Methods and compositions for treating yellow fever

Abstract

To provide antibodies that specifically bind the yellow fever virus and / or neutralize the virus.SOLUTION: The invention provides an antibody or antigen-binding portion thereof, where the antibody or antigen-binding portion thereof specifically binds to an E-DII epitope of the yellow fever virus, and where the E-DII epitope comprises an asparagine at position 106, a lysine at position 93, and a lysine at position 104 of yellow fever virus E-protein.SELECTED DRAWING: None

Description

[Background technology]

[0001] Related Applications This application claims the benefit of priority under 35 U.S.C. §119 of U.S. Provisional Application No. 62 / 656,352, filed April 11, 2018, the entire contents of which are incorporated herein by reference.

[0002] background Yellow fever is an acute viral hemorrhagic disease caused by the yellow fever virus (YFV). In humans, the main vector of YFV transmission is the mosquito Aedes aegypti, which transmits several other viruses, including dengue and Zika viruses. YFV is endemic in tropical and subtropical regions of Africa and Central and South America. Infected individuals exhibit a wide range of symptoms, from asymptomatic infection to acute viral hemorrhagic disease with a 50% case fatality rate (10-15%). Live attenuated vaccines have been on the market since the 1930s. However, efforts to contain and control YFV outbreaks have been hampered by a global supply shortage. The most recent manifestation of this has been seen in Brazil, where YFV has already infected over 700 people and claimed over 200 lives since the outbreak began in 2017. Currently, there are no YFV therapies available to treat those who have not been vaccinated or for whom the vaccine has not provided protection. There is a need for YFV therapies. Summary of the Invention

[0003] The present disclosure is based, at least in part, on the discovery of an antibody that specifically binds to and / or neutralizes YFV. Compositions and methods related to such antibodies or antigen-binding portions thereof are provided. For example, the antibody or antigen-binding portion thereof can, in some embodiments, inhibit YFV from infecting cells. In some embodiments, the antibody or antigen-binding portion thereof in any one of the compositions or methods provided herein specifically binds to occupy key epitope residues (N106, K93 and K104) in E protein that are the most solvent exposed and accessible to antibodies. In some embodiments, the antibody or antigen-binding portion thereof in any one of the compositions or methods provided herein has energetically favorable paratope (CDR) interactions around these key epitope residues and / or is characterized by a threshold minimum binding energy.

[0004] Thus, one aspect of the disclosure provides an antibody or antigen-binding portion thereof that specifically binds to an E protein or envelope protein domain II (E-DII) epitope of a yellow fever virus. In certain embodiments of any one of the compositions or methods provided herein, the E protein or E-DII epitope includes an asparagine at position 106, a lysine at position 93, and a lysine at position 104 of a yellow fever virus E protein.

[0005] In certain embodiments of any one of the provided methods or compositions, the antibody or antigen-binding portion thereof comprises three CDRs of the heavy chain variable region (VH). In certain embodiments of any one of the provided methods or compositions, the three CDRs are those found in any one of the VH sequences described herein, such as in Table 1 (e.g., SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, and 36). In certain embodiments of any one of the provided methods or compositions, the antibody or antigen-binding portion thereof comprises three CDRs of the light chain variable region (VL). In certain embodiments of any one of the methods or compositions provided, the three CDRs are those found in any one of the VL sequences described herein, such as in Table 2 (SEQ ID NOs: 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73). In certain embodiments of any one of the provided methods or compositions, the antibody or antigen-binding portion thereof comprises three CDRs that are any one of the VH sequences described herein, such as in Table 1 (e.g., SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 11 3, 34, 35, and 36), and three CDRs that are any one of the VL sequences described herein, such as in Table 2 (SEQ ID NOs: 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73).In certain embodiments of any one of the methods or compositions provided, the antibody or antigen-binding portion thereof comprises the three CDRs of the VH sequence and the three CDRs of the VL sequence, which is any one of the specific combinations of VH and VL sequences as set forth in Table 3. Thus, in certain embodiments of any one of the methods or compositions provided herein, the antigen-binding portion is an antigen-binding portion of such an antibody.

[0006] In one aspect, provided herein is a nucleic acid encoding the three CDRs of any one of the VH sequences provided herein, such as directly provided above, and / or the three CDRs of any one of the VL sequences provided herein, such as directly provided above.

[0007] In certain embodiments of any one of the methods or compositions provided, the antibody or antigen-binding portion thereof comprises any one of the VH amino acid sequences provided herein, such as set forth in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, and 36. In certain embodiments of any one of the methods or compositions provided, the antibody or antigen-binding portion thereof comprises any one of the VL amino acid sequences provided herein, such as set forth in any one of SEQ ID NOs: 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73. In certain embodiments of any one of the methods or compositions provided, the antibody or antigen-binding portion thereof comprises a VH antigen as provided herein, such as set forth in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, and 36. In certain embodiments of any one of the provided methods or compositions, the antibody or antigen-binding portion thereof comprises any one of the VH and VL amino acid sequences provided herein, such as those set forth in any one of SEQ ID NOs: 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73. In certain embodiments of any one of the provided methods or compositions, the antibody or antigen-binding portion thereof comprises any one of the specific combinations of VH and VL amino acid sequences provided herein, such as those set forth in Table 3.

[0008] In one aspect, provided herein is a nucleic acid encoding any one of the VH sequences provided herein, such as directly provided above, and / or any one of the VL sequences provided herein, such as directly provided above.

[0009] In certain embodiments of any one of the methods or compositions provided herein, the antibody or antigen-binding portion thereof comprises three CDRs that have at least 90%, 95%, 96%, 97%, 98% or 99% identity to the three CDRs of any one of the VH sequences described herein, such as in Table 1 (e.g., SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, and 36). In certain embodiments of any one of the methods or compositions provided, the antibody or antigen-binding portion thereof comprises three CDRs having at least 90%, 95%, 96%, 97%, 98% or 99% identity to the three CDRs of any one of the VL sequences described herein, such as in Table 2 (SEQ ID NOs: 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73). In certain embodiments of any of the provided methods or compositions, the antibody or antigen-binding portion thereof has at least 90%, 95%, 96%, 97%, 98% or 99% identity with any one of the VH sequences described herein, such as in Table 1 (e.g., SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, and 36). and three CDRs that have at least 90%, 95%, 96%, 97%, 98% or 99% identity to any one of the VL sequences described herein, such as in Table 2 (SEQ ID NOs: 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73).In certain embodiments of any one of the methods or compositions provided, the antibody or antigen-binding portion thereof comprises three CDRs having at least 90%, 95%, 96%, 97%, 98% or 99% identity with the VH sequence and three CDRs having at least 90%, 95%, 96%, 97%, 98% or 99% identity with the VL sequence in any one of the specific combinations of VH and VL sequences provided herein, such as those set forth in Table 3. Thus, in certain embodiments of any one of the methods or compositions provided herein, the antigen-binding portion is an antigen-binding portion of such an antibody.

[0010] In one aspect, provided herein is a nucleic acid encoding the three CDRs of any one of the VH sequences provided herein, such as directly provided above, and / or the three CDRs of any one of the VL sequences provided herein, such as directly provided above.

[0011] In certain embodiments of any one of the methods or compositions provided, the antibody or antigen-binding portion thereof comprises a VH amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to any one of the VH amino acid sequences provided herein, such as set forth in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, and 36. In certain embodiments of any one of the methods or compositions provided, the antibody or antigen-binding portion thereof comprises a VL amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to any one of the VL amino acid sequences provided herein, such as set forth in any one of SEQ ID NOs: 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73. In certain embodiments of any one of the methods or compositions provided, the antibody or antigen-binding portion thereof has at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to any one of the VH amino acid sequences provided herein, such as set forth in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, and 36. and VL amino acid sequences having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to any one of the VL amino acid sequences provided herein, such as set forth in any one of SEQ ID NOs: 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73.In certain embodiments of any one of the provided methods or compositions, the antibody or antigen-binding portion thereof comprises a VH sequence and a VL sequence that have at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, independently of each other, to a VH and VL sequence that is any one specific combination of VH and VL amino acid sequences provided herein, such as those listed in Table 3.

[0012] In one aspect, provided herein is a nucleic acid encoding a VH sequence as directly provided above and / or a VL sequence as directly provided above.

[0013] In certain embodiments of any one of the methods or compositions provided herein, the antigen-binding portion is an antigen-binding portion of any one of the antibodies provided herein.

[0014] Any one of the antibodies described herein may be a full-length antibody. The antibody or antigen-binding portion thereof may be human, humanized, or chimeric in certain embodiments of any one of the methods or compositions provided herein. The antibody or antigen-binding portion thereof may be a single-chain antibody in certain embodiments of any one of the methods or compositions provided herein. Any of the antibodies described herein may be either monoclonal or polyclonal. These two terms do not limit the source of the antibody or the manner in which it is made. The antibody or antigen-binding portion thereof may be a monoclonal antibody or antigen-binding portion thereof in certain embodiments of any one of the methods or compositions provided herein. Furthermore, the antigen-binding portion thereof may be an scFv, F(ab')2 fragment, dAb fragment, Fab fragment, Fab' fragment, Fv, or disulfide-linked Fv fragment in certain embodiments of any one of the methods or compositions provided herein. The antibody or antigen-binding portion thereof may be a single domain antibody, a diabody, a multispecific antibody, a bispecific antibody, or a dual-specific antibody. The antibody or antigen-binding portion thereof in certain embodiments of any one of the methods or compositions provided herein may be an isolated antibody or antigen-binding portion thereof.

[0015] Further disclosed is a method of treating yellow fever or a disease or condition associated with yellow fever virus by administering to a subject in need of such treatment a therapeutically effective amount of one or more antibodies or antigen-binding portions thereof that specifically bind to YFV. In certain embodiments of any one of the methods provided herein, the subject is a subject who has not been vaccinated against YFV or in whom YFV vaccination did not provide sufficient protection. In certain embodiments of any one of the methods or compositions provided herein, the antibody or antigen-binding portion thereof is any one or more of the antibodies or antigen-binding portions thereof described herein. In certain embodiments of any one of the methods or compositions provided herein, the antibody or antigen-binding portion thereof specifically binds to the E protein or E-DII epitope of YFV. In certain embodiments of any one of the methods or compositions provided herein, the antibody or antigen-binding portion thereof specifically binds to residues 93, 104, and 106 of the E protein or E-DII epitope of the yellow fever virus E-protein. In certain embodiments of any one of the methods or compositions provided herein, the amount of the antibody or antigen-binding portion thereof is effective to reduce one or more symptoms of yellow fever in a subject. Any one of the anti-YFV antibodies or antigen-binding portions thereof may be administered systemically, e.g., via an enteral route or via a parenteral route, in any one of the methods provided herein.

[0016] The subject treated in any one of the methods described herein may be a patient (e.g., a human patient) suffering from or suspected of having yellow fever or a disease or condition associated with yellow fever virus. In certain embodiments of any one of the methods provided herein, the subject is a human patient suffering from or suspected of having acute viral hemorrhagic disease.

[0017] Also provided herein in some aspects is (a) a pharmaceutical composition for use in treating yellow fever or a disease or condition associated with the yellow fever virus (e.g., acute viral hemorrhagic disease) in a subject, the pharmaceutical composition comprising any one or more of the antibodies or antigen-binding portions thereof described herein and a pharmaceutically acceptable carrier; and (b) use of the aforementioned antibodies or antigen-binding portions thereof in medicaments and / or manufacture of medicaments for the treatment of yellow fever or a disease or condition associated with the yellow fever virus in a subject.

[0018] Also provided herein in some aspects are methods for producing an antibody or antigen-binding portion thereof, nucleic acids encoding any one of the antibodies or antigen-binding portions thereof, vectors that can include any one or more of the nucleic acids provided herein, and associated host cells.

[0019] In one aspect, the method for producing an antibody or an antigen-binding portion thereof is any one of the methods described herein. In one embodiment, the method includes considering distinct domain-proximal structural regions (e.g., interchain interfaces close to the icosahedral symmetry axis) present in the virus assembly and selecting promising Fv scaffolds. Conventional epitope prediction methods use domain structures to predict epitope surface regions embedded within domain regions. However, neutralizing flavivirus antibodies can recognize four epitope surfaces (spanning two or more E protein chains). Therefore, valuable information is not incorporated in conventional methods. [Brief description of the drawings]

[0020] [Figure 1] Figure 1 shows an example of an in vivo test design for the efficacy of engineered mAbs against YF-17D-204 in a mouse infection model. The efficacy of the designed mAbs was tested in a lethal yellow fever infection model in AG129 mice. The protective efficacy of the mAbs was tested in prophylaxis or as a treatment.

[0021] [Diagram 2] Figures 2A-2B show the in vivo efficacy of the designed mAbs against yellow fever virus. Figure 2A shows the survival curves of YF-17D-infected AG129 mice treated with mAbs at a dose of 10 mg / kg as prophylaxis (-1) or therapy (+1 and +1, +4). Figure 2B shows the blood viral titers of yellow fever virus in treated animals compared to the control group at 4 and 6 days post-infection. Administration of the mAbs resulted in complete protection compared to the control group (Figure 2A). Administration of the mAbs also resulted in a reduction in viremia of more than 2 Log10 at 4 and 6 days post-infection (Figure 2B). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Detailed Description The following description is intended merely to illustrate various aspects of the present invention. As such, the specific aspects discussed herein should not be construed as limiting the scope of the present invention. It will be apparent to those skilled in the art that various modifications can be made or equivalents can be made without departing from the scope of the present invention.

[0023] Various aspects of the disclosure relate to antibodies, antigen-binding portions thereof, and pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors, and host cells for producing such antibodies and fragments. Provided herein are antibodies and / or antigen-binding portions thereof that specifically bind to and / or neutralize Yellow Fever Virus (YFV). In some embodiments, the antibodies or antigen-binding portions thereof bind to the E-DII epitope of Yellow Fever Virus and inhibit the virus from infecting cells. In some embodiments, the antibodies or antigen-binding portions thereof bind to the E-DII epitope, including the asparagine at position 106, the lysine at position 93, and / or the lysine at position 104 of the Yellow Fever Virus E protein. The antibodies and antigen-binding portions provided herein are, in some embodiments, used to treat or inhibit Yellow Fever Virus infection in a subject.

[0024] Antibodies (used interchangeably in the plural), as used herein, refer broadly to immunoglobulin (Ig) molecules, or any functional mutant, variant, or derivative thereof. It is desirable for the functional mutants, variants, and derivatives thereof, as well as antigen-binding portions, to retain the essential epitope-binding properties of the Ig molecule.

[0025] Antibodies can specifically bind to a target through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. Generally, an intact or full-length antibody comprises two heavy chains and two light chains. Each heavy chain comprises a heavy chain variable region (V H ) and the first, second and third constant regions (C H 1. C H 2 and C H Each light chain contains a light chain variable region (V L) and a constant region (CL). The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A full-length antibody can be of any class, such as IgD, IgE, IgG, IgA, or IgM (or subclasses thereof), and an antibody need not be of a particular class. Depending on the antibody amino acid sequence of the constant domain of its heavy chain, an immunoglobulin can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0026] The term "antigen-binding portion" refers to a portion or region of an intact or full-length antibody molecule capable of specifically binding to a target. Preferably, the antigen-binding portions provided herein retain the ability to specifically bind to YFV. An antigen-binding portion may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both. Each of the VH and VL typically contains three complementarity determining regions, CDR1, CDR2, and CDR3.

[0027] Examples of antigen-binding portions include, but are not limited to, (1) a Fab fragment, which may be a monovalent fragment having a VL-CL chain and a VH-CH chain; (2) an F(ab')2 fragment, which may be a bivalent fragment having two Fab fragments linked by a disulfide bridge at the hinge region, i.e., a dimer of Fab; (3) an Fv fragment having the VL and VH domains of a single arm of an antibody; (4) a single-chain Fv (scFv), which may be a single polypeptide chain consisting of the VH and VL domains via a peptide linker; (5) an (scFv)2, which may include two VH domains linked by a peptide linker and two VL domains connected to the two VH domains via disulfide bridges; (6) an Fd fragment consisting of a VH and a CHI domain; (7) a dAb fragment, which includes a single variable domain; and (8) an isolated complementarity determining region (CDR).

[0028] Furthermore, the two domains of the Fv fragment, VL and VH, can be encoded by separate genes, but they can be engineered into a single protein chain in which the VL and VH regions are paired to form a monovalent molecule using recombinant techniques (known as single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Other forms of single-chain antibodies, such as diabodies, are also encompassed. Bispecific antibodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains on another chain, creating two antigen-binding sites (see, e.g., Holliger, P. et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ et al. (1994) Structure 2: 1121-1123). Bispecific antibodies are also encompassed within the term "antigen-binding portion."

[0029] The term "human antibody" refers to an antibody having variable and constant regions that substantially correspond to or are derived from an antibody obtained from a human subject, e.g., encoded by a human germline immunoglobulin sequence or a variant thereof. The human antibodies described herein may contain one or more amino acid residues not encoded by a human germline immunoglobulin sequence (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). Such mutations may be present in one or more CDRs, particularly CDR3, or in one or more framework regions. In some embodiments, a human antibody may have at least one, two, three, four, five or more positions substituted with an amino acid residue not encoded by a human germline immunoglobulin sequence. However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been transferred to human framework sequences.

[0030] The term "recombinant human antibody," as used herein, refers to antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant combinatorial human antibody library (Hoogenboom HR, (1997) TIB Tech. 15:62-70; Azzazy H. and Highsmith WE, (2002) Clin. Biochem. 35:425-445; Gavilondo JV and Larrick JW (2002) BioTechniques 29: 128-145; Hoogenboom H. and Chames P. (2000) Immunology Today 21:371-378), antibodies isolated from animals (e.g., mice) transgenic for human immunoglobulin genes (e.g., Taylor, LD et al. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann SA. and Green J. (2002) Immunology Today 21:371-378), and antibodies isolated from animals (e.g., mice) transgenic for human immunoglobulin genes (e.g., Taylor, LD et al. (1992) Nucl. Acids Res. 20:6287-6295). LL (2002) Current Opinion in Biotechnology 13:593-597; Little M. et al. (2000) Immunology Today 21:364-370), or any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences, are intended to include all human antibodies prepared, expressed, produced, or isolated by recombinant means. Such recombinant human antibodies have variable and constant regions as defined above. However, in certain embodiments, such recombinant human antibodies may have been subjected to in vitro mutagenesis (or in vivo somatic mutagenesis, in cases where a transgenic animal of a human Ig sequence animal is used) such that the amino acid sequences of the VH and VL regions of the recombinant antibody may be sequences that are derived from and related to human germline VH and VL sequences, but which may not naturally exist within the human antibody germline repertoire in vivo.

[0031] Some embodiments of the present disclosure provide fully human antibodies capable of binding to the E-DII epitope of Yellow Fever virus. In some embodiments, the E-DII epitope includes an asparagine at position 106, a lysine at position 93, and a lysine at position 104 of the Yellow Fever virus E protein. The protein sequences of the various VH and VL regions are shown in Tables 1 and 2, respectively.

[0032] [Table 1-1]

[0033] [Table 1-2]

[0034] [Table 1-3]

[0035] [Table 2-1]

[0036] [Table 2-2]

[0037] [Table 2-3]

[0038] [Table 2-4]

[0039] An "isolated" material means one that has been altered by the hand of man from its natural state. When a material is "isolated" in nature, it means that it has been altered or removed from its original environment, or both. For example, a polypeptide that is naturally present in an organism is not "isolated," but is "isolated" if it is substantially separated from the coexisting materials of its natural state and / or exists in a substantially pure state.

[0040] The term "specifically binds" or "specific binding" refers to a non-random binding reaction between two molecules, such as the binding of an antibody or antigen-binding portion thereof to an epitope of an antigen. An antibody or antigen-binding portion thereof that "specifically binds" to a target or epitope is a term well understood in the art, and methods for determining such specific binding are also well known in the art. A molecule is said to exhibit "specific binding" if it reacts or binds to a particular target antigen or epitope more frequently, more rapidly, with a longer duration, and / or with a higher affinity than it reacts with another target / epitope. An antibody or antigen-binding portion thereof "specifically binds" to a target antigen if it binds with higher affinity, avidity, more readily, and / or with a longer duration than it binds to other substances. In some embodiments, an antibody is said to specifically bind an antigen if it selectively recognizes its target antigen in a complex mixture of proteins and / or macromolecules.

[0041] An "epitope" is a region of an antigen that is bound by an antibody. The term includes any polypeptide determinant capable of specific binding to an immunoglobulin. In certain embodiments, epitopic determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryls, or sulfonyls, and in certain embodiments may have specific three dimensional structural characteristics, and / or specific charge characteristics.

[0042] As used herein, the term "neutralization" refers to neutralization of activity, such as the biological activity of a target protein (e.g., yellow fever virus E protein). In one embodiment, a neutralizing antibody binds to the E-DII epitope of yellow fever virus E-protein and causes inhibition of the biological activity of yellow fever virus and / or inhibits the virus from infecting cells.

[0043] The subject may be a human, but also includes other mammals, particularly those useful as experimental models for human disease, such as mice, rats, rabbits, dogs, and the like.

[0044] The term "treat", "treatment" or "treating" refers to the act, application or therapy in which a subject, including a human, is given medical assistance with the goal of directly or indirectly improving the subject's condition. In particular, the term refers in some embodiments to reducing the incidence or alleviating one or more symptoms, eliminating recurrence, inhibiting recurrence, inhibiting the incidence, improving one or more symptoms, and / or improving the prognosis, or a combination thereof. One of skill in the art will appreciate that treatment does not necessarily result in the complete absence or elimination of symptoms. For example, with respect to yellow fever virus, "treatment" or "treating" may refer to reducing the severity or duration of acute viral hemorrhagic disease caused by yellow fever.

[0045] As used herein, "effective amount" or "effective dose" or "therapeutically effective amount" in the context of administration of a pharmacological formulation refers to the amount of a drug or pharmaceutical formulation (e.g., an antibody or antigen-binding fragment or portion thereof, or a composition comprising the same) that produces the intended pharmacological result or effect in treating, curing, suppressing or ameliorating a disease or a symptom of a disease, disorder or side effect, or reducing the rate of progression of a disease or disorder or any symptom thereof, compared to a corresponding subject not receiving such amount. The effective amount or dose of a pharmacological agent may vary depending on the particular active ingredient employed, the mode of administration, and / or the age, size, and condition of the subject being treated.

[0046] The present disclosure also provides a composition (e.g., a pharmaceutical composition) comprising an antibody or antigen-binding portion thereof that specifically binds to the E-DII epitope of the Yellow Fever Virus E-protein. In some embodiments, the E-DII epitope comprises an asparagine at position 106, a lysine at position 93, and a lysine at position 104 of the Yellow Fever Virus E-protein. The composition can be prepared from any one of the antibodies or antigen-binding portions described herein. As described herein, the antibody or antigen-binding portion thereof, and the nucleic acid or nucleic acid set encoding same, the vector comprising same, or the host cell comprising said vector, can be mixed with a pharmaceutically acceptable carrier (excipient) to form a pharmaceutical composition for use in treating a target disease. Pharmaceutically acceptable excipients (carriers), including buffers, are well known in the art. See, for example, Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. KE Hoover.

[0047] The pharmaceutical composition may contain a pharma- ceutically acceptable carrier, excipient, or stabilizer in the form of a lyophilized formulation or an aqueous solution. (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. KE Hoover). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride); hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol; low molecular weight polypeptides (less than about 10 residues); serum albumin, gelatin, or immunoglobulins. may include proteins such as globulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextran; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™ (polysorbates), PLURONICS™ (poloxamers), or polyethylene glycol (PEG).

[0048] As used in connection with the compositions of the present disclosure, the phrase "pharmaceutical acceptable" refers to molecular entities and other components of such compositions that are physiologically tolerable and typically do not produce undesirable reactions when administered to a subject.Preferably, as used herein, the term "pharmaceutical acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in mammals, and more specifically, in humans. "Acceptable" means that the carrier is compatible with the active components of the composition (e.g., nucleic acids, vectors, cells, or therapeutic antibodies) and does not adversely affect the subject to which the composition is administered.Any pharmaceutical composition used in the methods of the present invention may contain a pharmaceutical acceptable carrier, excipient, or stabilizer in lyophilized or aqueous form.

[0049] Pharmaceutically acceptable carriers, including buffers, are well known in the art and may include phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; amino acids; hydrophobic polymers; monosaccharides; disaccharides; and other carbohydrates; metal complexes; and / or non-ionic surfactants. See, e.g., Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. KE Hoover.

[0050] The pharmaceutical compositions may be presented in unit dosage form and may be prepared by any suitable method, many of which are well known, including the step of bringing the anti-YFV antibodies into association with the carrier which constitutes one or more accessory ingredients.

[0051] The compositions provided herein may be sterile aqueous preparations, which are preferably isotonic with the blood of the recipient in some embodiments.These aqueous preparations may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents.The sterile aqueous preparations may also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents.The acceptable vehicles and solvents that may be used include water, Ringer's solution, and isotonic sodium chloride solution.

[0052] As used herein, the term "vector" is intended to refer to a nucleic acid molecule that can transport another nucleic acid to which it is linked. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Furthermore, a vector may be capable of directing the expression of a gene to which it is operatively linked. Such vectors are referred to herein as "recombinant expression vectors" or "expression vectors".

[0053] As used herein, the term "recombinant host cell" or "host cell" is intended to refer to a cell into which exogenous DNA has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain changes may occur in successive generations, either by mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are included within the scope of the term "host cell" as used herein. EXAMPLES

[0054] example Anti-YFV monoclonal antibodies are designed using structure-guided analysis of YFV envelope protein epitopes, focusing on the envelope domain II fusion loop. Mutation-restricted epitopes on the surface of the yellow fever virus are identified. Then, suitable antibody scaffolds that satisfy the epitope-paratope constraints are identified to engineer antibodies that target and neutralize the virus.

[0055] Anti-YFV antibodies will be cloned, expressed and purified using known methods and screened for expression and biophysical properties. In silico designed antibodies will be cloned into mammalian expression vectors and purified for further analysis. Various analytical parameters indicative of expression as well as purity and stability will be evaluated. The efficacy of antibodies against Yellow Fever Virus will be evaluated using in vitro and in vivo infection models. The in vitro neutralization potency of engineered antibodies will be assessed using a plaque reduction neutralization test (PRNT) against the YF17D-204 vaccine strain of yellow fever virus, and selected antibodies will be further evaluated for efficacy in a mouse model of YFV infection.

[0056] Rational design of anti-YF mAb Potential anti-YFV monoclonal antibodies should neutralize a broad range of strains and target regions associated with robust protection. Epitopes play an important role in the efficacy of therapeutic antibodies. In YFV infection, the envelope (E) protein is the predominant target of neutralizing antibodies. To define potential epitopes, a structure-guided analysis of the whole YFV assembly was performed to identify spatially clustered, solvent accessible, and sequence conserved residues. This was done by mapping sequence conservation scores (calculated from alignment of YFV envelope protein sequences) onto a homology model of the whole assembly of the YFV E protein. Residues with >70% sequence conservation and >40% solvent accessible surface area were considered as putative epitope residues. According to this analysis, the region proximal to the domain II (E-DII) hydrophobic fusion loop appeared to be highly accessible and conserved among YFV strains.

[0057] To engineer antibodies against the E-DII epitope, a structure-guided search was performed for Fv scaffolds that recognize highly homologous epitope surfaces. Promising scaffolds were docked against the epitope (using the software ZRANK) and ranked based on shape complementarity (>0.6), buried surface area (1,000 Sq.A°), and likelihood to contact key E-DII epitope residues (N106, K93, and K104 of E-protein; numbers correspond to the primary sequence of the 17D YF vaccine strain). The CDR loops of the top-ranked scaffolds were then redesigned by Rosetta Antibody Design to optimally contact the epitope. Engineered antibodies were screened for in vitro neutralization of YFV.

[0058] The synthetic DNA sequences encoding the VH and VL domains provided in Tables 1 and 2 were cloned in frame with the IgG1 constant region into a mammalian expression vector and various combinations of heavy and light chains were combined into at least 110 YFV antibodies. Examples of such combinations are shown in Table 3 below.

[0059] [Table 3]

[0060] Examples of expression levels and biophysical properties A subset of YFV antibodies was purified from large-scale transfections in Expi293 cells. Proteins from cell culture supernatants were purified using HiTrap Protein A columns. After dialysis, protein concentrations were estimated by nanodrop using theoretical extinction coefficients. The yields of the various antibodies are summarized in Table 4.

[0061] [Table 4]

[0062] Additionally, the purity and stability of various antibodies were determined by (1) UV spectroscopy, (2) size exclusion chromatography, (3) microfluidic capillary electrophoresis, and (4) thermal shift dye dissociation assay, respectively. Specific examples of such biophysical analysis parameters are summarized in Table 5 below.

[0063] [Table 5]

[0064] Further characterization of the glycoforms of the various antibodies was performed by LC-FLD-MS with the aid of procainamide labeling. Specific examples of glycosylation profiles observed are as follows (Table 6):

[0065] [Table 6]

[0066] Specific examples of neutralizing ability of artificially produced antibodies against YF-17D To determine the neutralization potency of YFV antibodies against YFV (17D-204), a plaque reduction neutralization test (PRNT) was performed. Vero cells were infected with either virus alone, no virus, or virus preincubated with various dilutions of YFV antibodies. Plates were incubated for 7 days, fixed, and stained with crystal violet to visualize plaque formation. Neutralization curves were generated using Prism software (Figure 1), and 50% effective concentration values ​​(EC50) were calculated by nonlinear regression with variable slope (Table 6). The neutralization potency of the antibodies was directly proportional to the reduction of plaques, and the neutralization capacity is expressed as the concentration at which 50% of the virus particles are neutralized. The antibodies were highly potent in neutralizing the virus, and examples are shown in Table 7 below.

[0067] [Table 7]

[0068] Examples of in vivo efficacy Figure 1 provides an example of the in vivo efficacy of engineered antibodies against YF-17D-204 in a mouse infection model. The efficacy of the engineered antibodies was tested in a lethal model of yellow fever infection in AG129 mice. The protective efficacy of the antibodies was tested prophylactically or as a treatment, as shown in Figure 1. In the specific example shown in Figure 2, administration of antibody resulted in complete protection compared to the control group (Figure 2A). Administration of antibody also resulted in a reduction in viremia of more than 2 Log10 at days 4 and 6 post-infection (Figure 2B).

Claims

1. 1. An antibody or antigen-binding portion thereof, wherein the antibody or antigen-binding portion thereof specifically binds to an E-DII epitope of a yellow fever virus, and the E-DII epitope comprises an asparagine at position 106, a lysine at position 93, and a lysine at position 104 of a yellow fever virus E protein.

2. The antibody or antigen-binding portion thereof of claim 1, wherein the antibody or antigen-binding portion thereof comprises three CDRs of the heavy chain variable region (VH), and the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, and 36.

3. 3. The antibody or antigen-binding portion thereof of claim 1 or 2, wherein the antibody or antigen-binding portion thereof comprises three CDRs of the light chain variable region (VL), and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73.

4. The antibody or antigen-binding portion thereof of any one of claims 1 to 3, wherein the antibody or antigen-binding portion thereof comprises a VH, and the VH comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, and 36.

5. 5. The antibody or antigen-binding portion thereof of any one of claims 1 to 4, wherein the antibody or antigen-binding portion thereof comprises a VL, and the VL comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73.

6. The antibody or antigen-binding portion thereof according to any one of claims 1 to 5, wherein the antibody or antigen-binding portion thereof is a human antibody or antigen-binding portion thereof, a monoclonal antibody or antigen-binding portion thereof, a chimeric antibody or antigen-binding portion thereof, or a humanized antibody or antigen-binding portion thereof.

7. The antibody or antigen-binding portion thereof according to any one of claims 1 to 6, wherein the antibody or antigen-binding portion thereof is a single chain antibody or antigen-binding portion thereof, an F(ab')2 fragment, a dAb fragment, a Fab fragment, or an Fv fragment.

8. The antibody or antigen-binding portion thereof of any one of claims 1 to 7, wherein the antibody or antigen-binding portion thereof is an isolated antibody or antigen-binding portion thereof.

9. A composition comprising an antibody or antigen-binding portion thereof according to any one of claims 1 to 8 and a carrier.

10. The composition of claim 9, wherein the composition is a pharmaceutical composition comprising a pharma- ceutically acceptable carrier.

11. An antibody or antigen-binding portion thereof for use in medicine, wherein the antibody or antigen-binding portion thereof is according to any one of claims 1 to 8.

12. 10. A method for treating yellow fever, or a disease or condition associated with the yellow fever virus, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen-binding portion thereof according to any one of claims 1-8.

13. 10. Use of an antibody or antigen-binding portion thereof according to any one of claims 1 to 8 in the manufacture of a medicament for treating a disease or condition associated with yellow fever virus.

14. A nucleic acid comprising a nucleotide sequence encoding an antibody heavy chain variable region (VH), an antibody light chain variable region (VL), or both, wherein VH and VL are as defined in any one of claims 2 to 7.

15. A vector comprising the nucleic acid of claim 14.

16. The vector of claim 15 , wherein the vector is an expression vector.

17. A host cell comprising a nucleic acid according to claim 14 or a vector according to claim 15 or 16.

18. 1. A method for producing an antibody or antigen-binding portion thereof that binds to a Yellow Fever Virus E protein, comprising: The method comprises culturing the host cell of claim 17 under conditions that allow expression of the antibody.

19. 20. The method of claim 18, further comprising recovering the antibody or antigen-binding portion thereof.

20. 20. The method of claim 18 or 19, wherein the antibody or antigen-binding portion thereof specifically binds to the E-DII epitope of the yellow fever virus E protein.

21. 21. The method of claim 20, wherein the E-DII epitope comprises residues 93, 104, and 106 of the yellow fever virus E protein.

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