Patent Application
20240002479
The text of the computer readable sequence listing filed herewith, titled “38953-601_SEQUENCE_LISTING_ST25”, created Nov. 23, 2021, having a file size of 62,755 bytes, is hereby incorporated by reference in its entirety.
The disclosure provides antigen-binding agents that specifically bind to flavivirus NS1 protein.
Flaviviruses are emerging arthropod-borne viruses representing an immense global health problem. The prominent viruses of this group include dengue virus, yellow fever virus, Japanese encephalitis virus, West Nile virus, tick borne encephalitis virus, and Zika Virus. Flaviviruses are endemic in many parts of the world and are responsible for illnesses ranging from mild flu like symptoms to severe hemorrhagic, neurologic, and cognitive manifestations leading to death. Flaviviruses have the potential to emerge and outbreak in non-endemic geographical regions, but the development of vaccines has been challenging. There are currently no approved anti-flaviviral therapeutics available.
Dengue virus serotypes 1-4 (DENV1-4) are mosquito-borne flaviviruses causing 50-100 million disease cases and about 500,000 hospitalizations annually, with severe forms of disease manifesting in vascular leak as a result of endothelial dysfunction (1, 2). The trigger(s) of these pathologies are often broadly described as a “cytokine storm” resulting from uncontrolled viral replication and activation of target immune cells, with a direct pathogenic role now characterized for the DENV non-structural protein 1 (NS1) via interactions with endothelial and immune cells (3-5). There are currently no approved therapeutics for dengue, and the only licensed DENV vaccine, DENGVAXIA®, is now reserved strictly for patients with preexisting DENV immunity due to the risk of predisposing DENV-naïve patients to severe dengue disease, presumably via antibody-dependent enhancement (ADE) (6, 7). This risk has made a successful vaccine targeting the DENV envelope protein (E) challenging.
There remains a need for compositions and methods for treating and preventing flavivirus infections.
The disclosure provides an agent which binds to a flavivirus NS1 protein and comprises three complementarity determining regions (CDRs) of an antibody heavy chain variable region (VH) and three CDRs of an antibody light chain variable region, wherein: (a) CDR1 of the VH (HCDR1) comprises the amino acid sequence of SEQ ID NO: 1, CDR2 of the VH (HCDR2) comprises the amino acid sequence of SEQ ID NO: 2, and CDR3 of the VH (HCDR3) comprises the amino acid sequence of SEQ ID NO: 3; and (b) CDR1 of the LH (LCDR1) comprises the amino acid sequence of SEQ ID NO: 4, CDR2 of the LH (LCDR2) comprises the amino acid sequence of SEQ ID NO: 5, and CDR3 of the LH (LCDR3) comprises the amino acid sequence of SEQ ID NO: 6.
The disclosure also provides a binding agent that specifically binds to a region of a flavivirus NS1 protein comprising one or more of the following amino acid residues: (a) H269, L270, E281, G282, R299, T301, V303, T304, G305, T307, E326, D327, G328, W330, E343, N344, L345, V346, K347, S348, and/or M349 of Dengue virus 1 (NCBI Accession No. P17763.2); (b) H269, L270, D281, G282, R299, T301, A303, S304, G305, L307, E326, D327, G328, W330, E343, N344, L345, V346, N347, S348, and/or L349 of Dengue virus 2 (NCBI Accession No. P29990); (c) H269, L270, E281, G282, R299, T301, V303, S304, G305, L307, E326, D327, G328, W330, E343, N344, M345, V346, K347, S348, and/or L349 of Dengue virus 3 (NCBI Accession No. YP_001621843); (d) H269, L270, P281, G282, R299, T301, A303, S304, G305, L307, E326, D327, G328, W330, E343, N344, M345, V346, K347, S348, and/or Q349 of Dengue virus 4 (NCBI Accession No. P09866.2); (e) H269, S270, P281, G282, R299, T301, A303, S304, G305, V307, K326, D327, G328, W330, S343, N344, L345, V346, R347, S348, and/or M349 of Zika virus (NCBI Accession No. AZS35340); (f) S269, E270, P281, G282, R299, T301, A303, S304, G305, L307, K326, D327, G328, W330, A343, K344, L345, V346, K347, S348, and/or R349 of St. Louis encephalitis virus (NCBI Accession No. P09732.2); (g) D269, E270, P281, G282, R299, T301, E303, S304, G305, L307, D326, S327, G328, W330, K343, T344, L345, V346, Q347, S348, and/or G349 of West Nile virus (NCBI Accession No. Q9Q6P4); (h) D269, E270, P281, G282, R299, T301, D303, S304, G305, L307, E326, N327, G328, W330, T343, T344, L345, V346, R347, S348, and/or Q349 of Japanese encephalitis virus (NCBI Accession No. P27395); (i) K270, Y271, P282, G283, R300, T302, E304, S305, G306, V307, G327, T328, D329, W331, G343, G344, L345, V346, R347, S348, and/or M349 of Tick-borne encephalitis virus (NCBI Accession No. NP_043135); (j) M269, Q270, P281, G282, R299, T301, D303, S304, G305, V307, S326, D327, G328, W330, S343, H344, L345, V346, R347, S348, and/or W349 of Yellow fever virus (NCBI Accession No. NP_041726.1); (k) D269, E270, P281, G282, R299, T301, S303, S304, G305, L307, K326, N327, G328, W330, T343, T344, L345, V346, K347, S348, and/or S349 of Usutu virus (NCBI Accession No. AWC68492); (l) D271, Q272, P283, G284, R301, T303, E305, S306, G307, I309, G328, T329, D330, W332, G344, G345, L346, V347, R348, S349, and/or M350 of Powassan virus (NCBI Accession No. ACD88752); and/or (m) H270, N271, P282, G283, R300, T302, D304, S305, G306, I308, P327, D328, G329, W331, E343, A344, H345, L346, V347, K348, and/or S349 of Wesselsbron virus (NCBI Accession No. ABI54474).
The disclosure further provides a composition comprising a recombinant NS1 antigen and a pharmaceutically acceptable carrier, which recombinant NS1 antigen comprises one or more of the following amino acid residues: (a) H269, L270, E281, G282, R299, T301, V303, T304, G305, T307, E326, D327, G328, W330, E343, N344, L345, V346, K347, S348, and/or M349 of Dengue virus 1 (NCBI Accession No. P17763.2); (b) H269, L270, D281, G282, R299, T301, A303, S304, G305, L307, E326, D327, G328, W330, E343, N344, L345, V346, N347, S348, and/or L349 of Dengue virus 2 (NCBI Accession No. P29990); (c) H269, L270, E281, G282, R299, T301, V303, S304, G305, L307, E326, D327, G328, W330, E343, N344, M345, V346, K347, S348, and/or L349 of Dengue virus 3 (NCBI Accession No. YP_001621843); (d) H269, L270, P281, G282, R299, T301, A303, S304, G305, L307, E326, D327, G328, W330, E343, N344, M345, V346, K347, S348, and/or Q349 of Dengue virus 4 (NCBI Accession No. P09866.2); (e) H269, S270, P281, G282, R299, T301, A303, S304, G305, V307, K326, D327, G328, W330, 5343, N344, L345, V346, R347, S348, and/or M349 of Zika virus (NCBI Accession No. AZS35340); (f) S269, E270, P281, G282, R299, T301, A303, S304, G305, L307, K326, D327, G328, W330, A343, K344, L345, V346, K347, S348, and/or R349 of St. Louis encephalitis virus (NCBI Accession No. P09732.2); (g) D269, E270, P281, G282, R299, T301, E303, S304, G305, L307, D326, S327, G328, W330, K343, T344, L345, V346, Q347, S348, and/or G349 of West Nile virus (NCBI Accession No. Q9Q6P4); (h) D269, E270, P281, G282, R299, T301, D303, S304, G305, L307, E326, N327, G328, W330, T343, T344, L345, V346, R347, S348, and/or Q349 of Japanese encephalitis virus (NCBI Accession No. P27395); (i) K270, Y271, P282, G283, R300, T302, E304, S305, G306, V307, G327, T328, D329, W331, G343, G344, L345, V346, R347, S348, and/or M349 of Tick-borne encephalitis virus (NCBI Accession No. NP_043135); (j) M269, Q270, P281, G282, R299, T301, D303, S304, G305, V307, S326, D327, G328, W330, S343, H344, L345, V346, R347, S348, and/or W349 of Yellow fever virus (NCBI Accession No. NP_041726.1); (k) D269, E270, P281, G282, R299, T301, S303, S304, G305, L307, K326, N327, G328, W330, T343, T344, L345, V346, K347, S348, and/or S349 of Usutu virus (NCBI Accession No. AWC68492); (l) D271, Q272, P283, G284, R301, T303, E305, S306, G307, T309, G328, T329, D330, W332, G344, G345, L346, V347, R348, S349, and/or M350 of Powassan virus (NCBI Accession No. ACD88752); or (m) H270, N271, P282, G283, R300, T302, D304, S305, G306, I308, P327, D328, G329, W331, E343, A344, H345, L346, V347, K348, and/or S349 of Wesselsbron virus (NCBI Accession No. ABI54474).
Also provided is a composition comprising a nucleic acid sequence encoding a recombinant flavivirus NS1 antigen and pharmaceutically acceptable carrier, wherein the recombinant NS1 antigen comprises (a) H269, L270, E281, G282, R299, T301, V303, T304, G305, T307, E326, D327, G328, W330, E343, N344, L345, V346, K347, S348, and/or M349 of Dengue virus 1 (NCBI Accession No. P17763.2); (b) H269, L270, D281, G282, R299, T301, A303, S304, G305, L307, E326, D327, G328, W330, E343, N344, L345, V346, N347, S348, and/or L349 of Dengue virus 2 (NCBI Accession No. P29990); (c) H269, L270, E281, G282, R299, T301, V303, S304, G305, L307, E326, D327, G328, W330, E343, N344, M345, V346, K347, S348, and/or L349 of Dengue virus 3 (NCBI Accession No. YP_001621843); (d) H269, L270, P281, G282, R299, T301, A303, S304, G305, L307, E326, D327, G328, W330, E343, N344, M345, V346, K347, S348, and/or Q349 of Dengue virus 4 (NCBI Accession No. P09866.2); (e) H269, S270, P281, G282, R299, T301, A303, S304, G305, V307, K326, D327, G328, W330, S343, N344, L345, V346, R347, S348, and/or M349 of Zika virus (NCBI Accession No. AZS35340); (f) S269, E270, P281, G282, R299, T301, A303, S304, G305, L307, K326, D327, G328, W330, A343, K344, L345, V346, K347, S348, and/or R349 of St. Louis encephalitis virus (NCBI Accession No. P09732.2); (g) D269, E270, P281, G282, R299, T301, E303, S304, G305, L307, D326, S327, G328, W330, K343, T344, L345, V346, Q347, S348, and/or G349 of West Nile virus (NCBI Accession No. Q9Q6P4); (h) D269, E270, P281, G282, R299, T301, D303, S304, G305, L307, E326, N327, G328, W330, T343, T344, L345, V346, R347, S348, and/or Q349 of Japanese encephalitis virus (NCBI Accession No. P27395); (i) K270, Y271, P282, G283, R300, T302, E304, S305, G306, V307, G327, T328, D329, W331, G343, G344, L345, V346, R347, S348, and/or M349 of Tick-borne encephalitis virus (NCBI Accession No. NP_043135); (j) M269, Q270, P281, G282, R299, T301, D303, S304, G305, V307, S326, D327, G328, W330, S343, H344, L345, V346, R347, S348, and/or W349 of Yellow fever virus (NCBI Accession No. NP_041726.1); (k) D269, E270, P281, G282, R299, T301, S303, S304, G305, L307, K326, N327, G328, W330, T343, T344, L345, V346, K347, S348, and/or S349 of Usutu virus (NCBI Accession No. AWC68492); (l) D271, Q272, P283, G284, R301, T303, E305, S306, G307, T309, G328, T329, D330, W332, G344, G345, L346, V347, R348, S349, and/or M350 of Powassan virus (NCBI Accession No. ACD88752); or (m) H270, N271, P282, G283, R300, T302, D304, S305, G306, I308, P327, D328, G329, W331, E343, A344, H345, L346, V347, K348, and/or S349 of Wesselsbron virus (NCBI Accession No. ABI54474).
The disclosure provides methods of inducing an immune response against flaviviruses in a mammal using the aforementioned binding agents and compositions comprising same.
The present disclosure is predicated, at least in part, on the identification of three crystal structures of full-length dengue virus (DENV) NS1 protein complexed with a flavivirus cross-reactive NS1-specific monoclonal antibody, 2B7, revealing a protective mechanism by which two domains of NS1 are antagonized simultaneously. A single chain variable fragment (scFv) derived from the 2B7 antibody has been generated that binds to the NS1 protein in the same manner as a 2B7 Fab and full-length 2B7 antibody. As described herein, the 2B7 scFv blocks NS1 protein binding to cell surfaces and the triggering of the endothelial dysfunction associated with the most severe forms of Flavivirus diseases. The present disclosure provides a mechanistic explanation for 2B7 protection against NS1-induced pathology and demonstrates that the 2B7 antibody, and fragments, derivatives, or analogs thereof, may be used to treat infections by multiple different flaviviruses. However, the invention is not limited to any particular mechanism of action and an understanding of the mechanism is not necessary to practice the invention.
To facilitate an understanding of the present technology, a number of terms and phrases are defined below. Additional definitions are set forth throughout the detailed description.
The terms “nucleic acid,” “polynucleotide,” “nucleotide sequence,” and “oligonucleotide” are used interchangeably herein and refer to a polymer or oligomer of pyrimidine and/or purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (See Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982)). The terms encompass any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases. The polymers or oligomers may be heterogenous or homogenous in composition, may be isolated from naturally occurring sources, or may be artificially or synthetically produced. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states. In some embodiments, a nucleic acid or nucleic acid sequence comprises other kinds of nucleic acid structures such as, for instance, a DNA/RNA helix, peptide nucleic acid (PNA), morpholino nucleic acid (see, e.g., Braasch and Corey, Biochemistry, 41(14): 4503-4510 (2002) and U.S. Pat. No. 5,034,506), locked nucleic acid (LNA; see Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 97: 5633-5638 (2000)), cyclohexenyl nucleic acids (see Wang, J. Am. Chem. Soc., 122: 8595-8602 (2000)), and/or a ribozyme. The terms “nucleic acid” and “nucleic acid sequence” may also encompass a chain comprising non-natural nucleotides, modified nucleotides, and/or non-nucleotide building blocks that can exhibit the same function as natural nucleotides (e.g., “nucleotide analogs”).
The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.
The terms “immunogen” and “antigen” are used interchangeably herein and refer to any molecule, compound, or substance that induces an immune response in an animal (e.g., a mammal). An “immune response” can entail, for example, antibody production and/or the activation of immune effector cells. An antigen in the context of the disclosure can comprise any subunit, fragment, or epitope of any proteinaceous or non-proteinaceous (e.g., carbohydrate or lipid) molecule that provokes an immune response in a mammal. By “epitope” is meant a sequence of an antigen that is recognized by an antibody or an antigen receptor. Epitopes also are referred to in the art as “antigenic determinants.” In some embodiments, an epitope is a region of an antigen that is specifically bound by an antibody. In other embodiments, an epitope may include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl groups. In certain embodiments, an epitope may have specific three-dimensional structural characteristics (e.g., a “conformational” epitope) and/or specific charge characteristics. The antigen can be a protein or peptide of viral, bacterial, parasitic, fungal, protozoan, prion, cellular, or extracellular origin, which provokes an immune response in a mammal, preferably leading to protective immunity.
The term “binding agent,” as used herein, refers to a molecule, ideally a proteinaceous molecule, which specifically binds to another molecule, such as another protein. By “antigen-binding agent” is meant a molecule, such as a proteinaceous molecule, that specifically binds to an antigen. The antigen-binding agent comprises at least two components which, in combination, form the antigen-binding site of an antigen-binding agent. In some embodiments, a first component of an antigen-binding agent comprises an antibody heavy chain or a fragment thereof, and a second component of antigen-binding agent comprises an antibody light chain or fragment thereof.
The term “immunoglobulin” or “antibody,” as used herein, refers to a protein that is found in blood or other bodily fluids of vertebrates, which is used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses. Typically, an immunoglobulin or antibody is a protein that comprises at least one complementarity determining region (CDR). The CDRs form the “hypervariable region” of an antibody, which is responsible for antigen binding (discussed further below). A whole antibody typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each of the heavy chains contains one N-terminal variable (VH) region and three C-terminal constant (CH1, CH2, and CH3) regions, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region. The light chains of antibodies can be assigned to one of two distinct types, either kappa (κ) or lambda (λ), based upon the amino acid sequences of their constant domains. In a typical antibody, each light chain is linked to a heavy chain by disulphide bonds, and the two heavy chains are linked to each other by disulphide bonds. The light chain variable region is aligned with the variable region of the heavy chain, and the light chain constant region is aligned with the first constant region of the heavy chain. The remaining constant regions of the heavy chains are aligned with each other.
The variable regions of each pair of light and heavy chains form the antigen binding site of an antibody. The VH and VL regions have the same general structure, with each region comprising four framework (FW or FR) regions. The term “framework region,” as used herein, refers to the relatively conserved amino acid sequences within the variable region which are located between the CDRs. There are four framework regions in each variable domain, which are designated FR1, FR2, FR3, and FR4. The framework regions form the β sheets that provide the structural framework of the variable region (see, e.g., C. A. Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, N.Y. (2001)).
The framework regions are connected by three CDRs. As discussed above, the three CDRs, known as CDR1, CDR2, and CDR3, form the “hypervariable region” of an antibody, which is responsible for antigen binding. The CDRs form loops connecting, and in some cases comprising part of, the beta-sheet structure formed by the framework regions. While the constant regions of the light and heavy chains are not directly involved in binding of the antibody to an antigen, the constant regions can influence the orientation of the variable regions. The constant regions also exhibit various effector functions, such as participation in antibody-dependent complement-mediated lysis or antibody-dependent cellular toxicity via interactions with effector molecules and cells.
The terms “fragment of an antibody,” “antibody fragment,” and “antigen-binding fragment” of an antibody are used interchangeably herein to refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (see, generally, Holliger et al., Nat. Biotech., 23(9): 1126-1129 (2005)). Any antigen-binding fragment of the antibody described herein is within the scope of the invention. The antibody fragment desirably comprises, for example, one or more CDRs, the variable region (or portions thereof), the constant region (or portions thereof), or combinations thereof. Examples of antibody fragments include, but are not limited to, (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains, (ii) a F(ab′)2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, (iii) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (iv) a Fab′ fragment, which results from breaking the disulfide bridge of an F(ab′)2 fragment using mild reducing conditions, (v) a disulfide-stabilized Fv fragment (dsFv), and (vi) a domain antibody (dAb), which is an antibody single variable region domain (VH or VL) polypeptide that specifically binds antigen.
As used herein, when an antibody or other entity (e.g., antigen binding domain) “specifically recognizes” or “specifically binds” an antigen or epitope, it preferentially recognizes the antigen in a complex mixture of proteins and/or macromolecules, and binds the antigen or epitope with affinity which is substantially higher than to other entities not displaying the antigen or epitope. In this regard, “affinity which is substantially higher” means affinity that is high enough to enable detection of an antigen or epitope which is distinguished from entities using a desired assay or measurement apparatus. Typically, it means binding affinity having a binding constant (Ka) of at least 107 M−1 (e.g., >107 M−1, >108 M−1, >109 M−1, >1010 M−1, >1011 M−1, >1012 M−1, >1013 M−1, etc.). In certain such embodiments, an antibody is capable of binding different antigens so long as the different antigens comprise that particular epitope. In certain instances, for example, homologous proteins from different species may comprise the same epitope.
The term “monoclonal antibody,” as used herein, refers to an antibody produced by a single clone of B lymphocytes that is directed against a single epitope on an antigen. Monoclonal antibodies typically are produced using hybridoma technology, as first described in Köhler and Milstein, Eur. J. Immunol., 5: 511-519 (1976). Monoclonal antibodies may also be produced using recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), isolated from phage display antibody libraries (see, e.g., Clackson et al. Nature, 352: 624-628 (1991)); and Marks et al., J. Mol. Biol., 222: 581-597 (1991)), or produced from transgenic mice carrying a fully human immunoglobulin system (see, e.g., Lonberg, Nat. Biotechnol., 23(9): 1117-25 (2005), and Lonberg, Handb. Exp. Pharmacol., 181: 69-97 (2008)). In contrast, “polyclonal” antibodies are antibodies that are secreted by different B cell lineages within an animal. Polyclonal antibodies are a collection of immunoglobulin molecules that recognize multiple epitopes on the same antigen.
A “chimeric” antibody is an antibody or fragment thereof comprising both human and non-human regions (e.g., variable regions from a mouse antibody and constant regions from a human antibody). A “humanized” antibody is a monoclonal antibody comprising a human antibody scaffold and at least one CDR obtained or derived from a non-human antibody. Non-human antibodies include antibodies isolated from any non-human animal, such as, for example, a rodent (e.g., a mouse or rat). A humanized antibody can comprise, one, two, or three CDRs obtained or derived from a non-human antibody.
The term “recombinant,” as used herein, means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, polymerase chain reaction (PCR) and/or ligation steps resulting in a construct having a structural coding or non-coding sequence distinguishable from endogenous nucleic acids found in natural systems. DNA sequences encoding polypeptides can be assembled from cDNA fragments or from a series of synthetic oligonucleotides to provide a synthetic nucleic acid which is capable of being expressed from a recombinant transcriptional unit contained in a cell or in a cell-free transcription and translation system. Genomic DNA comprising the relevant sequences can also be used in the formation of a recombinant gene or transcriptional unit. Sequences of non-translated DNA may be present 5′ or 3′ from the open reading frame, where such sequences do not interfere with manipulation or expression of the coding regions and may act to modulate production of a desired product by various mechanisms. Alternatively, DNA sequences encoding RNA that is not translated may also be considered recombinant. Thus, the term “recombinant” nucleic acid also refers to a nucleic acid which is not naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of sequence through human intervention. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. Such is usually done to replace a codon with a codon encoding the same amino acid, a conservative amino acid, or a non-conservative amino acid. Alternatively, the artificial combination may be performed to join nucleic acid segments of desired functions to generate a desired combination of functions. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. When a recombinant polynucleotide encodes a polypeptide, the sequence of the encoded polypeptide can be naturally occurring (“wild type”) or can be a variant (e.g., a mutant) of the naturally occurring sequence. Thus, the term “recombinant” polypeptide does not necessarily refer to a polypeptide whose sequence does not naturally occur. Instead, a “recombinant” polypeptide is encoded by a recombinant DNA sequence, but the sequence of the polypeptide can be naturally occurring (“wild type”) or non-naturally occurring (e.g., a variant, a mutant, etc.). Thus, a “recombinant” polypeptide is the result of human intervention but may comprise a naturally occurring amino acid sequence.
A “portion” of an amino acid sequence comprises at least three amino acids (e.g., about 3 to about 1,200 amino acids). Preferably, a “portion” of an amino acid sequence comprises 3 or more (e.g., 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, or 50 or more) amino acids, but less than 1,200 (e.g., 1,000 or less, 800 or less, 700 or less, 600 or less, 500 or less, 400 or less, 300 or less, 200 or less, or 100 or less) amino acids. Preferably, a portion of an amino acid sequence is about 3 to about 500 amino acids (e.g., about 10, 100, 200, 300, 400, or 500 amino acids), about 3 to about 300 amino acids (e.g., about 20, 50, 75, 95, 150, 175, or 200 amino acids), or about 3 to about 100 amino acids (e.g., about 15, 25, 35, 40, 45, 60, 65, 70, 80, 85, 90, 95, or 99 amino acids), or a range defined by any two of the foregoing values. More preferably, a “portion” of an amino acid sequence comprises no more than about 500 amino acids (e.g., about 3 to about 400 amino acids, about 10 to about 250 amino acids, or about 50 to about 100 amino acids, or a range defined by any two of the foregoing values).
The disclosure provides a binding agent that specifically binds to a flavivirus NS1 protein. Flaviviruses are enveloped, positive-sense, single-stranded RNA viruses. The RNA genome of the flaviviruses contains the 5′ cap (7mG) and 3′ CU—OH conserved tail, which directly translates into a long polypeptide in the cytoplasm of infected cells. The polypeptide is cleaved and processed by host and viral proteases into three structural proteins: envelope protein (E), capsid protein (C) and precursor membrane protein (prM), and seven non-structural components (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) (Rastogi et al., Virol J., 13: 131 (2016)). Among the non-structural proteins, NS1 is a highly conserved, dimer protein with the molecular weight ranges from 46-55 kDa depending on the extent of glycosylation. NS1 exists as a monomer, a dimer (membrane-bound protein, mNS1), and a hexamer (secreted protein, sNS1).
Extracellular NS1 acts as a virulence factor inhibiting complement, activating platelets and immune cells, and directly interacting with endothelial cells (11-13). This results in disruption of the endothelial glycocalyx layer (EGL) and intercellular junctional complexes, which are both critical for maintaining endothelial barrier integrity (13-15). NS1-mediated endothelial dysfunction is observed for multiple medically relevant mosquito-borne flaviviruses, including Zika (ZIKV), West Nile (WNV), Japanese encephalitis (JEV), and yellow fever (YFV) viruses (16, 17). With a prominent role in flavivirus pathogenesis, NS1 has emerged as a promising vaccine candidate. Indeed, vaccination with NS1 protects against lethal DENV or ZIKV challenge in mice (3, 18-20). Flavivirus NS1 has three domains that may possess distinct functions (21): a small “β-roll” dimerization domain (e.g., amino acids 1-29 of West Nile virus (WNV) and dengue virus type 2 (DENV2)), a “wing” domain protruding from the central β-domain like a wing (e.g., amino acids 30-180 of WNV and DENV2), and the “β-ladder domain” (e.g., amino acids 181-352 of WNV and DENV2), which is the predominant structural feature of NS1 (Akey et al., Science, 343: 881-885 (2014)). Despite a plethora of structural data (21-25), the mechanistic basis for antibody-mediated protection against NS1-induced endothelial dysfunction and the specific functional domains of NS1 responsible for different pathogenic functions are unknown. The binding agents described herein may bind to an NS1 protein of any flavivirus, of which there are over 50 known species. The majority of known members in the genus Flavivirus are arthropod borne (e.g., mosquito- or tick-borne), and many are important human and veterinary pathogens. Examples of mosquito-borne flaviviruses include yellow fever virus, dengue virus, Japanese encephalitis virus, West Nile virus, and Zika virus. Examples of tick-borne flaviviruses that cause encephalitis and hemorrhagic diseases include tick-borne encephalitis (TBE), Kyasanur Forest Disease (KFD) virus, Alkhurma disease virus, and Omsk hemorrhagic fever virus. Flavivirus classification and phylogeny is described in detail in, e.g., Schweitzer et al., Laboratory Medicine, 40(8): 493-499 (2009); DOI: 10.1309/LM5YWS85NJPCWESW; and Kuno et al., Journal of Virology, 72(1) 73-83 (1998); DOI: 10.1128/JVI.72.1.73-83.1998. In some embodiments, the binding agent specifically binds to an NS1 protein from yellow fever virus, dengue virus, Japanese encephalitis virus, West Nile virus, Zika virus, St. Louis encephalitis virus, tick-borne encephalitis virus, Usutu virus, Powassan virus, or Wesselsbron virus.
For example, the binding agent may specifically bind an NS1 protein from dengue virus. Dengue virus (DENV) is a mosquito-borne flavivirus that is estimated to cause up to 390 million infections, 96 million disease cases, and ˜500,000 hospitalizations annually (1). Infection with any of the four DENV serotypes (serotype 1 (DENV1), serotype 2 (DENV2), serotype 3 (DENV3), or serotype 4 (DENV4)) results in a range of syndromes from inapparent infection to classic dengue fever (DF) to dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), which is characterized by vascular leakage and shock (2). Most primary DENV infections caused by any of the four serotypes are asymptomatic or lead to the self-limiting but debilitating DF; however, secondary infections with a different (heterologous) DENV serotype can lead to increased risk of severe dengue (3). Immune responses after primary DENV infection lead to protective immunity to homologous secondary infection but may either protect against or cause increased disease severity in a subsequent DENV infection with a different serotype. The latter is thought to be mediated by serotype cross-reactive T cells or antibody-dependent enhancement (ADE), whereby cross-reactive antibodies that target viral structural proteins facilitate DENV infection of Fcγ receptor-bearing cells, leading to increased viral load (4, 5). ADE and cross-reactive T cells are thought to trigger an exaggerated and skewed immune response to a previously infecting serotype, resulting in a “cytokine storm,” i.e., rapid-onset, high-level production of proinflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), in the blood that leads to endothelial permeability and vascular leak (6). However, the potential role of viral proteins in mediating vascular leakage has not been elucidated.
The binding agents provided herein desirably bind to a proteinaceous molecule, such as an antigen. In this case, therefore, a binding agent may be referred to as an “antigen-binding agent.” An antigen-binding agent may bind to a conformational epitope and/or a linear epitope present on a target antigen. The term “conformational epitope,” as used herein, refers to an antigenic protein composed of amino acid residues that are spatially near each other on the antigen's surface and are brought together by protein folding. In contrast, a “linear epitope” (also referred to as a “sequential epitope”) comprises a sequence of continuous amino acids that is sufficient for antibody binding. The binding agents described herein desirably specifically bind to particular amino acid residues of an NS1 protein from any suitable flavivirus. For example, the binding agent may bind to any one or combination of the amino acid residues from the flavivirus species set forth in Table 1.
However, the above NS1 amino acid residues from the identified species are merely exemplary, and the invention is not limited to the specific amino acid residues set forth in Table 1. Indeed, in other embodiments, the binding agent may specifically bind to any one or combination of the following NS1 amino acid residues (reference sequence DENV2 (NCBI Accession No. P29990)): K94, G95, I96, T265, G266, P267, W268, G271, K272, L273, F279, C280, T283, G295, P296, S297, L298, T300, T302, K306, I308, T309, W311, R322, Y323, R324, G325, C329, Y331, E340, K341, E342, V350, T351, and/or A352.
In some embodiments, the antigen-binding agent is an antibody, such as a monoclonal antibody, or an antigen-binding fragment thereof. For example, the antigen-binding agent may be a whole antibody. As defined herein, a whole antibody comprises two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each of the heavy chains contains one N-terminal variable (VH) region and three C-terminal constant (CH1, CH2, and CH3) regions, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) The heavy chain C-terminal constant region contains the fragment crystallizable (Fc) domain, which determines antibody class and is responsible for humoral and cellular effector functions. Antibodies are divided into five major classes (or “isotypes”), IgG, IgM, IgA, IgD and IgE, which differ in their function in the immune system. IgGs are the most abundant immunoglobulins in the blood, representing 60/% of total serum antibodies in humans. IgG antibodies may be subclassified as IgG1, IgG2, IgG3, and IgG4, named in order of their abundance in serum (IgG1 being the most abundant) (Vidarsson et al., Frontiers in Immunology, 5: 520 (2014)). A whole antibody provided herein may be of any suitable class and/or subclass.
As discussed above, an exemplary whole antibody that specifically binds to a flavivirus NS1 protein is denoted “2B7.” 2B7 is an IgG2b mouse monoclonal antibody (mAb) directed against dengue virus NS1 protein and is a strong inhibitor of NS1-induced endothelial hyperpermeability (3). The heavy chain variable region (VII) of the 2B7 antibody comprises an HCDR1 amino acid sequence of SEQ ID NO: 1, an HCDR2 amino acid sequence of SEQ ID NO: 2, and an HCDR3 amino acid sequence of SEQ ID NO: 3. The light chain variable region (VL) of the 2B7 antibody comprises an LCDR1 amino acid sequence of SEQ ID NO: 4, an LCDR2 amino acid sequence of SEQ ID NO: 5, and an LCDR3 amino acid sequence of SEQ ID NO: 6. The heavy chain variable region of 2B7 comprises the amino acid sequence of SEQ ID NO: 7 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8.
Thus, in some embodiments, the binding agent disclosed herein comprises three complementarity determining regions (CDRs) of an antibody heavy chain variable region (VH) and three CDRs of an antibody light chain variable region (VL), wherein: (a) CDR1 of the VH (HCDR1) comprises the amino acid sequence of SEQ ID NO: 1, CDR2 of the VH (HCDR2) comprises the amino acid sequence of SEQ ID NO: 2, and CDR3 of the VH (HCDR3) comprises the amino acid sequence of SEQ ID NO: 3; and (b) CDR1 of the LH (LCDR1) comprises the amino acid sequence of SEQ ID NO: 4, CDR2 of the LH (LCDR2) comprises the amino acid sequence of SEQ ID NO: 5, and CDR3 of the LH (LCDR3) comprises the amino acid sequence of SEQ ID NO: 6.
In some embodiments, one or more amino acids of the aforementioned heavy chain variable region, light chain variable region, and CDRs thereof, may be replaced or substituted with a different amino acid. An amino acid “replacement” or “substitution” refers to the replacement of one amino acid at a given position or residue by another amino acid at the same position or residue within a polypeptide sequence. Any suitable number of amino acids may be substituted. In this regard, the aforementioned amino acid sequences may comprise a substitution of one or more amino acids (e.g., 2 or more, 5 or more, or 10 or more amino acids). For example, 1-10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) of the aforementioned VH, VL, and/or CDR amino acid sequences may be substituted. In some embodiments, the amino acid substitution is conservative. The terms “conservative amino acid substitution” or “conservative mutation” refer to the replacement of one amino acid by another amino acid with a common physiochemical property. A functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz and Schirmer, Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such analyses, groups of amino acids may be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz and Schirmer, supra). Examples of conservative amino acid substitutions include, for example, lysine for arginine and vice versa such that a positive charge may be maintained, glutamic acid for aspartic acid and vice versa such that a negative charge may be maintained, serine for threonine such that a free —OH can be maintained, and glutamine for asparagine such that a free —NH can be maintained.
In other embodiments, the binding agent comprises an antibody heavy chain variable region and an antibody light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7 and the light chain variable region comprises the amino acid sequence of SEQ ID NO. 8. In other embodiments, the binding agent comprises a CDR amino acid sequence, a heavy chain variable region amino acid sequence, and/or a light chain variable region amino acid sequence that is at least 90% identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to any of the aforementioned amino acid sequences. Nucleic acid or amino acid sequence “identity,” as described herein, can be determined by comparing a nucleic acid or amino acid sequence of interest to a reference nucleic acid or amino acid sequence. The percent identity is the number of nucleotides or amino acid residues that are the same (i.e., that are identical) as between the sequence of interest and the reference sequence divided by the length of the longest sequence (i.e., the length of either the sequence of interest or the reference sequence, whichever is longer). A number of mathematical algorithms for obtaining the optimal alignment and calculating identity between two or more sequences are known and incorporated into a number of available software programs. Examples of such programs include CLUSTAL-W, T-Coffee, and ALIGN (for alignment of nucleic acid and amino acid sequences), BLAST programs (e.g., BLAST 2.1, BL2SEQ, and later versions thereof) and FASTA programs (e.g., FASTA3×, FAS™, and SSEARCH) (for sequence alignment and sequence similarity searches). Sequence alignment algorithms also are disclosed in, for example, Altschul et al., J. Molecular Biol., 215(3): 403-410 (1990), Beigert et al., Proc. Natl. Acad. Sci. USA, 106(10): 3770-3775 (2009), Durbin et al., eds., Biological Sequence Analysis: Probabilistic Models of Proteins and Nucleic Acids, Cambridge University Press, Cambridge, UK (2009), Soding, Bioinformatics, 21(7): 951-960 (2005), Altschul et al., Nucleic Acids Res., 25(17): 3389-3402 (1997), and Gusfield, Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge UK (1997)).
When the binding agent is an antibody, the antibody can be, or can be obtained from, a human antibody, a non-human antibody, or a chimeric antibody as defined herein. A human antibody, a non-human antibody, a chimeric antibody, or a humanized antibody can be obtained by any means, including via in vitro sources (e.g., a hybridoma or a cell line producing an antibody recombinantly) and in vivo sources (e.g., rodents). Methods for generating antibodies are known in the art and are described in, for example, Köler and Milstein, Eur. J. Immunol., 5: 511-519 (1976); Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988); and Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, N.Y. (2001)). In certain embodiments, a human antibody or a chimeric antibody can be generated using a transgenic animal (e.g., a mouse) wherein one or more endogenous immunoglobulin genes are replaced with one or more human immunoglobulin genes. Examples of transgenic mice wherein endogenous antibody genes are effectively replaced with human antibody genes include, but are not limited to, the Medarex HUMAB-MOUSE™, the Kirin TC MOUSE™, and the Kyowa Kirin KM-MOUSE™ (see, e.g., Lonberg, Nat. Biotechnol., 23(9): 1117-25 (2005), and Lonberg, Handb. Fxp. Pharmacol., 181: 69-97 (2008)). A humanized antibody can be generated using any suitable method known in the art (see, e.g., An, Z. (ed.), Therapeutic Monoclonal Antibodies: From Bench to Clinic, John Wiley & Sons, Inc., Hoboken, N.J. (2009)), including, e.g., grafting of non-human CDRs onto a human antibody scaffold (see, e.g., Kashmiri et al., Methods, 36(1): 25-34 (2005); Hou et al., J. Biochem., 144(1): 115-120 (2008); and Strohl, W. R., Strohl, L. M., Therapeutic Antibody Engineering: Current and Future Advances Driving the Strongest Growth Area in the Pharmaceutical Industry (Woodhead Publishing Series in Biomedicine), 1st ed. (2012)).
The antigen-binding agent can also be a fragment or fusion of portions of an antibody, such as any of those defined herein or known in the art (see, e.g., Holliger et al., Nat. Biotech., 23(9): 1126-1129 (2005); and U.S. Pat. No. 9,260,533). In some embodiments, the antigen-binding agent can be a single chain antibody fragment. Examples of single chain antibody fragments include, but are not limited to, (i) a single chain variable fragment (scFv), which is a monovalent molecule consisting of the two domains of the Fv fragment (i.e., VL and VH) joined by a synthetic linker which enables the two domains to be synthesized as a single polypeptide chain (see, e.g., Bird et al., Science, 242: 423-426 (1988); and Huston et al., Proc. Natl. Acad. Sci. USA, 85: 5879-5883 (1988); and Osbourn et al., Nat. Biotechnol., 16: 778 (1998)) and (ii) a diabody, which is a dimer of polypeptide chains, wherein each polypeptide chain comprises a VH connected to a VL by a peptide linker that is too short to allow pairing between the VH and VL on the same polypeptide chain, thereby driving the pairing between the complementary domains on different VH-VL polypeptide chains to generate a dimeric molecule having two functional antigen-binding sites. Single chain variable regions have been employed in various therapeutic applications (see, e.g., Strohl, W. R., Strohl, L. M. (eds.), Antibody Fragments as Therapeutics, In Woodhead Publishing Series in Biomedicine, Therapeutic Antibody Engineering, Woodhead Publishing, pp. 265-595 (2012)), and several therapeutic antibody fragments have been approved by the U.S. Food and Drug Administration (FDA).
In some embodiments, the antigen-binding agent is a single-chain variable fragment. A single-chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide, typically of ten to about 25 amino acids. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker. The scFv may be based on or derived from the 2B7 antibody. In this regard, an exemplary scFv comprises a single antibody VH and a single antibody VL, wherein (a) CDR1 of the VH (HCDR1) comprises the amino acid sequence of SEQ ID NO: 1, CDR2 of the VH (HCDR2) comprises the amino acid sequence of SEQ ID NO: 2, and CDR3 of the VH (HCDR3) comprises the amino acid sequence of SEQ ID NO: 3; and (b) CDR1 of the LH (LCDR1) comprises the amino acid sequence of SEQ ID NO: 4, CDR2 of the LH (LCDR2) comprises the amino acid sequence of SEQ ID NO: 5, and CDR3 of the LH (LCDR3) comprises the amino acid sequence of SEQ ID NO: 6. The VH and VL may be joined by any suitable peptide linker known in the art (see, e.g., Huston et al., Proc. Natl Acad. Sci. LISA, 85: 5879-5883 (1988)). For example, the scFv may be engineered to include a linker comprising four repeats of the amino acid sequence GGSG.
The antigen-binding agent may also be an intrabody or fragment thereof. An intrabody is an antibody which is expressed and which functions intracellularly. Intrabodies typically lack disulfide bonds and are capable of modulating the expression or activity of target genes through their specific binding activity. Intrabodies include single domain fragments such as isolated VH and VL domains and scFvs. An intrabody can include sub-cellular trafficking signals attached to the N or C terminus of the intrabody to allow expression at high concentrations in the sub-cellular compartments where a target protein is located. Upon interaction with a target gene, an intrabody modulates target protein function and/or achieves phenotypic/functional knockout by mechanisms such as accelerating target protein degradation and sequestering the target protein in a non-physiological sub-cellular compartment. Other mechanisms of intrabody-mediated gene inactivation can depend on the epitope to which the intrabody is directed, such as binding to the catalytic site on a target protein or to epitopes that are involved in protein-protein, protein-DNA, or protein-RNA interactions.
The binding agent provided herein is not limited to antibodies or antibody fragments, however. Indeed, the binding agent may be an “alternative protein scaffold” or a fragment thereof. The term “alternative protein scaffold” (also referred to as “antibody mimetic”) refers to a non-antibody polypeptide or polypeptide domain which displays an affinity and specificity towards an antigen of interest similar to that of an antibody. Exemplary alternative scaffolds include a β-sandwich domain such as from fibronectin (e.g., Adnectins), lipocalins (e.g., Anticalin®), a Kunitz domain, thioredoxin (e.g., peptide aptamer), protein A (e.g., AFFIBODY® molecules), an ankyrin repeat (e.g., DARPins), γ-β-crystallin or ubiquitin (e.g., AFFLIN™ molecules), CTLD3 (e.g., Tetranectin), multivalent complexes (e.g., ATRIMER™ molecules or SIMP™ molecules), and AVIMER™ molecules. Alternative protein scaffolds are further described in, for example, Binz et al., Nat. Biotechnol., 23: 1257-1268 (2005); Skerra, Curr. Opin. Biotech., 18: 295-304 (2007); Silverman et al., Nat. Biotechnol., 23: 1493-94 (2005), Silverman et al., Nat. Biotechnol., 24; 220 (2006); Simeon, R. and Chen, Z., Protein Cell., 9(1): 3-14 (2018); and U.S. Patent Application Publication 2009/0181855 A1.
The present disclosure also provides a conjugate comprising the binding agent described herein linked to a therapeutic agent. Antibody-drug conjugates (ADCs, also referred to as “immunoconjugates”) generally are used in the art to target and kill cancer cells; however, more recently ADCs have been generated using antibodies that recognize viral proteins (e.g., structural proteins) that target virus-infected cells (see, e.g., Lacek et al., J Biol Chem., 289(50): 35015-35028 (2014) and Gavrilyuk et al., Journal of Virology, 87(9): 4985-4993 (2013)). Thus, in some embodiments, the conjugate may comprise (1) an antibody, an alternative protein scaffold, or antigen-binding fragments thereof, and (2) a therapeutic protein or non-protein moiety (e.g., an antiviral agent or a cytotoxic agent). Any suitable method know in the art for generating ADCs may be used to generate the aforementioned conjugate (see, e.g., Argwarl, P., Bertozzi, C. R., Bioconjugate Chem., 26(2): 176-192 (2015); Hoffmann et al., Oncoimmunology, 7:3 (2018), DOI: 10.1080/2162402X.2017.1395127; and Yao et al., Int J Mol Sci., 17(2): 194 (2016)).
The disclosure further provides a recombinant NS1 antigen which comprises at least a portion of the NS1 β-ladder domain and at least a portion of the NS1 wing domain. In some embodiments, the recombinant NS1 antigen may comprise two overlapping peptides from the C-terminus β-ladder domain, corresponding to, for example, amino acid residues 260-316 and 288-344 of the DENV2 NS1 full-length amino acid sequence. With respect to the wing domain, the recombinant antigen may comprise the conserved motif W115XXW118G119, which is believed to interact with the cell surface.
The disclosure provides a nucleic acid sequence which encodes the binding agent or the recombinant NS1 antigen described herein, as well as a vector comprising the nucleic acid sequence. The vector can be, for example, a plasmid, a viral vector, phage, or bacterial vector. Suitable vectors and methods of vector preparation are well known in the art (see, e.g., Sambrook et al., Molecular Cloning, a Laboratory Manual, 4th edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2012), and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, N.Y. (1994)).
In addition to the nucleic acid encoding the binding agent or recombinant NS1 antigen, the vector desirably comprises expression control sequences, such as promoters, enhancers, polyadenylation signals, transcription terminators, internal ribosome entry sites (IRES), and the like, that provide for the expression of the nucleic sequence in a host cell. Exemplary expression control sequences are known in the art and described in, for example, Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990).
The disclosure also provides a composition comprising the above-described binding agent, conjugate, recombinant NS1 antigen, or nucleic acid sequences encoding any of the foregoing. The composition desirably is a pharmaceutically acceptable (e.g., physiologically acceptable) composition, which comprises a carrier, preferably a pharmaceutically acceptable (e.g., physiologically acceptable) carrier, and the binding agent, the conjugate recombinant antigen, or nucleic acid sequence. Any suitable carrier can be used within the context of the disclosure, and such carriers are well known in the art. For example, the composition may contain preservatives, such as, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. A mixture of two or more preservatives optionally may be used. In addition, buffering agents may be included in the composition. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. A mixture of two or more buffering agents optionally may be used. Methods for preparing compositions for pharmaceutical use are known to those skilled in the art and are described in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).
One of ordinary skill in the art will appreciate that the composition may comprise other therapeutic or biologically active agents. For example, factors that control inflammation, such as ibuprofen or steroids, can be part of the composition to reduce swelling and inflammation associated with in vivo administration of the composition. To enhance the immune response generated against a flavivirus, the composition also may comprise an immune stimulator, or a nucleic acid sequence that encodes an immune stimulator. Immune stimulators also are referred to in the art as “adjuvants,” and include, for example, cytokines, chemokines, or chaperones. Cytokines include, for example, Macrophage Colony Stimulating Factor (e.g., GM-CSF), Interferon Alpha (IFN-α), Interferon Beta (IFN-β), Interferon Gamma (IFN-γ), interleukins (IL-1, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, IL-16, and IL-18), the TNF family of proteins, Intercellular Adhesion Molecule-1 (ICAM-1), Lymphocyte Function-Associated antigen-3 (LFA-3), B7-1, B7-2, FMS-related tyrosine kinase 3 ligand, (Flt3L), vasoactive intestinal peptide (VIP), and CD40 ligand. Chemokines include, for example, B Cell-Attracting chemokine-1 (BCA-1), Fractalkine, Melanoma Growth Stimulatory Activity protein (MGSA), Hemofiltrate CC chemokine 1 (HCC-1), Interleukin 8 (IL-8), Interferon-stimulated T-cell alpha chemoattractant (I-TAC), Lymphotactin, Monocyte Chemotactic Protein 1 (MCP-1), Monocyte Chemotactic Protein 3 (MCP-3), Monocyte Chemotactic Protein 4 (CP-4), Macrophage-Derived Chemokine (MDC), a macrophage inflammatory protein (MIP), Platelet Factor 4 (PF4), RANTES, BRAK, eotaxin, exodus 1-3, and the like. Chaperones include, for example, the heat shock proteins Hsp170, Hsc70, and Hsp40.
The disclosure further provides method of inducing an immune response against a flavivirus in a mammal, which comprises administering to the mammal an effective amount of the above-described binding agent, recombinant NS1 antigen, or compositions comprising same, whereupon an immune response against the flavivirus is induced in the mammal. The disclosure also is directed to the use of the above-described binding agent, recombinant NS1 antigen, or compositions comprising same in a method of inducing an immune response against a flavivirus in a mammal. The immune response can be a humoral immune response, a cell-mediated immune response, or, desirably, a combination of humoral and cell-mediated immunity. Ideally, the immune response provides protection upon subsequent challenge with a flavivirus of any type. However, protective immunity is not required in the context of the invention. The inventive method further can be used for antibody production and harvesting in non-human mammals (e.g., rabbits or mice).
Any route of administration can be used to deliver the binding agent, conjugate, recombinant NS1 antigen, or composition to the mammal. Indeed, although more than one route can be used to administer the composition, a particular route can provide a more immediate and more effective reaction than another route. Preferably, the composition is administered via intramuscular injection or intranasal administration. The composition also can be applied or instilled into body cavities, absorbed through the skin (e.g., via a transdermal patch), inhaled, ingested, topically applied to tissue, or administered parenterally via, for instance, intravenous, peritoneal, or intraarterial administration.
The dose of binding agent or recombinant NS1 antigen included in the composition administered to the mammal will depend on a number of factors, including the age and gender of the mammal, the extent of any side-effects, the particular route of administration, and the like. The dose ideally comprises an “effective amount” of binding agent or recombinant NS1 antigen, i.e., a dose which provokes a desired immune response in the mammal. The desired immune response can entail production of antibodies, protection upon subsequent challenge, immune tolerance, immune cell activation, and the like. Preferably, the desired immune response results in sufficient immunity for the recipient for a desired period of time such that subsequent infection with any other flavivirus does not result in illness.
Administering the composition containing the binding agent or the recombinant NS1 antigen can be one component of a multistep regimen for inducing an immune response against a flavivirus in a mammal. In particular, the inventive method can represent one arm of a prime and boost immunization regimen. In this respect, the method comprises administering to the mammal a boosting composition after administering the composition comprising the binding agent, the conjugate, or the recombinant NS1 antigen to the mammal. In such embodiments, therefore, the immune response is “primed” upon administration of the composition containing the binding agent or the recombinant NS1 antigen and is “boosted” upon administration of the boosting composition. The boosting composition may also comprise the binding agent or the recombinant NS1 antigen.
Administration of the priming composition and the boosting composition can be separated by any suitable timeframe, e.g., 1 week or more, 2 weeks or more, 4 weeks or more, 8 weeks or more, 12 weeks or more, 16 weeks or more, 24 weeks or more, 52 weeks or more, or a range defined by any two of the foregoing values. The boosting composition desirably is administered to a mammal (e.g., a human) 2 weeks or more (e.g., 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 20 weeks, 24 weeks, 28 weeks, 35 weeks, 40 weeks, 50 weeks, 52 weeks, or a range defined by any two of the foregoing values) following administration of the priming composition. More than one dose of priming composition and/or boosting composition can be provided in any suitable timeframe. The dose of the priming composition and boosting composition administered to the mammal depends on a number of factors, including the extent of any side-effects, the particular route of administration, etc.
The binding agent, conjugate, recombinant NS1 antigen, compositions comprising any of the foregoing, and components thereof can be provided in a kit, e.g., a packaged combination of reagents in predetermined amounts with instructions for performing a method using the binding agent, conjugate, recombinant NS1 antigen, or composition. As such, the disclosure provides a kit comprising the binding agent, conjugate, recombinant NS1 antigen, or composition described herein and instructions for use thereof. The instructions can be in paper form or computer-readable form, such as a disk, CD, DVD, etc. Alternatively or additionally, the kit can comprise a calibrator or control, and/or at least one container (e.g., tube, microtiter plates, or strips) for conducting a method, and/or a buffer. Ideally, the kit comprises all components, i.e., reagents, standards, buffers, diluents, etc., which are necessary to perform the method. Other additives may be included in the kit, such as stabilizers, buffers (e.g., a blocking buffer or lysis buffer), and the like. The relative amounts of the various reagents can be varied to provide for concentrations in solution of the reagents which substantially optimize the method. The reagents may be provided as dry powders (typically lyophilized), including excipients which on dissolution will provide a reagent solution having the appropriate concentration.
The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.
This example demonstrates that the anti-DENV NS1 IgG2b mouse monoclonal antibody (mAb) 2B7 protects a mouse model against lethal dengue virus infection and NS1-mediated vascular leak and endothelial dysfunction.
The anti-DENV NS1 IgG2b mouse monoclonal antibody (mAb) 2B7 was previously identified as a strong inhibitor of NS1-induced endothelial hyperpermeability (3). In a DENV2 lethal challenge mouse model, 2B7 was protective in a dose-dependent manner compared to an IgG isotype control, as was a single chain variable fragment (scFv) of 2B7, suggesting that protection could be achieved in a manner independent of antibody Fc effector functions (
Next, the protective mechanism of 2B7 was investigated in vitro using human pulmonary microvascular endothelial cells (HPMEC) and measuring electrical resistance in a trans-endothelial electrical resistance (TEER) assay. Both 2B7 and its antigen-binding fragment (Fab), but not an IgG isotype control, were sufficient to abrogate NS1-induced endothelial hyperpermeability (
This example demonstrates that the crystal structure of the 2B7 antigen-binding fragment complexed with DENV NS1 reveals binding to the β-ladder domain.
As discussed above, NS1 has three distinct domains: N-terminal pi-roll, wing, and C-terminal β-ladder (21). An ELISA measuring binding of 2B7 to full-length NS1, a recombinant wing domain (residues 38-151 of SEQ ID NO: 9), or a recombinant β-ladder domain (residues 176-352 of SEQ ID NO: 9) indicated that 2B7 bound strongly to both full-length NS1 and the β-ladder, but not to the wing domain (
The interaction between 2B7 and NS1 was visualized in detail by solving crystal structures of the 2B7 Fab and scFv in complex with DENV1 NS1 (Fab 3.3 Å,
Each NS1 dimer binds two copies of the scFv/Fab fragment—one to each distal tip of the β-ladder (
This example demonstrates that the 2B7 antibody is cross-reactive with NS1 from multiple flavivirus species.
The amino acid residues in the 2B7 epitope can be divided into two classes: the epitope core region, composed of residues that are highly conserved across flaviviruses, and the epitope periphery, displaying varying levels of divergence among flaviviruses. (
NS1 mutants produced in 293T cells were initially screened for candidates that were secreted and displayed diminished binding to 2B7 (
These experiments determined that 2B7 does not alter human clotting time and binds to the surface of endothelial cells significantly more than an isotype control only when NS1 is present, suggesting that 2B7 would likely not enhance DENV disease through these specific mechanisms (
This example describes an investigation of the molecular basis of NS1-mediated endothelial dysfunction.
As NS1 is reported to mediate endothelial dysfunction through distinct steps including cell binding, EGL disruption, and endothelial hyperpermeability (13, 14, 16), the mode of 2B7 binding to NS1 provides an opportunity to investigate the molecular basis of NS1-mediated endothelial dysfunction. Although 2B7 binds to the β-ladder, its tilted orientation towards the NS1 hydrophobic surface (
Taken together, these data show that the NS1 wing domain, specifically the WWG motif, is important for initial attachment of NS1 to cells, whereas the tip of the β-ladder is critical for downstream events required for NS1-mediated endothelial dysfunction.
The above-described structural investigation of the protective mechanism of the 2B7 mAb against DENV infection and DENV NS1-mediated vascular leak in vivo, as well as pan-flavivirus NS1-triggered endothelial dysfunction in vitro, serves as a proof-of-concept that one cross-reactive antibody targeting flavivirus NS1 proteins can provide protection against NS1-mediated pathology from multiple flaviviruses. Further, the structure of the 2B7 scFv/Fab in complex with NS1 revealed that 2B7 obscures the β-ladder through direct binding and the wing domain through indirect steric hinderance of the NS1 dimer and/or hexamer form, suggesting that one anti-NS1 mAb can simultaneously antagonize the cellular interactions of two domains. DENV NS1 mutagenized in these domains revealed the importance of these domains in NS1-mediated endothelial dysfunction, implicating the wing as critical for initial binding to the endothelial cell surface and the β-ladder as essential for downstream NS1-mediated events including cathepsin L activation, both crucial steps for NS1-triggered pathology.
In summary, the structural and mechanistic investigations of 2B7-mediated protection reveal the critical and distinct roles of the NS1 wing domain in cell binding and the β-ladder in downstream signaling. This, coupled with the flavivirus cross-reactivity of 2B7, possessing no risk of ADE, highlights the possibility of treating multiple flavivirus infections with one therapeutic targeting flavivirus NS1.
All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
This application claims the benefit of U.S. Provisional Application No. 63/117,222 filed Nov. 23, 2020, the contents of which is herein incorporated by reference in their entirety.
This invention was made with government support under AI124493 and AI130130 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US21/60468 | 11/23/2021 | WO |
