Patent Grant
8815522
Influenza has a long history of pandemics, epidemics, resurgences and outbreaks. Avian influenza, including the H5N1 strain, is a highly contagious and potentially fatal pathogen, but it currently has only a limited ability to infect humans. However, avian flu viruses have historically observed to accumulate mutations that alter its host specificity and allow it to readily infect humans. In fact, two of the major flu pandemics of the last century originated from avian flu viruses that changed their genetic makeup to allow for human infection.
Influenza remains a significant challenge to medical systems. Furthermore, there is a significant concern that the current H5N1, H7N7, H9N2 and H2N2 avian influenza strains might accumulate mutations that alter their host specificity and allow them to readily infect humans. There remains a need for improved systems and reagents for decreasing and treating influenza in humans, including influenza epidemics and pandemics.
The present invention provides systems for identifying agents useful as influenza therapeutics, as well as such agents, compositions containing them, and methods of employing them. Among other things, the invention provides agents that mimic umbrella-topology glycans, and/or that compete with influenza hemagglutinin polypeptides for interaction with umbrella-topology glycans.
6-O sulfated GlcNAc;
Glc or GalNAc;
Non-fucosylated GlcNAc;
Gal without GalNAcβ1-4Gal linkage.
which upon linearization becomes:
where y—fractional saturation of the glycan binding sites in the HA units; [HA]—concentration of HA (in M); n—cooperativity factor; Kd′—apparent binding constant for the multivalent HA-glycan interactions. The value of y is calculated by normalizing the binding signals to the saturating binding signal which represent 100% occupancy on all HA units. n and Kd′ were calculated based on the linearized Hill model. Typically n≧1 indicates positive cooperativity where binding of one HA unit enhances the binding of the other in the precomplex. On the other hand n≦1 indicates negative cooperativity where binding of one HA unit in the precomplex has a negative effect on binding of other subunits. The apparent binding constant Kd′ is used mainly for quantifying relative binding affinities of different HAs to different glycans and hence its absolute value should be taken in the context of comparing the affinities. An assumption in the above model is that the glycans in each well of the plate are in excess of HA. To satisfy this assumption, binding signals for HA concentration range from 0.05 μg/ml to 5 μg/ml were used to calculate n and Kd′. All efficiently human-adapted H1 and H3 HAs bind with high affinity to long α2-6 glycans (6′SLN-LN). Human-adapted SC18 and TX91 H1N1 viruses transmit efficiently in the ferret model. Human-adapted Mos99 H3N2 virus is a vaccine strain. Although HA from NY18 (a single Asp225Gly mutant of SC18, a virus that transmits inefficiently) shows comparable α2-6 binding to the other efficiently human-adapted HAs at high concentration, it has a dramatically lower long α2-6 binding affinity. Conversely, HA from AV18 (Asp 190Glu/Asp225Gly double mutant of SC18, a virus that does not transmit) shows a reverse trend of high binding affinity to α2-3.
HA Sequence Element 1
HA Sequence Element 1 is a sequence element corresponding approximately to residues 97-185 (where residue positions are assigned using H3 HA as reference) of many HA proteins found in natural influenza isolates. This sequence element has the basic structure:
In some embodiments, X1 is about 35 to about 45, or about 35 to about 43, or about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, or about 43 amino acids long. In some embodiments, X2 is about 9 to about 15, or about 9 to about 14, or about 9, about 10, about 11, about 12, about 13, or about 14 amino acids long. In some embodiments, X3 is about 26 to about 28, or about 26, about 27, or about 28 amino acids long. In some embodiments, X4 has the sequence (G/A) (I/V). In some embodiments, X4 has the sequence GI; in some embodiments, X4 has the sequence GV; in some embodiments, X4 has the sequence AI; in some embodiments, X4 has the sequence AV. In some embodiments, HA Sequence Element 1 comprises a disulfide bond. In some embodiments, this disulfide bond bridges residues corresponding to positions 97 and 139 (based on the canonical H3 numbering system utilized herein).
In some embodiments, and particularly in H1 polypeptides, X1 is about 43 amino acids long, and/or X2 is about 13 amino acids long, and/or X3 is about 26 amino acids long.
In some embodiments, and particularly in H1 polypeptides, HA Sequence Element 1 has the structure:
In some embodiments, and particularly in H1 polypeptides, HA Sequence Element 1 has the structure:
In some embodiments, and particularly in H1 polypeptides, HA Sequence Element 1 includes the sequence:
In some embodiments, and particularly in H3 polypeptides, X1 is about 39 amino acids long, and/or X2 is about 13 amino acids long, and/or X3 is about 26 amino acids long.
In some embodiments, and particularly in H3 polypeptides, HA Sequence Element 1 has the structure:
In some embodiments, and particularly in H3 polypeptides, HA Sequence Element 1 has the structure:
In some embodiments, and particularly in H3 polypeptides, HA Sequence Element 1 includes the sequence:
In some embodiments, and particularly in H5 polypeptides, X1 is about 42 amino acids long, and/or X2 is about 13 amino acids long, and/or X3 is about 26 amino acids long.
In some embodiments, and particularly in H5 polypeptides, HA Sequence Element 1 has the structure:
In some embodiments, and particularly in H5 polypeptides, HA Sequence Element 1 has the structure:
In some embodiments, and particularly in H5 polypeptides, HA Sequence Element 1 is extended (i.e., at a position corresponding to residues 186-193) by the sequence:
In some embodiments, and particularly in H5 polypeptides, HA Sequence Element 1 includes the sequence:
HA Sequence Element 2 is a sequence element corresponding approximately to residues 324-340 (again using a numbering system based on H3 HA) of many HA proteins found in natural influenza isolates. This sequence element has the basic structure:
In some embodiments, and particularly in H1 polypeptides, HA Sequence Element 2 has the structure:
X1A is approximately 3 amino acids long; in some embodiments, X1A is G (L/I) F.
In some embodiments, and particularly in H3 polypeptides, HA Sequence Element 2 has the structure:
In some embodiments, and particularly in H5 polypeptides, HA Sequence Element 2 has the structure:
Affinity: As is known in the art, “affinity” is a measure of the tightness with a particular ligand (e.g., an HA polypeptide) binds to its partner (e.g., an HA receptor). Affinities can be measured in different ways.
Approximately: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
Biologically active: As used herein, the phrase “biologically active” refers to a characteristic of any agent that has activity in a biological system, and particularly in an organism. For instance, an agent that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active. In particular embodiments, where a protein or polypeptide is biologically active, a portion of that protein or polypeptide that shares at least one biological activity of the protein or polypeptide is typically referred to as a “biologically active” portion.
Characteristic portion: As used herein, the phrase a “characteristic portion” of a protein or polypeptide is one that contains a continuous stretch of amino acids, or a collection of continuous stretches of amino acids, that together are characteristic of a protein or polypeptide. Each such continuous stretch generally will contain at least two amino acids. Furthermore, those of ordinary skill in the art will appreciate that typically at least 5, 10, 15, 20 or more amino acids are required to be characteristic of a protein. In general, a characteristic portion is one that, in addition to the sequence identity specified above, shares at least one functional characteristic with the relevant intact protein.
Characteristic sequence: A “characteristic sequence” is a sequence that is found in all members of a family of polypeptides or nucleic acids, and therefore can be used by those of ordinary skill in the art to define members of the family.
Cone-topology: The phrase “cone-topology” is used herein to refer to a 3-dimensional arrangement adopted by certain glycans and in particular by glycans on HA receptors. As illustrated in
Corresponding to: As used herein, the term “corresponding to” is often used to designate the position/identity of an amino acid residue in an HA polypeptide. Those of ordinary skill will appreciate that, for purposes of simplicity, a canonical numbering system (based on wild type H3 HA) is utilized herein (as illustrated, for example, in
Degree of separation removed: As used herein, amino acids that are a “degree of separation removed” are HA amino acids that have indirect effects on glycan binding. For example, one-degree-of-separation-removed amino acids may either: (1) interact with the direct-binding amino acids; and/or (2) otherwise affect the ability of direct-binding amino acids to interact with glycan that is associated with host cell HA receptors; such one-degree-of-separation-removed amino acids may or may not directly bind to glycan themselves. Two-degree-of-separation-removed amino acids either (1) interact with one-degree-of-separation-removed amino acids; and/or (2) otherwise affect the ability of the one-degree-of-separation-removed amino acids to interact with direct-binding amino acids, etc.
Direct-binding amino acids: As used herein, the phrase “direct-binding amino acids” refers to HA polypeptide amino acids which interact directly with one or more glycans that is associated with host cell HA receptors.
Engineered: The term “engineered,” as used herein, describes a polypeptide whose amino acid sequence has been selected by man. For example, an engineered HA polypeptide has an amino acid sequence that differs from the amino acid sequences of HA polypeptides found in natural influenza isolates. In some embodiments, an engineered HA polypeptide has an amino acid sequence that differs from the amino acid sequence of HA polypeptides included in the NCBI database.
H1 polypeptide: An “H1 polypeptide,” as that term is used herein, is an HA polypeptide whose amino acid sequence includes at least one sequence element that is characteristic of H1 and distinguishes H1 from other HA subtypes. Representative such sequence elements can be determined by alignments such as, for example, those illustrated in
H3 polypeptide: An “H3 polypeptide,” as that term is used herein, is an HA polypeptide whose amino acid sequence includes at least one sequence element that is characteristic of H3 and distinguishes H3 from other HA subtypes. Representative such sequence elements can be determined by alignments such as, for example, those illustrated in
H5 polypeptide: An “H5 polypeptide,” as used herein, is an HA polypeptide whose amino acid sequence includes at least one sequence element that is characteristic of H5 and distinguishes H5 from other HA subtypes. Representative such sequence elements can be determined by alignments such as, for example, those illustrated in
Hemagglutinin (HA) polypeptide: As used herein, the term “hemagglutinin polypeptide” (or “HA polypeptide”) refers to a polypeptide whose amino acid sequence includes at least one characteristic sequence of HA. A wide variety of HA sequences from influenza isolates are known in the art; indeed, the National Center for Biotechnology Information (NCBI) maintains a database (www.ncbi.nlm.nih.gov/genomes/FLU/flu.html) that, as of the filing of the present application included 9796 HA sequences. Those of ordinary skill in the art, referring to this database, can readily identify sequences that are characteristic of HA polypeptides generally, and/or of particular HA polypeptides (e.g., H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16 polypeptides); or of HAs that mediate infection of particular hosts, e.g., avian, camel, canine, cat, civet, environment, equine, human, leopard, mink, mouse, seal, stone martin, swine, tiger, whale, etc. For example, in some embodiments, an HA polypeptide includes one or more characteristic sequence elements found between about residues 97 and 185, 324 and 340, 96 and 100, and/or 130 and 230 of an HA protein found in a natural isolate of an influenza virus. In some embodiments, an HA polypeptide has an amino acid sequence comprising at least one of HA Sequence Elements 1 and 2, as defined herein. In some embodiments, an HA polypeptide has an amino acid sequence comprising HA Sequence Elements 1 and 2, in some embodiments separated from one another by about 100 to about 200, or by about 125 to about 175, or about 125 to about 160, or about 125 to about 150, or about 129 to about 139, or about 129, about 130, about 131, about 132, about 133, about 134, about 135, about 136, about 137, about 138, or about 139 amino acids. In some embodiments, an HA polypeptide has an amino acid sequence that includes residues at positions within the regions 96-100 and/or 130-230 that participate in glycan binding. For example, many HA polypeptides include one or more of the following residues: Tyr98, Ser/Thr136, Trp153, His183, and Leu/Ile194. In some embodiments, an HA polypeptide includes at least 2, 3, 4, or all 5 of these residues. As used herein, the term “HA polypeptide” encompasses amino acid chains of any length (i.e. amino acid chains comprising at least 2 amino acids or longer).
Isolated: The term “isolated,” as used herein, refers to an agent or entity that has either (i) been separated from at least some of the components with which it was associated when initially produced (whether in nature or in an experimental setting); or (ii) produced by the hand of man. Isolated agents or entities may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were initially associated. In some embodiments, isolated agents are more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure.
Long oligosaccharide: For purposes of the present disclosure, an oligosaccharide is typically considered to be “long” if it includes at least one linear chain that has at least four saccharide residues.
Non-natural amino acid: The phrase “non-natural amino acid” refers to an entity having the chemical structure of an amino acid (i.e.:
and therefore being capable of participating in at least two peptide bonds, but having an R group that differs from those found in nature. In some embodiments, non-natural amino acids may also have a second R group rather than a hydrogen, and/or may have one or more other substitutions on the amino or carboxylic acid moieties.
Polypeptide: A “polypeptide,” generally speaking, is a string of at least two amino acids attached to one another by a peptide bond. In some embodiments, a polypeptide may include at least 3-5 amino acids, each of which is attached to others by way of at least one peptide bond. Those of ordinary skill in the art will appreciate that polypeptides sometimes include “non-natural” amino acids or other entities that nonetheless are capable of integrating into a polypeptide chain, optionally.
Pure: As used herein, an agent or entity is “pure” if it is substantially free of other components. For example, a preparation that contains more than about 90% of a particular agent or entity is typically considered to be a pure preparation. In some embodiments, an agent or entity is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure.
Short oligosaccharide: For purposes of the present disclosure, an oligosaccharide is typically considered to be “short” if it has fewer than 4, or certainly fewer than 3, residues in any linear chain.
Specificity: As is known in the art, “specificity” is a measure of the ability of a particular ligand (e.g., an HA polypeptide) to distinguish its binding partner (e.g., a human HA receptor, and particularly a human upper respiratory tract HA receptor) from other potential binding partners (e.g., an avian HA receptor).
Subject: As used herein, the term “subject” or “patient” refers to any organism to which a composition of this invention may be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans; insects; worms; etc.).
Substantially: As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.
Suffering from: An individual who is “suffering from” influenza infection has been diagnosed with or displays one or more symptoms of influenza infection.
Susceptible to: An individual who is “susceptible to” influenza infection has not been diagnosed with influenza infection and/or may not exhibit symptoms of influenza infection. In some embodiments, an individual who is susceptible to influenza infection will develop influenza infection. In some embodiments, an individual who is susceptible to influenza infection will not develop influenza infection.
Therapeutic agent: As used herein, the phrase “therapeutic agent” refers to any agent that elicits a desired biological or pharmacological effect when administered to a subject.
Therapeutically effective amount: As used herein, the term “therapeutically effective amount” means an amount of inventive glycan decoy that is sufficient, when administered to a subject suffering from or susceptible to influenza infection, to treat, diagnose, prevent, and/or delay the onset of influenza infection and/or symptom(s) thereof.
Treating: As used herein, the term “treat,” “treatment,” or “treating” refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and/or reduce incidence of one or more symptoms or features of a particular disease, disorder, and/or condition (e.g., influenza infection). Treatment may be administered to a subject who does not exhibit signs of a disease and/or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.
Umbrella-topology: The phrase “umbrella-topology” is used herein to refer to a 3-dimensional arrangement adopted by certain glycans and in particular by certain glycans on HA receptors. The present invention encompasses the recognition that binding to umbrella-topology glycans is characteristic of HA proteins that mediate infection of human hosts. As illustrated in
where:
Vaccination: As used herein, the term “vaccination” refers to the administration of a composition intended to generate an immune response, for example to a disease-causing agent. For the purposes of the present invention, vaccination can be administered before, during, and/or after exposure to a disease-causing agent, and in certain embodiments, before, during, and/or shortly after exposure to the agent. In some embodiments, vaccination includes multiple administrations, appropriately spaced in time, of a vaccinating composition.
Variant: As used herein, the term “variant” is a relative term that describes the relationship between a particular HA polypeptide of interest and a “parent” HA polypeptide to which its sequence is being compared. An HA polypeptide of interest is considered to be a “variant” of a parent HA polypeptide if the HA polypeptide of interest has an amino acid sequence that is identical to that of the parent but for a small number of sequence alterations at particular positions. Typically, fewer than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% of the residues in the variant are substituted as compared with the parent. In some embodiments, a variant has 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substituted residue(s) as compared with a parent. Often, a variant has a very small number (e.g., fewer than 5, 4, 3, 2, or 1) number of substituted functional residues (i.e., residues that participate in a particular biological activity). Furthermore, a variant typically has not more than 5, 4, 3, 2, or 1 additions or deletions, and often has no additions or deletions, as compared with the parent. Moreover, any additions or deletions are typically fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and commonly are fewer than about 5, about 4, about 3, or about 2 residues. In some embodiments, the parent HA polypeptide is one found in a natural isolate of an influenza virus (e.g., a wild type HA).
Vector: As used herein, “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In some embodiment, vectors are capable of extra-chromosomal replication and/or expression of nucleic acids to which they are linked in a host cell such as a eukaryotic or prokaryotic cell. Vectors capable of directing the expression of operatively linked genes are referred to herein as “expression vectors.”
Wild type: As is understood in the art, the phrase “wild type” generally refers to a normal form of a protein or nucleic acid, as is found in nature. For example, wild type HA polypeptides are found in natural isolates of influenza virus. A variety of different wild type HA sequences can be found in the NCBI influenza virus sequence database, http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html.
The present invention encompasses the recognition that interactions between influenza HA polypeptides and umbrella-topology glycans mediate influenza infections. The present further encompasses the recognition that agents that mimic umbrella-topology glycans can act as “receptor decoys” and can be useful as therapeutic agents in the treatment of influenza infections.
Among other things, the present invention provides systems for identifying agents that mimic umbrella-topology glycans and that compete interactions between HA polypeptides and HA receptors in subjects. The present invention further provides various agents and compositions containing them, as well as therapeutic strategies for the treatment of influenza infections. The present invention provides systems (e.g. detection assays) for characterizing HA-receptor interactions and agents that affect HA-receptor interactions.
HA Receptors
HA interacts with the surface of cells by binding to a glycoprotein receptor. Binding of HA to HA receptors is predominantly mediated by N-linked glycans on the HA receptors. Specifically, HA on the surface of flu virus particles recognizes sialylated glycans that are associated with HA receptors on the surface of the cellular host. After recognition and binding, the host cell engulfs the viral cell and the virus is able to replicate and produce many more virus particles to be distributed to neighboring cells. Some crystal structures of exemplary HA-glycan interactions have been identified and are presented in Table 1:
HA receptors are modified by either α2-3 or α2-6 sialylated glycans near the receptor's HA-binding site, and the type of linkage of the receptor-bound glycan can affect the conformation of the receptor's HA-binding site, thus affecting the receptor's specificity for different HAs.
For example, the glycan binding pocket of avian HA is narrow. According to the present invention, this pocket binds to the trans conformation of α2-3 sialylated glycans, and/or to cone-topology glycans, whether α2-3 or α2-6 linked.
HA receptors in avian tissues, and also in human deep lung and gastrointestinal (GI) tract tissues are characterized by α2-3 sialylated glycan linkages, and furthermore (according to the present invention), are characterized by glycans, including α2-3 sialylated and/or α2-6 sialylated glycans, which predominantly adopt cone topologies. HA receptors having such cone topology glycans may be referred to herein as CTHArs.
By contrast, human HA receptors in the bronchus and trachea of the upper respiratory tract are modified by α2-6 sialylated glycans. Unlike the α2-3 motif, the α2-6 motif has an additional degree of conformational freedom due to the C6-C5 bond (Russell et al., 2006, Glycoconj. J., 23:85; incorporated herein by reference). HAs that bind to such α2-6 sialylated glycans have a more open binding pocket to accommodate the diversity of structures arising from this conformational freedom. Moreover, according to the present invention, HAs may need to bind to glycans (e.g., α2-6 sialylated glycans) in an umbrella-topology, and particularly may need to bind to such umbrella-topology glycans with strong affinity and/or specificity, in order to effectively mediate infection of human upper respiratory tract tissues. HA receptors having such umbrella topology glycans may be referred to herein as UTHArs.
As a result of these spatially restricted glycosylation profiles, humans are not usually infected by viruses containing many wild type avian HAs (e.g., avian H5). Specifically, because the portions of the human respiratory tract that are most likely to encounter virus (i.e., the trachea and bronchi) lack receptors with cone-topology glycans (e.g., α2-3 sialylated glycans, and/or short glycans) and wild type avian HAs typically bind primarily or exclusively to receptors associated with cone-topology glycans (e.g., α2-3 sialylated glycans, and/or short glycans), humans rarely become infected with avian viruses. Only when in sufficiently close contact with virus that it can access the deep lung and/or gastrointestinal tract receptors having umbrella-topology glycans (e.g., long α2-6 sialylated glycans) do humans become infected.
Umbrella-Topology Glycans
As described in co-pending application Ser. No. 11/893,171, filed Aug. 14, 2007 (the entire contents of which are attached hereto as Appendix A), the present inventors have demonstrated that infection of mammalian (e.g., human) subjects by influenza viruses is mediated by interactions between viral HA polypeptides and umbrella-topology glycans on HA receptors in the subjects.
In some embodiments, umbrella-topology glycan preparations comprise a greater proportion of long (e.g. multiple lactosamine units) α2-6 oligosaccharide branches than short α2-6 (e.g. single lactosamine) branches. In some embodiments, umbrella-topology glycans comprise about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, or greater than about 50-fold more long α2-6 oligosaccharide branches than short α2-6 (e.g. single lactosamine) branches.
In some embodiments, umbrella-topology glycans are glycans exhibiting a three-dimensional structure such as the structure presented in
In some embodiments, glycan structural topology is classified based on parameter θ defined as angle between C2 of Sia, C1 of Gal, and C1 of GlcNAc. Values of θ>110° represent cone-like topology adopted by α2-3 and short α2-6 glycans. Values of θ<100° represent umbrella-like topology, such as topology adopted by long α2-6 glycans (
In certain embodiments, the unique characteristic of HA interactions with umbrella-topology glycans and/or glycan decoys is the HA contact with a glycan comprising sialic acid (SA) and/or SA analogs at the non-reducing end. In some embodiments, chain length of the oligosaccharide is at least a trisaccharide (excluding the SA or SA analog). In some embodiments, a combination of the numbered residues shown in the right-hand panel of
In some embodiments, a sialic acid analog interacts with one or more of any highly conserved sialic acid-anchoring amino acid in HA, including, but not limited to, Tyr98, Ser136, Thr136, Trp153, Thr155, Val 155, Ser 155, His183, and Leu194. These interactions may include non-covalent interactions (e.g. ionic, van der Waals, hydrophobic, etc.) or covalent interactions with one or more of the above amino acids. Exemplary sialic acid analogs are shown in Table 2. Binding of HA to glycans containing some of these sialic acid analogs have been reported (Kelm et al., 1992 Eur. J. Biochem., 205:146; incorporated herein by reference).
In some embodiments, umbrella-topology glycans are glycans that are preferentially found on mammalian upper respiratory epithelial cells. In some embodiments, umbrella-topology glycans are glycans that are preferentially found on human upper respiratory epithelial cells. In some embodiments, umbrella-topology glycans are glycans that are about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, or greater than about 50-fold more likely to be found on human upper respiratory epithelial cells than on other cells.
In certain embodiments, structural features of umbrella-like topology glycans can be defined according to the following rules:
An oligosaccharide of the following form:
where:
In some embodiments, an umbrella-topology glycan has the structural form:
Neu5Acα2-6Sug1-Sug2-Sug3
where:
Examples of oligosaccharide motifs commonly found in physiological N-linked, O-linked glycans and glycolipids that comprise the structural features described above are shown in
In some embodiments, glycan decoys may include the entire complex physiological N-linked, O-linked glycans (and glycoproteins) and glycolipids that comprise the above structural features. In some embodiments, three-dimensional structural topology of a decoy is classified using a parameter θ shown in
Analysis of HA-glycan co-crystals reveals that the position of Neu5Ac relative to the HA binding site is almost invariant. Contacts with Neu5Ac involve highly conserved residues such as Y98, S/T136, W153, H183 and L/1194. Contacts with other sugars involve different residues, depending on whether the sugar linkage is α2-3 or α2-6 and whether the glycan topology is cone or umbrella. For example, in the cone topology, the primary contacts are with Neu5Ac and with Gal sugars. E190 and Q226 play particularly important roles in this binding. Other positions (e.g., 137, 145, 186, 187, 193, 222) can participate in binding to cone structures. In some cases, different residues can make different contacts with different glycan structures. The type of amino acid in these positions can influence ability of an HA polypeptide to bind to receptors with different modification and/or branching patterns in the glycan structures. In the umbrella topology, contacts are made with sugars beyond Neu5Ac and Gal. In some embodiments, amino acid residues at one or more positions (e.g., 137, 145, 156, 159, 186, 187, 189, 190, 192, 193, 196, 222, 225, 226) can participate in binding to umbrella structures. In some cases, different residues can make different contacts with different glycan structures. The type of amino acid in these positions can influence ability of an HA polypeptide to bind to receptors with different modification and/or branching patterns in the glycan structures. In some embodiments, a D residue at position 190 and/or a D residue at position 225 contribute(s) to binding to umbrella topologies.
In some embodiments, umbrella-topology glycans are any substances that comprise an umbrella-topology glycan moiety. In some embodiments, an umbrella-topology glycan is or comprises an umbrella-topology glycan moiety (i.e. an umbrella-topology glycan moiety that is not associated with any other moiety). In some embodiments, the carrier moiety may comprise any type of substance, including, but not limited to, a peptide, polypeptide, protein, carbohydrate, lipid, nucleic acid, small molecule, cell, virus, etc. In some embodiments the ratio of umbrella glycan to carrier molecule (on a weight or number basis) is greater than the ratio of glycan to protein in a human HA receptor molecule, e.g., a naturally occurring human HA receptor which carries an umbrella topology glycan. For example, a carrier molecule can have more umbrella glycans than a naturally occurring HA receptor molecule, e.g., a naturally occurring human HA receptor which carries an umbrella topology glycan. In some embodiments the ratio of umbrella glycan to carrier molecule (on a weight or number basis) is less than the ratio of glycan to protein in a human HA receptor molecule, e.g., a naturally occurring human HA receptor which carries an umbrella topology glycan. In an embodiment the carrier molecule presents more than one umbrella glycan and the glycans, as presented, are spaced apart from one another similarly as is found on naturally occurring human HA receptors which carry an umbrella topology glycan. In other embodiments the glycans are more, or less, spaced apart, as compared to their presentation on naturally occurring human HA receptors which carry an umbrella topology glycan. In a preferred embodiment the decoy includes an umbrella glycan on an HA polypeptide that differs from a naturally occurring human HA by at least one amino acid residue.
In some embodiments, umbrella-topology glycans comprise a peptide or polypeptide moiety and at least one umbrella-topology glycan moiety. In certain embodiments, umbrella-topology glycans comprise an HA receptor and at least one umbrella-topology glycan moiety. In certain embodiments, umbrella-topology glycans comprise a characteristic portion of an HA receptor and at least one umbrella-topology glycan moiety. In certain embodiments, umbrella-topology glycans comprise an HA receptor derived and/or obtained from human upper respiratory tissues (e.g. trachea and/or bronchus) and at least one umbrella-topology glycan moiety. In certain embodiments, umbrella-topology glycans comprise a characteristic portion of an HA receptor derived and/or obtained from human upper respiratory tissues (e.g. trachea and/or bronchus) and at least one umbrella-topology glycan moiety.
Umbrella-Topology Glycan Decoys
In some embodiments, an “umbrella-topology glycan decoy” refers to any substance that shares sufficient structural similarity with umbrella-topology glycans to bind to HA polypeptides. In some embodiments, an “umbrella-topology glycan decoy” refers to any substance that is able to compete away the interaction between HA and umbrella-topology glycans. In some embodiments, an “umbrella-like topology glycan decoy” can be any substance that is able to contact any or all of the residues (including any combination of individual residues) in the glycan binding site of HA that are capable of interacting with umbrella-topology glycans (see, e.g.,
In some embodiments, an umbrella-topology glycan decoy is or comprises an umbrella-topology glycan mimic. In some embodiments, an umbrella-topology glycan mimic is or comprises a glycan. In some embodiments, an umbrella-topology glycan decoy is or comprises an umbrella-topology glycan moiety. In some embodiments, an umbrella-topology glycan decoy is or comprises an isolated umbrella-topology glycan moiety (i.e. an umbrella-topology glycan moiety that is not associated with any other moiety). In some embodiments, an umbrella-topology glycan moiety has a structure that is or is substantially similar to any of the structures shown in
In some embodiments, glycan decoys may include entire complex physiological N-linked and/or O-linked glycans, glycoproteins, and/or glycolipids that are defined by any or all of the structural features described in the section, above, entitled “Umbrella-Topology Glycans.” In some embodiments, three-dimensional structural topology of a decoy is classified using a parameter θ shown in
In some embodiments, an umbrella-topology glycan decoy is or comprises a small molecule. In some embodiments, an umbrella-topology glycan decoy is or comprises a peptide (e.g., peptide, polypeptide, protein, etc.). In some embodiments, an umbrella-topology glycan decoy is or comprises a lipid. In some embodiments, an umbrella-topology glycan decoy is or comprises a nucleic acid.
An umbrella-topology glycan decoy can be any substance that is capable of interacting with HA amino acid residues that are involved in or are capable of binding to umbrella-topology glycans. In certain embodiments, an umbrella-topology glycan decoy is or comprises any substance that is capable of interacting with HA amino acids 136, 137, 145, 153, 155, 156, 159, 186, 187, 189, 190, 192, 193, 194, 196, 222, 225, 226, 228, and/or combinations thereof. In certain embodiments, an umbrella-topology glycan decoy is or comprises any substance that is capable of interacting with HA amino acids 156, 159, 189, 192, 193, 196, and/or combinations thereof. In certain embodiments, an umbrella-topology glycan decoy is or comprises any substance that is capable of interacting with HA amino acids 186, 187, 189, 190, and/or combinations thereof. In certain embodiments, an umbrella-topology glycan decoy is or comprises any substance that is capable of interacting with HA amino acids 137, 145, 190, 226, 228, and/or combinations thereof. In certain embodiments, an umbrella-topology glycan decoy is or comprises any substance that is capable of interacting with HA amino acids 190, 222, 225, 226, and/or combinations thereof. In certain embodiments, an umbrella-topology glycan is or comprises any substance that is capable of interacting with HA amino acids 136, 153, 155, 194, and combinations thereof. In certain embodiments, an umbrella-topology glycan decoy is or comprises any substance that is capable of interacting with HA amino acids 190 and 226. In certain embodiments, an umbrella-topology glycan decoy is or comprises any substance that is capable of interacting with HA amino acids 222, 225, and 226. In certain embodiments, an umbrella-topology glycan decoy is or comprises any substance that is capable of interacting with HA amino acids 190, 192, 193, and 225. In certain embodiments, an umbrella-topology glycan decoy is or comprises any substance that is capable of interacting with HA amino acids 186, 193, and 222. Note that amino acid positions stated above are based on H3 HA numbering.
In some embodiments, an umbrella-topology glycan decoy is or comprises an umbrella-topology glycan mimic (e.g. glycan, peptide, small molecule, etc.) that is physically associated with a carrier moiety. In some embodiments, a carrier will carry a plurality of individual glycan mimics. Without wishing to be bound by any particular theory, the present inventors propose that multiple points of contact between glycans and their binding partners (e.g., HA polypeptides) may facilitate, or even be required, for specific and/or stable interaction.
In some embodiments, the umbrella-topology glycan mimic is covalently associated with the carrier. In some embodiments, the covalent association is direct (e.g. the umbrella-topology glycan mimic is directly attached to the carrier). In some embodiments, the covalent association is indirect (e.g. mediated by a linker). In certain embodiments, the linker is a cleavable linker (e.g. a linker that can be cleaved by enzymatic activity, chemical cleavage, heat-induced cleavage, pH-induced cleavage, light-induced cleavage, etc.). In certain embodiments, the linker is a non-cleavable linker. In certain embodiments, the linker comprises proteins or peptides, nucleic acids, carbohydrates, lipids, small molecules, etc.
In some embodiments, the umbrella-topology glycan mimic is non-covalently associated with the carrier. In some embodiments, a non-covalent interaction includes electrostatic interactions, affinity interactions, metal coordination, physical adsorption, host-guest interactions, hydrophobic interactions, pi-stacking interactions, hydrogen bonding interactions, van der Waals interactions, magnetic interactions, dipole-dipole interactions, and combinations thereof.
In some embodiments, the umbrella-topology glycan mimic is associated with the surface of the carrier. In some embodiments, the umbrella-topology glycan mimic is associated with the interior surface of the carrier (e.g. the interior surface of a particle, cell, etc.). In some embodiments, the umbrella-topology glycan mimic is encapsulated within the carrier (e.g. within the interior of a particle, in the cytoplasm of a cell, within a polymeric matrix, etc.).
In some embodiments, the carrier moiety may comprise any type of substance, including, but not limited to, a peptide (including a polypeptide, a protein, an antibody, etc.), carbohydrate (e.g., mono-, di-, or polysaccharide, glycan, etc.), lipid, nucleic acid, polymer, small molecule, dendrimer (e.g., starburst dendrimers containing umbrella-topology glycans), cell, virus, particle (e.g. microparticle, nanoparticle, picoparticle, polymeric particle, quantum dot, metal particle, bone-derived particle, ceramic-derived particle, liposome, micelle, etc.), etc.
In certain embodiments, umbrella-topology glycan decoys comprise a peptide or polypeptide carrier and at least one umbrella-topology glycan moiety. In certain embodiments, umbrella-topology glycan decoys comprise an HA receptor and at least one umbrella-topology glycan moiety. In certain embodiments, umbrella-topology glycan decoys comprise a characteristic portion of an HA receptor and at least one umbrella-topology glycan moiety. In certain embodiments, umbrella-topology glycan decoys comprise an HA receptor derived and/or obtained from mammalian (e.g., human) upper respiratory tissues (e.g. trachea and/or bronchus) and at least one umbrella-topology glycan moiety. In certain embodiments, umbrella-topology glycan decoys comprise a fragment (e.g., a characteristic portion) of an HA receptor derived and/or obtained from human upper respiratory tissues (e.g. trachea and/or bronchus) and at least one umbrella-topology glycan moiety. In some embodiments, HA receptor fragments are obtained by protease treatment of membrane glycoproteins from upper respiratory cells of humans or other animals (e.g., ferrets). Examples of upper respiratory cells include ciliated tracheal epithelial, bronchial epithelial, goblet cells from tracheal or bronchial regions, non-ciliated epithelial cells, and combinations thereof.
In some embodiments, a peptide carrier moiety may comprise at least about 5 amino acids, at least about 10 amino acids, at least about 20 amino acids, at least about 50 amino acids, at least about 100 amino acids, at least about 200 amino acids, or more amino acids. In certain embodiments, peptides are proteins having fewer than about 100 amino acids. In some embodiments, peptides range from about 5 to about 100, from about 5 to about 50, from about 5 to about 40, from about 5 to about 35, from about 5 to about 30, from about 5 to about 25, or from about 20 to about 25 amino acids in length. In some embodiments, a peptide sequence can be based on the sequence of a protein. In some embodiments, a peptide sequence can be a random arrangement of amino acids. Peptides from panels of peptides comprising random sequences and/or sequences which have been varied consistently to provide a maximally diverse panel of peptides may be used.
In certain embodiments, umbrella-topology glycan decoys comprise a glycopeptide (e.g., HA receptor, mucin, fetuin, IgG, etc) carrier that then carries N- or O-linked umbrella-topology glycans. In certain embodiments, umbrella-topology glycan decoys comprise a neo-glycoprotein carrier in which glycans are linked to a polypeptide backbone via reductive amination to a core polypeptide (e.g., bovine serum albumin).
In some embodiments, umbrella-topology glycan decoys comprise carriers with artificial polypeptide backbones such as, for example, poly-L-glutamic acid. In some specific embodiments, one or more (and in some embodiments each) glutamic acid may have an umbrella topology glycan conjugated via the alpha carboxyl group.
In certain embodiments, umbrella-topology glycan decoys comprise a nucleic acid carrier and at least one umbrella-topology glycan moiety. Nucleic acid carriers include, but are not limited to, viral fragments, including viral DNA and/or RNA; DNA and/or RNA chimeras; mRNA; plasmids; cosmids; genomic DNA; cDNA; gene fragments; various structural forms of DNA including single-stranded DNA, double-stranded DNA, supercoiled DNA and/or triple-helical DNA; Z-DNA; functional nucleic acids (e.g. RNAi-inducing entities, ribozymes, etc.), etc.
In certain embodiments, umbrella-topology glycan decoys comprise a small molecule carrier and at least one umbrella-topology glycan moiety. In general, a “small molecule” is understood in the art to be an organic molecule that is less than about 5 kilodaltons (Kd) in size. In some embodiments, the small molecule is less than about 4 Kd, 3 Kd, about 2 Kd, or about 1 Kd. In some embodiments, the small molecule is less than about 800 daltons (D), about 600 D, about 500 D, about 400 D, about 300 D, about 200 D, or about 100 D. In some embodiments, a small molecule is less than about 2000 g/mol, less than about 1500 g/mol, less than about 1000 g/mol, less than about 800 g/mol, or less than about 500 g/mol. In some embodiments, small molecules are non-polymeric.
In certain embodiments, umbrella-topology glycan decoys comprise a lipid carrier and at least one umbrella-topology glycan moiety. In some embodiments, exemplary lipids that may be used in accordance with the present invention include, but are not limited to, oils, fatty acids, saturated fatty acid, unsaturated fatty acids, essential fatty acids, cis fatty acids, trans fatty acids, glycerides, monoglycerides, diglycerides, triglycerides, hormones, steroids (e.g., cholesterol, bile acids), vitamins (e.g. vitamin E), phospholipids, sphingolipids, and lipoproteins.
In some embodiments, a lipid may comprise one or more fatty acid groups or salts thereof. In some embodiments, the fatty acid group may comprise digestible, long chain (e.g., C8-C50), substituted or unsubstituted hydrocarbons. In some embodiments, the fatty acid group may be a C10-C20 fatty acid or salt thereof. In some embodiments, the fatty acid group may be a C15-C20 fatty acid or salt thereof. In some embodiments, the fatty acid group may be a C15-C25 fatty acid or salt thereof. In some embodiments, the fatty acid group may be unsaturated. In some embodiments, the fatty acid group may be monounsaturated. In some embodiments, the fatty acid group may be polyunsaturated. In some embodiments, a double bond of an unsaturated fatty acid group may be in the cis conformation. In some embodiments, a double bond of an unsaturated fatty acid may be in the trans conformation.
In some embodiments, the fatty acid group may be one or more of butyric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic, arachidic, behenic, or lignoceric acid. In some embodiments, the fatty acid group may be one or more of palmitoleic, oleic, vaccenic, linoleic, alpha-linolenic, gamma-linoleic, arachidonic, gadoleic, arachidonic, eicosapentaenoic, docosahexaenoic, or erucic acid.
In certain embodiments, umbrella-topology glycan decoys comprise a carbohydrate carrier and at least one umbrella-topology glycan moiety. In some embodiments, a carbohydrate may be natural or synthetic. A carbohydrate may also be a derivatized natural carbohydrate. In certain embodiments, a carbohydrate may be a simple or complex sugar. In certain embodiments, a carbohydrate is a monosaccharide, including but not limited to glucose, fructose, galactose, and ribose. In certain embodiments, a carbohydrate is a disaccharide, including but not limited to lactose, sucrose, maltose, trehalose, and cellobiose. In certain embodiments, a carbohydrate is a polysaccharide, including but not limited to cellulose, microcrystalline cellulose, hydroxypropyl methylcellulose (HPMC), methylcellulose (MC), dextrose, dextran, glycogen, xanthan gum, gellan gum, starch, and pullulan. In certain embodiments, a carbohydrate is a sugar alcohol, including but not limited to mannitol, sorbitol, xylitol, erythritol, malitol, and lactitol.
In certain embodiments, umbrella-topology glycan decoys comprise a particle carrier and at least one umbrella-topology glycan moiety. In some embodiments, carriers may be particles, including, but not limited to, microparticles, nanoparticles, picoparticles, polymeric particles, quantum dots, metal particles, bone-derived particles, ceramic-derived particles, liposomes, micelles, etc. In some embodiments, particles are biodegradable and biocompatible. In general, a biocompatible substance is not toxic to cells. In some embodiments, a substance is considered to be biocompatible if its addition to cells results in less than a certain threshold of cell death (e.g., less than 75%, less than 50%, less than 25%, or less than 10% cell death). In some embodiments, a substance is considered to be biocompatible if its addition to cells does not induce adverse effects. In general, a biodegradable substance is one that undergoes breakdown under physiological conditions over the course of a therapeutically relevant time period (e.g., weeks, months, or years). In some embodiments, a biodegradable substance is a substance that can be broken down by cellular machinery. In some embodiments, a biodegradable substance is a substance that can be broken down by chemical processes.
In general, a particle in accordance with the present invention is any entity having a greatest dimension (e.g. diameter) of less than 500 microns (μm). In some embodiments, inventive particles have a greatest dimension of less than 100 μm. In some embodiments, inventive particles have a greatest dimension of less than 10 μm. In some embodiments, inventive particles have a greatest dimension of less than 1000 nanometers (nm). In some embodiments, inventive particles have a greatest dimension of less than 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm. Typically, inventive particles have a greatest dimension (e.g., diameter) of 300 nm or less. In some embodiments, inventive particles have a greatest dimension (e.g., diameter) of 250 nm or less. In some embodiments, inventive particles have a greatest dimension (e.g., diameter) of 200 nm or less. In some embodiments, inventive particles have a greatest dimension (e.g., diameter) of 150 nm or less. In some embodiments, inventive particles have a greatest dimension (e.g., diameter) of 100 nm or less. Smaller particles, e.g., having a greatest dimension of 50 nm or less are used in some embodiments of the invention. In some embodiments, inventive particles have a greatest dimension ranging between 5 nm and 1 μm. In some embodiments, inventive particles have a greatest dimension ranging between 25 nm and 200 nm.
In certain embodiments, particles can have a mean geometric diameter between about 1 μm and about 500 μm, between about 1 μm and about 200 μm, between about 1 μm and about 106 μm, between about 1 μm and about 100 μm, between about 1 μm and about 50 μm, between about 1 μm and about 53 μm, between about 5 μm and about 53 μm, between about 5 μm and about 50 μm, between about 53 μm and about 106 μm, between about 1 μm and about 5 μm, between about 1 μm and about 20 μm, between about 20 μm and about 53 μm, between about 53 μm and about 75 μm, between about 75 μm and about 106 μm, or about 30 μm.
In certain embodiments, particles can have a geometric size distribution between about 1 μm and about 250 μm, between about 1 μm and about 100 μm, between about 1 μm and about 50 μm, between about 5 μm and about 200 μm, between about 10 μm and about 500 μm, between about 10 μm and about 250 μm, between about 10 μm and about 100 μm, between about 100 μm and about 200 μm, between about 100 μm and about 150 μm, between about 53 μm and about 106 μm, between about 1 μm and about 5 μm, between about 1 μm and about 20 μm, between about 20 μm and about 53 μm, between about 53 μm and about 75 μm, or between about 75 μm and about 106 μm.
Aerodynamic diameter is a physical property of a particle in a viscous fluid such as air. In general, particles have irregular shapes with actual geometric diameters that can be difficult to measure. Aerodynamic diameter is an expression of a particle's aerodynamic behavior as if it were a perfect sphere with unit-density and diameter equal to the aerodynamic diameter. Such a model has the same terminal settling velocity. Aerodynamic diameter can be applied to particulate pollutants and inhaled drugs to predict where in the respiratory tract such particles will deposit. In some embodiments, drug particles for pulmonary delivery can be characterized by aerodynamic diameter instead of or in addition to geometric diameter. The velocity at which drug settles is proportional to the aerodynamic diameter, da:
da=(ρ/X)1/2×dg
where ρ=density and X=shape factor. For spherical species, X=1. Aerodynamic diameter is the diameter of a sphere of unit density (1 g/ml) that has the same gravitational settling velocity as the particle in question. Aerodynamic diameter is given as:
dpa=dps×sqrt(density of particle)
where dps=Stoke's diameter. In certain embodiments, particles can have mean aerodynamic diameters between about 1 μm and about 5 μm, between about 1 μm and about 35 μm, between about 5 μm and about 35 μm, between about 1 μm and about 35 μm, between about 35 μm and about 70 μm, between about 1 μm and about 75 μm, between about 35 μm and about 75 μm, between about 1 μm and about 50 μm, or about 5 μm.
Bulk density a property of particulate materials. In general, bulk density is used to refer to the mass of a population of particles divided by the volume they occupy. Typically, the volume includes the space between particles as well as the space inside the pores of individual particles. Bulk density is not an intrinsic property of a material, but can change depending on how the material is handled. For example, grain poured in cylinder will have a particular bulk density; if the cylinder is disturbed, grain particles will move and settle closer together, resulting in a higher bulk density. For this reason, the bulk density of powders is usually reported both as “freely settled” and “tapped” density, where the tapped density refers to the bulk density of the powder after a compaction process. Typically, the compactation process may involve vibration of the container which holds the population of particles. In certain embodiments, particles can have tap density ranges between about 0.01 g/cm3 and about 0.4 g/cm3, greater than 0.4 g/cm3, or less than 0.4 g/cm3.
In some embodiments, particles have a diameter of approximately 1000 nm. In some embodiments, particles have a diameter of approximately 750 nm. In some embodiments, particles have a diameter of approximately 500 nm. In some embodiments, particles have a diameter of approximately 450 nm. In some embodiments, particles have a diameter of approximately 400 nm. In some embodiments, particles have a diameter of approximately 350 nm. In some embodiments, particles have a diameter of approximately 300 nm. In some embodiments, particles have a diameter of approximately 275 nm. In some embodiments, particles have a diameter of approximately 250 nm. In some embodiments, particles have a diameter of approximately 225 nm. In some embodiments, particles have a diameter of approximately 200 nm. In some embodiments, particles have a diameter of approximately 175 nm. In some embodiments, particles have a diameter of approximately 150 nm. In some embodiments, particles have a diameter of approximately 125 nm. In some embodiments, particles have a diameter of approximately 100 nm. In some embodiments, particles have a diameter of approximately 75 nm. In some embodiments, particles have a diameter of approximately 50 nm. In some embodiments, particles have a diameter of approximately 25 nm.
In certain embodiments, particles are greater in size than the renal excretion limit (e.g. particles having diameters of greater than 6 nm). In specific embodiments, particles have diameters greater than 5 nm, greater than 10 nm, greater than 15 nm, greater than 20 nm, greater than 50 nm, greater than 100 nm, greater than 250 nm, greater than 500 nm, greater than 1000 nm, or larger. In certain embodiments, particles are small enough to avoid clearance of particles from the bloodstream by the liver (e.g. particles having diameters of less than 1000 nm). In specific embodiments, particles have diameters less than 1500 nm, less than 1000 nm, less than 750 nm, less than 500 nm, less than 250 nm, less than 100 nm, or smaller. In general, physiochemical features of particles, including particle size, can be selected to allow a particle to circulate longer in plasma by decreasing renal excretion and/or liver clearance. In some embodiments, particles have diameters ranging from 5 nm to 1500 nm, from 5 nm to 1000 nm, from 5 nm to 750 nm, from 5 nm to 500 nm, from 5 nm to 250 nm, or from 5 nm to 100 nm. In some embodiments, particles have diameters ranging from 10 nm to 1500 nm, from 15 nm to 1500 nm, from 20 nm to 1500 nm, from 50 nm to 1500 nm, from 100 nm to 1500 nm, from 250 nm to 1500 nm, from 500 nm to 1500 nm, or from 1000 nm to 1500 nm.
It is often desirable to use a population of particles that is relatively uniform in terms of size, shape, and/or composition so that each particle has similar properties. For example, at least 80%, at least 90%, or at least 95% of the particles may have a diameter or greatest dimension that falls within 5%, 10%, or 20% of the average diameter or greatest dimension. In some embodiments, a population of particles may be heterogeneous with respect to size, shape, and/or composition.
Zeta potential is a measurement of surface potential of a particle. In some embodiments, particles have a zeta potential ranging between −50 mV and +50 mV. In some embodiments, particles have a zeta potential ranging between −25 mV and +25 mV. In some embodiments, particles have a zeta potential ranging between −10 mV and +10 mV. In some embodiments, particles have a zeta potential ranging between −5 mV and +5 mV. In some embodiments, particles have a substantially negative zeta potential. In some embodiments, particles have a substantially positive zeta potential. In some embodiments, particles have a substantially neutral zeta potential (i.e. approximately 0 mV).
Particles can have a variety of different shapes including spheres, oblate spheroids, cylinders, ovals, ellipses, shells, cubes, cuboids, cones, pyramids, rods (e.g., cylinders or elongated structures having a square or rectangular cross-section), tetrapods (particles having four leg-like appendages), triangles, prisms, etc.
In some embodiments, particles are microparticles (e.g. microspheres). In general, a “microparticle” refers to any particle having a diameter of less than 1000 μm. In some embodiments, particles are nanoparticles (e.g. nanospheres). In general, a “nanoparticle” refers to any particle having a diameter of less than 1000 nm. In some embodiments, particles are picoparticles (e.g. picospheres). In general, a “picoparticle” refers to any particle having a diameter of less than 1 nm. In some embodiments, particles are liposomes. In some embodiments, particles are micelles.
Particles can be solid or hollow and can comprise one or more layers (e.g., nanoshells, nanorings, etc.). Particles may have a core/shell structure, wherein the core(s) and shell(s) can be made of different materials. Particles may comprise gradient or homogeneous alloys. Particles may be composite particles made of two or more materials, of which one, more than one, or all of the materials possesses magnetic properties, electrically detectable properties, and/or optically detectable properties.
In some embodiments, particles may optionally comprise one or more dispersion media, surfactants, release-retarding ingredients, or other pharmaceutically acceptable excipient. In some embodiments, particles may optionally comprise one or more plasticizers or additives.
In some specific embodiments, particular carriers utilized in umbrella-topology decoys of the present invention are selected from the group consisting of HA receptor fragments, mucins, fetuins, IgG, bovine serum albumin, gamma poly glutamic acid, polyacrylamide, chitosan, gold nanoparticles (i.e., functionalized with umbrella topology glycans), and combinations thereof.
In certain embodiments, umbrella-topology glycan decoys comprise a cell or virus carrier and at least one umbrella-topology glycan moiety. For example, an umbrella-topology glycan moiety may be associated with (e.g. covalently or non-covalently bound to) the surface of a cell or virus. In some embodiments, an umbrella-topology glycan moiety may be encapsulated within a cell or virus.
The present invention encompasses the recognition that agents that mimic such umbrella-topology glycans can act as decoys and compete interactions between HA polypeptides and their receptors. In some embodiments, glycan mimics compete with endogenous glycans for binding to HA polypeptides. In some embodiments, glycan mimics compete with endogenous umbrella-type glycans for binding to HA polypeptides. In some embodiments, glycan mimics compete with upper-respiratory glycans for binding to HA polypeptides. In some embodiments, glycan mimics compete with umbrella-topology upper-respiratory glycans for binding to HA polypeptides.
In some embodiments, glycan mimics bind to HA polypeptides, but do not compete with endogenous glycans for binding to HA polypeptides.
In some embodiments, umbrella-topology glycan decoys are characterized by a three-dimensional structure that is capable of binding to and/or associating with one or more HA polypeptides. In some embodiments, umbrella-topology glycan decoys comprise a multivalent arrangement of individual glycan moieties associated with a carrier. For example, a glycan decoy may comprise 2, 3, 4, 5, 10, 20, 50, 100, 200, 500, 1000, or more individual glycan moieties associated with a carrier (covalently or non-covalently, as described above) in an arrangement and/or orientation that allows the glycan decoy to bind to and/or associate with one or more HA polypeptides. In some embodiments, an umbrella-topology glycan decoy comprising a multivalent arrangement of individual glycan moieties resembles and/or mimics the HA polypeptide-binding site of a wild-type HA receptor. In some embodiments, an umbrella-topology glycan decoy comprising a multivalent arrangement of individual glycan moieties resembles and/or mimics the HA polypeptide-binding site of a wild-type HA receptor in its natural context (e.g. in various lung tissues).
HA polypeptides are described in further detail below, in the section entitled “Hemagglutinin (HA).”
Hemagglutinin (HA)
Influenza viruses are RNA viruses which are characterized by a lipid membrane envelope containing two glycoproteins, hemagglutinin (HA) and neuraminidase (NA), embedded in the membrane of the virus particular. There are 16 known HA subtypes and 9 NA subtypes, and different influenza strains are named based on the number of the strain's HA and NA subtypes. Based on comparisons of amino acid sequence identity and of crystal structures, the HA subtypes have been divided into two main groups and four smaller clades. The different HA subtypes do not necessarily share strong amino acid sequence identity, but the overall 3D structures of the different HA subtypes are similar to one another, with several subtle differences that can be used for classification purposes. For example, the particular orientation of the membrane-distal subdomains in relation to a central α-helix is one structural characteristic commonly used to determine HA subtype (Russell et al., 2004, Virology, 325:287; incorporated herein by reference).
HA exists in the membrane as a homotrimer of one of 16 subtypes, termed H1-H16. Only three of these subtypes (H1, H2, and H3) have thus far become adapted for human infection. One reported characteristic of HAs that have adapted to infect humans (e.g., of HAs from the pandemic H1N1 (1918) and H3N2 (1967-68) influenza subtypes) is their ability to preferentially bind to α2-6 sialylated glycans in comparison with their avian progenitors that preferentially bind to α2-3 sialylated glycans (Skehel and Wiley, 2000, Ann. Rev. Biochem., 69:531; Rogers and Paulson, 1983, Virology, 127:361; Rogers et al., 1983, Nature, 304:76; Sauter et al., 1992, Biochemistry, 31:9609; Connor et al., 1994, Virology, 205:17; and Tumpey et al., 2005, Science, 310:77; all of which are incorporated herein by reference). The present invention, however, encompasses the recognition that ability to infect human hosts correlates less with binding to glycans of a particular linkage, and more with binding to glycans of a particular topology. Thus, the present invention demonstrates that HAs that mediate infection of humans bind to umbrella-topology glycans, often showing preference for umbrella-topology glycans over cone-topology glycans (even though cone-topology glycans may be α2-6 sialylated glycans).
Several crystal structures of HAs from H1 (human and swine), H3 (avian) and H5 (avian) subtypes bound to sialylated oligosaccharides (of both α2-3 and α2-6 linkages) are available and provide molecular insights into the specific amino acids that are involved in distinct interactions of the HAs with these glycans (Eisen et al., 1997, Virology, 232:19; Ha et al., 2001, Proc. Natl. Acad. Sci., USA, 98:11181; Ha et al., 2003, Virology, 309:209; Gamblin et al., 2004, Science, 303:1838; Stevens et al., 2004, Science, 303:1866; Russell et al., 2006, Glycoconj. J., 23:85; and Stevens et al., 2006, Science, 312:404; all of which are incorporated herein by reference).
For example, the crystal structures of H5 (A/duck/Singapore/3/97) alone or bound to an α2-3 or an α2-6 sialylated oligosaccharide identifies certain amino acids that interact directly with bound glycans, and also amino acids that are one or more degree of separation removed (Stevens et al., 2001, Proc. Natl. Acad. Sci., USA, 98:11181; incorporated herein by reference). In some cases, conformation of these residues is different in bound versus unbound states. For instance, Glu190, Lys193, and Gln226 all participate in direct-binding interactions and have different conformations in the bound versus the unbound state. The conformation of Asn186, which is proximal to Glu190, is also significantly different in the bound versus the unbound state.
Binding Characteristics of HA Polypeptides
In certain embodiments, HA polypeptides bind to umbrella-topology glycans with high affinity. In certain embodiments, HA polypeptides bind to a plurality of different umbrella-topology glycans, often with high affinity and/or specificity. In accordance with the present invention, HA polypeptides include peptides that are able to bind to receptors in human upper respiratory epithelial tissues.
In some embodiments, HA polypeptides bind to umbrella-topology glycans (e.g., long α2-6 sialylated glycans such as, for example, Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAc-) with high affinity. For example, in some embodiments, HA polypeptides bind to umbrella-topology glycans with an affinity comparable to that observed for a wild type HA that mediates infection of a humans (e.g., H1N1 HA or H3N2 HA). In some embodiments, HA polypeptides bind to umbrella-topology glycans with an affinity that is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of that observed under comparable conditions for a wild type HA that mediates infection of humans. In some embodiments, HA polypeptides bind to umbrella-topology glycans with an affinity that is greater than that observed under comparable conditions for a wild type HA that mediates infection of humans.
In certain embodiments, binding affinity of HA polypeptides is assessed over a range of concentrations. Such a strategy provides significantly more information, particularly in multivalent binding assays, than do single-concentration analyses. In some embodiments, for example, binding affinities of HA polypeptides are assessed over concentrations ranging over at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more fold.
In certain embodiments, HA polypeptides show high affinity if they show a saturating signal in a multivalent glycan array binding assay such as those described herein. In some embodiments, HA polypeptides show high affinity if they show a signal above about 400000 or more (e.g., above about 500000, 600000, 700000, 800000, etc.) in such studies. In some embodiments, HA polypeptides show saturating binding to umbrella-topology glycans over a concentration range of at least 2 fold, 3 fold, 4 fold, 5 fold or more, and in some embodiments over a concentration range as large as 10 fold or more.
Furthermore, in some embodiments, HA polypeptides bind to umbrella-topology glycans more strongly than they bind to cone-topology glycans. In some embodiments, HA polypeptides show a relative affinity for umbrella-topology glycans versus cone-topology glycans that is about 10, 9, 8, 7, 6, 5, 4, 3, or 2.
In some embodiments, HA polypeptides bind to α2-6 sialylated glycans; in some embodiments, HA polypeptides bind preferentially to α2-6 sialylated glycans. In certain embodiments, HA polypeptides bind to a plurality of different α2-6 sialylated glycans. In some embodiments, HA polypeptides are not able to bind to α2-3 sialylated glycans, and in other embodiments, HA polypeptides are able to bind to α2-3 sialylated glycans.
In some embodiments, HA polypeptides bind to receptors found on human upper respiratory epithelial cells. In certain embodiments, HA polypeptides bind to HA receptors in the bronchus and/or trachea. In some embodiments, HA polypeptides are not able to bind receptors in the deep lung, and in other embodiments, HA polypeptides are able to bind receptors in the deep lung.
In some embodiments, HA polypeptides bind to at least about 10%, about 15%, about 20%, about 25%, about 30% about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more of the glycans found on HA receptors in human upper respiratory tract tissues (e.g., epithelial cells).
In some embodiments, HA polypeptides bind to one or more of the glycans illustrated in
The present invention provides isolated HA polypeptides with designated binding specificity, and also provides engineered HA polypeptides with designated binding characteristics with respect to umbrella-topology glycans.
In some embodiments, HA polypeptides with designated binding characteristics are H1 polypeptides. In some embodiments, HA polypeptides with designated binding characteristics are H2 polypeptides. In some embodiments, HA polypeptides with designated binding characteristics are H3 polypeptides. In some embodiments, HA polypeptides with designated binding characteristics are H4 polypeptides. In some embodiments, HA polypeptides with designated binding characteristics are H5 polypeptides. In some embodiments, HA polypeptides with designated binding characteristics are H6 polypeptides. In some embodiments, HA polypeptides with designated binding characteristics are H7 polypeptides. In some embodiments, HA polypeptides with designated binding characteristics are H8 polypeptides. In some embodiments, HA polypeptides with designated binding characteristics are H9 polypeptides. In some embodiments, HA polypeptides with designated binding characteristics are H10 polypeptides. In some embodiments, HA polypeptides with designated binding characteristics are H11 polypeptides. In some embodiments, HA polypeptides with designated binding characteristics are H12 polypeptides. In some embodiments, HA polypeptides with designated binding characteristics are H13 polypeptides. In some embodiments, HA polypeptides with designated binding characteristics are H14 polypeptides. In some embodiments, HA polypeptides with designated binding characteristics are H15 polypeptides. In some embodiments, HA polypeptides with designated binding characteristics are H16 polypeptides.
In some embodiments, HA polypeptides with designated binding characteristics are not H1 polypeptides, are not H2 polypeptides, and/or are not H3 polypeptides.
In some embodiments, HA polypeptides do not include the H1 protein from any of the strains: A/South Carolina/1/1918; A/Puerto Rico/8/1934; A/Taiwan/i/1986; A/Texas/36/1991; A/Beijing/262/1995; A/Johannesburg/92/1996; A/New Calcdonia/20/1999; A/Solomon Islands/3/2006.
In some embodiments, HA polypeptides are not the H2 protein from any of the strains of the Asian flu epidemic of 1957-58). In some embodiments, HA polypeptides do not include the H2 protein from any of the strains: A/Japan/305+/1957; A/Singapore/i/1957; A/Taiwan/1/1964; A/Taiwan/1/1967.
In some embodiments, HA polypeptides do not include the H3 protein from any of the strains: A/Aichi/2/1968; A/Phillipines/2/1982; A/Mississippi/i/1985; A/Leningrad/360/1986; A/Sichuan/2/1987; A/Shanghai/11/1987; A/Beijing/353/1989; A/Shandong/9/1993; A/Johannesburg/33/1994; A/Nanchang/813/1995; A/Sydney/5/1997; A/Moscow/10/1999; A/Panama/2007/1999; A/Wyoming/3/2003; A/Oklahoma/323/2003; A/California/7/2004; A/Wisconsin/65/2005.
Variant HA Polypeptides
In certain embodiments, an HA polypeptide is a variant of a parent HA polypeptide in that its amino acid sequence is identical to that of the parent HA but for a small number of particular sequence alterations. In some embodiments, the parent HA is an HA polypeptide found in a natural isolate of an influenza virus (e.g., a wild type HA polypeptide).
In some embodiments, HA polypeptide variants have different glycan binding characteristics than their corresponding parent HA polypeptides. In some embodiments, HA variant polypeptides have greater affinity and/or specificity for umbrella-topology glycans (e.g., as compared with for cone-topology glycans) than do their cognate parent HA polypeptides. In certain embodiments, such HA polypeptide variants are engineered variants.
In some embodiments, HA polypeptide variants with altered glycan binding characteristics have sequence alternations in residues within or affecting the glycan binding site. In some embodiments, such substitutions are of amino acids that interact directly with bound glycan; in other embodiments, such substitutions are of amino acids that are one degree of separation removed from those that interact with bound glycan, in that the one degree of separation removed-amino acids either (1) interact with the direct-binding amino acids; (2) otherwise affect the ability of the direct-binding amino acids to interact with glycan, but do not interact directly with glycan themselves; or (3) otherwise affect the ability of the direct-binding amino acids to interact with glycan, and also interact directly with glycan themselves. HA polypeptide variants contain substitutions of one or more direct-binding amino acids, one or more first degree of separation-amino acids, one or more second degree of separation-amino acids, or any combination of these. In some embodiments, HA polypeptide variants may contain substitutions of one or more amino acids with even higher degrees of separation.
In some embodiments, HA polypeptide variants with altered glycan binding characteristics have sequence alterations in residues that make contact with sugars beyond Neu5Ac and Gal (see, for example,
In some embodiments, HA polypeptide variants have at least one amino acid substitution, as compared with a wild type parent HA. In certain embodiments, HA polypeptide variants have at least two, three, four, five or more amino acid substitutions as compared with a cognate wild type parent HA; in some embodiments HA polypeptide variants have two, three, or four amino acid substitutions. In some embodiments, all such amino acid substitutions are located within the glycan binding site.
In some embodiments, HA polypeptide variants have sequence substitutions at positions corresponding to one or more of residues 137, 145, 156, 159, 186, 187, 189, 190, 192, 193, 196, 222, 225, 226, and 228. In some embodiments, HA polypeptide variants have sequence substitutions at positions corresponding to one or more of residues 156, 159, 189, 192, 193, and 196; and/or at positions corresponding to one or more of residues 186, 187, 189, and 190; and/or at positions corresponding to one or more of residues 190, 222, 225, and 226; and/or at positions corresponding to one or more of residues 137, 145, 190, 226 and 228. In some embodiments, HA polypeptide variants have sequence substitutions at positions corresponding to one or more of residues 190, 225, 226, and 228.
In certain embodiments, HA polypeptide variants, and particularly H5 polypeptide variants, have one or more amino acid substitutions relative to a wild type parent HA (e.g., H5) at residues selected from the group consisting of residues 98, 136, 138, 153, 155, 159, 183, 186, 187, 190, 193, 194, 195, 222, 225, 226, 227, and 228. In some embodiments, HA polypeptide variants, and particularly H5 polypeptide variants, have one or more amino acid substitutions relative to a wild type parent HA at residues selected from amino acids located in the region of the receptor that directly binds to the glycan, including but not limited to residues 98, 136, 153, 155, 183, 190, 193, 194, 222, 225, 226, 227, and 228. In some embodiments, an HA polypeptide variant, and particularly an H5 polypeptide variant, has one or more amino acid substitutions relative to a wild type parent HA at residues selected from amino acids located adjacent to the region of the receptor that directly binds the glycan, including but not limited to residues 98, 138, 186, 187, 195, and 228.
In some embodiments, HA polypeptide variants, and particularly H5 polypeptide variants, have one or more amino acid substitutions relative to a wild type parent HA at residues selected from the group consisting of residues 138, 186, 187, 190, 193, 222, 225, 226, 227 and 228. In other embodiments, HA polypeptide variants, and particularly H5 polypeptide variants, have one or more amino acid substitutions relative to a wild type parent HA at residues selected from amino acids located in the region of the receptor that directly binds to the glycan, including but not limited to residues 190, 193, 222, 225, 226, 227, and 228. In further embodiments, HA polypeptide variants, and particularly H5 polypeptide variants, have one or more amino acid substitutions relative to a wild type parent HA at residues selected from amino acids located adjacent to the region of the receptor that directly binds the glycan, including but not limited to residues 138, 186, 187, and 228.
In further embodiments, HA polypeptide variants, and particularly H5 polypeptide variants, have one or more amino acid substitutions relative to a wild type parent HA at residues selected from the group consisting of residues 98, 136, 153, 155, 183, 194, and 195. In some embodiments, HA polypeptide variants, and particularly H5 polypeptide variants, have one or more amino acid substitutions relative to a wild type parent HA at residues selected from amino acids located in the region of the receptor that directly binds to the glycan, including but not limited to residues 98, 136, 153, 155, 183, and 194. In some embodiments, HA polypeptide variants, and particularly H5 polypeptide variants, have one or more amino acid substitutions relative to a wild type parent HA at residues selected from amino acids located adjacent to the region of the receptor that directly binds the glycan, including but not limited to residues 98 and 195.
In certain embodiments, HA polypeptide variants, and particularly H5 polypeptide variants, have one or more amino acid substitutions relative to a wild type parent HA at residues selected from amino acids that are one degree of separation removed from those that interact with bound glycan, in that the one degree of separation removed-amino acids either (1) interact with the direct-binding amino acids; (2) otherwise affect the ability of the direct-binding amino acids to interact with glycan, but do not interact directly with glycan themselves; or (3) otherwise affect the ability of the direct-binding amino acids to interact with glycan, and also interact directly with glycan themselves, including but not limited to residues 98, 138, 186, 187, 195, and 228.
In some embodiments, HA polypeptide variants, and particularly H5 polypeptide variants, have one or more amino acid substitutions relative to a wild type parent HA at residues selected from amino acids that are one degree of separation removed from those that interact with bound glycan, in that the one degree of separation removed-amino acids either (1) interact with the direct-binding amino acids; (2) otherwise affect the ability of the direct-binding amino acids to interact with glycan, but do not interact directly with glycan themselves; or (3) otherwise affect the ability of the direct-binding amino acids to interact with glycan, and also interact directly with glycan themselves, including but not limited to residues 138, 186, 187, and 228.
In some embodiments, HA polypeptide variants, and particularly H5 polypeptide variants, have one or more amino acid substitutions relative to a wild type parent HA at residues selected from amino acids that are one degree of separation removed from those that interact with bound glycan, in that the one degree of separation removed-amino acids either (1) interact with the direct-binding amino acids; (2) otherwise affect the ability of the direct-binding amino acids to interact with glycan, but do not interact directly with glycan themselves; or (3) otherwise affect the ability of the direct-binding amino acids to interact with glycan, and also interact directly with glycan themselves, including but not limited to residues 98 and 195.
In certain embodiments, HA polypeptide variants, and particularly H5 polypeptide variants, have an amino acid substitution relative to a wild type parent HA at residue 159.
In some embodiments, HA polypeptide variants, and particularly H5 polypeptide variant, have one or more amino acid substitutions relative to a wild type parent HA at residues selected from 190, 193, 225, and 226. In some embodiments, HA polypeptide variants, and particularly H5 polypeptide variants, have one or more amino acid substitutions relative to a wild type parent HA at residues selected from 190, 193, 226, and 228.
In some embodiments, HA polypeptide variants, and particularly H5 variants, have one or more of the following amino acid substitutions: Ser137Ala, Lys 156Glu, Asn186Pro, Asp187Ser, Asp187Thr, Ala189Gln, Ala189Lys, Ala189Thr, Glu190Asp, Glu190Thr, Lys193Arg, Lys193Asn, Lys193His, Lys193Ser, Gly225Asp, Gln226Ile, Gln226Leu, Gln226Val, Ser227Ala, Gly228Ser.
In some embodiments, HA polypeptide variants, and particularly H5 variants, have one or more of the following sets of amino acid substitutions:
Glu190Asp, Lys193Ser, Gly225Asp and Gln226Leu;
Glu190Asp, Lys193Ser, Gln226Leu and Gly228Ser;
Ala189Gln, Lys193Ser, Gln226Leu, Gly228Ser;
Ala189Gln, Lys193Ser, Gln226Leu, Gly228Ser;
Asp187Ser/Thr, Ala189Gln, Lys193Ser, Gln226Leu, Gly228Ser;
Ala189Lys, Lys193Asn, Gln226Leu, Gly228Ser;
Asp187Ser/Thr, Ala189Lys, Lys193Asn, Gln226Leu, Gly228Ser;
Lys156Glu, Ala189Lys, Lys193Asn, Gln226Leu, Gly228Ser;
Lys193His, Gln226Leu/Ile/Val, Gly228Ser;
Lys193Arg, Gln226Leu/Ile/Val, Gly228Ser;
Ala189Lys, Lys193Asn, Gly225Asp;
Lys156Glu, Ala189Lys, Lys193Asn, Gly225Asp;
Ser137Ala, Lys156Glu, Ala189Lys, Lys193Asn, Gly225Asp;
Glu190Thr, Lys193Ser, Gly225Asp;
Asp187Thr, Ala189Thr, Glu190Asp, Lys193Ser, Gly225Asp;
Asn186Pro, Asp187Thr, Ala189Thr, Glu190Asp, Lys193Ser, Gly225Asp;
Asn186Pro, Asp187Thr, Ala189Thr, Glu190Asp, Lys193Ser, Gly225Asp, Ser227Ala.
In some embodiments, an HA polypeptide has at least one further substitution as compared with a wild type HA, such that affinity and/or specificity of the variant for umbrella-topology glycans is increased.
In some embodiments, HA polypeptides (including HA polypeptide variants) have sequences that include D190, D225, L226, and/or S228. In some embodiments, HA polypeptides have sequences that include D190 and D225; in some embodiments, HA polypeptides have sequences that include L226 and S228.
In some embodiments, HA polypeptide variants have an open binding site as compared with a parent HA, and particularly with a parent wild type HAs.
Portions or Fragments of HA Polypeptides
The present invention further provides characteristic portions of HA polypeptides and nucleic acids that encode them. In general, a characteristic portion is one that contains a continuous stretch of amino acids, or a collection of continuous stretches of amino acids, that together are characteristic of the HA polypeptide. Each such continuous stretch generally will contain at least two amino acids. Furthermore, those of ordinary skill in the art will appreciate that typically at least 5, 10, 15, 20 or more amino acids are required to be characteristic of a H5 HA polypeptide. In general, a characteristic portion is one that, in addition to the sequence identity specified above, shares at least one functional characteristic with the relevant intact HA polypeptide. In some embodiments, inventive characteristic portions of HA polypeptides share glycan binding characteristics with the relevant full-length HA polypeptides.
While some of the particular examples and descriptions of HA polypeptides provided herein are specifically directed to H5 HA, one of ordinary skill in the art will readily recognize that the invention is not limited to H5 HA, but that the invention and its principles are generally applicable to any HA genotype, subtype, variant, portion, fragment, etc.
Identifying Glycan Decoys
In some embodiments, the present invention provides systems and methods for identifying glycan decoys. In general, such systems and methods involve contacting HA polypeptides with one or more candidate substances that mimic umbrella-topology glycans (referred to herein as “candidate decoys”) and determine the ability of the agent(s) to bind to the HA polypeptide and/or to compete an interaction between the HA polypeptide and an HA receptor (or umbrella-topology glycans found on an HA receptor).
In some embodiments, glycan decoys are glycan mimics that bind to HA polypeptides. In some embodiments, glycan mimics compete with endogenous glycans for binding to HA polypeptides. In some embodiments, glycan mimics compete with endogenous umbrella-type glycans for binding to HA polypeptides. In some embodiments, glycan mimics compete with upper-respiratory glycans for binding to HA polypeptides. In some embodiments, glycan mimics compete with umbrella-topology upper-respiratory glycans for binding to HA polypeptides.
In some embodiments, peptide mimics bind to HA polypeptides, but do not compete with endogenous glycans for binding to HA polypeptides.
In some embodiments, a candidate decoy may be free in solution, fixed to a support, and/or expressed in and/or on the surface of a cell. The candidate decoy and/or the HA polypeptide may be labeled, thereby permitting detection of binding. The candidate decoy is frequently the labeled species, decreasing the chance that the labeling will interfere with and/or enhance binding. Competitive binding formats may be performed in which one of the substances is labeled, and one may measure the amount of free label versus bound label to determine the effect on binding.
In some embodiments, binding assays involve, for example, exposing a candidate decoy to an HA polypeptide and detecting binding between the candidate decoy and the HA polypeptide. A binding assay may be conducted in vitro (e.g., in a candidate tube, comprising substantially only the components mentioned; in cell-free extracts; and/or in substantially purified components). Alternatively or additionally, binding assays may be conducted in cyto and/or in vivo (e.g., within a cell, tissue, organ, and/or organism; described in further detail below).
In certain embodiments, at least one HA polypeptide is contacted with at least one candidate decoy and an effect detected. In some embodiments, for example, a HA polypeptide is contacted with a candidate decoy, and binding between the two entities is monitored. In some embodiments, an assay may involve contacting a candidate decoy with a characteristic portion of a HA polypeptide (including, but not limited to, the region of the HA polypeptide that contacts umbrella-topology glycans). Binding of the HA polypeptide to the candidate decoy is detected. It will be appreciated that fragments, portions, homologs, variants, and/or derivatives of HA polypeptides may be employed, provided that they comprise umbrella-topology glycan binding activity.
In some embodiments, assays may involve providing a candidate decoy (e.g. immobilized on a solid support, such as a glycan array, described in further detail below in the section entitled “Glycan Arrays”), and a non-immobilized HA polypeptide. The extent to which the candidate decoy and HA polypeptide bind to one another is determined. Alternatively, the HA polypeptide may be immobilized and the candidate decoy non-immobilized. Such assays may be used to identify candidate decoys capable of binding to HA polypeptides and/or fragments, portions, homologs, variants, and/or derivatives thereof.
In some embodiments, an antibody that recognizes an HA polypeptide (e.g. an α-HA antibody) is immobilized to a solid support (e.g. Protein-A beads). The antibody is contacted with the HA polypeptide, which binds to the immobilized antibody. The resulting complex is then brought into contact with the candidate decoy (purified proteins, cellular extract, combinatorial library, etc.). If the HA polypeptide interacts with the candidate decoy, the candidate decoy will become indirectly immobilized to the solid support. Presence of the candidate decoy on the solid support can be assayed by any standard technique known in the art (including, but not limited to, western blotting, mass spectrometry, etc.). This type of assay is known in the art as an “immunoprecipitation” assay.
In some embodiments, an HA polypeptide is immobilized on a solid support (e.g. agarose beads). In specific embodiments, the HA polypeptide is expressed as a GST-fusion protein in bacteria, yeast, insect cells, and/or higher eukaryotic cell line and/or purified from crude cell extracts using glutathione-agarose beads. As a control, binding of a candidate decoy, which is not a GST-fusion protein, to the glutathione-agarose beads is determined in the absence of HA polypeptide. The binding of the candidate decoy to the immobilized HA polypeptide is then determined. This type of assay is known in the art as a “GST pulldown” assay. Alternatively or additionally, the candidate substance may be immobilized and the HA polypeptide non-immobilized.
It is possible to perform this type of assay using different affinity purification systems for immobilizing one of the components, for example Ni-NTA agarose- and/or histidine-tagged components.
Binding of a HA polypeptide to the candidate decoy may be determined by a variety of methods well-known in the art. For example, the non-immobilized component may be labeled (with for example, a radioactive label, an epitope tag, an enzyme-antibody conjugate, etc.). Alternatively or additionally, binding may be determined by immunological detection techniques. For example, the reaction mixture may be subjected to Western blotting and the blot probed with an antibody that detects the non-immobilized component. Alternatively or additionally, enzyme linked immunosorbent assay (ELISA) may be utilized to assay for binding.
In some embodiments, screening methods of the present invention comprise: (1) obtaining a candidate substance; (2) contacting the candidate decoy with an HA polypeptide (and/or characteristic portion thereof) and an umbrella-topology glycan known to bind to the HA polypeptide; and (3) detecting inhibition of binding between the HA polypeptide and the known umbrella-topology glycan in the presence and/or absence of the candidate decoy.
In some embodiments, screening methods of the present invention comprise: (1) obtaining a candidate decoy; and (2) contacting the candidate decoy with a pre-formed HA polypeptide (and/or characteristic portion thereof)-umbrella-topology glycan complex; and (3) determining whether the candidate substance affects the HA polypeptide (and/or characteristic portion thereof)-umbrella-topology glycan complex.
In some embodiments, screening methods of the present invention comprise (1) obtaining a candidate decoy; (2) contacting the candidate decoy with HA polypeptide (and/or characteristic portion thereof) and an umbrella-topology glycan known to specifically bind to the HA polypeptide; and (3) detecting whether the candidate decoy can compete with the binding interaction between HA polypeptide (and/or characteristic portion thereof) and the known umbrella-topology glycan.
In some embodiments, a candidate decoy is determined to be an umbrella-topology glycan decoy if administering the candidate decoy to an HA polypeptide and an umbrella-topology glycan known to specifically bind to the HA polypeptide results in decreased binding between the HA polypeptide and the known umbrella-topology glycan. In some embodiments, a candidate decoy is determined to be an umbrella-topology glycan if administering the candidate decoy to an HA polypeptide and an umbrella-topology glycan known to specifically bind to the HA polypeptide results in an at least 2-fold decrease in binding between the HA polypeptide and the known umbrella-topology glycan. In some embodiments, a candidate decoy is determined to be an umbrella-topology glycan if administering the candidate decoy to an HA polypeptide and an umbrella-topology glycan known to specifically bind to the HA polypeptide results in an at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 500-fold, at least 1000-fold, at least 10,000-fold, or greater than 10,000-fold decrease in binding between the HA polypeptide and the known umbrella-topology glycan. In some embodiments, a candidate decoy is determined to be an HA polypeptide if administering the candidate decoy to the HA polypeptide and an umbrella-topology glycan known to specifically bind to the HA polypeptide results in an at least 25%, 50%, 75%, 100%, 200%, 500%, 1000%, or greater than 1000% decrease in binding between the HA polypeptide and the known umbrella-topology glycan.
In some embodiments, the present invention provides methods for screening for umbrella-topology glycan decoys wherein a candidate decoy is contacted with a cell (e.g. a cell that expresses or is bound to an HA polypeptide). The cell can then be assayed for the ability of an HA polypeptide to interact with one or more candidate umbrella-topology glycan decoys.
In certain embodiments, cells may be directly assayed for binding between HA polypeptides and candidate decoys. Immunohistochemical techniques, confocal techniques, and/or other techniques to assess binding are well known to those of skill in the art. Various cell lines may be utilized for such screening assays, including cells specifically engineered for this purpose. Examples of cells used in the screening assays include mammalian cells, fungal cells, bacterial cells, or viral cells. A cell may be a stimulated cell, such as a cell stimulated with a growth factor. One of skill in the art would understand that the invention disclosed herein contemplates a wide variety of in cyto assays for measuring the ability of HA polypeptides to bind to candidate decoys.
Depending on the assay, cell and/or tissue culture may be required. A cell may be examined using any of a number of different physiologic assays. Alternatively or additionally, molecular analysis may be performed, including, but not limited to, western blotting to monitor protein expression and/or test for protein-protein interactions; mass spectrometry to monitor other chemical modifications; etc.
The present invention provides methods for identifying substances that bind to HA polypeptides and, therefore, may be involved in influenza infection. One in cyto method of identifying substances that bind to HA polypeptides is the two-hybrid system assay (Fields et al., 1994, Trends in Genetics, 10:286; and Colas et al., 1998, TIBTECH, 16:355; both of which are incorporated herein by reference). In this assay, yeast cells express a first fusion protein comprising a test substance in accordance with the present invention (e.g. an HA polypeptide, gene encoding an HA polypeptide, and/or characteristic portions thereof) and a DNA-binding domain of a transcription factor such as Gal4 and/or LexA. The cells additionally contain a reporter gene whose promoter contains binding sites for the corresponding DNA-binding domain. By transforming the cells with a vector that expresses a second fusion protein comprising a candidate decoy fused to an activation domain (e.g. from Gal4 and/or herpes simplex virus VP16) expression of the reporter gene may be increased if the candidate decoy interacts with the HA polypeptide. In this way, it is possible rapidly to identify novel glycan decoys.
The present invention provides assays involving solid phase-bound HA polypeptides and detecting their interactions with one or more candidate decoys. Thus, an HA polypeptide may comprise a detectable marker, such as a radioactive, fluorescent, and/or luminescent label. Furthermore, candidate decoys can be coupled to substances which permit indirect detection (e.g. by means of employing an enzyme which uses a chromogenic substrate and/or by means of binding a detectable antibody). Changes in the conformation of HA polypeptides as the result of an interaction with a candidate decoys may be detected, for example, by the change in the emission of the detectable marker. Alternatively or additionally, solid phase-bound protein complexes may be analyzed by means of mass spectrometry.
In some embodiments, any of the binding assays described herein may be performed using a range of concentrations of HA polypeptide and/or candidate decoy. In some embodiments, the binding assays described herein are used to assess the ability of a candidate decoy to compete with a known umbrella-topology glycan over range of candidate decoy and known umbrella-topology glycan concentrations (e.g. candidate decoy: known umbrella-topology glycan ratios of less than about 1:1000, about 1:1000, about 1:100, about 1:50, about 1:20, about 1:10, about 1:5, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 5:1, about 10:1, about 20:1, about 50:1, about 100:1, about 1000:1, and/or greater than about 1000:1). In some embodiments, the binding assays described herein are used to assess the ability of a candidate decoy to compete with a known umbrella-topology glycan over range of HA polypeptide concentrations (e.g. greater than about 100 μg/ml, about 100 μg/ml, about 50 μg/ml, about 40 μg/ml, about 30 μg/ml, about 20 μg/ml, about 10 μg/ml, about 50 μg/ml, about 4 μg/ml, about 3 μg/ml, about 2 μg/ml, about 1.75 μg/ml, about 1.5 μg/ml, about 1.25 μg/ml, about 1.0 μg/ml, about 0.9 μg/ml, about 0.8 μg/ml, about 0.7 μg/ml, about 0.6 μg/ml, about 0.5 μg/ml, about 0.4 μg/ml, about 0.3 μg/ml, about 0.2 μg/ml, about 0.1 μg/ml, about 0.05 μg/ml, about 0.01 μg/ml, and/or less than about 0.01 μg/ml).
In some embodiments, any of the binding studies described herein can be executed in a high throughput fashion. Using high throughput assays, it is possible to screen up to several thousand candidate decoys in a single day. In some embodiments, each well of a microtiter plate can be used to run a separate assay against a selected candidate decoy, or, if concentration and/or incubation time effects are to be observed, every 5-10 wells can test a single candidate decoy. Thus, a single standard microtiter plate can assay up to 96 candidate decoy; if 1536 well plates are used, then a single plate can assay up to 1536 candidate decoys; and so forth. It is possible to assay many plates per day; assay screens for up to about 6,000, about 20,000, about 50,000, or more than about 100,000 different candidate decoys are possible using high throughput systems in accordance with the present invention.
Glycan Arrays
To rapidly expand the current knowledge of known specific glycan-glycan binding protein (GBP) interactions, the Consortium for Functional Glycomics (CFG; www.functionalglycomics.org), an international collaborative research initiative, has developed glycan arrays comprising several glycan structures that have enabled high throughput screening of GBPs for novel glycan ligand specificities. Glycan arrays comprise both monovalent and polyvalent glycan motifs (i.e., attached to polyacrylamide backbone), and each array comprises 264 glycans with low (10 μM) and high (100 μM) concentrations, and six spots for each concentration (see http://www.functionalglycomics.org/static/consortium/resources/resourcecoreh5.shtml).
Glycan arrays predominantly comprise synthetic glycans that capture the physiological diversity of N- and O-linked glycans. In addition to synthetic glycans, N-linked glycan mixtures derived from different mammalian glycoproteins may be also represented on the array.
As used herein, a glycan “array” refers to a set of one or more glycans, optionally immobilized on a solid support. In some embodiments, an “array” is a collection of glycans present as an organized arrangement or pattern at two or more locations that are physically separated in space. Typically, a glycan array will have at least 4, 8, 16, 24, 48, 96 or several hundred or thousand discrete locations. In general, glycan arrays may have any of a variety of formats. Various different array formats applicable to biomolecules are known in the art. For example, a huge number of protein and/or nucleic acid arrays are well known. Those of ordinary skill in the art will immediately appreciate standard array formats appropriate for glycan arrays of the present invention.
In some embodiments, glycan arrays are present in “microarray” formats. A microarray may typically have sample locations separated by a distance of 50 μm-200 μm or less and immobilized sample in the nano- to micro-molar range or nano- to pico-gram range. Array formats known in the art include, for example, those in which each discrete sample location has a scale of, for example, 10 μm.
In some embodiments, glycan arrays comprise a plurality of glycans spatially immobilized on a support. As used herein, “support” refers to any material that is suitable to be used to array glycan molecules. As will be appreciated by those of ordinary skill in the art, any of a wide variety of materials may be employed. To give but a few examples, support materials which may be of use in the invention include hydrophobic membranes, for example, nitrocellulose, PVDF or nylon membranes. Such membranes are well known in the art and can be obtained from, for example, Bio-Rad, Hemel Hempstead, UK.
In some embodiments, the support on which glycans are arrayed may comprise a metal oxide. Suitable metal oxides include, but are not limited to, titanium oxide, tantalum oxide, and aluminum oxide. Examples of such materials may be obtained from Sigma-Aldrich Company Ltd, Fancy Road, Poole, Dorset. BH12 4QH UK.
In some embodiments, such a support is or comprises a metal oxide gel. A metal oxide gel is considered to provide a large surface area within a given macroscopic area to aid immobilization of the carbohydrate-containing molecules.
Additional or alternative support materials which may be used in accordance with the present invention include gels, for example silica gels or aluminum oxide gels. Examples of such materials may be obtained from, for example, Merck KGaA, Darmstadt, Germany.
In some embodiments, glycan arrays are immobilized on a support that can resist change in size or shape during normal use. For example a support may be a glass slide coated with a component material suitable to be used to array glycans. Also, some composite materials can desirable provide solidity to a support.
As demonstrated herein, glycan arrays are useful for the characterization of HA-receptor interactions and/or agents (e.g. umbrella-topology glycan decoys) that affect HA-receptor interactions. In certain embodiments, HA polypeptides are tested on such arrays to assess their ability to bind to umbrella-topology glycans (e.g., to α2-6 sialylated glycans, and particularly to long α2-6 sialylated glycans arranged in an umbrella-topology), and/or to interact with umbrella-topology glycan decoys.
Indeed, the present invention provides arrays of α2-6 sialylated glycans, and optionally α2-3 sialylated glycans, that can be used to characterize HA polypeptide binding interactions and/or to identify and/or characterize umbrella-topology glycan decoys. In some embodiments, arrays contain glycans (e.g., α2-6 sialylated glycans, and particularly long α2-6 sialylated glycans) in an umbrella-topology. As will be clear to those of ordinary skill in the art, such arrays are useful for characterizing or detecting interaction with any HA polypeptides, including for example, those found in natural influenza isolates in addition to those designed and/or prepared by researchers.
In some embodiments, such arrays include glycans representative of about 10%, about 15%, about 20%, about 25%, about 30% about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more of the glycans (e.g., umbrella-topology glycans, which will often be α2-6 sialylated glycans, particularly long α2-6 sialylated glycans) found on human HA receptors, and particularly on human upper respiratory tract HA receptors. In some embodiments, arrays include some or all of the glycan structures depicted in
The present invention provides methods for identifying or characterizing umbrella-topology glycan decoys using glycan arrays. In some embodiments, for example, such methods comprise steps of (1) providing a sample containing at least one HA polypeptide, (2) contacting the sample with a glycan array comprising candidate umbrella-topology glycan decoys, and (3) detecting binding of HA polypeptide(s) to one or more candidate decoys on the array.
In some embodiments, for example, the present invention provides for identifying or characterizing umbrella-topology glycan decoys using glycan arrays comprising steps of (1) providing a plurality of samples, each containing a different dilution of at least one HA polypeptide, (2) contacting the plurality of samples with a glycan array comprising candidate umbrella-topology glycan decoys, and (3) detecting binding of different dilutions of HA polypeptides to one or more candidate decoys on the array.
In some embodiments, for example, the present invention provides for identifying or characterizing umbrella-topology glycan decoys using glycan arrays comprising steps of (1) providing a sample containing at least one HA polypeptide, (2) contacting the sample with a glycan array comprising a plurality of dilutions of at least one candidate umbrella-topology glycan decoy, and (3) detecting binding of HA polypeptide(s) to one or more of the plurality of dilutions of candidate decoys on the array.
In some embodiments, for example, the present invention provides for identifying or characterizing umbrella-topology glycan decoys using glycan arrays comprising steps of (1) providing a plurality of samples, each containing a different dilution of at least one HA polypeptide, (2) contacting the plurality of samples with a glycan array comprising a plurality of dilutions of at least one candidate umbrella-topology glycan decoy, and (3) detecting binding of different dilutions of HA polypeptides to one or more of the plurality of dilutions of candidate decoys on the array.
In some embodiments, methods such as those described above are used to assess the ability of a candidate decoy to compete with a known umbrella-topology glycan over range of candidate decoy and known umbrella-topology glycan concentrations (e.g. candidate decoy: known umbrella-topology glycan ratios of less than about 1:1000, about 1:1000, about 1:100, about 1:50, about 1:20, about 1:10, about 1:5, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 5:1, about 10:1, about 20:1, about 50:1, about 100:1, about 1000:1, and/or greater than about 1000:1). In some embodiments, methods such as those described above are used to assess the ability of a candidate decoy to compete with a known umbrella-topology glycan over range of HA polypeptide concentrations (e.g. greater than about 100 μg/ml, about 100 μg/ml, about 50 μg/ml, about 40 μg/ml, about 30 μg/ml, about 20 μg/ml, about 10 μg/ml, about 50 μg/ml, about 4 μg/ml, about 3 μg/ml, about 2 μg/ml, about 1.75 μg/ml, about 1.5 μg/ml, about 1.25 μg/ml, about 1.0 μg/ml, about 0.9 μg/ml, about 0.8 μg/ml, about 0.7 μg/ml, about 0.6 μg/ml, about 0.5 μg/ml, about 0.4 μg/ml, about 0.3 μg/ml, about 0.2 μg/ml, about 0.1 μg/ml, about 0.05 μg/ml, about 0.01 μg/ml, and/or less than about 0.01 μg/ml).
Suitable sources for samples containing HA polypeptides to be contacted with glycan arrays according to the present invention include, but are not limited to, pathological samples, such as blood, serum/plasma, peripheral blood mononuclear cells/peripheral blood lymphocytes (PBMC/PBL), sputum, urine, feces, throat swabs, dermal lesion swabs, cerebrospinal fluids, cervical smears, pus samples, food matrices, and tissues from various parts of the body such as brain, spleen, and liver. Alternatively or additionally, other suitable sources for samples containing HA polypeptides include, but are not limited to, environmental samples such as soil, water, and flora. Yet other samples include laboratory samples, for example of engineered HA polypeptides designed and/or prepared by researchers. Other samples that have not been listed may also be applicable.
A wide variety of detection systems suitable for assaying HA polypeptide binding to glycan arrays are known in the art. For example, HA polypeptides can be detectably labeled (directly or indirectly) prior to or after being contacted with the array; binding can then be detected by detection of localized label. In some embodiments, scanning devices can be utilized to examine particular locations on an array.
Alternatively or additionally, binding to arrayed glycans can be measured using, for example, calorimetric, fluorescence, or radioactive detection systems, or other labeling methods, or other methods that do not require labeling. In general, fluorescent detection typically involves directly probing the array with a fluorescent molecule and monitoring fluorescent signals. Alternatively or additionally, arrays can be probed with a molecule that is tagged (for example, with biotin) for indirect fluorescence detection (in this case, by testing for binding of fluorescently-labeled streptavidin). Alternatively or additionally, fluorescence quenching methods can be utilized in which the arrayed glycans are fluorescently labeled and probed with a test molecule (which may or may not be labeled with a different fluorophore). In such embodiments, binding to the array acts to squelch the fluorescence emitted from the arrayed glycan, therefore binding is detected by loss of fluorescent emission. Alternatively or additionally, arrayed glycans can be probed with a live tissue sample that has been grown in the presence of a radioactive substance, yielding a radioactively labeled probe. Binding in such embodiments can be detected by measuring radioactive emission.
Such methods are useful to determine the fact of binding and/or the extent of binding by HA polypeptides to glycan arrays. In some embodiments of the invention, such methods can further be used to identify and/or characterize agents that interfere with or otherwise alter glycan-HA polypeptide interactions.
Methods described below may be of particular use in, for example, identifying whether a glycan (e.g. putative umbrella-topology glycan decoy) thought to be capable of interacting with an HA polypeptide can actually do so, or to identify whether a decoy unexpectedly has the capability of interacting with an HA polypeptide.
In some embodiments, binding to glycan arrays may be utilized, for example, to determine kinetics of interaction between an HA polypeptide and a glycan. For example, methods for determining interaction kinetics may include steps of (1) contacting a glycan array with the HA polypeptide being tested; and, (2) measuring kinetics of interaction between the HA polypeptide and arrayed glycan(s).
The kinetics of interaction of a binding agent with any of the glycans in an array can be measured by real time changes in, for example, colorimetric or fluorescent signals, as detailed above. Such methods may be of particular use in, for example, determining whether a particular binding agent is able to interact with a specific carbohydrate with a higher degree of binding than does a different binding agent interacting with the same glycan and/or set of glycans.
It will be appreciated, of course, that glycan binding by HA polypeptides can be evaluated on glycan samples or sources not present in an array format per se. For example, HA polypeptides can be bound to tissue samples and/or cell lines to assess their glycan binding characteristics. Appropriate cell lines include, for example, any of a variety of mammalian cell lines, particularly those expressing HA receptors containing umbrella-topology glycans (e.g., at least some of which may be α2-6 sialylated glycans, and particularly long α2-6 sialylated glycans). In some embodiments, utilized cell lines express individual glycans with umbrella-topology. In some embodiments, utilized cell lines express a diversity of glycans. In some embodiments, cell lines are obtained from clinical isolates; in some they are maintained or manipulated to have a desired glycan distribution and/or prevalence. In some embodiments, tissue samples and/or cell lines express glycans characteristic of mammalian upper respiratory epithelial cells.
Pharmaceutical Compositions
The present invention provides umbrella-topology glycan decoys used in the treatment of influenza infection. In some embodiments, the present invention provides pharmaceutical compositions comprising glycan decoys and at least one pharmaceutically acceptable excipient. The present invention provides pharmaceutical compositions comprising a therapeutically effective amount of inventive glycan decoy(s) appropriately formulated for administration to a subject suffering from and/or susceptible to influenza infection. Such pharmaceutical compositions may optionally comprise one or more additional therapeutically-active substances.
In accordance with some embodiments, methods of treating influenza infection are provided. In some embodiments, inventive methods comprise administering a pharmaceutical composition comprising at least one glycan decoy of the present invention to a subject in need thereof. Inventive glycan decoys may be administered with other medications used to treat the symptoms of influenza infection. In some embodiments, the compositions are administered to humans.
In some embodiments, inventive pharmaceutical compositions comprise a plurality of individual umbrella-topology glycan mimics. In some embodiments, such individual umbrella-topology glycan mimics are provided in ratios corresponding to those at which such umbrella-topology glycans are found on HA receptors in nature, and in particular in the human respiratory tract (e.g., the human upper respiratory tract). For example, human bronchial epithelial cells typically show glycan distributions as depicted in
In some embodiments, inventive pharmaceutical compositions are substantially free of agents that mimic glycans other than umbrella-topology glycans. For example, in some embodiments, inventive pharmaceutical compositions are substantially free of agents that mimic cone-topology glycans.
The invention encompasses the preparation and/or use of pharmaceutical compositions comprising at least one glycan decoy for treatment of influenza infection as an active ingredient. Such a pharmaceutical composition may consist of the active ingredient alone, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise the active ingredient and one or more pharmaceutically acceptable excipients, one or more additional ingredients, and/or a combination of these. The active ingredient may be present in the pharmaceutical composition in the form of a physiologically acceptable ester and/or salt, such as in combination with a physiologically acceptable cation and/or anion, as is well known in the art.
Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions that 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. Modification of pharmaceutical compositions suitable for administration to humans in order to render the 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. Patients to which administration of the pharmaceutical compositions of the invention 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, and/or dogs; and/or birds, including commercially relevant birds such as chickens, ducks, geese, and/or turkeys.
Pharmaceutical compositions as described herein may be prepared by any method known or hereafter developed in the art of pharmaceutics. In general, such preparatory methods include the step of bringing the active ingredient into association with one or more excipients 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 of the invention may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical 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.
The relative amounts of the active ingredient, the pharmaceutically acceptable excipient(s), and/or any additional ingredients in a pharmaceutical composition of the invention 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. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient.
Pharmaceutical formulations of the present invention may additionally comprise 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. Remington's The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro, (Lippincott, Williams & Wilkins, Baltimore, Md., 2006) discloses various excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional excipient is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention.
In some embodiments, the pharmaceutically acceptable excipient is at least 95%, 96%, 97%, 98%, 99%, or 100% pure. In some embodiments, the excipient is approved for use in humans and for veterinary use. In some embodiments, the excipient is approved by United States Food and Drug Administration. In some embodiments, the excipient is pharmaceutical grade. In some embodiments, the excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.
Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical 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 the inventive formulations. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and perfuming agents can be present in the composition, according to the judgment of the formulator.
Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc., and combinations thereof.
Exemplary granulating and/or dispersing agents include, but are not limited to, potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (VEEGUM), sodium lauryl sulfate, quaternary ammonium compounds, etc., and combinations thereof.
Exemplary surface active agents and/or emulsifiers include, but are not limited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and VEEGUM [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylene sorbitan monolaurate [TWLEN 20], polyoxyethylene sorbitan [TWEEN®60], polyoxyethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate [SPAN®40], sorbitan monostearate [SPAN®60], sorbitan tristearate [SPAN®65], glyceryl monooleate, sorbitan monooleate [SPAN®80]), polyoxyethylene esters (e.g. polyoxyethylene monostearate [MYRJ®45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and SOLUTOL), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. CREMOPHOR), polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether [BRIJ®30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLURONIC®F 68, POLOXAMER 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and/or combinations thereof.
Exemplary binding agents include, but are not limited to, starch (e.g. cornstarch and starch paste); gelatin; sugars (e.g. sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol,); natural and synthetic gums (e.g. acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (VEEGUM), and larch arabogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes; water; alcohol; etc.; and combinations thereof.
Exemplary preservatives may include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives. Exemplary antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and trisodium edetate. Exemplary antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal. Exemplary antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid. Exemplary alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol. Exemplary acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS“, PHENONIP”, methylparaben, GERMALL® 115, GERMABEN®II, NEOLONE™, KATHON, and EUXYL. In certain embodiments, the preservative is an anti-oxidant. In other embodiments, the preservative is a chelating agent.
Exemplary buffering agents include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, etc., and combinations thereof.
Exemplary lubricating agents include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc., and combinations thereof.
Exemplary oils include, but are not limited to, almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, chamomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils. Exemplary oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and combinations thereof.
Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient(s), 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. Besides inert diluents, oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. In certain embodiments for parenteral administration, an active ingredient can be mixed with solubilizing agents such as CREMOPHOR, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and combinations thereof.
Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. A sterile injectable preparation may be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.
Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, 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.
In order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
Compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing the active ingredients of this invention 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.
Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient such as sodium citrate or dicalcium phosphate and/or (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, (c) humectants such as glycerol, (d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, (e) solution retarding agents such as paraffin, (f) absorption accelerators such as quaternary ammonium compounds, (g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, (h) absorbents such as kaolin and bentonite clay, and/or (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may comprise buffering agents.
Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polethylene glycols and the like.
Active ingredients can be in micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active ingredient may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, dosage forms may comprise buffering agents. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes.
Dosage forms for topical and/or transdermal administration of a composition of this invention may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and/or patches. Generally, 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. Additionally, the present invention contemplates the use of transdermal patches, which often have the added advantage of providing controlled delivery of an active ingredient to the body. Such dosage forms may be prepared, for example, by dissolving and/or dispensing the active ingredient in the proper medium. Alternatively or additionally, the rate may be controlled by either providing a rate controlling membrane and/or by dispersing the active ingredient in a polymer matrix and/or gel.
Suitable devices for use in delivering intradermal pharmaceutical compositions described herein include short needle devices such as those described in U.S. Pat. Nos. 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 vaccines 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. Pat. Nos. 5,480,381; 5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189; 5,704,911; 5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335; 5,503,627; 5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880; 4,940,460; and PCT publications WO 97/37705 and WO 97/13537. 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. Alternatively or additionally, 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 1.0% to about 10% (w/w) active ingredient, although the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the excipients and/or additional ingredients described herein.
A pharmaceutical composition of the invention may be prepared, packaged, and/or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 μm to about 7 μm or from about 1 μm to about 6 μm. 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 μm and at least 95% of the particles by number have a diameter less than 7 μm. Alternatively, at least 95% of the particles by weight have a diameter greater than 1 μm and at least 90% of the particles by number have a diameter less than 6 μm. Dry powder compositions may include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.
Low boiling propellants generally include liquid propellants having a boiling point of below 65° F. at atmospheric pressure. Generally the propellant may constitute 50% to 99.9% (w/w) of the composition, and the active ingredient may constitute 0.1% to 20% (w/w) of the composition. The propellant may further comprise additional ingredients such as a liquid non-ionic and/or solid anionic surfactant and/or a solid diluent (which may have a particle size of the same order as particles comprising the active ingredient).
Pharmaceutical compositions of the invention formulated for pulmonary delivery may provide the 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 the 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 m to about 200 μm.
Formulations described herein as being useful for pulmonary delivery are useful for intranasal delivery of a pharmaceutical composition of the invention. Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 μm to 500 μm. 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 nares.
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 the active ingredient, and may comprise one or more of the excipients and/or additional ingredients described herein. A pharmaceutical composition of the invention 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 contain, 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 excipients and/or additional ingredients described herein. Alternately, formulations suitable for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising the 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 m to about 200 μm, and may further comprise one or more of the excipients and/or additional ingredients described herein.
A pharmaceutical composition of the invention 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 the excipients and/or 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 invention.
General considerations in the formulation and/or manufacture of pharmaceutical agents may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference).
Methods for Treating Influenza Infections
The present invention provides methods of treating influenza infection. In certain embodiments, such methods involve administering one or more inventive umbrella-topology glycan decoys to a subject in need thereof. In some embodiments, glycan decoys inhibit the ability of HA (e.g. HA expressed on the surface of influenza virus) to bind to umbrella-topology glycans (e.g. glycans associated with human upper respiratory epithelial tissues, such as trachea and bronchus).
In some embodiments, the invention provides pharmaceutical compositions comprising at least one glycan decoy and at least one pharmaceutically acceptable excipient. In other aspects, a pharmaceutical composition comprising a glycan decoy is administered to a cell, such as one in a patient suffering from and/or susceptible to influenza infection. In some embodiments, glycan decoys of the present invention are used in the treatment of influenza infection. In specific embodiments, inventive glycan decoys are used to inhibit the ability of influenza virus particles (e.g. comprising HA polypeptides) to bind to HA receptors (e.g. comprising umbrella-topology glycans).
In some embodiments, glycan decoys of the present invention are used in the treatment of one or more of the following symptoms: fever, sore throat, muscle pains, severe headache, coughing, weakness, general discomfort, pneumonia, nausea, and/or vomiting. In certain embodiments, these symptoms are caused by influenza infection.
In some embodiments, inventive pharmaceutical compositions are administered to a subject suffering from or susceptible to an influenza infection. In some embodiments, a subject is considered to be suffering from an influenza infection in the subject is displaying one or more symptoms commonly associated with influenza infection. In some embodiments, the subject is known or believed to have been exposed to the influenza virus. In some embodiments, a subject is considered to be susceptible to an influenza infection if the subject is known or believed to have been exposed to the influenza virus. In some embodiments, a subject is known or believed to have been exposed to the influenza virus if the subject has been in contact with other individuals known or suspected to have been infected with the influenza virus and/or if the subject is or has been present in a location in which influenza infection is known or thought to be prevalent.
In some embodiments, the present invention provides a method of treating influenza infection comprising steps of (1) providing a patient exhibiting symptoms of influenza infection, and (2) administering a therapeutic amount of one or more glycan decoys to the patient. In some embodiments, the present invention provides a method of treating influenza infection comprising steps of (1) providing a patient suffering from influenza infection, and (2) administering a therapeutic amount of one or more glycan decoys to the patient. In some embodiments, the present invention provides a method of treating influenza infection comprising steps of (1) providing a patient susceptible to influenza infection, and (2) administering a therapeutic amount of one or more glycan decoys to the patient.
In some embodiments, the present invention provides methods of treating influenza infection comprising steps of (1) providing a patient exhibiting symptoms of, suffering from, and/or susceptible to influenza infection, and (2) administering a substance that competes away the binding of HA polypeptides (e.g. HA polypeptides associated with influenza virus particles) with umbrella-topology glycans in human upper respiratory tissues.
In some embodiments, the present invention provides a method of preventing and/or delaying the onset of influenza infection comprising steps of (1) providing a patient susceptible to influenza infection, and (2) administering a therapeutic amount of one or more glycan decoys to the patient.
In some embodiments, the present invention provides methods of preventing and/or delaying the onset of influenza infection comprising steps of (1) providing a patient susceptible to influenza infection, and (2) administering a substance that competes away the binding of HA polypeptides (e.g. HA polypeptides associated with influenza virus particles) with umbrella-topology glycans in human upper respiratory tissues.
Administration
Inventive umbrella-topology glycan decoys are useful in the treatment of influenza infection. Thus, pharmaceutical compositions containing one or more inventive glycan decoys may be administered to one or more individuals suffering from, susceptible to, and/or exhibiting symptoms of influenza infection. The present invention therefore encompasses methods of inhibiting binding between HA polypeptides (e.g. HA polypeptides associated with influenza virus particles) and umbrella-topology glycans (e.g. glycans of human upper respiratory epithelial tissues), as well as methods of treating influenza infection in subjects.
In some embodiments, a therapeutically effective amount of a pharmaceutical composition comprising a glycan decoy is delivered to a subject and/or organism prior to, simultaneously with, and/or after diagnosis with influenza infection. In some embodiments, a therapeutic amount of a pharmaceutical composition is delivered to a patient and/or organism prior to, simultaneously with, and/or after onset of symptoms of influenza infection. In some embodiments, the amount of pharmaceutical composition is sufficient to treat, alleviate, ameliorate, relieve, delay onset of, inhibit progression of, reduce severity of, and/or reduce incidence of one or more symptoms or features of influenza infection.
Pharmaceutical compositions, according to the method of the present invention, may be administered using any amount and any route of administration effective for treatment. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the infection, the particular composition, its mode of administration, its mode of activity, and the like. Compositions of the invention 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 invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject or organism will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific active ingredient employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific active ingredient employed; the duration of the treatment; drugs used in combination or coincidental with the specific active ingredient employed; and like factors well known in the medical arts.
Pharmaceutical compositions of the present invention may be administered by any route. In some embodiments, pharmaceutical compositions of the present invention are administered by a variety of routes, including oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, parenteral, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (e.g. by powders, ointments, creams, gels, and/or drops), transdermal, mucosal, nasal, buccal, enteral, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol. In some embodiments, inventive compositions are administered intravenously. In some embodiments, inventive compositions are administered orally. In some embodiments, inventive compositions can be delivered using a pump (e.g., external pump or pump that is implanted within the body of a subject, such as a mechanical pump and/or an osmotic pump).
In general the most appropriate route of administration will depend upon a variety of factors including the nature of the composition (e.g., its stability in the environment of the gastrointestinal tract), the condition of the subject (e.g., whether the subject is able to tolerate oral administration), etc.
In certain embodiments, a composition may be administered in amounts ranging from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic effect. The desired dosage may be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations).
It will be appreciated that therapeutic agents and pharmaceutical compositions of the present invention can be employed in combination therapies. In some embodiments, the present invention encompasses “therapeutic cocktails” comprising inventive compositions. 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 be appreciated that the therapies employed may achieve a desired effect for the same purpose. For example, a glycan decoy may be administered with another agent that is used to treat symptoms of influenza infection. In some embodiments, the therapies employed may achieve different effects (e.g., control of any adverse side effects).
Pharmaceutical compositions of the present invention may be administered either alone or in combination with one or more other therapeutic agents. By “in combination with,” it is not intended to imply that the agents must be administered at the same time and/or formulated for delivery together, although these methods of delivery are within the scope of the invention. Compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent. Additionally, the invention encompasses the delivery of the inventive pharmaceutical compositions in combination with agents that may improve their bioavailability, reduce and/or modify their metabolism, inhibit their excretion, and/or modify their distribution within the body.
The particular combination of therapies (therapeutics and/or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and/or the desired therapeutic effect to be achieved. It will be appreciated that the therapies employed may achieve a desired effect for the same disorder (for example, an inventive agent may be administered concurrently with another therapeutic agent used to treat influenza infection), and/or they may achieve different effects (e.g., control of any adverse side effects). In some embodiments, compositions of the invention are administered with a second therapeutic agent that is approved by the U.S. Food and Drug Administration.
In will further be appreciated that therapeutically active agents utilized in combination may be administered together in a single composition or administered separately in different compositions.
In some embodiments, inventive glycan decoys may be administered in combination with one or more other glycan decoys.
In general, it is expected that 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 pharmaceutical compositions of the present invention may be administered alone and/or in combination with other agents that are used to treat influenza infection. In some embodiments, such agents interfere with expression or activity of an HA polypeptide. In certain embodiments, such agents comprise antibodies, such as antibodies that recognize virus particles containing a particular HA polypeptide (e.g., an HA polypeptide that binds to umbrella-topology glycans). In certain embodiments, such agents comprise nucleic acids (e.g., nucleic acid sequences complementary to HA sequences, which can be used for RNAi). In some embodiments, such agents may comprise TAMIFLU®.
In some embodiments, pharmaceutical compositions of the present invention may be administered alone and/or with other agents that are used to prevent and/or delay the onset of influenza infection. In some embodiments, such agents may comprise influenza vaccines. In certain embodiments, influenza vaccines are compositions comprising one or more of the following: (1) inactivated virus, (2) live attenuated influenza virus, for example, replication-defective virus, (3) HA polypeptide or characteristic or biologically active portion thereof, (4) nucleic acid encoding HA polypeptide or characteristic or biologically active portion thereof, (5) DNA vector that encodes HA polypeptide or characteristic or biologically active portion thereof, and/or (6) expression system, for example, cells expressing one or more influenza proteins to be used as antigens.
In certain embodiments, inactivated flu vaccines comprise one of three types of antigen preparation: inactivated whole virus, sub-virions where purified virus particles are disrupted with detergents or other reagents to solubilize the lipid envelope (“split” vaccine), or purified HA polypeptide (“subunit” vaccine). In certain embodiments, virus can be inactivated by treatment with formaldehyde, beta-propiolactone, ether, ether with detergent (such as Tween-80), cetyl trimethyl ammonium bromide (CTAB) and Triton N101, sodium deoxycholate and tri(n-butyl) phosphate. Inactivation can occur after or prior to clarification of allantoic fluid (from virus produced in eggs); the virions are isolated and purified by centrifugation (Nicholson et al., eds., Textbook of Influenza, Blackwell Science, Malden, M A, 1998). To assess the potency of the vaccine, the single radial immunodiffusion (SRD) test can be used (Schild et al., 1975, Bull. World Health Organ., 52:43-50 & 223-31; and Mostow et al., 1975, J. Clin. Microbiol., 2:531; both of which are incorporated herein by reference). In some embodiments, pharmaceutical compositions of the present invention may be administered in combination with live, attenuated flu vaccines. In some embodiments, vaccines comprise one or more adjuvants.
One of ordinary skill in the art will understand that the examples presented above are not meant to be limiting. The principles presented in the examples above can be generally applied to any combination therapies for treatment of influenza infection.
Diagnostics/Kits
The invention provides a variety of kits comprising one or more of umbrella-topology glycan decoys of the invention. For example, the invention provides kits comprising at least one glycan decoy and instructions for use. A kit may comprise multiple different glycan decoys. A kit may comprise any of a number of additional components or reagents in any combination. All of the various combinations are not set forth explicitly but each combination is included in the scope of the invention.
In some embodiments, novel glycan decoys to be utilized in inventive kits may be chemically synthesized in a laboratory. Alternatively or additionally, novel glycan decoys may be isolated from pathological samples, including, but not limited to, blood, serum/plasma, peripheral blood mononuclear cells/peripheral blood lymphocytes (PBMC/PBL), sputum, urine, feces, throat swabs, dermal lesion swabs, cerebrospinal fluids, cervical smears, pus samples, food matrices, and tissues from various parts of the body such as brain, spleen, and liver. In some embodiments, novel glycan decoys may be isolated from environmental samples, including, but not limited to, soil, water, and flora. Other samples that have not been listed may also be applicable.
According to certain embodiments of the invention, a kit may include, for example, (i) an umbrella-topology glycan decoy; (ii) instructions for administering the glycan decoy to a subject suffering from, susceptible to, and/or exhibiting symptoms of influenza infection.
According to certain embodiments of the invention, a kit for identifying glycan decoys may include, for example, (i) HA polypeptide; (ii) at least one candidate glycan decoy, such as a glycan array comprising a plurality of candidate glycan decoys; (iii) a positive control (e.g. umbrella-topology glycan known to bind to HA polypeptides); (iv) a negative control (e.g. cone-topology glycan); (v) instructions for determining whether the HA polypeptide is able to bind to a candidate decoy or whether a candidate decoy is able to compete away the binding between the HA polypeptide and the positive control.
Kits typically include instructions for use of inventive glycan decoys. Instructions may, for example, comprise protocols and/or describe conditions for identification of glycan decoys, administration of glycan decoys to a subject in need thereof, etc. Kits generally include one or more vessels or containers so that some or all of the individual components and reagents may be separately housed. Kits may also 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. An identifier, e.g., a bar code, radio frequency identification (RFID) tag, etc., may be present in or on the kit or in or one or more of the vessels or containers included in the kit. An identifier can be used, e.g., to uniquely identify the kit for purposes of quality control, inventory control, tracking, movement between workstations, etc.
In certain embodiments, inventive glycan arrays and/or kits are used to perform dose response studies to assess binding of HA polypeptides to umbrella-topology glycans and/or glycan decoys (e.g. candidate umbrella-topology glycan decoys) at multiple doses (e.g., as described herein). Such studies give particularly valuable insight into the binding characteristics of tested HA polypeptides, and are particularly useful to assess specific binding. Dose response binding studies of this type find many useful applications. To give but one example, they can be helpful in tracking the evolution of binding characteristics in a related series of HA polypeptide variants, whether the series is generated through natural evolution, intentional engineering, or a combination of the two.
The representative Examples that follow are intended to help illustrate the invention, and are not intended to, nor should they be construed to, limit the scope of the invention. Indeed, various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including the examples which follow and the references to the scientific and patent literature cited herein. It should further be appreciated that the contents of those cited references are incorporated herein by reference to help illustrate the state of the art.
The following Examples contain important additional information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and the equivalents thereof. It will be appreciated, however, that these examples do not limit the invention. Variations of the invention, now known and/or further developed, are considered to fall within the scope of the present invention as described herein and as hereinafter claimed.
Crystal structures of HAs from H1 (PDB IDS: 1RD8, 1RU7, 1RUY, 1RV0, 1RVT, 1RVX, 1RVZ), H3 (PDB IDs: 1MQL, 1MQM, 1MQN) and H5 (1JSN, 1JSO, 2FK0) and their complexes with α2-3 and/or α2-6 sialylated oligosaccharides have provided molecular insights into residues involved in specific HA-glycan interactions. Analyses of these HA-glycan co-crystal structures indicated that the orientation of the Neu5Ac sugar (SA) is fixed relative to the HA glycan binding site. A highly-conserved set of amino acids (including Tyr98, Ser/Thr136, Trp153, His183, Leu/Ile194, numbered based on H3 HA) across different HA subtypes are involved in anchoring the SA. The specificity of HA to α2-3 or α2-6 is therefore governed by interactions of the HA glycan binding site with the glycosidic oxygen atom and sugars beyond SA.
The conformation of the Neu5Acα2-3Gal linkage is such that Gal and sugars beyond Gal (at the reducing end, and in both linear and branched α2-3), occupy a “cone-like” region of space, and is governed by the glycosidic torsion angles at this linkage; hence, the present inventors define it as a “cone-like glycan topology” (
Interrogation of HA-glycan co-crystal structures highlights the fact that the human adapted H1 and H3 HAs have mutated from their presumed avian counterparts to gain additional contacts with α2-6 in the umbrella-like topology. Defining HA-glycan interactions based on trans and cis conformations (adopted by α2-3 and α2-6 linkages, respectively) is inadequate as this does not fully capture the structural features and conformational flexibility of the diverse sialylated glycans observed in human tissues. However, the “cone-like” and “umbrella-like” classification for HA binding fully captures the structural diversity and conformational plurality of sialylated glycans. Significantly, this classification is able to distinguish α2-3 and α2-6 binding of avian HAs from that of α2-6 binding of human adapted H1 and H3 HAs.
Characteristics of HA binding to cone-like and umbrella-like topologies were further corroborated using a novel data mining approach to analyze the extensive glycan array data available for H1, H3, and H5 HAs. Data mining analysis provided correlations between glycan features (Table 3 and
Classifier rules were obtained based on the features defined in
Consistent with these observations, distinct α2-3 classifiers (
In the following, H5 HA is used as an illustrative example to describe identification of mutations that could provide optimal contacts with long α2-6 adopting umbrella-like topology. Since H1, H2, and H5 HA belong to the same structural clade, they would have almost identical backbone structures and loop topologies. Based on this classification, mutations in H5 HA that would provide “H1-like” or “H2-like” contacts with long α2-6 glycans are defined.
H1-Like Mutations
Analysis of H1 HA-α2-6 glycan co-crystal structures showed that HA contacts can be divided into two regions—a base region involving contacts with Neu5Acα2-6Galβ1- motif and an extension region involving contacts with -GlcNAcβ1-3Galβ1-4Glcβ1- motif. In addition to highly conserved SA anchors (as described above), HA residues involved in base contacts are Lys222, Asp225 and Gln226. HA residues involved in extension contacts are Asp190, Gln192 and Ser193. Superimposition of the glycan binding sites of H1 and H5 HAs indicates that H5 HA already has a few residues such as Lys222 and Gln226 positioned to interact with base region. Lys193 (in H5 HA) could potentially have unfavorable steric contacts with the extension region when compared to Ser193 (in H1 HA). Consistent with this observation, the H5 HA double mutant Glu190Asp/Gly225Asp (which has Asp190 and Asp225, as observed in H1 HA) does not show any binding signal for α2-6 glycans in the glycan array due to possible steric interference from Lys193. Thus, the present invention encompasses the recognition that a combination of mutations at positions 190, 192, 193 and 225 would provide optimal “H1-like” contacts with long α2-6.
H2-Like Mutations
Given the high sequence similarity of H5 to H2 HA, this provides another perspective for defining H5 HA mutations for human receptor specificity. There are no crystal structures of H2 HA to date. Preliminary comparison of avian and human adapted H2 HA sequences indicates two primary amino acid changes (Gln226Leu and Gly228Ser) from avian to human H2 HA. Therefore, the present invention encompasses the recognition that a combination of these mutations taken together with other mutations at the above residue positions would provide optimal “H2-like” contacts with long α2-6.
Since human upper respiratory epithelia are primary targets for influenza A virus human infection and human-human transmission, the present inventors set out to identify what the different types of sialylated glycans are in these tissues. The approach was to bridge lectin staining of different tissue sections with glycan profiling of representative human cell lines using MALDI-MS and MS/MS (TOF-TOF) analyses. To elaborate the diversity of the α2-6 glycans predominantly expressed in the upper respiratory tissues, representative human tracheal sections were co-stained with a panel of plant lectins. Sambucus nigra agglutinin (SNA-I) is a prototypic lectin that specifically binds to α2-6 glycans. Jacalin and Concanavalin A (Con A) bind to specific motifs in O-linked (-Galβ1-3GalNAcα- and -GlcNAcβ1-3GalNAcα-) and N-linked (trimannosyl core) glycans, respectively. Lectin binding studies showed diversity in the distribution of α2-3 and α2-6 in upper respiratory tissues. Staining studies indicate predominant distribution of α2-6 sialylated glycans as a part of both N-linked (ciliated cells) and O-linked glycans (in the goblet cells) on the apical side of the tracheal epithelium (
MALDI-MS glycan profiling analyses showed a substantial diversity (
Data in
Data in
The apical side of tracheal tissue predominantly expresses α2-6 glycans with long branch topology. The alveolar tissue on the other hand predominantly expresses α2-3 glycans. HAs from the hallmark pandemic SC18 and the human vaccine strain A/Moscow/10/99H3N2 (Mos99) were chosen as typical representative human adapted H1 and H3 candidates. H1 HA binds significantly to localized regions on the apical surface of the trachea and its binding reduces gradually with dilution from 40 μg/ml to 10 μg/ml (
The data in
As described herein, the present invention encompasses the recognition that binding by HA polypeptides to glycans having a particular topology, herein termed “umbrella” topology, correlates with ability of the HA polypeptides to mediate infection of human hosts. The present Example describes results of direct binding studies with different HA polypeptides that mediate infection of different hosts, and illustrates the correlation between human infection and umbrella-topology glycan binding.
Direct binding assays typically utilize glycan arrays in which defined glycan structures (e.g., monovalent or multivalent) are presented on a support (e.g., glass slides or well plates), often using a polymer backbone. In so-called “sequential” assays, trimeric HA polypeptide is bound to the array and then is detected, for example using labeled (e.g., with FITC or horseradish peroxidase) primary and secondary antibodies. In “multivalent” assays, trimeric HA is first complexed with primary and secondary antibodies (typically in a 4:2:1 HA:primary:secondary ratio), such that there are 12 glycan binding sites per pre-complexed HA, and is then contacted with the array. Binding assays are typically carried out over a range of HA concentrations, so that information is obtained regarding relative affinities for different glycans in the array.
For example, direct binding studies were performed with arrays having different glycans such as 3′SLN, 6′SLN, 3′SLN-LN, 6′SLN-LN, and 3′SLN-LN-LN, where LN represents Galβ1-4GlcNAc, 3′ represents Neu5Acα2-3, and 6′ represents Neu5Acα2-6). Specifically, biotinylated glycans (50 μl of 120 μmol/ml) were incubated overnight (in PBS at 4° C.) with a streptavidin-coated High Binding Capacity 384-well plate that was previously rinsed three times with PBS. The plate was then washed three times with PBS to remove excess glycan, and was used without further processing.
During the early stages of development of dose-dependent direct binding assays using the biotin-avidin glycan array plate, different concentrations of His-tagged HA protein, primary antibody (mouse anti-6×His tag) and secondary antibody (HRP conjugated goat anti-mouse IgG) were incubated to achieve the ratio of 4:2:1 HA:primary:secondary. However, in this assay, it was challenging to detect binding signals at low HA concentrations since the binding kinetics limit the formation of HA precomplex at these concentrations (below 2.5 μg/ml). In order to accurately quantify relative binding affinities, binding of the precomplex was measured at a lower concentration range (0.05 μg/ml to 5 μg/ml). Towards addressing this issue, the binding assay was modified subsequently by preparation of the precomplex at the highest HA concentration followed by serial dilution of the stock. Briefly, the mixture (i.e., precomplexed HA) was then made up to a final volume of 250 μl with 1% BSA in PBS. 50 μl of the precomplexed HA was then added to the glycan-coated wells in the 384-well plate, and was incubated at room temperature for 2 hours. The wells were subsequently washed three times with PBS containing 0.05% TWEEN-20, and then three times with PBS. Direct binding studies were also done for representative H5N1 viruses to compare their HA binding specificity with that of human adapted HAs. Virus stocks (from CDC) were propagated in the allantoic cavity of 10-day-old embryonated hens' eggs at 37° C. Allantoic fluids were harvested 24 hours post inoculation and inactivated by treatment with B-propiolactone (BPL; 1/1,000) for 3 days at 4° C. Virus binding to the glycan-coated wells was performed as described (Yamada et al, 2006, Nature, 444:378; incorporated herein by reference) by adding appropriate amount of virus to each well after diluting in PBS containing 1% bovine serum albumin and incubating overnight at 4° C. After rinsing excess virus with PBS containing 0.05% Tween-20 and PBS, wells were incubated with antibody against the virus (ferret-α-influenza A raised against A/Hong Kong/213/03H5N1) for 5 hours at 4° C. Following extensive washing of the antibody, the plate was incubated with HRP-linked goat-α-ferret antibody (Rockland Immunochemicals) for 2 hours at 4° C. After extensive washes with PBS containing 0.05% Tween-20 and PBS, in all cases, HRP activity was estimated using Amplex Red Peroxidase Kit (Invitrogen, CA) according to the manufacturer's instructions. Serial dilutions of HA precomplexes were studied. Appropriate negative (non-sialylated glycans) and background (no glycans or no HA) controls were included, and all assays were done in triplicate. Results are presented in
The present invention encompasses the recognition that one characteristic of the binding pattern of known human adapted H1, H2, and H3 HAs is their binding at saturating levels to the long α2-6 (6′SLN-LN) over a range of dilution from 40 μg/ml to 2.5 μg/ml (
Human infection of influenza viruses involves binding of viral surface hemagglutinin (HA) to α2-6 sialylated glycan (α2-6) receptors on the epithelial cell surface of the human upper respiratory tissues. The relationship between the glycan binding preference of HA and transmission efficiency has been demonstrated in a ferret model using the highly pathogenic and virulent 1918 H1N1 viruses (Tumpey et al., 2007, Science, 315:655; incorporated herein by reference). In this study, it was demonstrated that while the 1918 H1N1 pandemic virus (A/South Carolina/1/1918 (SC18) with α2-6 binding preference) transmitted efficiently, a single amino acid HA mutation (SC18+1 mutation) resulted in a mixed α2-3 sialylated glycan (α2-3)/α2-6 binding virus (NY18) that transmitted inefficiently. Furthermore, an additional HA mutation (NY18+1 mutation) that switched the glycan binding preference of from α2-6 to α2-3 resulted in a virus (AV18) that was unable to transmit in the ferrets. While A/New York/1/18 (NY18) H1N1 virus, which shows mixed α2-3/α2-6 binding, transmitted inefficiently; surprisingly, A/Texas/36/91 (Tx91; also a mixed α2-3/α2-6 binding H1N1 virus) was able to transmit efficiently. These differences in virus transmission as the result of single amino acid HA mutations, in turn raise important questions about HA-glycan specificity in the context of glycan topology not only for efficient human adaptation (as discussed in Examples 1-5) but also for efficient transmission. Building on the previous examples, a dose-dependent glycan binding assay that is able to quantitatively measure the binding affinity and establish specificity was developed. The following demonstrates that while the typical glycan array assay shows no qualitative difference between the long α2-6 binding of SC18 and NY18, the dose-dependent assay of the same shows significant quantitative differences between their binding affinities to long α2-6. Furthermore, these differences are reflected as well in the distinct binding pattern of SC18 and NY18 HA to physiological glycans present in human upper respiratory tissues. Together, the present inventors have demonstrated that transmission differences between SC18 and NY18 viruses correlates with their quantitative binding affinity to long α2-6 glycans with characteristic umbrella-like topology.
Materials and Methods
Cloning, Baculovirus Synthesis, Expression and Purification of HA
The soluble form of HA was expressed using the Baculovirus Expression Vector System (BEVS). SC18 (A/South Carolina/1/1918; SC18) baculovirus (generated from pAcGP67-SC18-HA plasmid; Stevens et al., 2004, Science, 303:1866; Stevens et al., 2006, Nat. Rev. Microbiol., 4:857; both of which are incorporated herein by reference)) was a gift from Dr. James Stevens. pAcGp67-NY18-HA and pAcGp67-AV18-HA plasmids were generated from pAcGP67-SC18-HA by [Asp225Gly] and [Asp 190Glu, Asp225Gly] mutations respectively. Mutagenesis was carried out using QuikChange Multi Site-Directed Mutagenesis Kit (Stratagene). The primers used for mutagenesis were designed using the web based program, PrimerX (http://bioinformatics.org/primerx/), and synthesized by IDT DNA technologies (Coralville, Iowa). NY18 and AV18 baculoviruses were created from a pAcGP67-NY18-HA and pAcGP67-AV18-HA constructs using Baculogold system (BD Biosciences, San Jose, Calif.) according to manufacturer's instructions. The baculoviruses were used to infect 300 ml suspension cultures of Sf9 cells (BD Biosciences, San Jose, Calif.) cultured in BD BaculoGold Max-XP Insect Cell medium (BD Biosciences, San Jose, Calif.). These cultures were monitored for signs of infection and harvested 4-5 days post-infection. BEVS produces trimeric HA which provides multivalent binding to glycans. The soluble form of HA was purified from the supernatant of infected cells using the protocol described previously (Stevens et al., 2004, Science, 303:1866; incorporated herein by reference). Briefly, the supernatant was concentrated using Centricon Plus-70 centrifugal filters (Millipore, Billerica, Mass.) and the trimeric HA recovered from the concentrated cell supernatant using affinity chromatography with columns packed with Ni-NTA beads (Qiagen, Valencia, Calif.). Eluted fractions which contained HA were pooled and dialyzed overnight with a 10 mM Tris-HCl, 50 mM NaCl buffer (pH 8.0). Ion exchange chromatography was then performed on the dialyzed samples using a Mono-Q HR10/10 column (GE healthcare, Piscataway, N.J.). The fractions containing HA were pooled together and subjected to ultrafiltration using Amicon Ultra 100 K NMWL membrane filters (Millipore, Billerica, Mass.). The protein was then concentrated and reconstituted in PBS. The purified protein was quantified using Bio-Rad's protein assay (Bio-Rad, Hercules, Calif.).
Dose Dependent Direct Binding of H1N1 HA
To investigate the multivalent HA-glycan interactions, a streptavidin plate array comprising representative biotinylated α2-3 and α2-6 (short and long) was used. The spatial arrangement of the glycans in the wells of this plate array favors binding to only one of the three HA monomers in the HA unit. The glycan spacing and the HA unit size limit HA unit binding to a single glycan in the ELISA type binding assay which involved the sequential application of HA unit, primary antibody, and secondary antibody (see below). Conversely, the precomplexation of HA units (see below) with primary and secondary antibodies (HA:primary:secondary=4:2:1 ratio) facilitates a specific spatial arrangement of 4 HA units. This precomplex unit, therefore enabled the investigation of the effects of the relative spatial positioning of multiple HA units on the glycan binding affinity via multivalency (see
Streptavidin-coated High Binding Capacity 384-well plates (Pierce) were loaded to the full capacity of each well by incubating the well with 50 μl of 2.4 μM of biotinylated glycans overnight at 4° C. The biotinylated glycans (3′SLN, 6′SLN, 3′SLN-LN, 6′SLN-LN and 3′SLN-LN-LN) were obtained from the Consortium of Functional Glycomics through their resource request program. LN corresponds to lactosamine (Galβ1-4GlcNAc) and 3′SLN and 6′SLN respectively correspond to Neu5Acα2-3 and Neu5Acα2-6 linked to LN. Excess glycans were removed through extensive washing with PBS and the plates then used without additional processing. For the ELISA-type sequential binding the glycan-coated wells were incubated for 2 hours with HA at a concentration of 40 μg/ml (typical high concentrations used in glycan array experiments) in PBS containing 1% BSA and subsequently washed extensively with PBS-Tween followed by PBS. This was followed by an incubation with the primary antibody (Mouse anti-6×His tag IgG) at a concentration of 5 μg/ml in PBS containing 1% BSA for 3 hours at room temperature and repeating the above wash steps. The wells were finally incubated with a 2 μg/ml solution of the secondary antibody (HRP conjugated goat anti-Mouse IgG) for 1 hour at room temperature. In the case of dose dependent precomplexed HA experiments, appropriate amounts of His-tagged HA protein, primary antibody (Mouse anti-6×His tag IgG), and secondary antibody (HRP conjugated goat anti-Mouse IgG (Santacruz Biotechnology)) in the ratio 4:2:1 and incubated on ice for 20 minutes. Precomplexed HA was made up to a final volume of 250 μl with 1% BSA in PBS buffer and 50 μl of this solution was added to each of the glycan-coated wells in the 384-well plate and incubated at room temperature for 2 hours. The wells were then extensively washed with PBS-Tween followed by PBS. The binding signal was determined based on HRP activity using Amplex Red Peroxidase Assay (Invitrogen, CA) according to the manufacturer instructions. Negative controls were included and all assays were performed in triplicate.
To quantify the binding affinity (for the above pre-complexation assay), binding parameter Kd′ is defined and calculated based on the following model. The typical form of the Hill equation is used to represent multivalent binding:
which upon linearization becomes
where y—fractional saturation of the glycan binding sites in the HA units; [HA]—concentration of HA (in M); n—cooperativity factor; Kd′—apparent binding constant for the multivalent HA-glycan interactions. The value of y is calculated by normalizing the binding signals to the saturating binding signal which represent 100% occupancy on all HA units. n and Kd′ were calculated based on the linearized Hill model (shown in Table 5). In the above model, it is assumed that glycans in each well of the plate are in excess of HA. To satisfy this assumption, binding signals for HA concentration range from 0.05 μg/ml to 5 μg/ml were used to calculate n and Kd′.
Binding of H1 HA to Human Upper Respiratory Tissues
Formalin-fixed and paraffin-embedded normal human tracheal tissue sections were purchased from US Biological. Tissue sections were deparaffinized, rehydrated, and pre-blocked with 1% BSA in PBS for 30 minutes at room temperature. HA-antibody pre-complexes were generated by incubating recombinant SC18 and NY18 proteins with primary (mouse anti-6×His tag, Abcam) and secondary (Alexa Fluor 488 goat anti-mouse IgG, Invitrogen) antibodies in a ratio of 4:2:1 respectively for 20 minutes on ice. The complexes formed were then diluted in 1% BSA-PBS to different final HA concentrations. Tissue binding studies were performed by incubating tissue sections with diluted HA-antibody complexes for 3 hours at room temperature. To visualize cell nuclei, sections were counterstained with propidium iodide (Invitrogen; 1:100 in TBST) for 20 minutes at RT. For co-staining assays, tissues were deparaffinized, rehydrated, and co-incubated with FITC labeled Jacalin (Vector labs; 10 μg/ml in PBS with 0.05% Tween 20 (PBST)) and HA-antibody precomplexes (generated as described above using Alexa Fluor 546 goat anti-mouse as secondary antibody and diluted in PBST) for 3 hours at room temperature. Sections were then washed and viewed under a Zeiss LSM510 laser scanning confocal microscope.
Results
Molecular Interactions of SC18, NY18 and AV18 HAs with α2-3 and α2-6
Based on analysis of HA-glycan co-crystal structures, HA-glycan interactions are characterized based on cone-like and umbrella-like glycan topology (Chandrasekharan et al., 2007, Nat. Biotech., in press). The x-ray crystal structure of SC18 HA has been solved (Stevens et al., 2004, Science, 303:1866; and Gamblin et al., 2004, Science, 303:1838; both of which are incorporated herein by reference) but the co-crystal structure of this HA with sialylated glycans remains unsolved. The structures of NY18 and AV18 HA were obtained by making the single Asp225Gly (for NY18 HA) and the additional NY18+Asp 190Glu mutation (for AV18) in silico. Co-crystal structures of the related A/Swine/Iowa/15/30 (ASI30) and A/Puerto Rico/8/34 (APR34) H1N1 HAs with α2-3 and α2-6 glycans (Gamblin et al., 2004, Science, 303:1838; incorporated herein by reference) enabled the investigation of molecular interactions between SC18, NY18, and AV18 with α2-3 and α2-6 oligosaccharides.
The umbrella-like topology is preferred by α2-6 longer oligosaccharides (e.g., tetrasaccharide and higher). The LSTc pentasaccharide (Neu5Acα2-6Galβ1-4GlcNAcβ1-3Galβ1-4Glc) used in the APR34 and AS130 HA-α2-6 co-crystal structures represents this long α2-6. Superimposition of these co-crystal structures and SC18 HA demonstrated that the contacts with long α2-6 oligosaccharide are divided into two regions. The base region involves contacts with Neu5Acα2-6Galβ1- motif and the extension region involves contacts with -GlcNAcβ1-3Galβ1-4Glcβ1- motif. A highly conserved set of amino acids (Tyr95, Ser/Thr136, Trp153, His183, Leu/Ile194) are involved in anchoring Neu5Ac (
Distinct from the umbrella-like topology, the cone-like topology can be adopted both by α2-3 and α2-6 (preferentially by short trisaccharide units such as Neu5Acα2-6Galβ1-4Glc(NAc)). Unlike the two distinct regions of HA contacts found with the umbrella-like topology, the contacts with the cone-like topology primarily involve substitutions which center around a trisaccharide Neu5Acα2-3/6Galβ1-3/4Glc(NAc) motif as observed in the HA-α2-3 co-crystal structures. Specifically, Glu190 and Gln226, which are highly conserved across various avian HAs, are involved in making optimal contacts with the cone-like topology (
Dose Dependent Direct HA Binding to Representative α2-3 and α2-6 Oligosaccharides
Novel glycan array platforms have been developed to investigate HA-glycan interactions (Stevens et al., 2006, Nat. Rev. Microbiol., 4:857; and Kumari et al., 2007, J. Virol., 4:42; both of which are incorporated herein by reference). These arrays have provided important insights into α2-3 versus α2-6 binding specificity of various wild type and mutant HAs. In these glycan array studies high and low binding signals to the glycans within a single assay using saturating HA concentration determine the glycan binding preference for a given HA. It is generally accepted that the glycan binding affinity for a single binding site on any glycan binding protein (e.g., a lectin) is low (e.g., in the high μM to mM range). Since each HA unit is a trimeric protein, there are three glycan binding sites per unit (Skehel and Wiley, 2000, Ann. Rev. Biochem., 69:531; incorporated herein by reference). In addition, multiple HA units assemble on a virus surface. The glycan binding sites within a HA unit and the spatial arrangement of multiple HA units across a virus surface could play an important role in multivalent HA-glycan interactions. The present invention encompasses the recognition that the concentration of the HA units relative to the glycans consequently may govern HA-glycan binding specificity.
To understand differences in the glycan binding specificity of SC18, NY18, and AV18 HAs for a specific spatial arrangement of the HA units and the glycans, systematic direct HA binding studies on a plate array were performed (see Methods). Minimal binding signals were observed for SC18 HA in the sequential ELISA-type binding assay (
A dose-dependent direct binding assay by serial dilution of precomplexed SC18, NY18, and AV18 HAs was performed to establish their relative glycan binding affinities (
Table 5 shows the relative binding affinities of SC18, NY18, and AV18 HAs to representative long α2-3 and α2-6 glycans. The parameters n and Kd′ were determined as described in Methods and elaborated in
The R-square value >0.95 for all the glycan binding data indicates a good fit to the linearized Hill model. The value of n≧1 reflects the positive cooperativity in the HA-glycan interactions as a result of the spatial arrangement of multiple HA units and the binding of a HA unit to a single glycan. While SC18 HA showed high binding affinity to the single long α2-6 glycan (Kd′=5.5 pM), AV18 HA showed the reverse trend with almost no α2-6 binding and high α2-3 binding affinity (Kd′=5 pM). On the other hand, while the long α2-6 binding of NY18 HA was almost identical to that of SC18 HA at saturating HA concentrations (
Binding of SC18 and NY18 to Human Upper Respiratory Tissues
The present invention encompasses the recognition that differences in the binding affinities of α2-6 and α2-3 of SC18 and NY18 HA render them ideal lectin-like probes to investigate HA binding to diverse physiological glycans present in human upper respiratory tissues. Human tracheal sections were used to explore the binding pattern of SC18 and NY18 HA to physiological glycans. Although both SC18 and NY18 bind to the apical side of the trachea (
Glycan arrays comprising many distinct oligosaccharide motifs have been employed to investigate the glycan binding of wild type and mutant H1, H3, and H5 HAs (Stevens et al., 2006, Nat. Rev. Microbiol., 4:857; Kumari et al., 2007, J. Virol., 4:42; Stevens et al., 2006, Science, 312:404; all of which are incorporated herein by reference). In spite of these advances, interpretation of glycan receptor specificity which leads to human adaptation has remained challenging. This is due in large part to a general notion in the field that glycan-protein interactions are relatively weak and non-specific. Consequently, the glycan binding specificity for HA has largely been established using very high protein concentrations. High HA concentration may be useful to establish binders versus non-binders but the present invention encompasses the recognition that there are limitations in defining appropriate quantitative measurements to establish relative glycan binding affinities of HA. One example, at high concentrations, the typically used first order binding kinetics assumptions (pertaining to HA: glycan ratio) would not be satisfied. To accurately quantify the relative glycan binding affinities between HAs, the present invention encompasses the recognition that it is desirable to be at the “appropriate” HA concentration range. The present invention encompasses the recognition that several parameters (e.g., the spatial arrangement of multiple HA units, the surface glycan density, and the ratio of HA to glycan) should be taken into account when determining the “appropriate” HA concentration range for a given glycan array assay. Current approaches using high HA concentrations on glycan arrays do not account for these areas of concern. In this study, the specific spatial arrangement of the glycans (spacing imposed by biotin-avidin system) and the HA (imposed by precomplexation of HA units) in tandem with the dose-dependent glycan binding assay was able to overcome these challenges. The concentration range used to determine the binding affinity parameters (n and Kd′) in this study is such that the glycan is in excess of the HA as would be expected in physiological conditions. The present invention encompasses the recognition that the approach described in this study can be readily extended to any glycan array platform employed to investigate protein-glycan interactions in general.
High or saturating HA concentrations display no differences between the binding signals of SC18 and NY18 to long α2-6 glycan. The only difference between SC18 and NY18 is the additional binding to α2-3 glycans for NY18 HA. As the α2-3 binding of SC18, NY18, and AV18 shows inverse correlation with the transmissibility, without wishing to be bound by any one theory, a possible conclusion from these data is that the loss of α2-3 binding specificity is a factor for transmission. However, A/Texas/36/91 (TX91; i.e., a mixed α2-6/α2-3 binding virus similar to NY18) transmitted efficiently in ferrets. The dose-dependent direct binding of TX91 and A/Moscow/10/99H3N2 (Mos99; i.e., a mixed α2-6/α2-3 binding human adapted H3 HA) compared to that of SC18 and NY18 shows that the high affinity binding to long α2-6 glycan is a common feature for SC18, Tx91, and Mos99, but not for NY18 (
Differential binding of SC18 and NY18 to the diverse physiological glycans present in the human upper respiratory tissues corroborates further these direct binding studies. The present invention encompasses the recognition that the narrow binding of SC18 to goblet cells should spark future investigations into the cellular tropism of human adapted viruses. This observation is consistent with Matrosovich et al. (Matrosovich et al., 2004, Proc. Natl. Acad. Sci., USA, 101:4620; incorporated herein by reference), where they demonstrated that human adapted H1 and H3 virus strains preferentially infect non-ciliated cells and avian viruses preferentially infect ciliated cells. They also suggested that such differences in the cellular tropism of the viruses could explain the differences in their efficiency of human-human transmission.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention, described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the appended claims.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the appended claims.
In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Thus, for example, reference to “a nanoparticle” includes a plurality of such nanoparticle, and reference to “the cell” includes reference to one or more cells known to those skilled in the art, and so forth. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.
Where elements are presented as lists, e.g., in Markush group format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, etc. For purposes of simplicity those embodiments have not been specifically set forth in haec verba herein. It is noted that the term “comprising” is intended to be open and permits the inclusion of additional elements or steps.
Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.
In addition, it is to be understood that any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the invention (e.g., any glycan decoy, any glycan mimic, any glycan moiety, any HA polypeptide, any amino acid position, any influenza strain, any method of identifying novel glycan decoys, any pharmaceutical composition, any method of administration, etc.) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.
The publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims.
The present application claims priority to U.S. Provisional patent application Ser. No. 61/018,783, filed Jan. 3, 2008, the entire contents of which are incorporated herein by reference.
This invention was made with United States government support awarded by the National Institute of General Medical Sciences (glue grant contract number U54 GM62116) and by the National Institutes of Health (contract number GM57073). The United States Government has certain rights in the invention.
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| Number | Date | Country | |
|---|---|---|---|
| 20100004195 A1 | Jan 2010 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61018783 | Jan 2008 | US |
