WO2018096396A1

WO2018096396A1 - Albumin variants and uses thereof

Info

Publication number: WO2018096396A1
Application number: PCT/IB2017/001543
Authority: WO
Inventors: Jan Terje ANDERSEN; Inger Sandlie; Jeannette NILSEN
Original assignee: University Of Oslo
Priority date: 2016-11-22
Filing date: 2017-11-21
Publication date: 2018-05-31

Abstract

The present invention relates to albumin variants with an improved affinity for the neonatal Fc receptor (FcRn) and increased half-life and uses thereof, and in particular to the use of such albumin variants as carriers for molecules of interest (e.g., immunogens, drugs, etc.). In some embodiments, the present invention relates to vaccines and other therapeutic molecules (e.g., vaccines for mucosal delivery) comprising albumin/immunogen conjugates.

Description

ALBUMIN VARIANTS AND USES THEREOF

CROSS REFERENCE TO RELATED APPLICATIONS

The present invention claims the priority benefit of U.S. Provisional Patent

Application 62/425,433, filed November 22, 2016, which is incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to albumin variants with an improved affinity for the neonatal Fc receptor (FcRn) and increased plasma half-life and uses thereof, and in particular to the use of such albumin variants as carriers for molecules of interest (e.g., immunogens, drugs, etc.). In some embodiments, the present invention relates to vaccines and other therapeutic molecules (e.g., vaccines for mucosal delivery) comprising albumin/immunogen conjugates.

BACKGROUND OF THE INVENTION

Albumin is a protein naturally found in the blood plasma of mammals where it is the most abundant protein. It has important roles in maintaining the desired osmotic pressure of the blood and also in transport of various substances in the blood stream. Albumins have been characterized from many species including human beings, pig, mouse, rat, rabbit and goat and it has been found that albumins from different sources share a high degree of structural relationship.

Albumin binds in vivo to the neonatal Fc receptor (FcRn) and this interaction is known to be important for the plasma half-life of albumin (Chaudhury et al 2003; Montoyo et al., 2009). FcRn is a membrane bound protein, and has been found to salvage albumin as well as IgG from intracellular degradation (Roopenian D. C. and Akilesh, S. (2007), Nat.Rev. Immunol 7, 715-725.). Thus, FcRn is a bifunctional molecule that contributes to maintaining the high level of IgG and albumin in serum of mammals such as humans.

While the FcRn-IgG interaction has been characterized in the prior art, the FcRn- albumin is less well characterized. Data indicate that IgG and albumin bind noncooperatively to distinct sites on FcRn (Andersen et al. (2006), Eur. J. Immunol 36, 3044-3051 ; Chaudhury et al. (2006), Biochemistry 45, 4983-4990). It is known that mouse FcRn binds IgG from mice and humans whereas human FcRn appears to be more discriminating (Ober et al. (2001) Int Immunol 13, 1551-1559) and does not bind mouse IgG (Ober et al. (2001) Int Immunol 13, 1551-1559). Furthermore, human FcRn binds albumin from both mouse and human, whereas mouse FcRn does not bind human albumin (Andersen et al (2010) JBC).

Human serum albumin (HSA) has been well characterized as a polypeptide of 585 amino acids, the sequence of which can be found in Peters, T., Jr. (1996) All about Albumin: Biochemistry, Genetics and Medical, Applications, Academic Press, Inc., Orlando. It has a characteristic binding to its receptor FcRn, where it binds at pH 6.0 but not at pH 7.4. The plasma half-life of HSA has been found to be approximately 19 days. A natural variant having lower plasma half-life has been identified (Biochim Biophys Acta. 1991, 1097:49-54) having the substitution D494N. This substitution generated an N-glycosylation site in this variant, which is not present in the wild type HSA. It is not known whether the glycosylation or the amino acid change is responsible for the change in plasma half-life.

Albumin has a long plasma half-life and because of this property it has been used for drug delivery. Albumin has been conjugated to pharmaceutically beneficial compounds (WO0069902A), and it was found that conjugate had maintained the long plasma half-life of albumin so the resulting plasma half-life of the conjugate has generally been found to be considerably longer than the plasma half-life of the beneficial therapeutic compound alone.

Further, albumin has been fused to therapeutically beneficial peptides (WO 01/79271 A and WO 03/59934 A) with the typical result that the fusion has the activity of the therapeutically beneficial peptide and a long plasma half-life considerably longer than the plasma half-life of the therapeutically beneficial peptides alone.

Albumin has the ability to bind a number of ligands, and this property has been utilized to extend the plasma half-life of drugs having the ability to bind to albumin. This has been achieved by attaching a pharmaceutical beneficial compound to a moiety having albumin binding properties.

It is not clear what determines the plasma half-life of the formed conjugates or fusion polypeptides but it appears to be given by the albumin and the selected pharmaceutically beneficial compound/peptide they are composed of. It would be desirable to be able to control the plasma half-life of a given albumin conjugate or albumin fusion polypeptide so that a longer or shorter plasma half-life than given by the components of the conjugate/fusion can be achieved, in order to be able to design a particular drug or vaccine according to the particulars of the indication intended to be treated. SUMMARY OF THE INVENTION

The plasma half-life of human serum albumin (HSA) is dependent on a delicate recycling mechanism involving rescue from degradation by endosomal binding to FcRn in cells of the endothelium and subsequent release back to the bloodstream. In one aspect, the present disclosure provides novel HSA variants with improved affinity for FcRn. Such HSA variants include, but are not limited to, the following amino acid substitutions relative to the wild type HSA sequence: N111E/V547A/K573P, T83N/V547A/K573P,

T83N/E505Q/T527M/K573P, T83N/N111E/V547A/K573P, Nl 11E/E505Q/T527M/K573P, and T83N/N111E/E505Q/T527M/K573P. However, the plasma half-life of HSA variants does not solely rely on FcRn affinity. A variety of other factors may influence the actual performance of HSA variants in the bloodstream, for example size, charge, conformational robustness, degradation, stability etc.

In another aspect, the present disclosure provides novel HSA variants with increased plasma half-life relative to the wild type HSA (e.g., as measured in an improved mouse model). Such HSA variants include, but are not limited to, the following amino acid substitutions relative to the wild type HSA sequence: T83N/E505Q/T527M/K573P,

Nl 11E/E505Q/T527M/K573P, T83N/N111E/E505Q/T527M/K573P,

E501P/N503H/E505D/T506S and E501P/N503H/E505D/T506S/K573P.

Notably, cross-species artifacts may obscure the actual performance of HSA variants in non-human models. The mouse-model used herein utilizes FcRn-knockout mice expressing human FcRn instead. As such, this model is more reliable than conventional mice for testing of the plasma half-life of HSA variants. As used herein, the term "plasma half- life" refers to the time it takes for a HSA variant to reach 50% of its initial concentration in the plasma or other blood product of a mammal.

For example, in some embodiments, the present invention provides a variant HSA that binds to FcRn with increased affinity relative to wild type HSA or mouse serum albumin (MSA), wherein the polypeptide comprises at least one variant amino acid. In some embodiments, the polypeptide binds to FcRn with a Kd of 10 or less, 5 or less, 2 or less, or 1 or less (e.g., measured under acidic conditions).

In some embodiments, the variant polypeptide is at least 80%, 90%, or 95% identical to SEQ ID NO: l . In some embodiments, the variant amino acid is, for example, one or more of Nl 11E/V547A/K573P, T83N/V547A/K573P, T83N/E505Q/T527M/K573P,

T83N/N111E/V547A/K573P, Nl 1 IE/ E505Q/T527M/K573P, and

T83N/N111E/E505Q/T527M/K573P. In some embodiments, the HSA variant polypeptide comprises the amino acid sequence of SEQ ID NO:2 or sequences with more than 98% sequence identity thereto, wherein position 83 comprises a threonine residue; position 111 comprises a glutamic acid residue; position 505 comprises a glutamic acid residue; position 527 comprises a threonine residue; position 547 comprises an alanine residue; and position 573 comprises a proline residue. In some embodiments, the HSA variant polypeptide comprises the amino acid sequence of SEQ ID NO:2 or sequences with more than 98% sequence identity thereto, wherein position 83 comprises an asparagine residue; position 111 comprises an aparagine residue; position 505 comprises a glutamic acid residue; position 527 comprises a threonine residue; position 547 comprises an alanine residue; and position 573 comprises a proline residue. In some embodiments, the HSA variant polypeptide comprises the amino acid sequence of SEQ ID NO:2 or sequences with more than 98% sequence identity thereto, wherein position 83 comprises an asparagine residue; position 111 comprises an asparagine residue; position 505 comprises a glutamine residue; position 527 comprises a methionine residue; position 547 comprises a valine residue; and position 573 comprises a proline residue. In some embodiments, the HSA variant polypeptide comprises the amino acid sequence of SEQ ID NO:2 or sequences with more than 98% sequence identity thereto, wherein position 83 comprises an asparagine residue; position 111 comprises a glutamic acid residue; position 505 comprises a glutamic acid residue; position 527 comprises a threonine residue; position 547 comprises an alanine residue; and position 573 comprises proline. In some embodiments, the HSA variant polypeptide comprises the amino acid sequence of SEQ ID NO:2 or sequences with more than 98% sequence identity thereto, wherein position 83 comprises a threonine residue; position 111 comprises a glutamic acid residue; position 505 comprises a glutamine residue; position 527 comprises a methionine residue; position 547 comprises a valine residue; and position 573 comprises a proline residue. In some embodiments, the HSA variant polypeptide comprises the amino acid sequence of SEQ ID NO:2 or sequences with more than 98% sequence identity thereto, wherein position 83 comprises an asparagine residue; position 111 comprises a glutamic acid residue; position 505 comprises a glutamine residue; position 527 comprises a methionine residue; position 547 comprises a valine residue; and position 573 comprises a proline residue. In some embodiments, the HSA variant polypeptide comprises the amino acid sequence of SEQ ID NO:2 or sequences with more than 98% sequence identity thereto, wherein position 501 comprises a proline residue; position 503 comprises a histidine residue; position 505 comprises an aspartic acid residue; and position 506 comprises a serine residue. In some embodiments, the HSA variant polypeptide comprises the amino acid sequence of SEQ ID NO:2 or sequences with more than 98% sequence identity thereto, wherein position 501 comprises a proline residue; position 503 comprises a histidine residue; position 505 comprises an aspartic acid residue; position 506 comprises a serine residue; and position 573 comprises a proline residue. In some embodiments, the HSA variant polypeptide comprises an amino acid sequence selected from SEQ ID NOs:3 to 10.

In some embodiments, the present invention provides a fusion of the albumin variant and a molecule (e.g., small molecule, polypeptide or peptide) of interest (e.g., immunogen (e.g., antigen), diagnostic agent, or drug) conjugated to an amino acid of the albumin. In some embodiments, the altered binding to FcRn as compared to wild type albumin is an increased binding affinity. In some embodiments, the immunogen is covalently attached to an amino acid comprising a thiol group.

In some embodiments, the present invention provides a nucleic acid encoding the albumin variant, fragment thereof, or fusion thereof as described above. In some embodiments, the present invention provides host cells comprising the nucleic acids.

In some embodiments, the present invention provides a composition (e.g., vaccine composition) comprising the albumin variant or fusion protein described above and a pharmaceutically acceptable carrier. In some embodiments, the composition is formulated as a vaccine for mucosal administration.

In yet other embodiments, the present invention provides a composition comprising a fusion protein of a variant HSA polypeptide and a molecule (e.g., small molecule, peptide or polypeptide). In some embodiments, the molecule is a drug or a diagnostic agent.

In some embodiments, the present invention provides a method of inducing an immune response (e.g., mucosal immune response) in a subject comprising administering to the subject an fusion protein comprising an albumin variant, fragment thereof, or fusion thereof; and an immunogen as described above. In some embodiments, the present invention provides for the use of the fusion protein comprising albumin variant, fragment thereof, fusion thereof or conjugate thereof; and an immunogen as described above to treat a subject. Further embodiments provide a vaccine composition comprising: a) a fusion protein comprising a variant albumin polypeptide and a conjugate (e.g. an immunogen or drug); and b) a pharmaceutically acceptable carrier. Additional embodiments provide methods and uses of inducing an immune response in a subject, comprising administering to the subject the aforementioned vaccine composition under conditions such that said subject generates an immune response to the immunogen. In some embodiments, the vaccine composition is aerosolized. In some embodiments, the vaccine composition is delivered to a mucosal surface of the subject (e.g., instranasally).

Still other embodiments provide a method of delivering a pharmaceutical agent, comprising administering a pharmaceutical composition comprising the fusion proteins described herein to a subject in need thereof.

Further embodiments provide the use of the fusion proteins described herein in the production of a medicament.

Additional embodiments are described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. HSA wild type and engineered variants produced in HEK293E cells and purified on a Capture Select human serum albumin affinity matrix were analyzed by 12% (w/v) SDS-PAGE.

FIG. 2A-L. SPR binding kinetics of HSA wild type and DI/DIII variants to human

FcRn. Representative sensorgrams showing binding at pH 6.0 of injected human FcRn to HSA wild type (A), K573P (B), Nl 11E/K573P (C), T83N/N111E/K573P (D), K573P/V547A (E), N111E/K573P/V547A (F), T83N/K573P/V547A (G) , T83N/N111E/K573P/V547A (H), K573P/E505Q/T527M (I), N111E/K573P/E505Q/T527M (J),

T83N/K573P/E505Q/T527M (K) and T83N/N111E/K573P/E505Q/T527M (L) immobilized on a CM5 chip (-500 RU). Injections were performed with a flow rate of 40 μΐ/min at 25°c.

FIG. 3. SEQ ID NO: l, wild-type HSA.

FIG. 4. SEQ ID NO:2, A truncated version of SEQ ID NO: 1 wherein the positions 83, 111, 505, 527, 547 and 573 are indicated with bold x. Each bold x thus represents an amino acid residue in the sequence, but not necessarily the same amino acid residue.

FIG. 5. SEQ ID NOs: 3-10. Sequences of variant HSA polypeptides with mutations in bold. FIG. 6. WT HSA and HSA DIDIII variants produced in HEK293E cells and purified on a Capture Select human serum albumin affinity matrix were analyzed by 12% (w/v) SDS- PAGE.

FIG. 7A-H. SPR binding kinetics of HSA DIDIII variants to human FcRn.

Representative sensorgrams showing binding at pH 5.5 of injected human FcRn to WT HSA (A), KP (B), NE/VA/KP (C), TN/VA/KP (D), TN/NE/VA/KP (E), NE/EQ/TM/KP (F), TN/EQ/TM/KP (G) and TN/NE/EQ/TM/KP (H) immobilized on a CM5 chip (-500 RU). Injections were performed with a flow rate of 40 μΐ/min at 25°c.

FIG. 8. Elimination curves for WT HSA and engineered variants (KP, TN/EQ/TM/KP and TN/NE/EQ/TM/KP) in Tg32 human FcRn transgenic mice.

FIG. 9. Elimination curves for WT HSA and engineered variants (KP and

NE/EQ/TM/KP) in Tg32 human FcRn transgenic mice.

FIG 10. ELISA results showing binding of WT HSA and HSA variants (KP, EP/NH/ED/TS and EP/NH/ED/TS/KP) to human FcRn at pH 5.5.

FIG 11. Elimination curves for WT HSA and engineered variants (KP, EP/NH/ED/TS and EP/NH/ED/TS/KP) in Tg32 human FcRn transgenic mice.

DEFINITIONS

The term "albumin" as used herein means a protein having substantially the same three dimensional structure as HSA. Examples of albumin proteins according to the invention include, but are not limited to, human serum albumin, primate serum albumin, such as chimpanzee serum albumin, gorilla serum albumin, rodent serum albumin such as rabbit serum albumin, mouse serum albumin and rat serum albumin, bovine serum albumin, equine serum albumin, donkey serum albumin, hamster serum albumin, goat serum albumin, sheep serum albumin, dog serum albumin, guinea pig serum albumin, chicken serum albumin and pig serum albumin. HSA as disclosed in SEQ ID NO: 1 or any naturally occurring allele thereof, is the preferred albumin according to the invention and has a molecular weight of 67 kDa. The skilled person will appreciate that natural alleles may exist having essentially the same properties as HSA but having one or a few changes compared to SEQ ID NO: 1, and the inventors also contemplate the use of such natural alleles.

The term "fragments of albumin" as used herein means a part of albumin having retained the ability to bind to FcRn. Fragments may consist of one uninterrupted sequence derived from HSA or it may comprise two or more sequences derived from HSA. The fragments according to the invention have a size of more than approximately 20 amino acid residues, preferably more than 30 amino acid residues, more preferred more than 40 amino acid residues, more preferred more than 50 amino acid residues, more preferred more than 75 amino acid residues, more preferred more than 100 amino acid residues, more preferred more than 200 amino acid residues, more preferred more than 300 amino acid residues, even more preferred more than 400 amino acid residues and most preferred more than 500 amino acid residues.

The term "wildtype" when used in reference to a protein refers to proteins encoded by the genome of a cell, tissue, or organism.

The term "variant" and "mutant" when used in reference to a polypeptide refer to an amino acid sequence that differs by one or more amino acids from another, usually related polypeptide. The variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties. One type of conservative amino acid substitutions refers to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is

phenylalanine, tyrosine, and tryptophan; unnatural amino acids like p-aminophenylalanine, a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine- tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. More rarely, a variant may have "non-conservative" changes (e.g., replacement of a glycine with a tryptophan). Similar minor variations may also include amino acid deletions or insertions (i.e., additions), or both. Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing biological activity may be found using computer programs well known in the art, for example, DNAStar software. Variants can be tested in functional assays. Preferred variants have less than 10%, and preferably less than 5%, and still more preferably less than 2% changes (whether substitutions, deletions, and so on). For an amino acid substitution, the following nomenclature is used: Original amino acid, position, substituted amino acid. Accordingly, the substitution of lysine with alanine at position 573 is designated as "K573A" and the substitution of lysine with proline at position 573 is designated as K573P. Multiple mutations are separated by addition marks ("+") or "/", e.g., "Gly205Arg + Ser411Phe" or "G205R/S411F", representing mutations at positions 205 and 411 substituting glycine (G) with arginine (R), and serine (S) with phenylalanine (F), respectively.

The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "identity". For purposes of the present invention, the degree of identity between two amino acid sequences is determined using the Needleman- Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as

implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends in Genetics 16: 276-277), preferably version 3.0.0 or later. The optional parameters 11644.000-EP7 used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

(Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment).

The expression "amino acid position corresponding to" a position in a reference sequence and similar expression is intended to identify the amino acid residue that in the primary or spatial structure corresponds to the particular position in the reference sequence. The skilled person will appreciate that this can be done by aligning a given sequence with the reference sequence and identifying the amino acid residue that aligns with the particular position in the reference sequence. For example in order to find the amino acid residue in a given albumin sequence that corresponds to position 573 in HSA, the given albumin sequence is aligned with HSA and the amino acid that aligns with position 573 in HSA is identified as the amino acid in the given albumin sequence that corresponds to position 573 in HSA. In order to identify or correlate the amino acid positions in truncated version with the wild type HSA, the amino acid sequence of the truncated version is aligned with the SEQ ID NO: 1 for maximum identity. Then the positions in SEQ ID NO: 1 dictate the positions in the truncated version. For example, if a truncated version of SEQ ID NO: 1 lacks the three N- terminal amino acid residues, the amino acid residue in position 300 is alanine for both SEQ ID NO: 1 and for the truncated version. In some embodiments, in order to identify or correlate the amino acid positions in a fusion protein with the wild type HSA, the amino acid sequence of the fusion protein is aligned with the SEQ ID NO: 1 for maximum identity. Then, the positions in SEQ ID NO: 1 dictates the positions in the fusion protein. For example, if a fusion protein comprises SEQ ID NO:2, the amino acid residue in position 300 is alanine for both SEQ ID NO: 1 and for the fusion protein. The expression Xnnn is intended to mean an amino acid residue X located in a position corresponding to position nnn in HSA and the expression XnnnY is intended to mean a substitution of any amino acid X located in a position corresponding to position nnn in HSA with the amino acid residue Y.

As used herein, the term "affinity" refers to a measure of the strength of binding between two members of a binding pair, for example, an albumin and FcRn. K_<j is the dissociation constant and has units of molarity. The affinity constant is the inverse of the dissociation constant. An affinity constant is sometimes used as a generic term to describe this chemical entity. It is a direct measure of the energy of binding. The natural logarithm of K is linearly related to the Gibbs free energy of binding through the equation AGo = -RT LN(K) where R= gas constant and T=temperature in degrees Kelvin. Affinity may be determined experimentally, for example by surface plasmon resonance (SPR) using commercially available Biacore SPR units (GE Healthcare).

As used herein, the term "conjugate" as in "a fusion protein comprising an albumin and a conjugate" refers to any molecule attached (e.g., covalently as in a fusion protein or non-covalently (e.g., via hydrophobic interactions)) to a albumin. Examples include, but are not limited to, peptides, polypeptides, immunogens, drugs, proteins, lipids, small molecules, nucelotides, radioactive tracers etc.

As used herein, the term "under conditions such that said subject generates an immune response" refers to any qualitative or quantitative induction, generation, and/or stimulation of an immune response (e.g., innate or acquired).

A used herein, the term "immune response" refers to a response by the immune system of a subject. For example, immune responses include, but are not limited to, a detectable alteration (e.g., increase) in Toll receptor activation, lymphokine (e.g., cytokine (e.g., Thl or Th2 type cytokines) or chemokine) expression and/or secretion, macrophage activation, dendritic cell activation, T cell activation (e.g., CD4+ or CD8+ T cells), NK cell activation, and/or B cell activation (e.g., antibody generation and/or secretion). Additional examples of immune responses include binding of an immunogen (e.g., antigen (e.g., immunogenic polypeptide)) to an MHC molecule and inducing a cytotoxic T lymphocyte ("CTL") response, inducing a B cell response (e.g., antibody production), and/or T-helper lymphocyte response, and/or a delayed type hypersensitivity (DTH) response against the antigen from which the immunogenic polypeptide is derived, expansion (e.g., growth of a population of cells) of cells of the immune system (e.g., T cells, B cells (e.g., of any stage of development (e.g., plasma cells), and increased processing and presentation of antigen by antigen presenting cells. An immune response may be to immunogens that the subject's immune system recognizes as foreign (e.g., non-self antigens from microorganisms (e.g., pathogens), or self-antigens recognized as foreign). Thus, it is to be understood that, as used herein, "immune response" refers to any type of immune response, including, but not limited to, innate immune responses (e.g., activation of Toll receptor signaling cascade) cell- mediated immune responses (e.g., responses mediated by T cells (e.g., antigen-specific T cells) and non-specific cells of the immune system) and humoral immune responses (e.g., responses mediated by B cells (e.g., via generation and secretion of antibodies into the plasma, lymph, and/or tissue fluids). The term "immune response" is meant to encompass all aspects of the capability of a subject's immune system to respond to antigens and/or immunogens (e.g., both the initial response to an immunogen (e.g., a pathogen) as well as acquired (e.g., memory) responses that are a result of an adaptive immune response).

As used herein, the term "immunity" refers to protection from disease (e.g., preventing or attenuating (e.g., suppression) of a sign, symptom or condition of the disease) upon exposure to a microorganism (e.g., pathogen) capable of causing the disease. Immunity can be innate (e.g., non-adaptive (e.g., non-acquired) immune responses that exist in the absence of a previous exposure to an antigen) and/or acquired (e.g., immune responses that are mediated by B and T cells following a previous exposure to antigen (e.g., that exhibit increased specificity and reactivity to the antigen)).

As used herein, the term "immunogen" refers to an agent (e.g., a microorganism (e.g., bacterium, virus or fungus) and/or portion or component thereof (e.g., a protein antigen)) that is capable of eliciting an immune response in a subject. In some embodiments, immunogens elicit immunity against the immunogen (e.g. , microorganism (e.g., pathogen or a pathogen product)).

The term "test compound" refers to any chemical entity, pharmaceutical, drug, and the like that can be used to treat or prevent a disease, illness, sickness, or disorder of bodily function, or otherwise alter the physiological or cellular status of a sample. Test compounds comprise both known and potential therapeutic compounds. A test compound can be determined to be therapeutic by screening using the screening methods of the present invention. A "known therapeutic compound" refers to a therapeutic compound that has been shown (e.g. , through animal trials or prior experience with administration to humans) to be effective in such treatment or prevention.

The term "sample" as used herein is used in its broadest sense. In one sense it can refer to a tissue sample. In another sense, it is meant to include a specimen or culture obtained from any source, as well as biological. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include, but are not limited to blood products, such as plasma, serum and the like. These examples are not to be construed as limiting the sample types applicable to the present invention. A sample suspected of containing a human chromosome or sequences associated with a human chromosome may comprise a cell, chromosomes isolated from a cell (e.g., a spread of metaphase chromosomes), genomic DNA (in solution or bound to a solid support such as for Southern blot analysis), RNA (in solution or bound to a solid support such as for Northern blot analysis), cDNA (in solution or bound to a solid support) and the like. A sample suspected of containing a protein may comprise a cell, a portion of a tissue, an extract containing one or more proteins and the like.

DETAILED DESCRIPTION OF THE INVENTION

The principle binding site for FcRn on albumin was first shown to be located within the C-terminal Dili (Andersen et al, Nat Commun. 2012 Jan 3;3:610; Chaudhury et al. Biochemistry. 2006 Apr 18;45(15):4983-90). Then, targeting of three fully conserved histidine residues within Dili of human albumin (His464, His510 and His535) by site- directed mutagenesis revealed that all are crucial for binding (Andersen et al., 2012, supra). A docking model of the human FcRn-human albumin complex was built, where in addition to Dili, two exposed loops within the N-terminal DI were shown to be in proximity to the receptor (Andersen et al, 2012, supra). In agreement with these predictions, two recently published co-crystal structures of human FcRn in complex with human albumin confirmed the contributions from both DI and Dili (Oganesyan et al., J Biol Chem. 2014 Mar

14;289(l l):7812-24.; Schmidt et al, Structure. 2013 Nov 5;21(11): 1966-78). One of the co- crystal structures contains wild-type albumin and the other an engineered human albumin variant (HSA13) with four amino acid substitutions (V418M, T420A, E505G, and V547A). The latter has improved affinity for FcRn at both pH 6 and pH 7.4. The two co-crystal structures show highly similar modes of binding, but with some differences that are likely due to the introduced mutations in HSA13 Dili. Furthermore, both co-crystal structures show the two exposed loops in DI in contact with FcRn.

Several studies have shown that human FcRn can transport both monomeric IgG and IgG-containing immune complexes across mucosal epithelial barriers in both directions (Zhu et al, J Immunol. 2005 Jul 15;175(2):967-76; Yoshida et al, Immunity. 2004 Jun;20(6):769- 83; Spiekermann et al, J Exp Med. 2002 Aug 5;196(3):303-10. Erratum in: J Exp Med. 2003 Jun 2;197(11): 1601; Dickinson et al, J Clin Invest. 1999 Oct; 104(7): 903-11; Zhu et al, J Immunol. 2001 Mar l ;166(5):3266-76)).

Using polarized Madin-Darby canine kidney (MDCK) cells that over-express FcRn it was demonstrated that the receptor transports IgG by transcytosis from either the apical or the basolateral side (Zhu et al, J Immunol. 2001 Mar l;166(5):3266-76; Jerdeva et al, Traffic. 2010 Sep;l 1(9): 1205-20).

These findings raise the question of whether or not FcRn is capable of mediating transcytosis of albumin, and whether the stoichiometry of the interactions with FcRn plays a role, as albumin binds FcRn in a 1 : 1 manner, while IgG is homodimeric and has two binding sites for FcRn. So far, one study using MDCK cells indicate that albumin is not transcytosed (Tesar et al, Traffic. 2006 Sep;7(9): 1127-42).

Yeast display has been used to develop human albumin variants with a range of affinities toward human FcRn. One such variant (E505G/V547A) gained more than 10-fold improved affinity at pH 6.0 with a minor increase at neutral pH, which extended the plasma half-life in human FcRn transgenic mice and cynomolgus monkeys by 1.5-fold and 1.3-fold, respectively (Schmidt et al, Structure. 2013 Nov 5;21(11): 1966-78).

Furthermore, using an approach based on structural analysis and cross-species binding analyses, a single substituted human albumin variant (K573P) was identified with 12-fold improved affinity towards human FcRn at acidic pH without detectable binding at neutral pH (Andersen et al, J Biol Chem. 2014 May 9;289(19): 13492-502.). When evaluated in mice transgenic for human FcRn and cynomolgus monkeys the engineered variant showed 1.4 and 1.6-fold extended plasma half-life, respectively.

As described above, embodiments of the present invention provide fusion proteins comprising a molecule (e.g., immunogen, drug, or diagnostic agent) and an albumin variant with enhanced affinity for FcRn relative to wild type albumin. The engineered albumin variants and derived fragments with altered FcRn binding properties have improved immunogenicity, as a consequence of 1) improved transcytosis by FcRn; 2) improved biodistribution/ plasma half-life as a function of the molecular weight above the renal clearance threshold; 3) increased FcRn mediated rescue from degradation; 4) increased presentation on MHC class I and II due to FcRn mediated enhanced intracellular transport and processing by dendritic cells; 5) suitability for mucosal delivery; and 6) increased thermal stability as albumin is a very stable molecule.

Vaccine subunits fused to such albumin variants do not interfere with FcRn binding.

As FcRn functions in rescue from degradation, drives antigen presentation on MHC class I and II and allows for mucosal delivery, the pharmacokinetics and immunogenicity of the vaccines of embodiments of the present invention are improved relative to immunogens not bound to the variant albumin polypeptides. Thus, embodiments of the present invention provide improved vaccine compositions and uses thereof that overcome limitations of existing vaccines.

Experiments conducted during the course of development of embodiments of the present invention variant HSA polypeptides with altered (e.g., enhanced) affinity for FcRn (e.g., Nl l lE/V547A/K573P, T83N/V547A/K573P, T83N/E505Q/T527M/K573P,

T83N/N1 11E/V547A/K573P, Nl 1 IE/ E505Q/T527M/K573P, or

T83N/N1 11E/E505Q/T527M/K573P).

Embodiments of the present invention provide vaccines for use in mucosal delivery. The role of FcRn in vaccination and mucosal delivery has been demonstrated and described in the literature for Fc-fusions. Infectious agents such as viruses and bacteria enter the body at mucosal surfaces. Intramuscular or subcutaneous vaccination usually provides only minimal protection at sites of infection owing to suboptimal delivery and activation of the mucosal immune system. There is a close association between mucosal epithelial cells and the immune effector cells within the lamina propria, and delivery of vaccines through the mucosal surface may therefore be an ideal approach. The mucosa is a selective barrier that prevents efficient entry. Embodiments of the present invention provide compositions and methods for circumvent this problem by targeting mucosal vaccines to FcRn expressed at the mucosal epithelium. This provides secure specific transport of the intact subunit vaccines across the epithelial barrier to the mucosal immune system for subsequent induction of immune cell activation and memory.

Vaccines of embodiments of the present invention designed for mucosal delivery utilize the FcRn mediated transcytosis pathway for mucosal delivery of therapeutics or subunit vaccines (antigen/immunogen) based on fusion (chemically or genetically) to full length albumin, or albumin mutants or fragment with altered FcRn binding properties. Such vaccines find use in prevention and treatment of infection (e.g., by microorganisms), as well as in the prevention of virus induced cancers. In other embodiments, the fusions are utilized to deliver therapeutics to specific mucosal body sites. Thus, embodiments of the present invention provide methods and compositions for local delivery to the infected/inflamed site or to the site of cancer.

The present invention encompasses variants that comprise mutations (e.g., substitutions, deletions or additions) at positions other than positions described, so long as the substitution at the described position is maintained. Accordingly, in some embodiments, the albumin variants are at least 80%, 90%, 95%, 97% or 99% identical to a wild type serum albumin (e.g., wild type HSA, SEQ ID NO: l), with the proviso that the albumin variant comprises one of the mutations or deletions described herein. In some embodiments, the present invention provides fragments of the variant albumin. As above, the fragments are preferably at least 80%, 90%, 95%, 97% or 99% identical to a portion of SEQ ID NO: 1 (i.e., the parent albumin of the fragment). In some embodiments, the present invention provides fusion proteins comprising heterologous polypeptide sequence fused to a variant albumin or fragment thereof. As above, the variant albumins and fragments that form a portion of the fusion protein are preferably at least 80%, 90%, 95%, 97% or 99% identical to SEQ ID NO: l or a portion thereof (i.e., the parent albumin of the fragment), and comprise a substitution mutation as described herein.

In some embodiments, the variant albumins, fragments and fusions thereof have an increased affinity for human or mouse FcRn as compared to the corresponding wild type sequence. The skilled person will understand that any suitable method might be useful to determine whether the affinity of a variant albumin to FcRn is higher or lower than the affinity of the parent albumin to FcRn, e.g. determination and comparison of the binding constants Kd. Thus, according to the invention variant albumins having a Kd that is lower than the Kd for natural HSA is considered to have a longer plasma half-life than HSA and variant albumins having a Kd that is higher than the Kd for natural HSA is consider to have a shorter plasma half-life than HSA.

In some embodiments, the substitutions result in higher affinity for FcRn (e.g., lower Kd). For example, in some embodiments, variants have a Kd of 10, or lower, 5 or lower, 2 or lower, or 1 or lower. In some embodiments, substitutions are conservative or non- conservative changes. In some embodiments, one or more variants at a given positions that have similar side chains to the variants described herein are specifically contemplated (e.g., conservative changes relative to the variants described herein). Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic- hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Exemplary conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

Genetically encoded amino acids can be divided into four families: (1) acidic (aspartate, glutamate); (2) basic (lysine, arginine, histidine); (3) nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); and (4) uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In similar fashion, the amino acid repertoire can be grouped as (1) acidic (aspartate, glutamate); (2) basic (lysine, arginine, histidine), (3) aliphatic (glycine, alanine, valine, leucine, isoleucine, serine, threonine), with serine and threonine optionally be grouped separately as aliphatic hydroxyl; (4) aromatic (phenylalanine, tyrosine, tryptophan); (5) amide (asparagine, glutamine); and (6) sulfur containing (cysteine and methionine) (e.g., Shyer ed.,

Biochemistry, pg. 17-21, 2nd ed., WH Freeman and Co., 1981).

In some embodiments, a variant includes "nonconservative" changes (e.g., replacement of a glycine with a tryptophan). Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological activity can be found using computer programs.

The albumin or fragment thereof according to the invention may be conjugated to an immunogen (e.g., antigen) using techniques known within the art. The present invention is not limited to a particular immunogen. Any immunogen or antigenic fragment may be utilized. Examples include, but are not limited, immunogens derived from microorganisms (e.g., pathogenic microorganisms), tumors (e.g., for cancer vaccines) and the like.

The variant albumins, fragments thereof, and fusions of the present invention can be prepared using techniques well known to the skilled person. One convenient way is by cloning nucleic acid encoding the parent albumin, fragment thereof or fusion polypeptide comprising the substitution mutations described herein. The fusion proteins comprising variant albumins, fragments thereof, and fusions of the present invention may also be connected to a signal sequence in order to have the polypeptide secreted into the growth medium during culturing of the transformed host organism. It is generally advantageous to have the variant polypeptide secreted into the growth medium in order to ease recovery and purification.

Techniques for preparing variant polypeptides have also been disclosed in WO 2009019314 (included by reference) and these techniques may also be applied to the present invention. Albumins have been successfully expressed as recombinant proteins in a range of hosts including fungi (including but not limited to Aspergillus (WO06066595),

Klyveromyces (Fleer 1991, Bio/technology 9, 968-975), Pichia Pichia (Kobayashi 1998 Therapeutic Apheresis 2, 257-262) and Saccharomyces (Sleep 1990, Bio/technology 8, 42- 46)), bacteria (Pandjaitab 2000, J. Allergy Clin. Immunol 105, 279-285)), animals (Barash 1993, Transgenic Research 2, 266-276) and plants (including but not limited to potato and tobacco (Sijmons 1990, Bio/technology 8, 217 and Farran 2002, Transgenic Research 11, 337-346). The HSA domain III derivative, fragment, or variant thereof of the invention is preferably produced recombinantly in a suitable host cell. In principle any host cell capable of producing a polypeptide in suitable amounts may be used and it is within the skills of the average practitioner to select a suitable host cell according to the invention. A preferred host organism is yeast, preferably selected among Saccharomycacae, more preferred

Saccharomyces cerevisiae.

The fusion proteins comprising variant albumins, fragments thereof, and fusions of the present invention may be recovered and purified from the growth medium using a combination of known separation techniques such as filtrations, centrifugations,

chromatography, affinity separation techniques etc. It is within the skills of the average practitioner to purify the variant albumins, fragments thereof, and fusions of the invention using a particular combination of such known separation steps. As an example of purification techniques that may be applied to the variants of the present invention can be mentioned the teaching of WO0044772.

In some embodiments, fusion proteins are expressed from fusion nucleic acids using molecular biology techniques known in the art. The one or more immunogen polypeptides may be fused to the N-terminus, the C-terminus of the albumin variant or fragment thereof, inserted into a loop in the albumin variant or fragment thereof structure or any combination thereof. It may or it may not comprise linker sequences separating the various components of the fusion polypeptide. Teachings relating to fusions of albumin or a fragment thereof are known in the art and the skilled person will appreciate that such teachings can also be applied to the present invention. PCT Publications WO 01/79271A and WO 03/59934A, both of which are incorporated herein by reference, also contain examples of polypeptides that may be fused to the albumin variants and fragments thereof of the present invention and these examples apply also for the present invention.

The albumin variants or fragments thereof or fusion polypeptides comprising the albumin variants or fragments thereof according to the invention have the benefit that their plasma half-life is altered compared to the parent albumin variants or fragments thereof or fusion polypeptides comprising the albumin variants or fragments thereof. This has the advantage that the plasma half-life of conjugates comprising albumin variants or fragments thereof or fusion polypeptides comprising the albumin variants or fragments thereof according to the invention can be selected in accordance with the particular therapeutic purpose.

In other embodiments, albumin variants are conjugated to immunogens. Techniques for conjugating immunogens to the albumin derivative, fragment, or variant thereof are known in the art. WO2009019314 discloses examples of techniques suitable for conjugating a therapeutically compound to a polypeptide which techniques can also be applied to the present invention. Further WO2009019314 discloses examples of compounds and moieties that may be conjugated to substituted transferrin and these examples may also be applied to the present invention. The teaching of WO2009019314 is included herein by reference.

HSA contains in its natural form one free thiol group that conveniently may be used for conjugation. As a particular embodiment within this aspect the variant albumins, fragments thereof, and fusions of the present invention may comprise further modifications provided to generate additional free thiol groups on the surface. This has the benefit that the pay load of the albumin derivative, fragment, or variant thereof is increased so that more than one molecule of the immunogen can be conjugated to each albumin derivative, fragment, or variant thereof, or two or more different immunogens may be conjugated to each molecule of the variant albumins, fragments thereof, and fusions. Teaching of particular residues that may be modified to provide for further free thiol groups on the surface can be found in the co- pending patent application (EP 2009 152 625.1), which is incorporated by reference.

In some embodiments, the present invention provides vaccine compositions comprising an albumin variant or wild type albumin described herein and an immunogen. The present invention is not limited by the particular formulation of a composition comprising an albumin/immunogen fusion. Indeed, a vaccine composition of the present invention may comprise one or more different agents in addition to the fusion protein. These agents or cofactors include, but are not limited to, adjuvants, surfactants, additives, buffers, solubilizers, chelators, oils, salts, therapeutic agents, drugs, bioactive agents, antibacterials, and antimicrobial agents (e.g., antibiotics, antivirals, etc.). In some embodiments, a vaccine composition comprising a fusion protein comprises an agent and/or co-factor that enhance the ability of the immunogen to induce an immune response (e.g., an adjuvant). In some preferred embodiments, the presence of one or more co-factors or agents reduces the amount of immunogen required for induction of an immune response (e.g., a protective immune respone (e.g., protective immunization)). In some embodiments, the presence of one or more co-factors or agents can be used to skew the immune response towards a cellular (e.g., T cell mediated) or humoral (e.g., antibody mediated) immune response. The present invention is not limited by the type of co-factor or agent used in a therapeutic agent of the present invention.

Adjuvants are described in general in Vaccine Design—the Subunit and Adjuvant Approach, edited by Powell and Newman, Plenum Press, New York, 1995. The present invention is not limited by the type of adjuvant utilized (e.g., for use in a composition (e.g., pharmaceutical composition). For example, in some embodiments, suitable adjuvants include an aluminium salt such as aluminium hydroxide gel (alum) or aluminium phosphate. In some embodiments, an adjuvant may be a salt of calcium, iron or zinc, or may be an insoluble suspension of acylated tyrosine, or acylated sugars, cationically or anionically derivatised polysaccharides, or polyphosphazenes.

In general, an immune response is generated to an antigen through the interaction of the antigen with the cells of the immune system. Immune responses may be broadly categorized into two categories: humoral and cell mediated immune responses (e.g., traditionally characterized by antibody and cellular effector mechanisms of protection, respectively). These categories of response have been termed Thl -type responses (cell- mediated response), and Th2-type immune responses (humoral response).

Stimulation of an immune response can result from a direct or indirect response of a cell or component of the immune system to an intervention (e.g., exposure to an immunogen). Immune responses can be measured in many ways including activation, proliferation or differentiation of cells of the immune system (e.g., B cells, T cells, dendritic cells, APCs, macrophages, NK cells, NKT cells etc.); up-regulated or down-regulated expression of markers and cytokines; stimulation of IgA, IgM, or IgG titer; splenomegaly (including increased spleen cellularity); hyperplasia and mixed cellular infiltrates in various organs. Other responses, cells, and components of the immune system that can be assessed with respect to immune stimulation are known in the art.

Although an understanding of the mechanism is not necessary to practice the present invention and the present invention is not limited to any particular mechanism of action, in some embodiments, compositions and methods of the present invention induce expression and secretion of cytokines (e.g., by macrophages, dendritic cells and CD4+ T cells).

Modulation of expression of a particular cytokine can occur locally or systemically. It is known that cytokine profiles can determine T cell regulatory and effector functions in immune responses. In some embodiments, Thl-type cytokines can be induced, and thus, the immunostimulatory compositions of the present invention can promote a Thl type antigen- specific immune response including cytotoxic T-cells (e.g., thereby avoiding unwanted Th2 type immune responses (e.g., generation of Th2 type cytokines (e.g., IL-13) involved in enhancing the severity of disease (e.g., IL-13 induction of mucus formation))).

Cytokines play a role in directing the T cell response. Helper (CD4+) T cells orchestrate the immune response of mammals through production of soluble factors that act on other immune system cells, including B and other T cells. Most mature CD4+T helper cells express one of two cytokine profiles: Thl or Th2. Thl -type CD4+ T cells secrete IL-2, IL-3, IFN-γ, GM-CSF and high levels of TNF-a. Th2 cells express IL-3, IL-4, IL-5, IL-6, IL- 9, IL-10, IL-13, GM-CSF and low levels of TNF-a. Thl type cytokines promote both cell- mediated immunity, and humoral immunity that is characterized by immunoglobulin class switching to IgG2a in mice and IgGl in humans. Thl responses may also be associated with delayed-type hypersensitivity and autoimmune disease. Th2 type cytokines induce primarily humoral immunity and induce class switching to IgGl and IgE. The antibody isotypes associated with Thl responses generally have neutralizing and opsonizing capabilities whereas those associated with Th2 responses are associated more with allergic responses.

Several factors have been shown to influence skewing of an immune response towards either a Thl or Th2 type response. The best characterized regulators are cytokines. IL-12 and IFN-γ are positive Thl and negative Th2 regulators. IL-12 promotes IFN- γ production, and IFN-γ provides positive feedback for IL-12. IL-4 and IL-10 appear important for the establishment of the Th2 cytokine profile and to down-regulate Thl cytokine production.

Thus, in preferred embodiments, the present invention provides a method of stimulating a Thl -type immune response in a subject comprising administering to a subject a composition comprising an immunogen. However, in other embodiments, the present invention provides a method of stimulating a Th2-type immune response in a subject (e.g., if balancing of a T cell mediated response is desired) comprising administering to a subject a composition comprising an immunogen. In further preferred embodiments, adjuvants can be used (e.g., can be co-administered with a composition of the present invention) to skew an immune response toward either a Thl or Th2 type immune response. For example, adjuvants that induce Th2 or weak Thl responses include, but are not limited to, alum, saponins, and SB-As4. Adjuvants that induce Thl responses include but are not limited to MPL, MDP, ISCOMS, IL-12, IFN- γ, and SB-AS2.

Several other types of Thl -type immunogens can be used (e.g., as an adjuvant) in compositions and methods of the present invention. These include, but are not limited to, the following. In some embodiments, monophosphoryl lipid A (e.g., in particular 3-de-O- acylated monophosphoryl lipid A (3D-MPL)), is used. 3D-MPL is a well known adjuvant manufactured by Ribi Immunochem, Montana. Chemically it is often supplied as a mixture of 3-de-O-acylated monophosphoryl lipid A with either 4, 5, or 6 acylated chains. In some embodiments, diphosphoryl lipid A, and 3-O-deacylated variants thereof are used. Each of these immunogens can be purified and prepared by methods described in GB 2122204B, hereby incorporated by reference in its entirety. Other purified and synthetic

lipopolysaccharides have been described (See, e.g., U.S. Pat. No. 6,005,099 and EP 0 729 473; Hilgers et al, 1986, Int. Arch. Allergy. Immunol, 79(4):392-6; Hilgers et al, 1987, Immunology, 60(1): 141-6; and EP 0 549 074, each of which is hereby incorporated by reference in its entirety). In some embodiments, 3D-MPL is used in the form of a particulate formulation (e.g., having a small particle size less than 0.2 μιτι in diameter, described in EP 0 689 454, hereby incorporated by reference in its entirety).

In some embodiments, saponins are used as an immunogen (e.g.,Thl-type adjuvant) in a composition of the present invention. Saponins are well known adjuvants (See, e.g., Lacaille-Dubois and Wagner (1996) Phytomedicine vol 2 pp 363-386). Examples of saponins include Quil A (derived from the bark of the South American tree Quillaja

Saponaria Molina), and fractions thereof (See, e.g., U.S. Pat. No. 5,057,540; Kensil, Crit Rev Ther Drug Carrier Syst, 1996, 12 (1-2): 1-55; and EP 0 362 279, each of which is hereby incorporated by reference in its entirety). Also contemplated to be useful in the present invention are the haemolytic saponins QS7, QS17, and QS21 (HPLC purified fractions of Quil A; See, e.g., Kensil et al. (1991). J. Immunology 146,431-437, U.S. Pat. No. 5,057,540; WO 96/33739; WO 96/11711 and EP 0 362 279, each of which is hereby incorporated by reference in its entirety). Also contemplated to be useful are combinations of QS21 and polysorbate or cyclodextrin (See, e.g., WO 99/10008, hereby incorporated by reference in its entirety.

In some embodiments, an immunogenic oligonucleotide containing unmethylated CpG dinucleotides ("CpG") is used as an adjuvant. CpG is an abbreviation for cytosine- guanosine dinucleotide motifs present in DNA. CpG is known in the art as being an adjuvant when administered by both systemic and mucosal routes (See, e.g., WO 96/02555, EP 468520, Davis et al, J.Immunol, 1998, 160(2):870-876; McCluskie and Davis, J.Immunol, 1998, 161(9):4463-6; and U.S. Pat. App. No. 20050238660, each of which is hereby incorporated by reference in its entirety). For example, in some embodiments, the immunostimulatory sequence is Purine-Purine-C-G-pyrimidine-pyrirnidine; wherein the CG motif is not methylated.

Although an understanding of the mechanism is not necessary to practice the present invention and the present invention is not limited to any particular mechanism of action, in some embodiments, the presence of one or more CpG oligonucleotides activate various immune subsets including natural killer cells (which produce IFN-γ) and macrophages. In some embodiments, CpG oligonucleotides are formulated into a composition of the present invention for inducing an immune response. In some embodiments, a free solution of CpG is co-administered together with an antigen (e.g., present within a solution (See, e.g., WO 96/02555; hereby incorporated by reference). In some embodiments, a CpG oligonucleotide is covalently conjugated to an antigen (See, e.g., WO 98/16247, hereby incorporated by reference), or formulated with a carrier such as aluminium hydroxide (See, e.g., Brazolot- Millan et al, Proc.Natl.AcadSci., USA, 1998, 95(26), 15553-8).

In some embodiments, adjuvants such as Complete Freunds Adjuvant and Incomplete Freunds Adjuvant, cytokines (e.g., interleukins (e.g., IL-2, IFN-γ, IL-4, etc.), macrophage colony stimulating factor, tumor necrosis factor, etc.), detoxified mutants of a bacterial ADP- ribosylating toxin such as a cholera toxin (CT), a pertussis toxin (PT), or an E. Coli heat- labile toxin (LT), particularly LT-K63 (where lysine is substituted for the wild-type amino acid at position 63) LT-R72 (where arginine is substituted for the wild-type amino acid at position 72), CT-S109 (where serine is substituted for the wild-type amino acid at position 109), and PT-K9/G129 (where lysine is substituted for the wild-type amino acid at position 9 and glycine substituted at position 129) (See, e.g., WO93/13202 and W092/19265, each of which is hereby incorporated by reference), and other immunogenic substances (e.g., that enhance the effectiveness of a composition of the present invention) are used with a composition comprising an immunogen of the present invention. Additional examples of adjuvants that find use in the present invention include poly(di(carboxylatophenoxy)phosphazene (PCPP polymer; Virus Research Institute, USA); derivatives of lipopolysaccharides such as monophosphoryl lipid A (MPL; Ribi

ImmunoChem Research, Inc., Hamilton, Mont.), muramyl dipeptide (MDP; Ribi) and threonyl-muramyl dipeptide (t-MDP; Ribi); OM-174 (a glucosamine disaccharide related to lipid A; OM Pharma SA, Meyrin, Switzerland); and Leishmania elongation factor (a purified Leishmania protein; Corixa Corporation, Seattle, Wash.).

Adjuvants may be added to a composition comprising an immunogen, or, the adjuvant may be formulated with carriers, for example liposomes, or metallic salts (e.g., aluminium salts (e.g., aluminium hydroxide)) prior to combining with or co-administration with a composition.

In some embodiments, a composition comprising an immunogen comprises a single adjuvant. In other embodiments, a composition comprises two or more adjuvants (See, e.g., WO 94/00153; WO 95/17210; WO 96/33739; WO 98/56414; WO 99/12565; WO 99/11241 ; and WO 94/00153, each of which is hereby incorporated by reference in its entirety).

In some embodiments, a composition comprising an immunogen comprises one or more mucoadhesives (See, e.g., U.S. Pat. App. No. 20050281843, hereby incorporated by reference in its entirety). The present invention is not limited by the type of mucoadhesive utilized. Indeed, a variety of mucoadhesives are contemplated to be useful in the present invention including, but not limited to, cross-linked derivatives of poly(acrylic acid) (e.g., carbopol and polycarbophil), polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides (e.g., alginate and chitosan), hydroxypropyl methylcellulose, lectins, fimbrial proteins, and carboxymethylcellulose. Although an understanding of the mechanism is not necessary to practice the present invention and the present invention is not limited to any particular mechanism of action, in some embodiments, use of a mucoadhesive (e.g., in a composition comprising an immunogen) enhances induction of an immune response in a subject (e.g., administered a composition of the present invention) due to an increase in duration and/or amount of exposure to an immunogen that a subject experiences when a mucoadhesive is used compared to the duration and/or amount of exposure to an immunogen in the absence of using the mucoadhesive.

The in vivo pharmacokinetics of small therapeutic and diagnostic molecules are hampered by their very short plasma half-life since they are very rapidly cleared from the blood as a function of kidney clearance and degradation within cells that cover the circulation. If such molecules are genetically or chemically fused to the human albumin variants described above, they may acquire improved biodistribution as a consequence of increased size and altered plasma half-life. Although albumin fusions are in clinical trials, they are all fused to complete wild type albumin. Accordingly, in some embodiments, the present invention provides fusions of the variant HSA polypeptides described herein to diagnostic or therapeutic molecules (e.g., small molecule, peptide, or polypeptide drugs or diagnostic agents). The human albumin mutants may be used to tailor biodistribution and plasma half-life of drugs (e.g., drugs used for instance for tumor targeting).

In some embodiments, a composition of the present invention may comprise sterile aqueous preparations. Acceptable vehicles and solvents include, but are not limited to, water, Ringer's solution, phosphate buffered saline 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 mineral or non-mineral oil may be employed including synthetic mono-ordi-glycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. Carrier formulations suitable for mucosal, subcutaneous, intramuscular, intraperitoneal, intravenous, or administration via other routes may be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa.

A composition comprising an immunogen of the present invention can be used therapeutically (e.g., to enhance an immune response) or as a prophylactic (e.g., for immunization (e.g., to prevent signs or symptoms of disease)). A composition comprising an immunogen of the present invention can be administered to a subject via a number of different delivery routes and methods.

For example, the compositions of the present invention can be administered to a subject (e.g., mucosally (e.g., nasal mucosa, vaginal mucosa, etc.)) by multiple methods, including, but not limited to: being suspended in a solution and applied to a surface; being suspended in a solution and sprayed onto a surface using a spray applicator; being mixed with a mucoadhesive and applied (e.g., sprayed or wiped) onto a surface (e.g., mucosal surface); being placed on or impregnated onto a nasal and/or vaginal applicator and applied; being applied by a controlled-release mechanism; being applied as a liposome; or being applied on a polymer.

In some embodiments, compositions of the present invention are administered mucosally (e.g., using standard techniques; See, e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Company, Easton, Pa., 19th edition, 1995 (e.g., for mucosal delivery techniques, including intranasal, pulmonary, vaginal and rectal techniques), as well as European Publication No. 517,565 and Ilium et al, J. Controlled Rel., 1994, 29: 133-141 (e.g., for techniques of intranasal administration), each of which is hereby incorporated by reference in its entirety). Alternatively, the compositions of the present invention may be administered dermally or trans dermally, using standard techniques (See, e.g., Remington: The Science arid Practice of Pharmacy, Mack Publishing Company, Easton, Pa., 19th edition, 1995). The present invention is not limited by the route of administration.

Although an understanding of the mechanism is not necessary to practice the present invention and the present invention is not limited to any particular mechanism of action, in some embodiments, mucosal vaccination is the preferred route of administration as it has been shown that mucosal administration of antigens has a greater efficacy of inducing protective immune responses at mucosal surfaces (e.g., mucosal immunity), the route of entry of many pathogens. In addition, mucosal vaccination, such as intranasal vaccination, may induce mucosal immunity not only in the nasal mucosa, but also in distant mucosal sites such as the genital mucosa (See, e.g., Mestecky, Journal of Clinical Immunology, 7:265-276, 1987). More advantageously, in further preferred embodiments, in addition to inducing mucosal immune responses, mucosal vaccination also induces systemic immunity. In some embodiments, non-parenteral administration (e.g., muscosal administration of vaccines) provides an efficient and convenient way to boost systemic immunity (e.g., induced by parenteral or mucosal vaccination (e.g., in cases where multiple boosts are used to sustain a vigorous systemic immunity)).

In some embodiments, a composition comprising an immunogen of the present invention may be used to protect or treat a subject susceptible to, or suffering from, disease by means of administering a composition of the present invention via a mucosal route (e.g., an oral/alimentary or nasal route). Alternative mucosal routes include intravaginal and intrarectal routes. In preferred embodiments of the present invention, a nasal route of

administration is used, termed "intranasal administration" or "intranasal vaccination" herein. Methods of intranasal vaccination are well known in the art, including the administration of a droplet or spray form of the vaccine into the nasopharynx of a subject to be immunized. In some embodiments, a nebulized or aerosolized composition is provided. Enteric

formulations such as gastro resistant capsules for oral administration, suppositories for rectal or vaginal administration also form part of this invention. Compositions of the present invention may also be administered via the oral route. Under these circumstances, a composition comprising an immunogen may comprise a pharmaceutically acceptable excipient and/or include alkaline buffers, or enteric capsules. Formulations for nasal delivery may include those with dextran or cyclodextran and saponin as an adjuvant. Compositions of the present invention may also be administered via a vaginal route. In such cases, a composition comprising an immunogen may comprise pharmaceutically acceptable excipients and/or emulsifiers, polymers (e.g., CARBOPOL), and other known stabilizers of vaginal creams and suppositories. In some embodiments, compositions of the present invention are administered via a rectal route. In such cases, compositions may comprise excipients and/or waxes and polymers known in the art for forming rectal suppositories.

In some embodiments, the same route of administration (e.g., mucosal administration) is chosen for both a priming and boosting vaccination. In some embodiments, multiple routes of administration are utilized (e.g., at the same time, or, alternatively, sequentially) in order to stimulate an immune response.

For example, in some embodiments, a composition comprising an immunogen is administered to a mucosal surface of a subject in either a priming or boosting vaccination regime. Alternatively, in some embodiments, the composition is administered systemically in either a priming or boosting vaccination regime. In some embodiments, a composition comprising an immunogen is administered to a subject in a priming vaccination regimen via mucosal administration and a boosting regimen via systemic administration. In some embodiments, a composition comprising an immunogen is administered to a subject in a priming vaccination regimen via systemic administration and a boosting regimen via mucosal administration. Examples of systemic routes of administration include, but are not limited to, a parenteral, intramuscular, intradermal, transdermal, subcutaneous, intraperitoneal or intravenous administration. A composition comprising an immunogen may be used for both prophylactic and therapeutic purposes.

In some embodiments, compositions of the present invention are administered by pulmonary delivery. For example, a composition of the present invention can be delivered to the lungs of a subject (e.g., a human) via inhalation (e.g., thereby traversing across the lung epithelial lining to the blood stream (See, e.g., Adjei, et al. Pharmaceutical Research 1990; 7:565-569; Adjei, et al. Int. J. Pharmaceutics 1990; 63: 135-144; Braquet, et al. J.

Cardiovascular Pharmacology 1989 143-146; Hubbard, et al. (1989) Annals of Internal Medicine, Vol. Ill, pp. 206-212; Smith, et al. J. Clin. Invest. 1989;84: 1145-1146; Oswein, et al. "Aerosolization of Proteins", 1990; Proceedings of Symposium on Respiratory Drug Delivery II Keystone, Colorado; Debs, et al. J. Immunol. 1988; 140:3482-3488; and U.S. Pat. No. 5,284,656 to Platz, et al, each of which are hereby incorporated by reference in its entirety). A method and composition for pulmonary delivery of drugs for systemic effect is described in U.S. Pat. No. 5,451,569 to Wong, et al, hereby incorporated by reference; See also U.S. Pat. No. 6,651,655 to Licalsi et al, hereby incorporated by reference in its entirety)).

Further contemplated for use in the practice of this invention are a wide range of mechanical devices designed for pulmonary and/or nasal mucosal delivery of pharmaceutical agents including, but not limited to, nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. Some specific examples of commercially available devices suitable for the practice of this invention are the Ultravent nebulizer (Mallinckrodt Inc., St. Louis, Mo.); the Acorn II nebulizer (Marquest Medical Products, Englewood, Colo.); the Ventolin metered dose inhaler (Glaxo Inc., Research Triangle Park, N.C.); and the Spinhaler powder inhaler (Fisons Corp., Bedford, Mass.). All such devices require the use of formulations suitable for dispensing of the therapeutic agent. Typically, each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to the usual diluents, adjuvants, surfactants, carriers and/or other agents useful in therapy. Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated.

Thus, in some embodiments, a composition comprising an immunogen of the present invention may be used to protect and/or treat a subject susceptible to, or suffering from, a disease by means of administering the composition by mucosal, intramuscular,

intraperitoneal, intradermal, transdermal, pulmonary, intravenous, subcutaneous or other route of administration described herein. Methods of systemic administration of the vaccine preparations may include conventional syringes and needles, or devices designed for ballistic delivery of solid vaccines (See, e.g., WO 99/27961, hereby incorporated by reference), or needleless pressure liquid jet device (See, e.g., U.S. Pat. No. 4,596,556; U.S. Pat. No.

5,993,412, each of which are hereby incorporated by reference), or transdermal patches (See, e.g., WO 97/48440; WO 98/28037, each of which are hereby incorporated by reference). The present invention may also be used to enhance the immunogenicity of antigens applied to the skin (transdermal or transcutaneous delivery, See, e.g., WO 98/20734 ; WO 98/28037, each of which are hereby incorporated by reference). Thus, in some embodiments, the present invention provides a delivery device for systemic administration, pre-filled with the vaccine composition of the present invention.

The present invention is not limited by the type of subject administered (e.g., in order to stimulate an immune response (e.g., in order to generate protective immunity (e.g., mucosal and/or systemic immunity))) a composition of the present invention. Indeed, a wide variety of subjects are contemplated to be benefited from administration of a composition of the present invention. In preferred embodiments, the subject is a human. In some embodiments, human subjects are of any age (e.g., adults, children, infants, etc.) that have been or are likely to become exposed to a microorganism (e.g., E. coli). In some

embodiments, the human subjects are subjects that are more likely to receive a direct exposure to pathogenic microorganisms or that are more likely to display signs and symptoms of disease after exposure to a pathogen (e.g., immune suppressed subjects). In some embodiments, the general public is administered (e.g., vaccinated with) a composition of the present invention (e.g., to prevent the occurrence or spread of disease). For example, in some embodiments, compositions and methods of the present invention are utilized to vaccinate a group of people (e.g., a population of a region, city, state and/or country) for their own health (e.g., to prevent or treat disease). In some embodiments, the subjects are non- human mammals (e.g., pigs, cattle, goats, horses, sheep, or other livestock; or mice, rats, rabbits or other animal). In some embodiments, compositions and methods of the present invention are utilized in research settings (e.g., with research animals).

A composition of the present invention may be formulated for administration by any route, such as mucosal, oral, transdermal, intranasal, parenteral or other route described herein. The compositions may be in any one or more different forms including, but not limited to, tablets, capsules, powders, granules, lozenges, foams, creams or liquid preparations.

Topical formulations of the present invention may be presented as, for instance, ointments, creams or lotions, foams, and aerosols, and may contain appropriate conventional additives such as preservatives, solvents (e.g., to assist penetration), and emollients in ointments and creams.

Topical formulations may also include agents that enhance penetration of the active ingredients through the skin. Exemplary agents include a binary combination of N- (hydroxy ethyl) pyrrolidone and a cell-envelope disordering compound, a sugar ester in combination with a sulfoxide or phosphine oxide, and sucrose monooleate, decyl methyl sulfoxide, and alcohol.

Other exemplary materials that increase skin penetration include surfactants or wetting agents including, but not limited to, poly oxy ethylene sorbitan mono-oleoate

(Polysorbate 80); sorbitan mono-oleate (Span 80); p-isooctyl polyoxyethylene-phenol polymer (Triton WR-1330); poly oxy ethylene sorbitan tri-oleate (Tween 85); dioctyl sodium sulfosuccinate; and sodium sarcosinate (Sarcosyl NL-97); and other pharmaceutically acceptable surfactants.

In certain embodiments of the invention, compositions may further comprise one or more alcohols, zinc-containing compounds, emollients, humectants, thickening and/or gelling agents, neutralizing agents, and surfactants. Water used in the formulations is preferably deionized water having a neutral pH. Additional additives in the topical formulations include, but are not limited to, silicone fluids, dyes, fragrances, pH adjusters, and vitamins. Topical formulations may also contain compatible conventional carriers, such as cream or ointment bases and ethanol or oleyl alcohol for lotions. Such carriers may be present as from about 1% up to about 98% of the formulation. The ointment base can comprise one or more of petrolatum, mineral oil, ceresin, lanolin alcohol, panthenol, glycerin, bisabolol, cocoa butter and the like.

In some embodiments, pharmaceutical compositions of the present invention may be formulated and used as foams. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product. The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, preferably do not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents (e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like) that do not deleteriously interact with the immunogen or other components of the formulation. In some embodiments, immunostimulatory

compositions of the present invention are administered in the form of a pharmaceutically acceptable salt. When used the salts should be pharmaceutically acceptable, but non- pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof. Such salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric, citric, methane sulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, and benzene sulphonic. Also, such salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.

Suitable buffering agents include, but are not limited to, acetic acid and a salt (1-2% w/v); citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v); and phosphoric acid and a salt (0.8-2% w/v). Suitable preservatives may include benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).

In some embodiments, vaccine compositions are co-administered with one or more antibiotics. For example, one or more antibiotics may be administered with, before and/or after administration of the composition. The present invention is not limited by the type of antibiotic co-administered. Indeed, a variety of antibiotics may be co-administered including, but not limited to, β-lactam antibiotics, penicillins (such as natural penicillins,

aminopenicillins, penicillinase-resistant penicillins, carboxy penicillins, ureido penicillins), cephalosporins (first generation, second generation, and third generation cephalosporins), and other β-lactams (such as imipenem, monobactams,), β -lactamase inhibitors, vancomycin, aminoglycosides and spectinomycin, tetracyclines, chloramphenicol, erythromycin, lincomycin, clindamycin, rifampin, metronidazole, polymyxins, doxycycline, quinolones (e.g., ciprofloxacin), sulfonamides, trimethoprim, and quinolines.

There are an enormous amount of antimicrobial agents currently available for use in treating bacterial, fungal and viral infections. For a comprehensive treatise on the general classes of such drugs and their mechanisms of action, the skilled artisan is referred to Goodman & Gilman's "The Pharmacological Basis of Therapeutics" Eds. Hardman et al , 9th Edition, Pub. McGraw Hill, chapters 43 through 50, 1996, (herein incorporated by reference in its entirety). Generally, these agents include agents that inhibit cell wall synthesis (e.g., penicillins, cephalosporins, cycloserine, vancomycin, bacitracin); and the imidazole antifungal agents (e.g., miconazole, ketoconazole and clotrimazole); agents that act directly to disrupt the cell membrane of the microorganism (e.g., detergents such as polmyxin and colistimethate and the antifungals nystatin and amphotericin B); agents that affect the ribosomal subunits to inhibit protein synthesis (e.g., chloramphenicol, the tetracyclines, erthromycin and clindamycin); agents that alter protein synthesis and lead to cell death (e.g., aminoglycosides); agents that affect nucleic acid metabolism (e.g., the rifamycins and the quinolones); the antimetabolites (e.g., trimethoprim and sulfonamides); and the nucleic acid analogues such as zidovudine, gangcyclovir, vidarabine, and acyclovir which act to inhibit viral enzymes essential for DNA synthesis. Various combinations of antimicrobials may be employed.

The present invention also includes methods involving co-administration of a vaccine composition comprising an immunogen with one or more additional active and/or immunostimulatory agents (e.g., a composition comprising a different immunogen, an antibiotic, anti-oxidant, etc.). Indeed, it is a further aspect of this invention to provide methods for enhancing prior art immunostimulatory methods (e.g., immunization methods) and/or pharmaceutical compositions by co-administering a composition of the present invention. In co-administration procedures, the agents may be administered concurrently or sequentially. In one embodiment, the compositions described herein are administered prior to the other active agent(s). The pharmaceutical formulations and modes of administration may be any of those described herein. In addition, the two or more co-administered agents may each be administered using different modes (e.g., routes) or different formulations. The additional agents to be co-administered (e.g., antibiotics, adjuvants, etc.) can be any of the well-known agents in the art, including, but not limited to, those that are currently in clinical use.

In some embodiments, a composition comprising an immunogen is administered to a subject via more than one route. For example, a subject that would benefit from having a protective immune response (e.g., immunity) towards a pathogenic microorganism may benefit from receiving mucosal administration (e.g., nasal administration or other mucosal routes described herein) and, additionally, receiving one or more other routes of

administration (e.g., parenteral or pulmonary administration (e.g., via a nebulizer, inhaler, or other methods described herein). In some preferred embodiments, administration via mucosal route is sufficient to induce both mucosal as well as systemic immunity towards an immunogen or organism from which the immunogen is derived. In other embodiments, administration via multiple routes serves to provide both mucosal and systemic immunity. Thus, although an understanding of the mechanism is not necessary to practice the present invention and the present invention is not limited to any particular mechanism of action, in some embodiments, it is contemplated that a subject administered a composition of the present invention via multiple routes of administration (e.g., immunization (e.g., mucosal as well as airway or parenteral administration of the composition) may have a stronger immune response to an immunogen than a subj ect administered a composition via just one route. Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the compositions, increasing convenience to the subject and a physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer based systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides.

Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Pat. No. 5,075,109, hereby incorporated by reference. Delivery systems also include non- polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-di-and tri-glycerides; hydrogel release systems;

sylastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include, but are not limited to: (a) erosional systems in which an agent of the invention is contained in a form within a matrix such as those described in U.S. Pat. Nos. 4,452,775, 4,675,189, and 5,736,152, each of which is hereby incorporated by reference and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Pat. Nos. 3,854,480, 5,133,974 and 5,407,686, each of which is hereby incorporated by reference. In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation.

In some embodiments, a vaccine composition of the present invention is formulated in a concentrated dose that can be diluted prior to administration to a subject. For example, dilutions of a concentrated composition may be administered to a subject such that the subject receives any one or more of the specific dosages provided herein. In some embodiments, dilution of a concentrated composition may be made such that a subject is administered (e.g., in a single dose) a composition comprising 0.5-50% of a nanoemulsion and immunogen present in the concentrated composition. Concentrated compositions are contemplated to be useful in a setting in which large numbers of subjects may be administered a composition of the present invention (e.g., an immunization clinic, hospital, school, etc.). In some embodiments, a composition comprising an immunogen of the present invention (e.g., a concentrated composition) is stable at room temperature for more than 1 week, in some embodiments for more than 2 weeks, in some embodiments for more than 3 weeks, in some embodiments for more than 4 weeks, in some embodiments for more than 5 weeks, and in some embodiments for more than 6 weeks. In some embodiments, following an initial administration of a composition of the present invention (e.g., an initial vaccination), a subject may receive one or more boost administrations (e.g., around 2 weeks, around 3 weeks, around 4 weeks, around 5 weeks, around 6 weeks, around 7 weeks, around 8 weeks, around 10 weeks, around 3 months, around 4 months, around 6 months, around 9 months, around 1 year, around 2 years, around 3 years, around 5 years, around 10 years) subsequent to a first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, and/or more than tenth administration. Although an

understanding of the mechanism is not necessary to practice the present invention and the present invention is not limited to any particular mechanism of action, in some embodiments, reintroduction of an immunogen in a boost dose enables vigorous systemic immunity in a subject. The boost can be with the same formulation given for the primary immune response, or can be with a different formulation that contains the immunogen. The dosage regimen will also, at least in part, be determined by the need of the subject and be dependent on the judgment of a practitioner.

Dosage units may be proportionately increased or decreased based on several factors including, but not limited to, the weight, age, and health status of the subject. In addition, dosage units may be increased or decreased for subsequent administrations (e.g., boost administrations).

It is contemplated that the compositions and methods of the present invention will find use in various settings, including research settings. For example, compositions and methods of the present invention also find use in studies of the immune system (e.g., characterization of adaptive immune responses (e.g., protective immune responses (e.g., mucosal or systemic immunity))). Uses of the compositions and methods provided by the present invention encompass human and non-human subjects and samples from those subjects, and also encompass research applications using these subjects. Compositions and methods of the present invention are also useful in studying and optimizing albumin variant, immunogens, and other components and for screening for new components. Thus, it is not intended that the present invention be limited to any particular subject and/or application setting.

The present invention further provides kits comprising the vaccine compositions comprised herein. In some embodiments, the kit includes all of the components necessary, sufficient or useful for administering the vaccine. For example, in some embodiments, the kits comprise devices for administering the vaccine (e.g., needles or other injection devices), temperature control components (e.g., refrigeration or other cooling components), sanitation components (e.g., alcohol swabs for sanitizing the site of injection) and instructions for administering the vaccine.

EXPERIMENTAL

Example 1

Construction and production of HSA variants - The cDNA fragments encoding WT HSA, HSA with Dili substitutions (K573P/ K573P/V547A and K573P/E505Q/T527M) and HSA with a combination of Dili and DI substitutions (Nl 11E/K573P, T83N/N111E/K573P, N111E/K573P/V547A, T83N/K573P/V547A, T83N/N111E/K573P/V547A,

Nl 11E/K573P/E505/QT527M, T83N/K573P/E505Q/T527M and

T83N/N111E/K573P/E505Q/T527M) were ordered from Genscript and cloned into a pcDNA3 vector (Invitrogen). Adherent human embryonic kidney (HEK) 293E cells were grown in RPMI to 95% confluency, before the DNA constructs were introduced by transient transfection using polyethylenimine Max (Polysciences), and supematants were subsequently harvested every second day for 12 days. Purification of HSA variants were done using

Capture Select Human Serum Albumin affinity matrix (Invitrogen) packed in a 5 ml column (Atoll). The column was pre-equilibrated with 100 ml of lxPBS supplemented with 0.05% sodium azide (pH 7.2), before supematants were applied with a flow-rate of 1-2 ml/min. Washing was performed with 100 ml of lxPBS/0.05% sodium azide and bound protein was eluted with 45 ml of 0.1 M Gly-HCl (pH 3.0) and neutralized with 5 ml 1 M Tris-HCl (pH 8.0). Eluted fractions were concentrated and buffer-exchanged using Amicon Ultra columns (Millipore) with 30 kDa cut-off. Protein concentrations were determined using a DS-11 spectrophotometer (DeNovix Inc.) and 2 μg of each protein product were analyzed on a 12% NuPAGE SDS-PAGE (Thermo Fisher Scientific).

SPR - SPR analysis was performed using a Biacore 3000 instrument (GE Healthcare).

Following the manufacturer^' s protocol, CM5 sensor chips were coupled with HSA variants (-500 RU) using amine coupling chemistry, by injecting 6 μg/ml HSA in 10 mM sodium acetate, pH 4.5 (GE Healthcare), and unreacted moieties were subsequently blocked with 1 M ethanolamine. Phosphate buffer (67 mM phosphate buffer, 0.15 M NaCl, 0.005% Tween 20) pH 6.0, was used as running buffer and for preparation of serial dilutions of souble human FcRn-his (0.03-1 μΜ), which were injected at 40 μΐ/min at 25°C. All binding curves were zero adjusted and the reference cell value was subtracted, before binding kinetics were estimated using the Langmuir 1 : 1 ligand binding model provided by the BIAevaluation 4.1 software. Results are shown in Table 1 and Figures 1 and 2. Table 1. SPR derived kinetics for binding of HSA variants to human FcRn

The kinetic rate constants were obtained using a simple first-order (1 : 1) Langmuir binding model.

Example 2

EXPERIMENTAL PROCEDURES

Construction and production of human serum albumin (HSA) variants - The cDNA fragments encoding WT HSA, HSA with Dili substitution (K573P,

E501P/N503H/E505D/T506S and E501P/N503H/E505D/T506S/K573P) and HSA with a combination of DI and Dili substitutions (N111E/V547A/K573P, T83N/V547A/K573P, T83N/N111E/V547A/K573P, Nl 11E/E505Q/T527M/K573P, T83N/E505Q/T527M/K573P and T83N/N111E/E505Q/T527M/K573P) were ordered from Genscript and cloned into a pcDNA3 vector (Invitrogen). Adherent human embryonic kidney (HEK) 293E cells were grown in freestyle 293 expression medium to 95% confluency, before the DNA constructs were introduced by transient transfection using polyethylenimine Max (Polysciences), and supematants were subsequently harvested every second day for 12 days. Purification of HSA was done using Capture Select Human Serum Albumin affinity matrix (Invitrogen) packed in a 5 ml column (Atoll). The column was pre-equilibrated with 100 ml of lx PBS

supplemented with 0.05% sodium azide (pH 7.2), before supernatant was applied with a flow-rate of 1-2 ml/min. Washing was performed with 100 ml of lxPBS/0.05% sodium azide and bound protein was eluted with 45 ml of 0.1M Gly-HCl (pH 3.0) and neutralized with 5 ml 1M Tris-HCl (pH 8.0). Eluted fractions were concentrated and buffer-exchanged using Amicon Ultra columns (Millipore) with 30 kDa cut-off. Protein concentrations were determined using a DS-11 spectrophotometer (DeNovix Inc.) and 2 μg of each protein product were analyzed on a 12% NuPAGE SDS-PAGE (Thermo Fisher Scientific).

SPR - SPR analysis was performed using a Biacore 3000 instrument (GE

Healthcare). Following the manufacturer s protocol, CM5 sensor chips were coupled with HSA variants (-500 RU) using amine coupling chemistry, by injecting 6 μg/ml HSA in 10 mM sodium acetate, pH 4.5 (GE Healthcare), and unreacted moieties were subsequently blocked with 1 M ethanolamine. Phosphate buffer (67 mM phosphate buffer, 0.15 M NaCl, 0.005% Tween 20) at pH 5.5, was used as running buffer and for preparation of serial dilutions of souble human FcRn-his (0.03-1 μΜ), which were injected at 40 μΐ/min at 25°C. HBS-P buffer (0.01 M Hepes, 0.15 M NaCl, 0.005% surfactant P20) at pH 7.4 was used for regeneration of the flow cells. All binding curves were zero adjusted and the reference cell value was subtracted, before binding kinetics were estimated using the Langmuir 1 : 1 ligand binding model provided by the BIAevaluation 4.1 software.

FcRn-albumin binding ELISA - 96-well ELISA plates (Costar) were coated with 100 μΐ/well of a human IgGl mutant variant (M252Y/S254T/T256E/H433K/N434F) with specificity for 4-hydroxy-3-iodo-5- nitrophenylacetic acid (10 μg/ml) in PBS pH 7.4 and incubated over night at 4°C. The wells were blocked with 200 μΐ PBS, 4% skimmed milk (PBS/M) for 1 hour at RT, and then washed three times with 200 μΐ PBS, 0.005% Tween 20 (PBS/T) pH 5.5. 100 μΐ/well his-tagged human FcRn (10 μg/ml) in PBS/T/M pH 5.5 was added and incubated for 1 hour at RT, before repeating the washing step above. Dilution series of HSA (10- 0.005 μg/ml) were prepared in PBS/T/M pH 5.5, and 100 μΐ/well was added in duplicates and incubated for 1 hour at RT. The wells were washed as before. Bound HSA was detected with 100 μΐ/well of alkaline phosphatase-conjugated polyclonal anti-HSA antibody from goat diluted 1 :3000 in PBS/T/M (Bethyl Laboratories, Inc), and incubated for 1 hour at RT. The wells were washed as before, and developed by adding 100 μΐ of the p- nitropenylphospate substrate (Sigma- Aldrich) (10 μg/ml) in diethanolamine buffer. The absorbance was measured at 405 nm using the Sunrise spectrophotometer (TECAN). Plasma half-life studies - Human FcRn transgenic mice were used; B6.Cg-Fcgrt ^cr Tg(FCGRT)32Dcr/DcrJ (abbreviated Tg32). The mice have disrupted alleles for mouse FcRn HC (Fcgrt^tmlOcr) and expresses the genomic transgene of human FcRn HC (FCGRT) under the control of the native human FcRn promoter. Tg32 mice (hemizygous for FCGRT, aged 7- 9 weeks, weight between 21 and 26 g, 5 mice/group) received 1 mg/kg of HSA in 5 ml/kg lx PBS by intravenous injection, and blood samples (25 μΐ) were collected from the retro-orbital sinus at 1, 2, 3, 5, 7, 9, 12 and 16 days after injection. The blood samples were collected into 1 μΐ of 1% K3-EDTA to prevent coagulation and then centrifuged at 17000 χ g for 5 min at 4°C. Plasma was isolated, diluted 1 : 10 in 50% glycerol in PBS and then stored at -20 °C until analysis. The mouse studies were performed at The Jackson Laboratory (JAX service; Bar Harbor, ME). The animal experiments and protocols used were reviewed and approved by The Jackson Laboratory Animal Care and Use Committee.

ELISA to quantify HSA in plasma - 96-well ELISA plates (Costar) were coated with 100 μΐ/well of polyclonal goat anti-HSA (Sigma- Aldrich) (1.0 μg/ml) in PBS, and incubated over night at 4°C. Plates were blocked with 200 μΐ/well PBS, 4% skimmed milk (PBS/M) over night at 4°C and washed four times with PBS, 0.05% Tween20 (PBS/T). Serial dilutions of HSA (500.0-0.2 ng/ml) in PBS/T/M were added in parallel with plasma samples diluted 1 :200 in PBS/T/M, followed by 1 h incubation at RT. The wells were washed as before, and bound HSA was detected with 100 μΐ/well of alkaline phosphatase-conjugated polyclonal anti-HSA antibody from goat (1 :3000) in PBS/T/M (Bethyl Laboratories, Inc), and incubated for 1 h at RT. The wells were washed as before, and developed by adding 100 μΐ of the p- nitropenylphospate substrate (Sigma- Aldrich) (10 μg/ml) in diethanolamine buffer. The absorbance was measured at 405 nm using the Sunrise spectrophotometer (TECAN).

Half-life calculation - The plasma concentration of HSA variants was presented as percentage remaining in the circulation at different time points after injection compared to the concentration on day 1 (100%). Non-linear regression analysis was performed to fit a straight line through the data using Prism 7. The β-phase half-life was calculated using the formula: ti/2 = log 0.5/(log A_e/A₀) x t, where ty₂ is the half-life of the HSA variant evaluated, A_e is the amount of HSA remaining, A₀ is the amount of HSA on day 1 and t is the elapsed time.

Table 2. HSA variants and mutations introduced

N E/VA/KP N 111E/V547A/K573P

TN/VA/KP T83N/V547A/K573P

TN/N E/VA/KP T83N/N 111E/V547A/K573P

N E/EQ/TM/KP N 111E/E505Q/T527M/K573P

TN/EQ/TM/KP T83N/E505Q/T527M/K573P

TN/N E/EQ/TM/KP T83N/N 111E/E505Q/T527M/K573P

EP/N H/ED/TS E501P/N503H/E505D/T506S

EP/N H/ED/TS/KP E501P/N503H/E505DT/506S/K573P

RESULTS

Results are shown in Figures 6-11 and Tables 3-6. Figure 6 shows WT HSA and HSA DIDIII variants produced in HEK293E cells and purified on a Capture Select human serum albumin affinity matrix that were analyzed by 12% (w/v) SDS-PAGE. Figure 7 shows SPR binding kinetics of HSA DIDIII variants to human FcRn. Representative sensorgrams showing binding at pH 5.5 of injected human FcRn to WT HSA (A), KP (B), NE/VA/KP (C), TN/VA/KP (D), TN/NE/VA/KP (E), NE/EQ/TM/KP (F), TN/EQ/TM/KP (G) and

TN/NE/EQ/TM/KP (H) immobilized on a CM5 chip (-500 RU). Injections were performed with a flow rate of 40 μΐ/min at 25°C. Figure 8 shows elimination curves for WT HSA and engineered variants (KP, TN/EQ/TM/KP and TN/NE/EQ/TM/KP) in Tg32 human FcRn transgenic mice. The plasma levels of HSA are presented as percentage remaining in the circulation compared to day 1. The curves represent the mean HSA concentration ± SD of five mice. Figure 9 shows elimination curves for WT HSA and engineered variants (KP and NE/EQ/TM/KP) in Tg32 human FcRn transgenic mice. The plasma levels of HSA are presented as percentage remaining in the circulation compared to day 1. The curves represent the mean HSA concentration ± SD of ten mice from two experiments. Figure 10 shows ELISA results showing binding of WT HSA and HSA variants (KP, EP/NH/ED/TS and EP/NH/ED/TS/KP) to human FcRn at pH 5.5. The ELISA was performed twice and the mean ± SD of duplicates in one representative experiment is shown. Figure 11 shows elimination curves for WT HSA and engineered variants (KP, EP/NH/ED/TS and EP/NH/ED/TS/KP) in Tg32 human FcRn transgenic mice. The plasma levels of HSA are presented as percentage remaining in the circulation compared to day 1. The curves represent the mean HSA concentration ± SD of five mice. Table 3. SPR derived kinetics for binding of HSA variants to human FcRn

The HSA variants were immobilized on CM5 chips (~500 RU) and serial dilutions of human FcRn were injected.

*' The kinetic rate constants were obtained using a simple first-order (1 : 1) Langmuir binding model.

c χ values resulting from curve fitting using the first-order (1: 1) Langmuir interaction model, χ is a measure of the average squared residual (the difference between the experimental data and the fitted curve).

Table 4. β-phase plasma half-life of HSA variants in human FcRn transgenic mice

Table 5. β-phase plasma half-life of HSA variants in human FcRn transgenic mice

Table 6. β-phase plasma half-life of HSA variants in human FcRn transgenic mice

All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.

Claims

1. A human serum albumin (HSA) variant polypeptide that binds to FcRn with increased affinity relative to wild type HSA, wherein said polypeptide comprises at least one variant amino acid selected from the group consisting of Nl 11E/V547A/K573P, T83N/V547A/K573P, T83N/E505Q/T527M/K573P, T83N/N111E/V547A/K573P, Nl 11E/E505Q/T527M/K573P, T83N/N111E/E505Q/T527M/K573P, E501P/N503H/E505D/T506S and

E501P/N503H/E505D/T506S/K573P .

2. A protein comprising the amino acid sequence of SEQ ID NO:2 or sequences with more than 98% sequence identity thereto, wherein

position 111 comprises a glutamic acid residue;

position 547 comprises an alanine residue; and

position 573 comprises a proline residue.

3. A protein comprising the amino acid sequence of SEQ ID NO:2 or sequences with more than 98% sequence identity thereto, wherein

position 83 comprises an asparagine residue;

position 547 comprises an alanine residue; and

position 573 comprises a proline residue.

4. A protein comprising the amino acid sequence of SEQ ID NO:2 or sequences with more than 98%) sequence identity thereto, wherein

position 83 comprises an asparagine residue;

position 505 comprises a glutamine residue;

position 527 comprises a methionine residue; and

position 573 comprises a proline residue.

5. A protein comprising the amino acid sequence of SEQ ID NO:2 or sequences with more than 98%o sequence identity thereto, wherein position 83 comprises an asparagine residue;

position 111 comprises a glutamic acid residue;

position 547 comprises an alanine residue; and

position 573 comprises proline.

6. A protein comprising the amino acid sequence of SEQ ID NO:2 or sequences with more than 98% sequence identity thereto, wherein

position 111 comprises a glutamic acid residue;

position 505 comprises a glutamine residue;

position 527 comprises a methionine residue; and

position 573 comprises a proline residue.

7. A protein comprising the amino acid sequence of SEQ ID NO:2 or sequences with more than 98% sequence identity thereto, wherein

position 83 comprises an asparagine residue;

position 111 comprises a glutamic acid residue;

position 505 comprises a glutamine residue;

position 527 comprises a methionine residue; and

position 573 comprises a proline residue.

8. A protein comprising the amino acid sequence of SEQ ID NO:2 or sequences with more than 98%) sequence identity thereto, wherein

position 501 comprises a proline residue;

position 503 comprises a histidine residue;

position 505 comprises an aspartic acid residue; and

position 506 comprises a serine residue.

9. A protein comprising the amino acid sequence of SEQ ID NO:2 or sequences with more than 98%o sequence identity thereto, wherein

position 501 comprises a proline residue;

position 503 comprises a histidine acid residue; position 505 comprises a aspartic acid residue;

position 506 comprises a serine residue; and

position 573 comprises a proline residue.

10. A protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:3 to 10.

11. A protein conjugate comprising the protein of any one of claims 1 to 10 or a fragment thereof fused to a conjugate.

12. The protein conjugate of claim 11, wherein said conjugate is covalently attached to an amino acid comprising a thiol group.

13. The protein conjugate of claims 11 or 12, wherein said conjugate is an immunogen.

14. The protein conjugate of claim 11 or 12, wherein said conjugate is a drug or diagnostic agent.

15. The protein conjugate of claim 14, wherein said drug or diagnostic agent is a small molecule, peptide, or polypeptide.

16. A nucleic acid encoding the protein of any one of claims 1 to 10.

17. A host cell comprising the nucleic acid of claim 16.

18. A pharmaceutical composition comprising the protein conjugate of any one of claims 11 to 15 and a pharmaceutically acceptable carrier.

19. The pharmaceutical composition of claim 18, wherein said composition is a vaccine composition.

20. A method of inducing an immune response in a subject, comprising administering to the subject a vaccine composition of claim 19 under conditions such that said subject generates an immune response.

21. The method of claim 20, wherein said vaccine is delivered to a mucosal surface of a subject.

22. The method of claim 20, wherein said vaccine is delivered intranasally or through inhalation.

23. A vaccine composition of claim 19 for use in eliciting an immune response in a subject.

24. Use of the vaccine composition of claim 19 in the preparation of a medicament.