular target e.g., an intracellular polypeptide, such as to inhibit, label or activate the target, e.g., for treatment of a disorder, for labeling to monitor expression or as a diagnostic, for research purposes.
- Inhibition can be, e.g., by steric inhibition, e.g., by blocking protein interaction with a substrate, or inhibition can be, e.g., by causing target protein degradation.
- An AAM moiety for delivery into a cell can be, e.g., an agent for treatment, prophylaxis, diagnosis, imaging, or labeling.
- the AAM moiety has a desirable activity in a target cell, but the Surf+ Penetrating Polypeptide that delivers the AAM moiety is inert, i.e., the Surf+ Penetrating Polypeptide has no observable biological function in the cell other than to deliver the agent to the interior of the cell.
- the Surf+ Penetrating Polypeptide has at least one desired biological activity, e.g., the polypeptide modifies (e.g., enhances) the effect of the AAM moiety on a target molecule, or the Surf+ Penetrating Polypeptide binds to and affects the activity of a second target molecule that is separate from the first molecule targeted by the high affinity binding ligand.
- the polypeptide modifies (e.g., enhances) the effect of the AAM moiety on a target molecule, or the Surf+ Penetrating Polypeptide binds to and affects the activity of a second target molecule that is separate from the first molecule targeted by the high affinity binding ligand.
- AAM moiety itself has charge, size and charge distribution characteristics. However, such charge or charge distribution characteristics are not considered when describing the charge
- antibody or “antibody molecule” refers to a protein that includes sufficient sequence (e.g., antibody variable region sequence) to mediate binding to a target, and in embodiments, includes at least one immunoglobulin variable region or an antigen binding fragment thereof.
- An antibody molecule can be, for example, a full-length, mature antibody, or an antigen binding fragment thereof.
- An antibody molecule also known as an antibody or an immunoglobulin, encompass monoclonal antibodies (including full- length monoclonal antibodies), polyclonal antibodies, multispecific antibodies formed from at least two different epitope binding fragments (e.g., bispecific antibodies), human antibodies, humanized antibodies, camelised antibodies, chimeric antibodies, single-chain Fvs (scFv), Fab fragments, F(ab')2 fragments, antibody fragments that exhibit the desired biological activity (e.g.
- antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain at least one antigen-binding site.
- Immunoglobulin molecules can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), subisotype (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or allotype (e.g., Gm, e.g., Glm(f, z, a or x), G2m(n), G3m(g, b, or c), Am, Em, and Km(l, 2 or 3)).
- isotype e.g., IgG, IgE, IgM, IgD, IgA and IgY
- subisotype e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2
- allotype e.g., Gm, e.g., Glm(f, z, a or x),
- Antibodies may be derived from any mammal, including, but not limited to, humans, monkeys, pigs, horses, rabbits, dogs, cats, mice, etc., or other animals such as birds (e.g. chickens).
- the antibody molecule can be a single domain antibody, e.g., a nanobody, such as a camelid, or a llama- or alpaca- derived single domain antibody, or a shark antibody (IgNAR).
- the single domain antibody comprises, e.g., only a variable heavy domain (VHH).
- An antibody molecule can also be a genetically engineered single domain antibody.
- the antibody molecule is a human, humanized, chimeric, camelid, shark or in vitro generated antibody.
- fragments include (i) an Fab fragment having a VL, VH, constant light chain domain (CL) and constant heavy chain domain 1 (CHI) domains; (ii) an Fd fragment having VH and CHI domains; (iii) an Fv fragment having VL and VH domains of a single antibody; (iv) a dAb fragment (Ward, E.S.
- Fv, scFv or diabody molecules may be stabilized by the incorporation of disulphide bridges linking the VH and VL domains (Reiter, Y. et al, Nature Biotech, 14, 1239- 1245, 1996).
- Minibodies comprising a scFv joined to a CH3 domain may also be made (Hu, S. et al, Cancer Res., 56, 3055-3061 , 1996).
- binding fragments are Fab', which differs from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CHI domain, including one or more cysteines from the antibody hinge region, and Fab'-SH, which is a Fab' fragment in which the cysteine residue(s) of the constant domains bear a free thiol group.
- Fab' which differs from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CHI domain, including one or more cysteines from the antibody hinge region
- Fab'-SH which is a Fab' fragment in which the cysteine residue(s) of the constant domains bear a free thiol group.
- antibody molecule includes intact molecules as well as functional fragments thereof. Constant regions of the antibody molecules can be altered, e.g., mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function).
- antibodies for use in the present disclosure are labelled, modified to increase half-life, and the like.
- the antibody is chemically modified, such as by PEGylation, or by incorporation in a liposome.
- Antibody molecules can also be single domain antibodies.
- Single domain antibodies can include antibodies whose complementary determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, light chains devoid of heavy chains, single domain antibodies derived from conventional 4-chain antibodies, and engineered antibodies and single domain scaffolds other than those derived from antibodies.
- Single domain antibodies may be any of the art, or any future single domain antibodies.
- Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, fish, shark, goat, rabbit, and bovine.
- a single domain antibody can be derived from a variable region of the immunoglobulin found in fish, such as, for example, that which is derived from the immunoglobulin isotype known as Novel Antigen Receptor (NAR) found in the serum of shark.
- NAR Novel Antigen Receptor
- Methods of producing single domain antibodies derived from a variable region of NAR are described in WO 03/014161 and Streltsov (2005) Protein Sci. 14:2901-2909.
- a single domain antibody is a naturally occurring single domain antibody known as a heavy chain antibody devoid of light chains. Such single domain antibodies are disclosed in WO 9404678, for example.
- variable domain derived from a heavy chain antibody naturally devoid of light chain is known herein as a VHH or nanobody to distinguish it from the conventional VH of four chain immunoglobulins.
- VHH molecule can be derived from antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca and guanaco. Other species besides Camelidae may produce heavy chain antibodies naturally devoid of light chain; and such VHHs are within the scope of the disclosure.
- the VH and VL regions can be subdivided into regions of hypervariability, termed “complementarity determining regions” (CDR), interspersed with regions that are more conserved, termed “framework regions” (FR).
- CDR complementarity determining regions
- FR framework regions
- the extent of the framework region and CDRs has been precisely defined by a number of methods (see, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; and the AbM definition used by Oxford Molecular' s AbM antibody modelling software.
- Each VH and VL typically includes three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
- the VH or VL chain of the antibody molecule can further include all or part of a heavy or light chain constant region, to thereby form a heavy or light
- the antibody molecule is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains.
- the heavy and light immunoglobulin chains can be connected by disulfide bonds.
- the heavy chain constant region typically includes three constant domains, CHI, CH2 and CH3.
- the light chain constant region typically includes a CL domain.
- the variable region of the heavy and light chains contains a binding domain that interacts with an antigen.
- the constant regions of the antibody molecules typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
- immunoglobulin comprises various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon ( ⁇ , ⁇ , ⁇ , ⁇ , ⁇ ) with some subclasses among them (e.g., ⁇ - ⁇ 4). It is the nature of this chain that determines the "class” of the antibody as IgG, IgM, IgA IgD, or IgE, respectively.
- the immunoglobulin subclasses isotypes) e.g., IgGl, IgG2, IgG3, IgG4, IgAl, etc.
- Light chains are classified as either kappa or lambda ( ⁇ , ⁇ ). Each heavy chain class may be bound with either a kappa or lambda light chain.
- antigen-binding fragment refers to one or more fragments of a full- length antibody that retain the ability to specifically bind to a target of interest.
- binding fragments encompassed within the term "antigen-binding fragment" of a full length antibody include (i) a Fab fragment, a monovalent fragment having VL, VH, CL and CHI domains; (ii) a F(ab') 2 fragment, a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment having VH and CHI domains; (iv) an Fv fragment having VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, (1989) Nature 341 :544-546), which has a VH domain; and (vi) an isolated complementarity determining region (CDR) that retains functionality.
- a Fab fragment a monovalent fragment having VL, VH, CL and CHI domains
- F(ab') 2 fragment a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region
- the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules known as single chain Fv (scFv).
- scFv single chain Fv
- antigen-binding site refers to the part of an antibody molecule that comprises determinants that form an interface that binds to a target antigen, or an epitope thereof.
- the antigen-binding site typically includes one or more loops (of at least four amino acids or amino acid mimics) that form an interface that binds to the target antigen or epitope thereof.
- the antigen-binding site of an antibody molecule includes at least one or two CDRs, or more typically at least three, four, five or six CDRs.
- the antibody may comprise replacing one or more amino acid residue(s) with a non-naturally occurring or non-standard amino acid, modifying one or more amino acid residue into a non-naturally occurring or non-standard form, or inserting one or more non- naturally occurring or non-standard amino acid into the sequence. Examples of numbers and locations of alterations in sequences are described elsewhere herein.
- Naturally occurring amino acids include the 20 "standard" L-amino acids identified as G, A, V, L, I, M, P, F, W, S, T, N, Q, Y, C, K, R, H, D, E by their standard single- letter codes.
- Non-standard amino acids include any other residue that may be incorporated into a polypeptide backbone or result from modification of an existing amino acid residue.
- Non-standard amino acids may be naturally occurring or non- naturally occurring.
- Several naturally occurring non-standard amino acids are known in the art, such as 4-hydroxyproline, 5-hydroxylysine, 3-methylhistidine, N- acetylserine, etc. (Voet & Voet, Biochemistry, 2nd Edition, (Wiley) 1995).
- Those amino acid residues that are derivatised at their N-alpha position will only be located at the N-terminus of an amino-acid sequence.
- an amino acid is an L-amino acid, but it may be a D-amino acid.
- Alteration may therefore comprise modifying an L-amino acid into, or replacing it with, a D-amino acid.
- Methylated, acetylated and/or phosphorylated forms of amino acids are also known, and amino acids in the present disclosure may be subject to such modification.
- the antibodies used in the claimed methods are generated using random mutagenesis of one or more selected VH and/or VL genes to generate mutations within the entire variable domain.
- random mutagenesis of one or more selected VH and/or VL genes to generate mutations within the entire variable domain.
- Such a technique is described by Gram et al, 1992, Proc. Natl. Acad. Sci., USA, 89:3576-3580 who used error- prone PCR.
- one or two amino acid substitutions are made within an entire variable domain or set of CDRs.
- Another method that may be used is to direct mutagenesis to CDR regions of
- VH or VL genes Such techniques are disclosed by Barbas et al, 1994, Proc. Natl. Acad. Sci., USA, 91 :3809-3813 and Schier et al, 1996, J. Mol. Biol. 263:551-567.
- Suitable antibodies for use as an AAM moiety can be prepared using methods well known in the art. For example, antibodies can be generated recombinantly, made using phage display, produced using hybridoma technology, etc. Non-limiting examples of techniques are described briefly below.
- Monoclonal antibodies can be obtained, for example, from a cell obtained from an animal immunized against the target antigen, or one of its fragments. Suitable fragments and peptides or polypeptides comprising them may be used to immunise animals to generate antibodies against the target antigen.
- the monoclonal antibodies can, for example, be purified on an affinity column on which the target antigen or one of its fragments containing the epitope recognized by said monoclonal antibodies, has previously been immobilized. More particularly, the monoclonal antibodies can be purified by chromatography on protein A and/or G, followed or not followed by ion-exchange chromatography aimed at eliminating the residual protein contaminants as well as the DNA and the lipopolysaccaride (LPS), in itself, followed or not followed by exclusion chromatography on SepharoseTM gel in order to eliminate the potential aggregates due to the presence of dimers or of other multimers. In one embodiment, the whole of these techniques can be used
- human hybridomas can be made as described by Kontermann, R & Dubel, S, Antibody Engineering, Springer- Verlag New York, LLC; 2001, ISBN: 3540413545.
- Phage display another established technique for generating antagonists has been described in detail in many publications, such as Kontermann & Dubel, supra and WO92/01047 (discussed further below), and US patents US 5,969,108, US 5,565,332, US 5,733,743, US 5,858,657, US 5,871,907, US 5,872,215, US 5,885,793, US 5,962,255, US 6,140,471, US 6,172,197, US 6,225,447, US 6,291,650, US 6,492,160 and US 6,521,404.
- mice in which the mouse antibody genes are inactivated and functionally replaced with human antibody genes while leaving intact other components of the mouse immune system, can be used for isolating human antibodies Mendez, M. et al. (1997) Nature Genet, 15(2): 146-156.
- Humanised antibodies can be produced using techniques known in the art such as those disclosed in, for example, WO91/09967, US 5,585,089, EP592106, US 5,565,332 and WO93/17105.
- WO2004/006955 describes methods for humanising antibodies, based on selecting variable region framework sequences from human antibody genes by comparing canonical CDR structure types for CDR sequences of the variable region of a non- human antibody to canonical CDR structure types for corresponding CDRs from a library of human antibody sequences, e.g. germline antibody gene segments.
- Human antibody variable regions having similar canonical CDR structure types to the non- human CDRs form a subset of member human antibody sequences from which to select human framework sequences.
- the subset members may be further ranked by amino acid similarity between the human and the non-human CDR sequences.
- top ranking human sequences are selected to provide the framework sequences for constructing a chimeric antibody that functionally replaces human CDR sequences with the non-human CDR counterparts using the selected subset member human frameworks, thereby providing a humanized antibody of high affinity and low immunogenicity without need for comparing framework sequences between the non-human and human antibodies.
- Chimeric antibodies made according to the method are also disclosed.
- Synthetic antibody molecules may be created by expression from genes generated by means of oligonucleotides synthesized and assembled within suitable expression vectors, for example as described by Knappik et al. J. Mol. Biol. (2000) 296, 57-86 or Krebs et al. Journal of Immunological Methods 254 2001 67-84.
- any such antibody can be subsequently produced using recombinant techniques.
- a nucleic acid sequence encoding the antibody may be expressed in a host cell. Such methods include expressing nucleic acid sequence encoding the heavy chain and light chain from separate vectors, as well as expressing the nucleic acid sequences from the same vector. These and other techniques using a variety of cell types are well known in the art.
- antibodies that specifically bind to any target can be made. Once made, antibodies can be tested to confirm that they bind to the desired target antigen and to select antibodies having desired properties. Such desired properties include, but are not limited to, selecting antibodies having the desired affinity and cross-reactivity profile. Given that large numbers of candidate antibodies can be made, one of skill in the art can readily screen a large number of candidate antibodies to select those antibodies suitable for the intended use. Moreover, the antibodies can be screened using functional assays to identify antibodies that bind the target and have a particular function, such as the ability to inhibit an activity of the target or the ability to bind to the target without inhibiting its activity. Thus, one can readily make antibodies that bind to a target and are suitable for an intended purpose.
- the nucleic acid (e.g., the gene) encoding an antibody can be cloned into a vector that expresses all or part of the nucleic acid.
- the nucleic acid can include a fragment of the gene encoding the antibody, such as a single chain antibody (scFv), a F(ab') 2 fragment, a Fab fragment, or an Fd fragment.
- Antibodies may also include modifications, e.g., modifications that alter Fc function, e.g., to decrease or remove interaction with an Fc receptor or with Clq, or both.
- the human IgG4 constant region can have a Ser to Pro mutation at residue 228 to fix the hinge region.
- the human IgGl constant region can be mutated at one or more residues, e.g., one or more of residues 234 and 237, e.g., according to the numbering in U.S. Patent No. 5,648,260.
- Other exemplary modifications include those described in U.S. Patent No. 5,648,260.
- the antibody production system may be designed to synthesize antibodies in which the Fc region is glycosylated.
- the Fc domain of IgG molecules is glycosylated at asparagine 297 in the CH2 domain.
- This asparagine is the site for modification with biantennary-type oligosaccharides. This glycosylation participates in effector functions mediated by Fey receptors and complement Clq (Burton and Woof (1992) Adv. Immunol. 51 : 1-84; Jefferis et al. (1998) Immunol. Rev. 163:59-76).
- the Fc domain can be produced in a mammalian expression system that appropriately glycosylates the residue corresponding to asparagine 297.
- the Fc domain can also include other eukaryotic post-translational modifications.
- Antibodies can be modified, e.g., with a moiety that improves its stabilization and/or retention in circulation, e.g., in blood, serum, lymph, bronchoalveolar lavage, or other tissues, e.g., by at least 1.5, 2, 5, 10, or 50 fold.
- an antibody generated by a method described herein can be associated with a polymer, e.g., a substantially non-antigenic polymer, such as a polyalkylene oxide or a polyethylene oxide.
- a polymer e.g., a substantially non-antigenic polymer, such as a polyalkylene oxide or a polyethylene oxide.
- Suitable polymers will vary substantially by weight. Polymers having molecular number average weights ranging from about 200 to about 35,000 daltons (or about 1,000 to about 15,000, and 2,000 to about 12,500) can be used.
- an antibody generated by a method described herein can be conjugated to a water soluble polymer, e.g., a hydrophilic polyvinyl polymer, e.g. polyvinylalcohol or polyvinylpyrrolidone.
- a water soluble polymer e.g., a hydrophilic polyvinyl polymer, e.g. polyvinylalcohol or polyvinylpyrrolidone.
- a non-limiting list of such polymers include polyalkylene oxide homopolymers such as polyethylene glycol (PEG) or polypropylene glycols, polyoxyethylenated polyols, copolymers thereof and block copolymers thereof, provided that the water solubility of the block copolymers is maintained.
- Additional useful polymers include polyoxyalkylenes such as
- polyoxyethylene, polyoxypropylene, and block copolymers of polyoxyethylene and polyoxypropylene Pluronics
- polymethacrylates carbomers; branched or unbranched polysaccharides that comprise the saccharide monomers D-mannose, D- and L-galactose, fucose, fructose, D-xylose, L-arabinose, D-glucuronic acid, sialic acid, D-galacturonic acid, D-mannuronic acid (e.g.
- polymannuronic acid or alginic acid
- D-glucosamine D-galactosamine
- D-glucose and neuraminic acid including homopolysaccharides and heteropolysaccharides such as lactose, amylopectin, starch, hydroxyethyl starch, amylose, dextrane sulfate, dextran, dextrins, glycogen, or the polysaccharide subunit of acid mucopolysaccharides, e.g. hyaluronic acid; polymers of sugar alcohols such as polysorbitol and polymannitol; heparin or heparan.
- Antibody-mimic molecules are antibody-like molecules comprising a protein scaffold or other non-antibody target binding region with a structure that facilitates binding with target molecules, e.g., polypeptides.
- an antibody mimic comprises a scaffold
- the scaffold structure of an antibody-mimic is reminiscent of antibodies, but antibody-mimics do not include the CDR and framework structure of immunoglobulins.
- a pool of scaffold proteins having different amino acid sequence can be made and screened to identify the antibody-mimic molecule having the desired features (e.g., ability to bind a particular target; ability to bind a particular target with a certain affinity; ability to bind a particular target to produce a certain result, such as to inhibit activity of the target).
- desired features e.g., ability to bind a particular target; ability to bind a particular target with a certain affinity; ability to bind a particular target to produce a certain result, such as to inhibit activity of the target.
- antibody-mimics molecules that bind a target and that have a desired function can be readily made and tested in much the same way that antibodies can be.
- an antibody-mimic moiety molecule can comprise binding site portions that are derived from a member of the immunoglobulin superfamily that is not an immunoglobulin (e.g., a T-cell receptor or a cell-adhesion protein such as CTLA-4, N-CAM, and telokin).
- a member of the immunoglobulin superfamily that is not an immunoglobulin (e.g., a T-cell receptor or a cell-adhesion protein such as CTLA-4, N-CAM, and telokin).
- antibody-mimic moiety molecules of the disclosure also comprise a binding site with a protein topology that is not based on the immunoglobulin fold (e.g., such as ankyrin repeat proteins or fibronectins) but which nonetheless are capable of specifically binding to a target antigen or epitope.
- Antibody-mimic moiety molecules may be identified by selection or isolation of a target-binding variant from a library of binding molecules having artificially diversified binding sites. Diversified libraries can be generated using completely random approaches (e.g., error-prone PCR, exon shuffling, or directed evolution) or aided by art-recognized design strategies. For example, amino acid positions that are usually involved when the binding site interacts with its cognate target molecule can be randomized by insertion of degenerate codons, trinucleotides, random peptides, or entire loops at corresponding positions within the nucleic acid which encodes the binding site (see e.g., U.S. Pub. No. 20040132028).
- the location of the amino acid positions can be identified by investigation of the crystal structure of the binding site in complex with the target molecule.
- Candidate positions for randomization include loops, flat surfaces, helices, and binding cavities of the binding site.
- amino acids within the binding site that are likely candidates for diversification can be identified by their homology with the immunoglobulin fold. For example, residues within the CDR-like loops of fibronectin may be randomized to generate a library of fibronectin binding molecules (see, e.g., Koide et al, J. Mol. Biol, 284: 1141-1151 (1998)). Other portions of the binding site which may be randomized include flat surfaces.
- an antibody-mimic molecule of the disclosure comprises a binding site from a fibronectin binding molecule.
- Fibronectin binding molecules e.g., molecules comprising the Fibronectin type I, II, or III domains
- display CDR- like loops which, in contrast to immunoglobulins, do not rely on intra-chain disulfide bonds.
- the Fnlll loops comprise regions that may be subjected to random mutation and directed evolutionary schemes of iterative rounds of target binding, selection, and further mutation in order to develop useful therapeutic tools.
- Fibronectin-based "addressable" therapeutic binding molecules (“FATBIM”) may be developed to specifically or preferentially bind the target antigen or epitope. Methods for making fibronectin binding polypeptides are described, for example, in WO 01/64942 and in U.S. Pat. Nos. 6,673,901, 6,703,199, 7,078,490, and 7,119,171, which are
- FATBIMs include, for example, the species of fibronectin-based binding molecules termed Adnectins ® .
- Adnectins ® also called Adnectins ®
- an Adnectin ® comprises far fewer amino acid residues than does an antibody, and in other embodiments, the Adnectin ® is approximately the size as a single variable domain of an antibody. In one embodiment, the Adnectin ® comprises approximately 90 amino acids, e.g., 94 amino acids, and has a molecular mass of about 10 kDa, which is fifteen times smaller than an IgG type antibody, and comparable to the size of a single variable domain of an antibody.
- an Adnectin ® is based on the structure of human fibronectin, and more specifically on the structure of the tenth extracellular type III domain of human fibronectin.
- This domain has a structure analogous to antibody variable domains, with seven beta sheets forming a barrel and three exposed loops on each side, which are analogous to the three complementarity determining regions.
- Adnectins ® typically lack binding sites for metal ions and a central disulfide bond.
- Adnectins ® can be engineered to have specificity for different target proteins by modifying the loops between the second and third beta sheets, and between the sixth and seventh beta sheets (i.e., by modifying loops BC and FG of the tenth extracellular type III domain of fibronectin).
- Adnectins ® are described in, e.g., U.S. Patent No. 7,115,396.
- the disclosure provides a complex comprising a Surf+ Penetrating Polypeptide associated with an Adnectin (e.g., a antibody-mimic based on the structure of human fibronectin), wherein the Adnectin binds to an intracellularly expressed target.
- complexes of the disclosure comprise an AAM moiety portion comprising a scaffold structure based on fibronectin, such as the tenth extracellular type III domain of fibronectin.
- an antibody-mimic molecule of the disclosure comprises a binding site from an affibody.
- Affibody ® molecules are derived from the immunoglobulin binding domains of staphylococcal Protein A (SPA) (see e.g., Nord et al, Nat. BiotechnoL, 15: 772-777 (1997)).
- An Affibody ® is an antibody mimic that has unique binding sites that bind specific targets.
- Affibody ® molecules can be small (e.g., consisting of three alpha helices with 58 amino acids and having a molar mass of about 6 kDa), have an inert format (no Fc function), and have been successfully tested in humans as targeting moieties.
- Affibody ® molecules have been shown to withstand high temperatures (90 °C) or acidic and alkaline conditions (pH 2.5 or pH 11, respectively).
- Affibody® binding sites employed in the disclosure may be synthesized by mutagenizing an SPA-related protein (e.g., Protein Z) derived from a domain of SPA (e.g., domain B) and selecting for mutant SPA- related polypeptides having binding affinity for a target antigen or epitope.
- SPA-related protein e.g., Protein Z
- domain B domain of SPA
- Other methods for making affibody binding sites are described in U.S. Pat. Nos. 6,740,734 and 6,602,977 and in WO 00/63243, each of which is incorporated herein by reference.
- the disclosure provides a complex comprising a Surf+ Penetrating Polypeptide associated with an Affibody, wherein the Affibody binds to an intraceullarly expressed target.
- an antibody-mimic molecule of the disclosure comprises a binding site from an anticalin.
- Anticalins ® are antibody functional mimetics derived from human lipocalins. Lipocalins are a family of naturally-occurring binding proteins that bind and transport small hydrophobic molecules such as steroids, bilins, retinoids, and lipids. The main structure of Anticalins ® is similar to wild type lipocalins. The central element of this protein architecture is a beta-barrel structure of eight antiparallel strands, which supports four loops at its open end. These loops form the natural binding site of the lipocalins and can be reshaped in vitro by extensive amino acid replacement, thus creating novel binding specificities.
- Anticalins ® possess high affinity and specificity for their prescribed ligands as well as fast binding kinetics, so that their functional properties are similar to those of antibodies. Anticalins ® however, have several advantages over antibodies, including smaller size, composition of a single polypeptide chain, and a simple set of four hypervariable loops that can be easily manipulated at the genetic level. Anticalins ® , for example, are about eight times smaller than antibodies with a size of about 180 amino acids and a mass of about 20 kDa. Anticalins ® have better tissue penetration than antibodies and are stable at temperatures up to 70 °C, and also unlike antibodies, Anticalins ® can be produced in bacterial cells ⁇ e.g., E. coli cells) in large amounts.
- Anticalins ® are able to selectively bind to small molecules as well. Anticalins ® are described in, e.g., U.S. Patent No. 7,723,476.
- the disclosure provides a complex comprising a Surf+
- Penetrating Polypeptide associated with an Affibody wherein the Affibody binds to an intraceullarly expressed target.
- an antibody-mimic molecule of the disclosure comprises a binding site from a cysteine -rich polypeptide.
- Cysteine-rich domains employed in the practice of the present disclosure typically do not form an alpha- helix, a beta-sheet, or a beta-barrel structure.
- the disulfide bonds promote folding of the domain into a three-dimensional structure.
- cysteine-rich domains have at least two disulfide bonds, more typically at least three disulfide bonds.
- An exemplary cysteine-rich polypeptide is an A domain protein.
- A-domains (sometimes called "complement-type repeats") contain about 30-50 or 30-65 amino acids.
- the domains comprise about 35-45 amino acids and in some cases about 40 amino acids. Within the 30-50 amino acids, there are about 6 cysteine residues. Of the six cysteines, disulfide bonds typically are found between the following cysteines: CI and C3, C2 and C5, C4 and C6.
- the A domain constitutes a ligand binding moiety.
- the cysteine residues of the domain are disulfide linked to form a compact, stable, functionally independent moiety. Clusters of these repeats make up a ligand binding domain, and differential clustering can impart specificity with respect to the ligand binding.
- Exemplary proteins containing A-domains include, e.g., complement components (e.g., C6, C7, C8, C9, and Factor I), serine proteases (e.g., enteropeptidase, matriptase, and corin), transmembrane proteins (e.g., ST7, LRP3, LRP5 and LRP6) and endocytic receptors (e.g. Sortilin-related receptor, LDL- receptor, VLDLR, LRP 1 , LRP2, and ApoER2).
- complement components e.g., C6, C7, C8, C9, and Factor I
- serine proteases e.g., enteropeptidase, matriptase, and corin
- transmembrane proteins e.g., ST7, LRP3, LRP5 and LRP6
- endocytic receptors e.g. Sortilin-related receptor, LDL- receptor, VLDLR, LRP 1 , LRP
- an antibody-mimic molecule of the disclosure comprises a binding site from a repeat protein.
- Repeat proteins are proteins that contain consecutive copies of small (e.g., about 20 to about 40 amino acid residues) structural units or repeats that stack together to form contiguous domains. Repeat proteins can be modified to suit a particular target binding site by adjusting the number of repeats in the protein.
- Exemplary repeat proteins include designed ankyrin repeat proteins (i.e., a DARPins) (see e.g., Binz et al, Nat. BiotechnoL, 22: 575-582 (2004)) or leucine-rich repeat proteins (i.e., LRRPs) (see e.g., Pancer et al, Nature, 430: 174-180 (2004)).
- DARPins are genetically engineered antibody mimetic proteins that typically exhibit highly specific and high-affinity target protein binding. DARPins were first derived from natural ankyrin proteins. In certain embodiments, DARPins comprise three, four or five repeat motifs of an ankyrin protein. In certain embodiments, a unit of an ankyrin repeat consists of 30-34 amino acid residues and functions to mediate protein-protein interactions. In ceratin embodiments, each ankyrin repeat exhibits a helix-turn-helix conformation, and strings of such tandem repeats are packed in a nearly linear array to form helix-turn-helix bundles connected by relatively flexible loops.
- an ankyrin repeat protein is stabilized by intra- and inter-repeat hydrophobic and hydrogen bonding interactions.
- the repetitive and elongated nature of the ankyrin repeats provides the molecular bases for the unique characteristics of ankyrin repeat proteins in protein stability, folding and unfolding, and binding specificity. While not wishing to be bound by theory, it is believed that the ankyrin repeat proteins do not recognize specific sequences, and interacting residues are discontinuously dispersed into the whole molecules of both the ankyrin repeat protein and its target protein.
- ankyrin repeat domain for use as a DARPin to target any number of proteins.
- the molecular mass of a DARPin domain is typically about 14 or 18 kDa for four- or five-repeat DARPins,
- DARPins are described in, e.g., U.S. Patent No. 7,417,130. All so far determined tertiary structures of ankyrin repeat units share a characteristic composed of a beta-hairpin followed by two antiparallel alpha-helices and ending with a loop connecting the repeat unit with the next one. Domains built of ankyrin repeat units are formed by stacking the repeat units to an extended and curved structure. LRRP binding sites from part of the adaptive immune system of sea lampreys and other jawless fishes and resemble antibodies in that they are formed by recombination of a suite of leucine-rich repeat genes during lymphocyte maturation. Methods for making DARpin or LRRP binding sites are described in WO 02/20565 and WO 06/083275, each of which is incorporated herein by reference.
- antibody mimics include all or a portion of an antibody like molecule, comprising the CH2 and CH3 domains of an immunoglulin, engineered with non-CDR loops of constant and/or variable domains, thereby mediating binding to an epitope via the non-CDR loops.
- Exemplary technology includes technology from F-Star, such as antigen binding Fc molecules (termed FcabTM) or full length antibody like molecules with dual functionality (mAb 2 TM).
- FcabTM antigen binding Fc
- antigen binding Fc are a "compressed” version of these antibody like molecules.
- These molecules include the CH2 and CH3 domains of the Fc portion of an antibody, naturally folded as a homodimer (50kDa).
- Antigen binding sites are engineered into the CH3 domains, but the molecules lack traditional antibody variable regions.
- mAb 2 TM molecules Similar antibody like molecules are referred to as mAb 2 TM molecules.
- Full length IgG antibodies with additional binding domains (such as two) engineered into the CH3 domains.
- additional binding domains such as two
- these molecules may be bispecific or multispecific or otherwise facilitate tissue targeting.
- an antibody-mimic molecule of the disclosure comprises binding sites derived from Src homology domains (e.g. SH2 or SH3 domains), PDZ domains, beta-lactamase, high affinity protease inhibitors, or small disulfide binding protein scaffolds such as scorpion toxins.
- Src homology domains e.g. SH2 or SH3 domains
- PDZ domains e.g., PDZ domains
- beta-lactamase e.g., high affinity protease inhibitors
- small disulfide binding protein scaffolds such as scorpion toxins.
- binding sites may be derived from a binding domain selected from the group consisting of an EGF-like domain, a Kringle-domain, a PAN domain, a Gla domain, a SRCR domain, a Kunitz/Bovine pancreatic trypsin Inhibitor domain, a Kazal-type serine protease inhibitor domain, a Trefoil (P-type) domain, a von Willebrand factor type C domain, an Anaphylatoxin-like domain, a CUB domain, a thyroglobulin type I repeat, LDL-receptor class A domain, a Sushi domain, a Link domain, a Thrombospondin type I domain, an Immunoglobulin-like domain, a C-type lectin domain, a MAM domain, a von Willebrand factor type A domain, a
- Somatomedin B domain a WAP -type four disulfide core domain, a F5/8 type C domain, a Hemopexin domain, a Laminin-type EGF-like domain, a C2 domain, and other such domains known to those of ordinary skill in the art, as well as derivatives and/or variants thereof.
- Exemplary antibody-mimic moiety molecules, and methods of making the same, can also be found in Stemmer et al., "Protein scaffolds and uses thereof, U.S. Patent Publication No. 20060234299 (Oct. 19, 2006) and Hey, et al, Artificial, Non- Antibody Binding Proteins for Pharmaceutical and Industrial
- an antibody-mimic molecule comprises a Kunitz domain.
- Kunitz domains are conserved protein domains that inhibit certain proteases, e.g., serine proteases. Kunitz domains are relatively small, typically being about 50 to 60 amino acids long and having a molecular weight of about 6 kDa.
- Kunitz domains typically carry a basic charge and are characterized by the placement of two, four, six or eight or more that form disulfide linkages that contribute to the compact and stable nature of the folded peptide. For example, many Kunitz domains have six conserved cysteine residues that form three disulfide linkages.
- the disulfide-rich ⁇ / ⁇ fold of a Kunitz domain can include two, three (typically), or four or more disulfide bonds.
- Kunitz domains have a pear-shaped structure that is stabilized the, e.g., three disulfide bonds, and that contains a reactive site region featuring the principal determinant PI residue in a rigid confirmation.
- These inhibitors competitively prevent access of a target protein (e.g., a serine protease) for its physiologically relevant macromolecular substrate through insertion of the PI residue into the active site cleft.
- the PI residue in the proteinase-inhibitory loop provides the primary specificity determinant and dictates much of the inhibitory activity that particular Kunitz protein has toward a targeted proteinase.
- the N-terminal side of the reactive site (P) is energetically more important that the P' C-terminal side.
- lysine or arginine occupy the PI position to inhibit proteinases that cleave adjacent to those residues in the protein substrate.
- Other residues, particularly in the inhibitor loop region, contribute to the strength of binding.
- about 10-12 amino acid residues in the target protein and 20-25 residues in the proteinase are in direct contact in the formation of a stable proteinase-inhibitor complex and provide a buried area of about 600 to 900 A.
- Kunitz domains can be designed to target and inhibit or activate a protein of choice, e.g., an intracellular protein of choice. Kunitz domains are described in, e.g., U.S. Patent No. 6,057,287.
- an antibody-mimic molecule of the disclosure is an Affilin ® .
- Affilin ® molecules are small antibody-mimic proteins which are designed for specific affinities towards proteins and small compounds. New Affilin ® molecules can be very quickly selected from two libraries, each of which is based on a different human derived scaffold protein. Affilin ® molecules do not show any structural homology to immunoglobulin proteins.
- an antibody-mimic moiety molecule of the disclosure is an Avimer.
- Avimers are evolved from a large family of human extracellular receptor domains by in vitro exon shuffling and phage display, generating
- Avimers consist of two or more peptide sequences of 30 to 35 amino acids each, connected by linker peptides.
- the individual sequences are derived from A domains of various membrane receptors and have a rigid structure, stabilised by disulfide bonds and calcium.
- Each A domain can bind to a certain epitope of the target protein.
- the combination of domains binding to different epitopes of the same protein increases affinity to this protein, an effect known as avidity (hence the name).
- Other potential advantages include simple and efficient production of multitarget-specific molecules in Escherichia coli, improved
- the disclosure provides complexes in which the AAM moiety portion is an antibody-mimic that binds to an intracellular target, such as any of the foregoing classes antibody-mimics.
- any of these antibody-mimics may be complexed with a Surf+ Penetrating Polypeptide or a portion comprising a Surf+ Penetrating Polypeptide, including any of the sub-categories or specific examples of Surf+ Penetrating Polypeptides.
- the present disclosure provides complexes comprising (i) a Surf+ Penetrating Polypeptide portion and (ii) an AAM moiety portion (e.g., at least one AAM moiety) associated with the Surf+ Penetrating Polypeptide portion.
- the complexes are useful, for example, for delivery into a cell, and thus facilitate delivery of the AAM moiety into a cell where it can bind its intracellular target.
- AAM moiety portion e.g., at least one AAM moiety
- the present disclosure provides complexes comprising (i) a Surf+ Penetrating Polypeptide portion and (ii) an AAM moiety portion (e.g., at least one AAM moiety) associated with the Surf+ Penetrating Polypeptide portion.
- the AAM moiety portion binds to an intracellular target and the Surf+ Penetrating Polypeptide portion facilitates entry of the complex, and thus entry of the AAM moiety, into cells. Once inside the cell, the AAM moiety portion can bind the intracellularly expressed target.
- the association between the AAM moiety and the Surf+ Penetrating Polypeptide is disruptable. Thus, in certain embodiments, once the complex enters the cell, the association can be disrupted and the AAM moiety alone can bind or continue binding to the target. However, the association need not be disrupted, and the complex may remain intact after entry into the cell.
- Complexes of the disclosure may, in certain embodiments, include portions in addition to the Surf+ Penetrating Polypeptide portion and the AAM moiety portion.
- the complexes may include one or more linkers, the complexes may include sequence that helps localize the complex to a sub-cellular location, and/or the complex may include tags to facilitate detection and/or purification of the complex or a portion of the complex. These additional sequences may be located at the N- terminus, at the C-terminus or internally.
- additional portions may be interconnected to the Surf+ Polypeptide portion to the AAM moiety portion or to both.
- Complexes of the disclosure comprises a Surf+ Penetrating Polypeptide that penetrates cells associated with an AAM moiety that binds to an intraceular target.
- a Surf+ Penetrating Polypeptide that penetrates cells associated with an AAM moiety that binds to an intraceular target.
- these complexes penetrate cells and bind to the intracellular target via the AAM moiety.
- the complex penetrates cells and the AAM moiety is able to bind to its intracellular target.
- an AAM moiety may bind to an intracellular target, such as a polypeptide or peptide, and alter the activity of the target and/or the activity of the cell via one or more of the following mechanisms (i) inhibit one or more functions of the target; (ii) activate one or more functions of the target; (iii) increase or decrease the activity of the target; (iv) promote or inhibit degradation of the target; (v) change the localization of the target; and (vi) prevent binding between the target and another protein.
- an intracellular target such as a polypeptide or peptide
- the Surf+ Penetrating Polypeptide and AAM moiety portions of the complex are associated covalently.
- these two portions may be fused (e.g., the complex comprises a fusion protein).
- Covalent interactions may be direct or indirect (via a linker). Additional interactions, such as non-covalent interactions, may also be involved in the association between the two portions.
- covalent interactions are mediated by one or more linkers.
- the linker is a cleavable linker.
- the cleavable linker comprises an amide, an ester, or a disulfide bond.
- the linker may be an amino acid sequence that is cleavable by a cellular enzyme.
- the enzyme is a protease. In other embodiments, the enzyme is an esterase. In some embodiments, the enzyme is one that is more highly expressed in certain cell types than in other cell types. For example, the enzyme may be one that is more highly expressed in tumor cells than in non- tumor cells. Exemplary sequences that can be used in linkers and enzymes that cleave those linkers are presented in Table 2. Table 2. Exemplary cleavable linker sequences.
- X denotes the Surf+ Penetrating Polypeptide or AAM moiety.
- linkers include flexible linkers, such as one or more repeats of glycine and serine (Gly/Ser linkers).
- the flexible linker comrises glycine, alanine and/or serine amino acid residues.
- Simple amino acids e.g., amino acids with simple side chains (e.g., H, CH 3 or CH 2 OH) and/or unbranched
- provide greater flexibility e.g., two-dimensional or three-dimensional flexibility
- alternating the glycine, alanine and/or serine residues may provide even greater flexibility with in the linker.
- the amino acids can
- Exemplary flexible linkers include linkers comprising repeats of gly-gly-gly-gly-ser, gly-ser, ala-ser, and ala-gly. Other combinations are also possible.
- the Surf+ Penetrating Polypeptide and the AAM moiety are fused by using a construct that comprises an intein, which is self-spliced out to join the Surf+ Penetrating Polypeptide and the AAM moiety via a peptide bond.
- the Surf+ Penetrating Polypeptide and the AAM moiety are synthesized by using a viral 2 A peptide construct that comprises the Surf+ Penetrating Polypeptide and the AAM moiety for bicistronic expression.
- the Surf+ Penetrating Polypeptide and the AAM moiety genes may be expressed on the bicistronic construct, and the 2A peptide results in cotranslational "cleavage" of the two proteins (Trichas et al, BMC Biology 6:40, 2008).
- the disclosure contemplates complexes in which the Surf+ Penetrating
- Polypeptide and the AAM moiety portions are associated by a covalent or non- covalent linkage. In either case, the association may be direct or via one or more additional intervening liners or moieties.
- a Surf+ Penetrating Polypeptide and an AAM moiety are associated through chemical or proteinaceous linkers or spacers.
- exemplary linkers and spacers include, but are not restricted to, substituted or unsubstituted alkyl chains, polyethylene glycol derivatives, amino acid spacers, sugars, or aliphatic or aromatic spacers common in the art.
- Suitable linkers include, for example, homobifunctional and
- heterobifunctional cross-linking molecules The homobifunctional molecules have at least two reactive functional groups, which are the same.
- the reactive functional groups on a homobifunctional molecule include, for example, aldehyde groups and active ester groups.
- Homobifunctional molecules having aldehyde groups include, for example, glutaraldehyde and subaraldehyde.
- Homobifunctional linker molecules having at least two active ester units include esters of dicarboxylic acids and N-hydroxysuccinimide.
- N-succinimidyl esters include disuccinimidyl suberate and dithio-bis- (succinimidyl propionate), and their soluble bis-sulfonic acid and bis-sulfonate salts such as their sodium and potassium salts.
- Heterobifunctional linker molecules have at least two different reactive groups.
- heterobifunctional reagents containing reactive disulfide bonds include N-succinimidyl 3-(2-pyridyl-dithio)propionate (Carlsson et al., 1978.
- heterobifunctional reagents comprising reactive groups having a double bond that reacts with a thiol group include succinimidyl 4-(N- maleimidomethyl)cyclohexahe- 1 -carboxylate and succinimidyl m- maleimidobenzoate.
- heterobifunctional molecules include succinimidyl 3- (maleimido)propionate, sulfosuccinimidyl 4-(p-maleimido-phenyl)butyrate, sulfosuccinimidyl 4-(N-maleimidomethyl-cyclohexane)- 1 -carboxylate,
- affinity molecule binding pairs which selectively interact with acceptor groups.
- One entity of the binding pair can be fused or otherwise linked to the Surf+ Penetrating Polypeptide and the other entity of the binding pair can be fused or otherwise linked to the AAM moiety.
- Exemplary affinity molecule binding pairs include biotin and streptavidin, and derivatives thereof; metal binding molecules; and fragments and combinations of these molecules.
- Exemplary affinity binding pairs include StreptTag (WSHPQFEK) (SEQ ID NO: 657) / SBP (streptavidin binding protein), cellulose binding domain/ cellulose, chitin binding domain/ chitin, S-peptide/ S-fragment of RNAseA, calmodulin binding peptide/ calmodulin, and maltose binding protein/ amylose.
- the Surf+ Penetrating Polypeptide and the AAM moiety are linked by ubiquitin (and ubiquitin-like) conjugation.
- the disclosure also provides nucleic acids encoding a Surf+ Penetrating Polypeptide and an AAM moiety, such as an antibody molecule, or a non-antibody molecule scaffold, such as a DARPin, an Adnectin ® , an Anticalin ® , or a Kunitz domain polypeptide.
- the complex of a Surf+ Penetrating Polypeptide and an AAM moiety can be expressed as a fusion protein, optionally separated by a peptide linker.
- the peptide linker can be cleavable or not cleavable.
- a nucleic acid encoding a fusion protein can express the fusion in any orientation.
- the nucleic acid can express an N-terminal Surf+ Penetrating Polypeptide fused to a C-terminal AAM moiety ⁇ e.g., antibody), or can express an N-terminal AAM moiety fused to a C- terminal Surf+ Penetrating Polypeptide.
- a nucleic acid encoding an Surf+ Penetrating Polypeptide can be on a vector that is separate from a vector that carries a nucleic acid encoding a AAM moiety.
- the Surf+ Penetrating Polypeptide and the AAM moiety can be expressed separately, and complexed (including chemically linked) prior to introduction to a cell for intracellular delivery.
- the isolated complex can be formulated for administration to a subject, as a pharmaceutical composition.
- the disclosure also provides host cells comprising a nucleic acid encoding the Surf+ Penetrating Polypeptide or the AAM moiety, or comprising the complex as a fusion protein.
- the host cells can be, for example, prokaryotic cells (e.g., E. coli) or eukaryotic cells.
- the recombinant nucleic acids encoding an complex, or the portions thereof may be operably linked to one or more regulatory nucleotide sequences in an expression construct.
- Regulatory nucleotide sequences will generally be appropriate for a host cell used for expression. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells.
- said one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters as known in the art are contemplated by the disclosure.
- the promoters may be either naturally occurring promoters, or hybrid promoters that combine elements of more than one promoter.
- An expression construct may be present in a cell on an episome, such as a plasmid, or the expression construct may be inserted in a chromosome.
- the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selectable marker genes are well known in the art and will vary with the host cell used.
- this disclosure relates to an expression vector comprising a nucleotide sequence encoding a complex of the disclosure (e.g., a complex comprising a Surf+ Penetrating Polypeptide portion and an AAM moiety portion) polypeptide and operably linked to at least one regulatory sequence. Regulatory sequences are art- recognized and are selected to direct expression of the encoded polypeptide.
- regulatory sequence includes promoters, enhancers, and other expression control elements. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, CA (1990). It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed. Moreover, the vector's copy number, the ability to control that copy number and the expression of any other protein encoded by the vector, such as antibiotic markers, should also be considered.
- the disclosure also provides host cells comprising or transfected with a nucleic acid encoding the complex as a fusion protein.
- the host cells can be, for example, prokaryotic cells (e.g., E. coli) or eukaryotic cells. Other suitable host cells are known to those skilled in the art.
- a recombinant expression vector may carry additional nucleic acid sequences, such as sequences that regulate replication of the vector in a host cells (e.g., origins of replication) and selectable marker genes.
- the selectable marker gene facilitates selection of host cells into which the vector has been introduced.
- Exemplary selectable marker genes include the ampicillin and the kanamycin resistance genes for use in E. coli.
- a host cell transfected with an expression vector can be cultured under appropriate conditions to allow expression of the polypeptide to occur.
- the polypeptide may be secreted and isolated from a mixture of cells and medium containing the polypeptides.
- the polypeptides may be retained in the cytoplasm or in a membrane fraction and the cells harvested, lysed and the protein isolated.
- a cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art.
- polypeptides can be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins, including ion-exchange
- the polypeptide is a fusion protein containing a domain which facilitates its purification.
- a nucleic acid encoding a Surf+ Penetrating Polypeptide can be on a vector that is separate from a vector that carries a nucleic acid encoding an AAM moiety.
- the portions of the complex can be expressed separately, and complexed prior to introduction to a cell for intracellular delivery.
- the isolated complex can be formulated for administration to a subject, as a pharmaceutical composition.
- Recombinant nucleic acids of the disclosure can be produced by ligating the cloned gene, or a portion thereof, into a vector suitable for expression in either prokaryotic cells, eukaryotic cells (yeast, avian, insect or mammalian), or both.
- Expression vehicles for production of a recombinant polypeptide include plasmids and other vectors.
- suitable vectors include plasmids of the types:
- the preferred mammalian expression vectors contain both prokaryotic sequences to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells.
- pcDNAI/amp examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Some of these vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells.
- viruses such as the bovine papilloma virus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells.
- BBV-1 bovine papilloma virus
- pHEBo Epstein-Barr virus
- pHEBo Epstein-Barr virus
- pREP-derived and p205 Epstein-Barr virus
- baculovirus expression systems include pVL- derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUWl), and pBlueBac-derived vectors (such as the ⁇ -gal containing pBlueBac III).
- fusion genes are well known. Essentially, the joining of various DNA fragments coding for different polypeptide sequences is performed in accordance with conventional techniques, employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
- the fusion gene can be synthesized by conventional techniques including automated DNA
- PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al, John Wiley & Sons: 1992).
- fusion polypeptides or protein of the present disclosure can be made in numerous ways. For example, a Surf+ Penetrating
- Polypeptide and an AAM moiety can be made separately, such as recombinantly produced in two separate cell cultures from nucleic acid constructs encoding their respective proteins. Once made, the proteins can be chemically conjugated directly or via a linker.
- the fusion polypeptide can be made as an inframe fusion in which the entire fusion polypeptide, optionally including one or more linker, tag or other moiety, is made from a nucleic acid construct that includes nucleotide sequence encoding both a Surf+ Penetrating Polypeptide portion and an AAM moiety portion of the complex.
- a complex of the disclosure is formed under conditions where the linkage ⁇ e.g., by a covalent or non-covalent linkage) is formed, while the activity of the AAM moiety is maintained.
- any linkage to the AAM moiety can be at a site on the protein that is distant from the target-interacting region of the AAM moiety.
- an enzyme that cleaves a linker between the a Surf+ Penetrating Polypeptide and an AAM moiety does not have an effect on the AAM moiety, such that the structure of the AAM moiety remains intact and the AAM moiety retains its target binding activity.
- the Surf+ Penetrating Polypeptide and AAM moiety portions of the complex are separated, e.g., within the cell, under conditions where the linkage ⁇ e.g., a covalent or non-covalent linkage) is dissociated, while the activity of the AAM moiety is maintained.
- the Surf+ Penetrating Polypeptide and AAM moiety can be joined by a cleavable peptide linker that is subject to a protease that does not interfere with activity of the AAM moiety.
- the Surf+ Penetrating Polypeptide portion and AAM moiety portion are separated in the endosome due to the lower pH of the endosome.
- the linker is cleaved or broken in response to the lower pH, but the activity of the AAM moiety is not affected.
- the AAM moiety binds and inhibits (or activates) activity of the intracellular target while the AAM moiety is still complexed with the Surf+ Penetrating Polypeptide.
- the complex does not dissociate in the cell, prior to the activity of the AAM moiety on the target protein.
- the Surf+ Penetrating Polypeptide and AAM moiety dissociate following delivery into the cell and, for example, the AAM moiety may interact with its intracellular target after dissociation from the Surf+ Penetrating Polypeptide.
- Polypeptides may be modified chemically or biologically. For example one or more amino acids may be added, deleted, or changed from the primary sequence. This includes changes intended to supercharge a polypeptide (e.g., to increase surface positive charge, net charge or charge/molecular weight). However, modifications to the Surf+ Penetrating Polypeptides also include variation that is not intended to supercharge the protein.
- modifications may be modifications to a complex of the disclosure, and the modification may be appended directly or indirectly to either or both of the Surf+ Penetrating Polypeptide portion or the AAM moiety portion.
- a polyhistidine tag or other tag may be added to the complex or to either polypeptide portion of the complex to aid in the purification of the complex or of either portion of the complex.
- Other peptides, protein or small molecules may be added onto the complex to alter the biological, biochemical, and/or biophysical properties of the complex.
- a targeting peptide may be added to the primary sequence of the Surf+ Penetrating Polypeptides or complex.
- Surf+ Penetrating Polypeptides or complex modifications include, but are not limited to, post-translational or post-production modifications (e.g. , glycosylation, phosphorylation, acylation, lipidation, farnesylation, acetylation, proteolysis, etc.).
- the Surf+ Penetrating Polypeptides or complex may be modified to reduce its immunogenicity.
- the Surf+ Penetrating Polypeptides or complex may be modified to improve half-life or bioavailability.
- the complex or either portion of the complex may be conjugated to a soluble polymer or carbohydrate, e.g., to increase serum half life of the Surf+ Penetrating Polypeptide, AAM moiety and/or complex.
- the Surf+ Penetrating Polypeptides, AAM moiety or complex may be conjugated to a polyethylene glycol (PEG) polymer, e.g., a monomethoxy PEG.
- PEG polyethylene glycol
- Other polymers useful as stabilizing materials may be of natural, semi-synthetic (modified natural) or synthetic origin. Exemplary natural polymers include naturally occurring
- polysaccharides such as, for example, arabinans, fructans, fucans, galactans, galacturonans, glucans, mannans, xylans (such as, for example, inulin), levan, fucoidan, carrageenan, galatocarolose, pectic acid, pectins, including amylose, pullulan, glycogen, amylopectin, cellulose, dextran, dextrin, dextrose, glucose, polyglucose, polydextrose, pustulan, chitin, agarose, keratin, chondroitin, dermatan, hyaluronic acid, alginic acid, xanthin gum, starch and various other natural homopolymer or heteropolymers, such as those containing one or more of the following aldoses, ketoses, acids or amines: erythose, threose, ribose, arabinose, xylose, lyx
- hydroxymethylcellulose hydroxypropylmethylcellulose, methylcellulose, and methoxycellulose.
- exemplary synthetic polymers include polyphosphazenes, hydroxyapatites, fluoroapatite polymers, polyethylenes (such as, for example, polyethylene glycol (including for example, the class of compounds referred to as PLURONICTM, commercially available from BASF, Parsippany, N. J.),
- polyoxyethylene and polyethylene terephthalate
- polypropylenes such as, for example, polypropylene glycol
- polyurethanes such as, for example, polyvinyl alcohol (PVA), polyvinyl chloride and polyvinylpyrrolidone
- polyamides including nylon, polystyrene, polylactic acids, fluorinated hydrocarbon polymers, fluorinated carbon polymers (such as, for example, polytetrafluoroethylene), acrylate, methacrylate, and polymethylmethacrylate, and derivatives thereof.
- the primary purpose of the modification is a purpose other than to further supercharge the complex versus that of the unmodified complex.
- the disclosure contemplates that any of the foregoing modifications may be to the Surf+ Penetrating Polypeptide portion of a complex or to the AAM moiety portion of a complex.
- the modification may be made prior to complex formation, concurrently with complex, such as fusion protein formation, or as a post-production step following complex (such as fusion protein) formation.
- localization domains to facilitate localization of the complex to the intended intracellular location.
- the localization domain may be appended directly or indirectly to the Surf+ Penetrating Polypeptide portion or to the AAM moiety portion.
- Exemplary localization domains include, for example, nuclear localization signal, a mitochondrial matrix localization signal, and the like.
- complexes of the disclosure can be modified to comprise a detectable moiety.
- Detectable moieties include fluorescent or otherwise detectable polypeptides, peptide, radioactive or other moieties which allow for detection of the complex or the portions of the complex.
- detectable moieties can be included in the polypeptide sequence of the complex, or operably linked thereto, such as in a fusion protein, or by covalent or non-covalent linkages.
- the disclosure contemplates that the detectable moiety may be appended directly or indirectly to the Surf+ Penetrating Polypeptide portion of the complex and/or the AAM moiety portion of the complex and/or to any linker portion.
- Exemplary fluorescent proteins include green fluorescent protein, blue fluorescent protein, cyan fluorescent protein or yellow fluorescent protein.
- Other exemplary fluorescent proteins include, but are not limited to, enhanced green fluorescent protein (EGFP), split GFP, AcGFP, TurboGFP, Emerald, Azami Green, ZsGreen, EBFP, Sapphire, T-Sapphire, ECFP, mCFP, Cerulean, CyPet, AmCyanl, Midori-Ishi Cyan, mTFPl (Teal), enhanced yellow fluorescent protein (EYFP), Topaz, Venus, mCitrine, YPet, PhiYFP, ZsYellowl, mBanana, Kusabira Orange, mOrange, dTomato, dTomato-Tandem, DsRed, DsRed2, DsRed-Express (Tl), DsRed-Monomer, mTangerine, mStrawberry, AsRed2, mRFPl, JRed, mCh
- suitable labels that can be used in accordance with the disclosure include, but are not limited to, fluorescent, chemiluminescent, chromogenic, phosphorescent, and/or radioactive labels.
- the complex when an epitope tag is included in a complex, the complex is detectable using an antibody that is immunoreactive with the epitope tag. Any complex of the disclosure can be readily tested to confirm that, following complex formation, the complex retains the ability to penetrate cells and the AAM moiety retains the ability to specifically bind its target. This testing can be done regardless of whether the complex is a fusion protein (directly or via a linker) or a chemical fusion or otherwise associated.
- the Surf+ Penetrating Polypeptide may be tested for cell penetration activity alone and the AAM moiety may be tested for specific binding (in vitro or ex vivo) to its target. After confirming that the selected Surf+ Penetrating Polypeptide does penetrate cells and the AAM moiety does bind its target, a complex is generated using any suitable method.
- the present disclosure provides complexes comprising (i) a Surf+ Penetrating Polypeptide portion and (ii) an AAM moiety portion, wherein the Surf+ Penetrating Polypeptide portion is associated with the AAM moiety portion.
- the present disclosure also provides methods for using such complexes.
- the AAM moiety binds to a target expressed in a cell and providing the AAM moiety as a complex promotes delivery of the AAM moiety into the cell (e.g., due to the cell penetrating ability of the Surf+ Penetrating Polypeptide). Once inside the cell, the AAM moiety can bind to its target. Such binding may occur while the AAM moiety remained complexed to the Surf+ Penetrating Polypeptide portion, or such binding may occur after cleavage or dissociation of the two portions of the complex.
- binding may initially occur while the AAM moiety is complexed to the Surf+ Penetrating Polypeptide, but the complex may then be disrupted or cleaved so that, subsequently, the AAM moiety alone is bound to the target (e.g., the target polypeptide or peptide expressed in the cell).
- target e.g., the target polypeptide or peptide expressed in the cell
- Any AAM moiety may be provided as a complex with a Surf+ Penetrating
- Polypeptide and delivered to a cell using the inventive system Given the ability to readily make and test antibodies and antibody-mimics, and thus, to generate AAM moieties capable of binding to a target and having a desired activity (e.g., inhibiting the function of the target, promoting the function of the target, binding without interfering or altering the function of the target), the present system may be used in combination with virtually any target, such as a polypeptide or peptide, expressed in a cell. Accordingly, the complexes of the disclosure have numerous applications, including research uses, therapeutic uses, diagnostic uses, imaging uses, and the like, and such uses are applicable over a wide range of targets and disease indications.
- Complexes of the disclosure are useful for delivering AAM moieties into cells where they are useful for labeling a target protein, such as for imaging cells, tissues and whole organisms. Labeling may be useful when performing research studies of protein expression, disease progression, cell fate, protein localization and the like. Labeling may be useful diagnostically or prognostically, such as in cases where target expression correlates with a particular condition. In certain
- an AAM moiety intended for labeling may be selected such that it does not interfere with the function of the target (e.g., a moiety that binds to a target but does not alter the activity of the target).
- complexes of the disclosure may be used in research setting to study target expression, presence/absence of target in a disease state, impact of inhibiting or promoting target activity, etc. Complexes of the disclosure are suitable for these studies in vitro or in vivo. By promoting delivery of the AAM moiety into cells, complexes of the disclosure help avoid false negative results obtained when an AAM moiety is unable to penetrate a cell (e.g., a non-experiment because the AAM moiety cannot contact a target expressed inside the cell).
- complexes of the disclosure have therapeutic uses by promoting delivery of therapeutic AAM moieties into cells in humans or animals (including animal models of a disease or condition).
- the use of complexes of the disclosure decrease failure of an AAM moiety due to inability to effectively penetrate cells or due to the inability to effectively penetrate cells at concentrations that are not otherwise toxic to the organism.
- the result is that the AAM moiety is delivered into a cell following contacting the cell with the complex (e.g., either contacting a cell in culture or administrated to a subject). Once inside the cells, the AAM moiety binds its intracellular target.
- the AAM moiety binds a target expressed in the nucleus or in the cytosol of a cell. In some embodiments, AAM moiety binds a membrane associated target, e.g., a target localized on the cytosolic side of the cell membrane, the cytosolic side of the nuclear membrane, or the cytosolic side of the mitochondrial membrane.
- a Surf+ Penetrating Polypeptide is complexed with an AAM moiety that binds an intracllular target in the nucleus of a cell, such as an NFAT (Nuclear Factor of Activated T cells) (e.g., NFAT-2), a STAT (Signal Transducer and Activator of Transcription) (e.g., STAT-3, STAT-5, or STAT-6) or RORgammaT (retinoic acid-related orphan receptor).
- NFAT Nuclear Factor of Activated T cells
- STAT Signal Transducer and Activator of Transcription
- STAT-3, STAT-5, or STAT-6 retinoic acid-related orphan receptor
- a Surf+ Penetrating Polypeptide is complexed with an AAM moiety that binds an intracellular target in the cytosol of the cell, such as FK506, calcineurin, or a Janus Kinase (e.g., JAK-1 or JAK-2.
- a Surf+ Penetrating Polypeptide is complexed with an
- AAM moiety that binds an intracellular target localized on the cytosoloic side of the cell membrane, such as ras, a PI3K (phosphoinositide-3-kinase), or fms-related tyrosine kinase 1 (vascular endothelial growth factor/vascular permeability factor receptor).
- ras a PI3K (phosphoinositide-3-kinase)
- fms-related tyrosine kinase 1 vascular endothelial growth factor/vascular permeability factor receptor
- a Surf+ Penetrating Polypeptide is complexed with an AAM moiety that binds an intracellular target localized on the cytosoloic side of the mitochondrial membrane, such as Bcl-2.
- the AAM moiety binds a kinase, a transcription factor or an oncoprotein.
- the AAM moiety can bind a kinase, such as a JAK kinase (e.g., JAK-1 or JAK-2) or b-raf (v-raf murine sarcoma viral oncogene homolog Bl ) or Erk (mitogen-activated protein kinase 1).
- the AAM moiety can bind a transcription factor, such as Hifl -alpha, a STAT (e.g., STAT-3, STAT-5 or STAT-6), or IRF-1 (Interferon Regulatory Factor 1).
- the AAM moiety binds an oncogene, such as ras, b-raf or Akt (v-akt murine thymoma viral oncogene homolog 1).
- a complex comprising (i) a Surf+ Penetrating
- Polypeptide portion and (ii) an AAM moiety portion in accordance with the present disclosure may be used for therapeutic purposes, or may be used for diagnostic purposes.
- the disease or condition that may be treated depends on the target (e.g., the target is one for which binding by an AAM moiety has a therapeutic benefit).
- a complex in accordance with the present disclosure may be used for treatment of any of a variety of diseases, disorders, and/or conditions, including but not limited to one or more of the following: autoimmune disorders; inflammatory disorders; and proliferative disorders, including cancers.
- the disease treated by the complex is a cardiovascular disorder, or an angiogenic disorder such as macular degeneration.
- the disease treated by the complex is an eye disease, such as age-related macular degeneration (AMD), diabetic macular edema (DME), retinitis pigmentosa, or uveitis.
- AMD age-related macular degeneration
- DME diabetic macular edema
- retinitis pigmentosa or uveitis.
- a complex is useful for treating one or more of the following: an infectious disease; a neurological disorder; a respiratory disorder; a digestive disorder; a musculoskeletal disorder; an endocrine, metabolic, or nutritional disorders; a urological disorder; psychological disorder; a skin disorder; a blood and lymphatic disorder; etc.
- the complex of the disclosure binds, via the AAM moiety, a protein set forth in Table 3 (each, an intracellular target).
- the AAM moiety portion of the complex binds (e.g., specifically binds) to the target expressed or otherwise located inside the cell (the intracellular target).
- targeting the protein may be useful in the research, diagnosis, prognosis, monitoring or treatment of the listed disease.
- Table 3 Exemplary intracellular target proteins.
- Ras GTPase signal cytosolic-side of transducing protein cell membrane cancer such as b-raf serine/threonine cytosol melanoma kinase
- MEK MAP/Erk kinase serine/threonine cytosol
- PI3K phosphatidyl lipid kinase cytosolic-side of inositol 3 kinase
- AKT serine/threonine cytosol
- inflammatory NEMO also known as regulatory binding cytosol diseases such as ⁇ (IKK gamma) protein/adaptor
- Myeloid regulatory binding cytosol diseases differentiation primary protein/adaptor Myeloid regulatory binding cytosol diseases differentiation primary protein/adaptor
- inflammatory NLRP3 inflammasome regulatory binding cytosol diseases (arthritis, component) protein/adaptor
- diseases such as diabetes type 2 and others.
- IRF-1 IRF-1 - transcription factor
- cancer fms-related tyrosine tyrosine kinase cytosolic-side of kinase 1 (vascular cell membrane endothelial growth
- cancer fms-related tyrosine tyrosine kinase cytosolic-side of kinase 3 cell membrane cancer kinase insert domain tyrosine kinase cytosolic-side of receptor (a type III cell membrane receptor tyrosine kinase)
- cancer macrophage stimulating 1 tyrosine kinase cytosolic-side of receptor c-met-related cell membrane tyrosine kinase
- intracellular targets e.g., generating complexes comprising an AAM moiety that binds to any intracellular target).
- a complex is administered to a cell or organism in an effective amount.
- effective amount means an amount of an agent to be delivered that is sufficient, when administered to a cell or a subject to have the desired effect.
- an effective amount may be the amount sufficient to promote delivery of the complex into a cell and to promote binding of the AAM moiety to its target.
- an effective amount is the amount sufficient to treat (e.g., alleviate, improve or delay onset of one or more symptoms of) a disease, disorder, and/or condition.
- the AAM moiety is bispecific, e.g., is a bispecific antibody, or bispecific fragment thereof.
- a complex comprising a bispecific antibody can bind two different target polypeptides at the same time, or at different times.
- a complex of the disclosure may be used in a clinical setting, such as for therapeutic purposes.
- Therapeutic complexes may include an AAM moiety that binds to and reduces the activity of one or more targets ⁇ e.g., polypeptide targets).
- AAM moieties are particularly useful for treating a disease, disorder, and/or condition associated with high levels of one or more particular targets, or high activity levels of one or more particular targets.
- the complex is detectable (e.g., one or both of the Surf+ Penetrating Polypeptide portion and the AAM moiety portion are modified with a detectable label).
- one or both portions of the complex may include at least one fluorescent moiety.
- the Surf+ Penetrating Polypeptide portion and the AAM moiety portion are modified with a detectable label.
- one or both portions of the complex may include at least one fluorescent moiety.
- the Surf+ Penetrating Polypeptide portion and the AAM moiety portion are modified with a detectable label.
- the Surf+ Penetrating Polypeptide portion and the AAM moiety portion are modified with a detectable label.
- one or both portions of the complex may include at least one fluorescent moiety.
- the Surf+ Penetrating Polypeptide portion and the AAM moiety portion are modified with a detectable label.
- the Surf+ Penetrating Polypeptide portion and the AAM moiety portion are modified with a detectable label.
- one or both portions of the complex may include at least
- Polypeptide portion has inherent fluorescent qualities.
- one or both portions of the complex may be associated with at least one fluorescent moiety ⁇ e.g., conjugated to a fluorophore, fluorescent dye, etc.).
- one or both portions of the complex may include at least one radioactive moiety ⁇ e.g., protein may comprise iodine-131 or Yttrium-90; etc).
- detectable moieties may be useful for detecting and/or monitoring delivery of the complex to a target site.
- a complex associated with a detectable label can be used in detection, imaging, disease staging, diagnosis, or patient selection.
- Suitable labels include fluorescent, chemiluminescent, enzymatic labels, colorimetric, phosphorescent, density-based labels, e.g., labels based on electron density, and in general contrast agents, and/or radioactive labels.
- the complexes featured in the disclosure may be used for research purposes, e.g., to efficiently deliver AAM moieties to cells in a research context.
- the complexes may be used as research tools to efficiently transduce cells with antibody molecules or with other AAM moieties.
- complexes may be used as research tools to efficiently introduce an AAM moiety into cells for purposes of studying the effect of the AAM moiety on cellular activity.
- a complex can be used to deliver an AAM moiety into a cell for the purpose of studying the biological activity of the target peptide or protein (e.g., what happens if the target is inhibited or agonized, etc.).
- a complex may be introduced into a cell for the purpose of studying the biological activity of the AAM moiety (e.g., does it inhibit target activity, does it promote target activity, etc.).
- the present disclosure provides complexes of the disclosure (e.g., a Surf+ Penetrating Polypeptide portions-associated with an AAM moiety portion).
- This section describes exemplary compositions, such as compositions of a complex of the disclosure formulated in a pharmaceutically acceptable carrier. Any of the complexes comprising any of the Surf+ Penetrating Polypeptides amd any of the AAM moieties described herein may be formulated in accordance with this section of the disclosure.
- compositions such as pharmaceutical compositions, comprising one or more such complexes, and one or more pharmaceutically acceptable excipients.
- Pharmaceutical compositions may optionally include one or more additional therapeutically active substances.
- a method of administering pharmaceutical compositions comprising one or more Surf+ Penetrating Polypeptide or one or more complexes of the disclosure e.g., a complex comprising a Surf+ Penetrating
- compositions are administered to humans.
- active ingredient generally refers to an AAM moiety portion complexed with a Surf+ Penetrating Polypeptide portion to be delivered as described herein.
- compositions are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts, as well as suitable or adaptable for research use. Modification of pharmaceutical
- compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation.
- Subjects or patients to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and/or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/or birds, including commercially relevant birds such as chickens, ducks, geese, and/or turkeys.
- Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit.
- a pharmaceutical composition in accordance with the disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
- a "unit dose" is a discrete amount of the
- composition comprising a predetermined amount of the active ingredient.
- the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one -third of such a dosage.
- compositions in accordance with the disclosure will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered.
- the composition may include between 0.1% and 100% (w/w) active ingredient.
- compositions may additionally include a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
- a pharmaceutically acceptable excipient includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
- Remington's The Science and Practice of Pharmacy 21 st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference) discloses various excipients used in
- a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, an excipient is approved for use in humans and for veterinary use. In some embodiments, an excipient is approved by United States Food and Drug
- an excipient is pharmaceutical grade. In some embodiments, an excipient meets the standards of the United States
- USP European Pharmacopoeia
- EP European Pharmacopoeia
- British Pharmacopoeia British Pharmacopoeia
- International Pharmacopoeia International Pharmacopoeia
- compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Such excipients may optionally be included in pharmaceutical formulations.
- Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and/or perfuming agents can be present in the composition, according to the judgment of the formulator.
- Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and/or elixirs.
- liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
- oral compositions can include adjuvants, tetrahydrofurfuryl
- compositions are mixed with solubilizing agents such as Cremophor ® , alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof.
- solubilizing agents such as Cremophor ® , alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof.
- Injectable preparations for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing agents, wetting agents, and/or suspending agents.
- Sterile injectable preparations may be sterile injectable solutions, suspensions, and/or emulsions in nontoxic parenterally acceptable diluents and/or solvents, for example, as a solution in 1,3-butanediol.
- the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S. P., and isotonic sodium chloride solution.
- Sterile, fixed oils are conventionally employed as a solvent or suspending medium.
- any bland fixed oil can be employed including synthetic mono- or diglycerides.
- Fatty acids such as oleic acid can be used in the preparation of injectables.
- Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, and/or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
- the rate of drug release can be controlled.
- biodegradable polymers include poly(orthoesters) and poly(anhydrides).
- Depot injectable formulations are prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.
- compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing compositions with suitable non-irritating excipients such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.
- suitable non-irritating excipients such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.
- Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
- an active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient.
- the dosage form may comprise buffering agents.
- Dosage forms for topical and/or transdermal administration of a composition may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and/or patches.
- an active ingredient is admixed under sterile conditions with a pharmaceutically acceptable excipient and/or any needed preservatives and/or buffers as may be required.
- the present disclosure contemplates the use of transdermal patches, which often have the added advantage of providing controlled delivery of a compound to the body.
- dosage forms may be prepared, for example, by dissolving and/or dispensing the compound in the proper medium.
- rate may be controlled by either providing a rate controlling membrane and/or by dispersing the compound in a polymer matrix and/or gel.
- compositions described herein include short needle devices such as those described in U.S. Patents 4,886,499; 5,190,521; 5,328,483; 5,527,288; 4,270,537; 5,015,235; 5,141,496; and 5,417,662.
- Intradermal compositions may be administered by devices which limit the effective penetration length of a needle into the skin, such as those described in PCT publication WO 99/34850 and functional equivalents thereof.
- Jet injection devices which deliver liquid compositions to the dermis via a liquid jet injector and/or via a needle which pierces the stratum corneum and produces a jet which reaches the dermis are suitable. Jet injection devices are described, for example, in U.S.
- Ballistic powder/particle delivery devices which use compressed gas to accelerate vaccine in powder form through the outer layers of the skin to the dermis are suitable.
- conventional syringes may be used in the classical mantoux method of intradermal administration.
- Formulations suitable for topical administration include, but are not limited to, liquid and/or semi liquid preparations such as liniments, lotions, oil in water and/or water in oil emulsions such as creams, ointments and/or pastes, and/or solutions and/or suspensions.
- Topically-administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of active ingredient may be as high as the solubility limit of the active ingredient in the solvent.
- a pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for pulmonary administration via the buccal cavity.
- a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 nm to about 7 nm or from about 1 nm to about 6 nm.
- Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder and/or using a self propelling solvent/powder dispensing container such as a device comprising the active ingredient dissolved and/or suspended in a low-boiling propellant in a sealed container.
- Such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nm and at least 95% of the particles by number have a diameter less than 7 nm. Alternatively, at least 95% of the particles by weight have a diameter greater than 1 nm and at least 90% of the particles by number have a diameter less than 6 nm.
- Dry powder compositions may include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.
- compositions formulated for pulmonary delivery may provide an active ingredient in the form of droplets of a solution and/or suspension.
- Such formulations may be prepared, packaged, and/or sold as aqueous and/or dilute alcoholic solutions and/or suspensions, optionally sterile, comprising active ingredient, and may conveniently be administered using any nebulization and/or atomization device.
- Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, and/or a preservative such as methylhydroxybenzoate.
- Droplets provided by this route of administration may have an average diameter in the range from about 0.1 nm to about 200 nm.
- Formulations described herein as being useful for pulmonary delivery are useful for intranasal delivery of a pharmaceutical composition.
- Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 ⁇ to 500 ⁇ .
- Such a formulation is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nose.
- Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of active ingredient, and may comprise one or more of the additional ingredients described herein.
- a pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets and/or lozenges made using conventional methods, and may, for example, 0.1% to 20% (w/w) active ingredient, the balance comprising an orally dissolvable and/or degradable composition and, optionally, one or more of the additional ingredients described herein.
- formulations suitable for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising active ingredient.
- Such powdered, aerosolized, and/or aerosolized formulations when dispersed, may have an average particle and/or droplet size in the range from about 0.1 nm to about 200 nm, and may further comprise one or more of any additional ingredients described herein.
- a pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for ophthalmic administration.
- Such formulations may, for example, be in the form of eye drops including, for example, a 0.1/1.0% (w/w) solution and/or suspension of the active ingredient in an aqueous or oily liquid excipient.
- Such drops may further comprise buffering agents, salts, and/or one or more other of any additional ingredients described herein.
- Other opthalmically- administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form and/or in a liposomal preparation. Ear drops and/or eye drops are contemplated as being within the scope of this disclosure.
- complexes of the disclosure and compositions of the disclosure, including pharmaceutical preparations are non-pyrogenic.
- the compositions are substantially pyrogen free.
- the formulations of the disclosure are pyrogen-free formulations which are substantially free of endotoxins and/or related pyrogenic substances.
- Endotoxins include toxins that are confined inside a microorganism and are released only when the microorganisms are broken down or die.
- Pyrogenic substances also include fever- inducing, thermostable substances (glycoproteins) from the outer membrane of bacteria and other microorganisms. Both of these substances can cause fever, hypotension and shock if administered to humans. Due to the potential harmful effects, even low amounts of endotoxins must be removed from intravenously administered pharmaceutical drug solutions.
- FDA endotoxin units
- the endotoxin and pyrogen levels in the composition are less then 10 EU/mg, or less then 5 EU/mg, or less then 1 EU/mg, or less then 0.1 EU/mg, or less then 0.01 EU/mg, or less then 0.001 EU/mg.
- the present disclosure provides methods for delivering an AAM moiety into a cell.
- Cells or tissues are contacted with a complex comprising an AAM moiety and a Surf+ Penetrating Polypeptide, thereby promoting delivery of the AAM moiety into the cell.
- the present disclosure provides methods comprising administering Surf+ Penetrating Polypeptide/AAM moiety complexes to a subject in need thereof, as well as methods of contacting cells or cells in culture with such complexes.
- the disclosure contemplates that any of the complexes of the disclosure (e.g., complexes including a Surf+ Penetrating Polypeptide Portion and a AAM moiety portion) may be administrated, such as described herein.
- Complexes of the disclosure, including as pharmaceutical compositions may be administered or otherwise used for research, diagnostic, imaging, prognostic, or therapeutic purposes, and may be used or administered using any amount and any route of administration effective for preventing, treating, diagnosing, researching or imaging a disease, disorder, and/or condition.
- compositions in accordance with the disclosure are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present disclosure will be decided by the attending physician within the scope of sound medical judgment.
- the specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.
- Surf+ Penetrating Polypeptide/ AAM moiety complexes comprising at least one agent to be delivered and/or pharmaceutical, prophylactic, diagnostic, research or imaging compositions thereof may be
- complexes of the disclosure comprising at least one agent to be delivered, and/or pharmaceutical, prophylactic, diagnostic, research or imaging compositions thereof are administered to humans.
- Complexes of the disclosure comprising at least one agent to be delivered and/or pharmaceutical, prophylactic, research diagnostic, or imaging compositions thereof in accordance with the present disclosure may be administered by any route and may be formulated in a manner suitable for the selected route of administration or in vitro application.
- complexes of the disclosure, and/or pharmaceutical, prophylactic, diagnostic, or imaging compositions thereof are administered by one or more of a variety of routes, including oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous,
- intraventricular, transdermal, intradermal, rectal, intravaginal, intraperitoneal, topical e.g. by powders, ointments, creams, gels, lotions, and/or drops
- topical e.g. by powders, ointments, creams, gels, lotions, and/or drops
- mucosal, nasal, buccal, enteral, vitreal, intratumoral, sublingual by intratracheal instillation, bronchial instillation, and/or inhalation; as an oral spray, nasal spray, and/or aerosol, and/or through a portal vein catheter.
- Other devices suitable for administration include, e.g., microneedles, intradermal specific needles, Foley's catheters (e.g., for bladder instillation), and pumps, e.g., for continuous release.
- complexes of the disclosure, and/or pharmaceutical, prophylactic, diagnostic, research or imaging compositions thereof are administered by systemic intravenous injection.
- complexes of the disclosure and/or pharmaceutical, prophylactic, research diagnostic, or imaging compositions thereof may be administered intravenously and/or orally.
- complexes of the disclosure, and/or pharmaceutical, prophylactic, research diagnostic, or imaging compositions thereof may be administered in a way which allows the complex to cross the blood-brain barrier, vascular barrier, or other epithelial barrier.
- Complexes of the disclosure comprising at least one AAM moiety to be delivered may be used in combination with one or more other therapeutic, prophylactic, diagnostic, research or imaging agents.
- compositions of the disclosure can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics, other reagents or medical procedures. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent.
- the disclosure encompasses the delivery of pharmaceutical, prophylactic, diagnostic, research or imaging compositions in combination with agents that improve their bioavailability, reduce and/or modify their metabolism, inhibit their excretion, and/or modify their distribution within the body.
- therapeutic, prophylactic, diagnostic, research or imaging active agents utilized in combination may be administered together in a single composition or administered separately in different compositions.
- agents utilized in combination with be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually.
- the particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve a desired effect for the same disorder (for example, a composition useful for treating cancer in accordance with the disclosure may be administered concurrently with a
- chemotherapeutic agent may achieve different effects (e.g., control of any adverse effects).
- kits for conveniently and/or effectively carrying out methods of the present disclosure.
- kits will comprise sufficient amounts and/or numbers of components to allow a user to perform multiple treatments of a subject(s) and/or to perform multiple experiments for desired uses (e.g., laboratory or diagnostic uses).
- a kit may be designed and intended for a single use.
- Components of a kit may be disposable or reusable.
- kits include one or more of (i) a Surf+ Penetrating
- kits may further include instructions for using the complex in a research, diagnostic or therapeutic setting.
- a kit includes one or more of (i) a Surf+ Penetrating Polypeptide portion as described herein and an AAM moiety portion to be delivered or a complex of such Surf+ Penetrating Polypeptide associated with such AAM moiety; (ii) at least one pharmaceutically acceptable excipient; (iii) a syringe, needle, applicator, etc. for administration of a pharmaceutical, prophylactic, diagnostic, or imaging composition to a subject; and (iv) instructions and/or a label for preparing the pharmaceutical composition and/or for administration of the composition to the subject.
- a kit includes one or more of (i) a pharmaceutical composition comprising a complex of the disclosure (e.g., a Surf+ Penetrating
- Polypeptide portion as described herein associated with an AAM moiety portion to be delivered (ii) a syringe, needle, applicator, etc. for administration of the
- kits need not include the syringe, needle, or applicator, but instead provides the composition in a vial, tube or other container suitable for long or short term storage until use.
- a kit includes one or more components useful for modifying proteins of interest, such as by supercharging the protein, to produce a Surf+ Penetrating Polypeptide. These kits typically include all or most of the reagents needed. In certain embodiments, such a kit includes computer software to aid a researcher in designing the engineered or otherwise modified Surf+ Penetrating
- such a kit includes reagents necessary for performing site-directed mutagenesis.
- kits may include additional components or reagents.
- a kit may include buffers, reagents, primers, oligonucleotides, nucleotides, enzymes, buffers, cells, media, plates, tubes, instructions, vectors, etc.
- kits comprises two or more containers.
- a kit may include one or more first containers which comprise a Surf+ Penetrating Polypeptide, and optionally, at least one AAM moiety molecule to be delivered, or a complex comprising a Surf+ Penetrating Polypeptide and at least one AAM moiety to be delivered for diagnosing or prognosing a disease, disorder or condition or for research use; and the kit also includes one or more second containers which comprise one or more other prophylactic or therapeutic agents useful for the prevention, management or treatment of the same disease, disorder or condition, or useful for the same research application.
- kits includes a number of unit dosages of a
- the unit dosage form is suitable for intravenous, intramuscular, intranasal, oral, topical or subcutaneous delivery.
- the disclosure herein encompasses solutions, preferably sterile solutions, suitable for each delivery route.
- a memory aid may be provided, for example in the form of numbers, letters, and/or other markings and/or with a calendar insert, designating the days/times in the treatment schedule in which dosages can be administered.
- Placebo dosages, and/or calcium dietary supplements either in a form similar to or distinct from the dosages of the pharmaceutical, prophylactic, diagnostic, or imaging compositions, may be included to provide a kit in which a dosage is taken every day.
- kits may further include a device suitable for administering the composition according to a specific route of administration or for practicing a screening assay.
- Kits may include one or more vessels or containers so that certain of the individual components or reagents may be separately housed.
- Exemplary containers include, but are not limited to, vials, bottles, pre-filled syringes, IV bags, blister packs (comprising one or more pills).
- a kit may include a means for enclosing individual containers in relatively close confinement for commercial sale (e.g., a plastic box in which instructions, packaging materials such as styrofoam, etc., may be enclosed). Kit contents can be packaged for convenient use in a laboratory.
- the kit may optionally contain a notice indicating appropriate use, safety considerations, and any limitations on use.
- the kit may optionally comprise one or more other reagents, such as positive or negative control reagents, useful for the particular diagnostic or laboratory use.
- kits sold for therapeutic and/or diagnostic use may also contain a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects (a) approval by the agency of manufacture, use or sale for human
- Example 1 A Microtubule Localizing Complex
- an antibody to tubulin is biotinylated at the sulfhydryl groups on one or more cysteines and conjugated to a supercharged streptavidin (+52SAV).
- +52SAV is an example of a Surf+ Penetrating Polypeptide. It has high net positive charge, surface positive charge and penetrates cells.
- +52SAV is a tetramer of four monomers, each of which has a net charge of +13. The mass of each monomer is 16.54 kDa and the charge/molecular weight ratio of the tetramer is 0.79.
- Each monomer of the +52SAV tetramer has the following amino acid sequence:
- the tubulin antibody complex For in vitro analysis of this complex, cells in culture are contacted with the +52SAV-tubulin antibody complex.
- the complex is internalized by the cells.
- the tubulin antibody binds its target (e.g., tubulin expressed by microtubules in the cell), which is detected by immunofluorescence with antibodies to the tubulin antibody after cell fixation and permeabilization.
- the +52SAV-tubulin antibody complex is injected subcutaneously into rats and, following a punch biopsy and/or harvest of various tissue samples, immunohistochemistry is performed with antibodies to the tubulin antibody to detect tissue penetration and biodistribution.
- Suitable controls are conducted and include the use of an anti-tubulin antibody alone to confirm that the AAM moiety alone does not efficiently penetrate non- permeabilized cells or does so at levels substantially less than that of the complex, as well as the use of the Surf+ Penetrating Polypeptide alone to confirm that it does not independently bind specifically to the intracellular target.
- +52SAV expression and purification His6x-tagged +52SAV was expressed in BL21(DE3) cells, grown in Terrific Broth media (Boston Bioproducts, Ashland, MA), and induced with 1 mM IPTG for 4 hours at 37°C. Cells were lysed with 5 mL of lysis buffer (IX Bugbuster® (EMD Chemicals, Rockland, MA), 20mM Hepes pH 7.5, 150mM NaCl, 25 U/mL Benzonase (EMD Chemicals, Rockland, MA),
- Biotinylation of antibody Disulfide bonds of commercially available anti- tubulin antibody (sheep polyclonal; Cytoskeleton, Inc., Denver, CO) were reduced by 1 hour incubation with lOmM beta-mercaptoethanol at 37°C. Residual beta- mercaptoethanol was removed from the antibody using ZebaTM Spin Desalting Columns (Pierce®, Thermo Fisher Scientific Inc., Rockford, IL) according to the manufacturer's instructions. The resulting reduced antibody was biotinylated on the free sulfhydryl groups using EZ-Link® BMCC-Biotin (Pierce®, Thermo Fisher
- +52SAV was incubated with biotinylated antibody and free biotin to generate a 1 : 1 molar ratio of antibody bound to +52SAV. This complex was then purified using a cation exchange resin (SP sepharose, fast flow; GE Healthcare).
- a cation exchange resin SP sepharose, fast flow; GE Healthcare.
- HeLa cells ATCC, Manassas, VA
- uptake and binding of tubulin antibody to intracellular microtubules will be assessed by dose ranging (0.05 to 2 ⁇ ) and time course incubation of the
- +52SAV may be replaced by a human Surf+ Penetrating Polypeptide, such as a fragment of a naturally occurring polypeptide set forth in Figure 1 or Figure 2, including specific domains identified by PDB number, or fragments thereof having surface positive charge, a mass of at least 4 kDa, and a charge/molecular weight ratio of at least 0.75.
- Amino acid sequence information for full length proteins identified in Figures 1 and 2 by GenBank Accession number are provided in Section 1 of the Sequence Listing.
- Amino acid sequence information for domains of protein identified in Figures 1 and 2 by PDB identifier are provided in Section 2 of the Sequence Listing.
- the commercially available anti-tubulin antibody may be replaced by a recombinantly produced anti-tubulin antibody.
- Use of a recombinantly produced antibody facilitates generating complexes as fusion proteins comprising a Surf+ Penetrating Polypeptide portion and an AAM moiety portion. Such replacement of the specific embodiments set forth in these examples with other suitable embodiments is specifically contemplated.
- cells in culture are contacted with the +52SAV-nucleoporin antibody complex.
- the complex is internalized by the cells.
- the nucleoporin antibody binds to the nuclear pore in the cell (e.g., binds to its target nucleoporin expressed by the nuclear pore), which is detected by immunofluorescence with antibodies to the nucleoporin antibody after cell fixation and permeabilization.
- the +52SAV-nucleoporin antibody complex is injected subcutaneously into rats and, following a punch biopsy and/or harvest of various tissue samples, immunohistochemistry is performed with antibodies to the
- nucleoporin antibody to detect tissue penetration and biodistribution. Methods for preparation and testing of the +52SAV-antibody complex will be followed as described above.
- +52SAV may be replaced by a human Surf+ Penetrating Polypeptide, such as a fragment of a naturally occurring polypeptide set forth in Figure 1 or Figure 2, including specific domains identified by PDB number, or fragments thereof having surface positive charge, a mass of at least 4 kDa, and a charge/molecular weight ratio of at least 0.75.
- Amino acid sequence information for full length proteins identified in Figures 1 and 2 by GenBank Accession number are provided in Section 1 of the Sequence Listing.
- Amino acid sequence information for domains of protein identified in Figures 1 and 2 by PDB identifier are provided in
- the commercially available antibody may be replaced by a recombinantly produced antibody.
- Use of a recombinantly produced antibody facilitates generating complexes as fusion proteins comprising a Surf+ Penetrating
- Polypeptide portion and an AAM moiety portion are specifically contemplated.
- antibody to p58 Golgi protein mouse monoclonal
- [58K-9]; Abeam) is biotinylated at the sulfhydryl groups at one or more cysteines and conjugated to a supercharged streptavidin (+52SAV).
- +52SAV-p58 Golgi antibody complex The complex is internalized by the cells. Once inside a cell, the p58 Golgi antibody binds to the perinuclear Golgi apparatus in the cell, which is detected by immunofluorescence with antibodies to the p58 Golgi antibody after cell fixation and permeabilization.
- the +52SAV-p58 Golgi antibody complex is injected subcutaneously into rats and, following a punch biopsy and/or harvest of various tissue samples, immunohistochemistry is performed with antibodies to the p58 Golgi antibody to detect tissue penetration and biodistribution. Methods for preparation and testing of the +52SAV-antibody complex will be followed as described above.
- +52SAV may be replaced by a human Surf+
- Penetrating Polypeptide such as a fragment of a naturally occurring polypeptide set forth in Figure 1 or Figure 2, including specific domains identified by PDB number, or fragments thereof having surface positive charge, a mass of at least 4 kDa, and a charge/molecular weight ratio of at least 0.75.
- Amino acid sequence information for full length proteins identified in Figures 1 and 2 by GenBank Accession number are provided in Section 1 of the Sequence Listing.
- Amino acid sequence information for domains of protein identified in Figures 1 and 2 by PDB identifier are provided in Section 1 of the Sequence Listing.
- the commercially available antibody may be replaced by a recombinantly produced antibody.
- Use of a recombinantly produced antibody facilitates generating complexes as fusion proteins comprising a Surf+ Penetrating Polypeptide portion and an AAM moiety portion. Such replacement of the specific embodiments set forth in these examples with other suitable embodiments is specifically contemplated.
- a neutralizing antibody to caspasel is biotinylated at the sulfhydryl groups at one or more cysteines and conjugated to a supercharged streptavidin (+52SAV).
- cells in culture are contacted with the +52SAV-caspase antibody complex.
- the complex is internalized by the cells.
- the functional activity of the caspasel antibody inside the cell is assayed by, for example, measuring the effect on inhibition of pro-IL- ⁇ processing and reduction in levels of secreted active IL- ⁇ , which can be monitored by an immunoassay of the cell supernatant such as an ELISA assay, for which a commercially available kit is available (Pierce®, Thermo Fisher Scientific Inc., Rockford, IL). Such an assay is used to confirm that once delivered into cells, the neutralizing antibody to caspasel maintains its function (e.g., the antibody inhibits an activity of caspasel).
- mice are injected intraarticularly with monosodium urate crystals plus CI 8 free fatty acids to induce joint swelling.
- Such joint swelling may be monitored by macroscopic scoring, by 99m Tc uptake, by local IL- ⁇ levels and/or by quantifying immune cell influx into the joint, and each of these methods have been previously described (Joosten LA, et al. (2010) Arthritis & Rheumatism 62:3237- 3248).
- the neutralizing caspasel antibody reduces IL- ⁇ levels
- the complex is evaluated for its ability to alleviate symptoms caused, in whole or in part, by elevated local IL- ⁇ levels.
- the +52SAV-caspase 1 antibody complex is injected intraarticularly with dose ranging and time course (including prior to, concomitant with and post injection of urate crystals plus CI 8 free fatty acids) studies. Following injection, treated mice are evaluated for inhibition of joint swelling in comparison to untreated mice.
- +52SAV may be replaced by a human Surf+ Penetrating Polypeptide, such as a fragment of a naturally occurring polypeptide set forth in Figure 1 or Figure 2, including specific domains identified by PDB number, or fragments thereof having surface positive charge, a mass of at least 4 kDa, and a charge/molecular weight ratio of at least 0.75.
- Amino acid sequence information for full length proteins identified in Figures 1 and 2 by GenBank Accession number are provided in Section 1 of the Sequence Listing.
- Amino acid sequence information for domains of protein identified in Figures 1 and 2 by PDB identifier are provided in Section 2 of the Sequence Listing.
- the commercially available antibody may be replaced by a recombinantly produced antibody.
- Use of a recombinantly produced antibody facilitates generating complexes as fusion proteins comprising a Surf+ Penetrating Polypeptide portion and an AAM moiety portion. Such replacement of the specific embodiments set forth in these examples with other suitable embodiments is specifically contemplated.
- Example 5 Complexes Comprising Naturally Occurring Surf+ Penetrating
- a naturally occurring human Surf+ Penetrating Polypeptide such as a cell penetrating fragment of HBEGF
- a naturally occurring human Surf+ Penetrating Polypeptide such as a cell penetrating fragment of HBEGF
- an AAM moiety such as an Adnectin ® , DARPin, nanobody, scFv or single VH or VL domain antibody.
- HBEGF and the AAM moiety can be directly linked, in this example the two moieties are interconnected via a linker, such as a (G 4 S) 3 (i.e., a (Gly-Gly-Gly-Gly-Ser) 3 ) linker.
- a suitable HBEGF fragment is set forth in PDB ID IXDT and is a polypeptide of about 79 amino acid residues (e.g., includes about amino acid residues 72-147 of the full length HBEGF protein).
- This HBEGF domain is an example of a naturally occurring human Surf+ Penetrating Polypeptide. It has surface positive charge,
- this polypeptide has a molecular weight of about 8.9 kDa, a net charge of +12, and a charge/molecular weight of 1.35.
- this HBEGF fragment is exemplary of Surf+ Penetrating Polypeptides having a charge/molecular weight of at least 0.75, but for which the charge/molecular weight of the full length naturally occurring protein is less than 0.75 (e.g., charge/molecular weight of full length HBEGF is about 0.52).
- Subdomains (e.g., smaller functional fragments) of HBEGF having surface positive charge, a mass of at least 4kDa, a charge/molecular weight ratio of at least 0.75, and cell penetrating capability may also be used.
- the complex includes one or more tags to facilitate detection and/or purification.
- a 10 amino acid sequence including the 6xHis tag is appended to the N-terminus of the fusion protein (MGHHHHHHGG) (SEQ ID NO: 659) and a 9 amino acid myc epitope tag plus two glycines as a linker sequence (GGEQKLISEEDL) (SEQ ID NO: 660)is appended to the C-terminus of the fusion protein.
- this His-HBEGF-linker-AAM moiety-myc fusion protein is contacted with and internalized by a cell. Accumulation in the cell is monitored by immunofluorescence with an anti-myc antibody (mouse monoclonal [9E10]; Abeam, Cambridge, MA).
- the AAM moiety may be an scFv that binds tubulin.
- the His-HBEGF-linker- tubulin scFv-myc fusion protein is contacted with and internalized by a cell and the myc-tagged tubulin scFv binds to microtubules in the cell, which can be subsequently detected by immunofluorescence with anti-myc tag antibody following fixation, permeabilization.
- the order of the fusion protein may be altered so that the Surf+ Penetrating Polypeptide portion of the complex is located C-terminally to the AAM moiety portion of the complex, e.g. myc-tubulin scFv-linker-HBEGF- His.
- HBEGF expression and purification the His-HBEGF-tubulin scFv-myc fusion protein was expressed in SHuffle® cells (New England Biolabs, Ipswich, MA), grown in ProgroTM media (Expression Technologies, San Diego, CA), and induced with 0.5mM IPTG for 19 hours at 22°C. Cells were lysed in lysis buffer as described above.
- the lysate supernatant was subjected to fractionationg on a HiTrapTM IMAC column (GE Healthcare, Piscataway, NJ), followed by a SP-HP cation exchange column (GE Healthcare, Piscataway, NJ), and finally a SuperdexTM 75 10/300 GL gel filtration column (GE Healthcare, Piscataway, NJ) to purify the fusion protein.
- the fusion protein is stored in high salt PBS buffer (8 mM sodium phosphate, 2 mM potassium phosphate, 2.7 mM KC1, 0.5 M NaCl, pH 7.4)
- HeLa cells are plated as above and subjected to dose ranging (0.05 to 2 ⁇ ) and time course studies for uptake of the
- His-HBEGF -tubulin scFv-myc fusion protein After incubation with the fusion protein, cells are fixed and permeabilized as described above. The fixed and permeabilized cells are incubated with a fluorescent labeled secondary antibody and visualized by fluorescent microscopy.
- the Surf+ Penetrating Polypeptide may be replaced by a human Surf+ Penetrating Polypeptide, such as a fragment of a naturally occurring polypeptide set forth in Figure 1 or Figure 2, including specific domains identified by PDB number, or fragments thereof having surface positive charge, a mass of at least 4 kDa, and a charge/molecular weight ratio of at least 0.75.
- Amino acid sequence information for full length proteins identified in Figures 1 and 2 by GenBank Accession number are provided in Section 1 of the Sequence Listing.
- the AAM moiety in the complex is an Adnectin ® sequence, such as the na ' ive, wild type Fn3 Adnectin ® , which has no target binding protein in the cells, but is studied for biophysical and biochemical properties in fusion with a Surf+ Penetrating Polypeptide of the disclosure and for monitoring uptake into cells.
- Adnectin ® sequence such as the na ' ive, wild type Fn3 Adnectin ® , which has no target binding protein in the cells, but is studied for biophysical and biochemical properties in fusion with a Surf+ Penetrating Polypeptide of the disclosure and for monitoring uptake into cells.
- a complex of a Surf+ Penetrating Polypeptide and the HA4 or 7c 12 Adnectin ® sequence is made and studied.
- These particular AAM moieties bind to the SH2 domain of the Abelson kinase, as described by Grebien, F et al (2011) Cell 147:306-319.
- the resulting complex is internalized by cells and binds (via the AAM moiety) to the cytoplasmic Bcr-Abl kinase fusion protein. Either complex is studied in vitro and/or in vivo, such as using assays described above.
- Example 7 Complexes Comprising an Antibody-Mimic Moiety
- the AAM moiety complexed to a Surf+ Penetrating Polypeptide is a designed ankyrin repeat protein, or DARPin, such as a na ' ive DARPin or the 2A1 and 2F6 DARPins that bind to the CC2-LZ domain of ⁇ ⁇ / ⁇ , as previously described (Wyler, E. et al (2007) Protein Science 16:2013-2022).
- DARPin ankyrin repeat protein
- DARPin such as a na ' ive DARPin or the 2A1 and 2F6 DARPins that bind to the CC2-LZ domain of ⁇ ⁇ / ⁇ , as previously described (Wyler, E. et al (2007) Protein Science 16:2013-2022).
- a His tag is optionally appended to the fusion protein to facilitate purification from E. coli
- a myc epitope tag is optionally appended to the DARPin sequence to monitor intracellular uptake, local
- HEK293T cells are transiently transfected with an NF-kB reporter plasmid, such as plgK-luc, and co-transfected with a ⁇ -galactosidase expressing reporter plasmid. After 24 hours, cells are stimulated with lOng/mL TNF-a and cell lysates are assayed for both reporter protein activities, where the ⁇ -galactosidase activity is used to normalize transfection and reporter protein activity.
- an NF-kB reporter plasmid such as plgK-luc
- a ⁇ -galactosidase expressing reporter plasmid After 24 hours, cells are stimulated with lOng/mL TNF-a and cell lysates are assayed for both reporter protein activities, where the ⁇ -galactosidase activity is used to normalize transfection and reporter protein activity.
- the His-Surf+ Penetrating Polypeptide-linker-DARPin-myc fusion protein is contacted with the cells for dose ranging and time course studies of inhibition of NEMO activity and reduced NF-kB activation following TNF-a stimulation, as previously described (Wyler, E. et al (2007) Protein Science 16:2013-2022).
- the present disclosure provides complexes and methods for delivering AAM moieties into cells.
- the target of the particular AAM moiety may itself be localized in, for example, the nucleus, peroxisome, cytoplasm, mitochondria, cytoplasmic face of the cell membrane, etc.
- the target of the particular AAM moiety is localized in the nucleus.
- a nuclear localization sequence for instance the peptide sequence DPKKKRKV (SEQ ID NO: 661), is included in the complex, such that the complex has any of the following exemplary structures to facilitate its targeting to the nucleus: His-Surf+ Penetrating Polypeptide-linker-NLS-AAM moiety-myc; His-Surf+ Penetrating Polypeptide-linker-AAM moiety-NLS-myc;
- His and/or myc tags may be present, absent or replaced with another tag.
- additional linkers may be present or absent.
- the target is localized in the peroxisome.
- a peroxisomal targeting sequence (PTS) is appended to the C-terminus of the AAM moiety (His-Surf+ Penetrating Polypeptide-linker-myc-AAM moiety-PTS). After contacting and penetration into the cell, the AAM moiety portion will transit to and accumulate inside peroxisomes. Accumulation in the cell is monitored by
- the PTS may be appended to another portion of the complex, such as to the Surf+ Penetrating
- the target is localized to the cytosolic face of the plasma membrane.
- a plasma membrane localization signal sequence is provided.
- AAM moiety His-Surf+ Penetrating Polypeptide-linker-AAM moiety-myc- membrane localization signal
- the AAM moiety will transit to and accumulates at the cytosolic face of the plasma membrane, which is monitored by immunofluorescence with an anti-myc antibody and detected by fluorescence microscopy of live or fixed cells.
- the plasma membrane localization signal may be appended to another portion of the complex, such as to the Surf+ Penetrating Polypeptide portion.
- the target is localized in the mitochondrial matrix.
- a mitochondrial matrix localization signal sequence (MLS) is appended to the N-terminus of the AAM moiety, which is followed by the linker sequence and then the Surf+ Penetrating Polypeptide (MLS-AAM moiety-myc-linker-Surf+
- the AAM moiety After contacting and penetration into the cell, the AAM moiety will transit to and accumulate inside the mitochondrial matrix. Accumulation in the cell is monitored by immunofluorescence with an anti-myc antibody and detected by fluorescence microscopy of live or fixed cells. Alternatively, the MLS may be appended to another portion of the complex, such as to the Surf+ Penetrating Polypeptide portion.
- Example 9 Surface-charged fusion proteins with single chain antibody (scFv) to huntingtin protein
- a complex comprising a supercharged GFP protein (another example of a Surf+ Penetrating Polypeptide, in this case a charge engineered protein) fused via a glycine-serine linker to an AAM moiety (in this case, an scFv that specifically binds huntingtin protein; an intracellular target) was expressed and purified.
- the complex was also tagged on the N-terminus with a Myc tag and on the C-terminus with a
- the complexes are fusion protein and can be represented as:
- +36GFP denotes the supercharged GFP portion
- C4 denotes the particular AAM moiety used in this example
- (G 4 S) 2 denotes the linker used to link the supercharged GFP portion to the AAM moiety (this linker is also referred to as GS10).
- the supercharged GFP portion has a net charge of +36.
- the amino acid sequence of +36GFP is set forth in SEQ ID NO: 663.
- the AAM moiety in this example is an scFv that specifically binds huntingtin protein; an intracellular target. This single chain Fv, also known as an intrabody because it is an scFv that binds an intracellular target, is denoted "C4".
- the C4 scFv binds to the first 17 amino acids of huntingtin protein and has been demonstrated to delay the aggregation phenotype when the gene is delivered in adeno-associated viral vectors (AAV2/1) in mice (J Neuopathol Exp Neurol. 2010. 69(10): 1078-1085).
- the scFv binds to the intracellular protein and prevents the bound protein from binding to another protein, in this case, another huntington protein molecule.
- the AAM is preventing the bound protein from binding its binding partner (a protein) which may be a different protein or another molecule of the same protein. Inability to get penetration of the protein has limited its use to such a viral- based approach.
- the complex is a fusion protein and the GFP and scFv portion are interconnected via a peptide linker.
- This fusion protein is a single polypeptide chain (e.g., the portions are connected to form a single polypeptide chain).
- the peptide linker is a ten amino acid linker, specifically (GGGGS) 2 .
- the GFP portion is N-terminal to the scFv.
- the GFP portion may be C-terminal to the scFv portion.
- the linker sequence and/or length can be varied, and the fusion protein may or may not have a tag.
- the amino acid sequence for the GFP-scFv fusion protein (Myc-+36GFP-(G 4 S) 2 - C4-His 6 ) is set forth in SEQ ID NO: 664.
- the amino acid sequence of the control complex (Myc-+36GFP-His 6 ) is set forth in SEQ ID NO: 665.
- Example 10 The binding of AMM moiety to its target is maintained when delivered into cells as a fusion protein with a Surf+ Penetrating Polypeptide
- Example 9 Myc-+36GFP-(G 4 S) 2 -C4-His 6
- the fusion protein have the ability to penetrate cells and yet retain the ability of the C4 (scFv; AAM moiety) to bind its intracellular target and disrupt the binding of this target to its binding partners (e.g., disrupt binding to another protein - whether that other protein be the same or different).
- C4 has been previously shown to block HTT aggregation when delivered by transient transfection using a viral system (Butler and Messer, PLosOne 2011, 6;e29199). This assay was employed to assess whether C4 maintains its activity when delivered into eels via a Surf+ Penetrating Polypeptide.
- a HTT exon 1 protein fragment containing 46 glutamine repeats and a red fluorescence protein tag (HDexl-RFP) was expressed in ST14A cells by transient transfection.
- ST14A cell are immortalized rat neuron progenitor cells, a cell line representative of immature CNS cells. If left untreated the protein forms punctate aggregates in the cells, which can be visualized by fluorescence microscopy.
- the assay is as followed:
- +36GFP-linker-C4 fusion protein reduces aggregation of HDexl-46QRFP (HTT46Q-RFP) by 30% at 48 hours relative to +36GFP alone at 2 micromolar.
- the number of aggregates formed by HTT46Q-RFP in the cells was determined by counting the number of aggregates seen when imaging for red fluorescence. Visual counting indicated 30% less aggregates in the +36GFP- linker-C4-treated cells, as compared to the +36GFP-treated cells.
- the 30% decrease in aggregation observed in this Example is significant.
- C4 was expressed via viral transfection as an intrabody with a PEST sequence that targets for proteosomal degradation
- aggregation was reduced 51% for HDexl-25Q and 78% for HDexl-72Q at 48 hours post-transfection (Butler and Messer, PLosOne 2011, 6;e29199).
- the intrabody is likely continuously expressed over the time course and the PEST sequence may further decrease aggregation by targeting HTT for proteosomal degradation.
- the 30% decrease observed in this Example is notable with a singular administration of protein in which the C4 scFv is fused to a Surf+
- Penetrating Polypeptide The use of a human Surf+ Penetrating Polypeptide is described below.
- H (SEQ ID NO: 665) Myc-(+36)GFP-(G 4 S) 2 -C4_scFv-His6 (where the underlined sequence is, from N- to C- terminus, GFP, linker, C4 scFv).
- Example 11 Fusion protein comprising a domain of FGF10 fused to an AAM moiety
- the Surf+ Penetrating Polypeptide is a domain of
- FGF10 having surface positive charge, an overall net positive charge, and a charge/molecular weight ratio greater than that of full length, unprocessed, naturally occurring FGF10.
- An exemplary AAM moiety which can be fused to the Surf+ Penetrating Polypeptide is an scFv.
- the FGF10 portion may be N- or C-terminal to the AAM moiety.
- the fusion proteins optionally include a linker that interconnects the FGF10 portion to the AAM moiety.
- Suitable linkers include a glycine/serine rich linker.
- the linker may also include a serum-stable proteolytic cleavage site, such as a site cleavable by cathepsin class proteases. Cleavable linkers permit the separation of the AAM moiety from the FGF10 portion following internalization.
- the following exemplary fusion protein is generated: Myc-FGF 10 portion-GS i 0 - AAM-His 6
- FGF10 portion is a domain of full length, naturally occurring human FGF10;
- AAM is the AMM moiety and can be an scFv;
- (GS)io is the linker amino acid sequence "GGGGSGGGGS";
- Myc is the tag "EQKLISEEDL”.
- the fusion protein is internalized by cells and binds (via the AAM moiety) to the target of interest.
- the fusion protein is studied in vitro and/or in vivo, such as using assays described herein.
- An exemplary fusion protein is a fusion protein made by fusing a domain of
- the FGF10 to a scFv specific for huntingtin protein.
- the fusion protein is tagged on the N-terminus with a Myc tag and on the C-terminus with a Hisx6 tag.
- a control lacking the AAM moiety is also made.
- the complexes can be represented as:
- FGF10 denotes the domain of FGF10
- C4 denotes the particular AAM moiety used in this example, as described above
- GSio denotes the linker (also known as (G 4 S) 2 used to link the FGF10 portion to the AAM moiety.
- the FGF10 portion has the amino acid sequence set forth in SEQ ID NO: 666.
- the AAM moiety in this example is an scFv specific for huntingtin protein.
- This scFv denoted “C4", targets the first 17 amino acids of huntingtin protein and has been demonstrated to delay the aggregation phenotype when the gene is delivered in adeno-associated viral vectors (AAV2/1) in mice (J Neuopathol Exp Neurol. 2010. 69(10): 1078-1085).
- Example 12 Fusion protein comprising a variant domain of FGF10 fused to an AAM moiety
- a fusion protein is made by fusing a variant domain of FGF10 having one or more amino acid additions, deletions, or substitutions relative to the naturally occurring domain, to an AAM moiety.
- the complex is tagged on the N-terminus with a Myc tag and on the C-terminus with a Hisx6 tag. A control lacking the AAM moiety is also made.
- the complexes can be represented as:
- FGF 10(mut4) denotes the variant domain of FGF 10
- C4 denotes the particular AAM moiety used in this example
- GSio denotes the linker used to link the variant FGF10 portion to the AAM moiety.
- the variant FGF10 portion has the amino acid sequence set forth in SEQ ID NO: 667.
- This variant FGF10 portion has been modified to minimize mitogenic effects and includes the following mutations:
- the amino acid sequence for the FGF10(mut4)-scFv fusion protein (Myc- FGF10(mut4)- GSi 0 -C4-His 6 ) is set forth in SEQ ID NO: 668.
- the amino acid sequence of the control complex (Myc-FGF10(mut4)-His6) is set forth in SEQ ID NO: 669.
- the AAM moiety in this example is an scFv specific for huntingtin protein.
- This scFv denoted “C4", targets the first 17 amino acids of huntingtin protein and has been demonstrated to delay the aggregation phenotype when the gene is delivered in adeno-associated viral vectors (AAV2/1) in mice (J Neuopathol Exp Neurol. 2010. 69(10): 1078-1085).
- sequence information is intended to provide a detailed description for the amino acid sequences referenced in Figures 1 and 2 by GenBank accession number and/or PDB identifier. As such, all such sequence information should be considered part of the detailed description of the invention and provides additional description for Surf+ Penetrating Polypeptides, as well as polypeptides suitable for use as a portion of a complex comprising a Surf+ Penetrating Polypeptide.
- the disclosure contemplates complexes comprising an amino acid sequence selected from amongst any of the amino acid sequences provided in this sequence listing, as well as functional fragments thereof (e.g., domains thereof having surface positive charge, a mass of at least 4 kDa, a charge/molecular weight ratio of at least 0.75).
- Such polypeptides are suitable for use in complexes of the disclosure.
- complexes of the disclosure comprise an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any of the foregoing.
- AMINO ACID SEQUENCE INFORMATION DISCLOSED IN GENBANK FOR NATURALLY OCCURRING PROTEINS IDENTIFIED BY GENBANK ACCESSION NUMBER IN FIGURE 1
- NP 001029058.1 stromal cell-derived factor 1 isoform gamma
- NP 001 108.2 pituitary adenylate cyclase-activating polypeptide precursor
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Abstract
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US14/385,072 US20150266939A1 (en) | 2012-03-15 | 2013-03-15 | Cell penetrating compositions for delivery of intracellular antibodies and antibody-like moieties and methods of use |
EP13760721.4A EP2825561A4 (en) | 2012-03-15 | 2013-03-15 | Cell penetrating compositions for delivery of intracellular antibodies and antibody-like moieties and methods of use |
CA2867188A CA2867188A1 (en) | 2012-03-15 | 2013-03-15 | Cell penetrating compositions for delivery of intracellular antibodies and antibody-like moieties and methods of use |
JP2015500675A JP2015512246A (en) | 2012-03-15 | 2013-03-15 | Cell permeation compositions for delivery of intracellular antibodies and antibody-like moieties and methods of use |
AU2013231851A AU2013231851A1 (en) | 2012-03-15 | 2013-03-15 | Cell penetrating compositions for delivery of intracellular antibodies and antibody-like moieties and methods of use |
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EP (1) | EP2825561A4 (en) |
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- 2013-03-15 US US14/385,072 patent/US20150266939A1/en not_active Abandoned
- 2013-03-15 JP JP2015500675A patent/JP2015512246A/en active Pending
- 2013-03-15 WO PCT/US2013/032686 patent/WO2013138795A1/en active Application Filing
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JP2015512246A (en) | 2015-04-27 |
EP2825561A1 (en) | 2015-01-21 |
US20150266939A1 (en) | 2015-09-24 |
CA2867188A1 (en) | 2013-09-19 |
AU2013231851A1 (en) | 2014-09-11 |
EP2825561A4 (en) | 2016-03-09 |
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