Methods and compositions for treating yellow fever

Abstract

The present disclosure is based, at least in part, on the discovery of antibodies that specifically bind to YFV and / or neutralize the virus. Compositions and methods related to such antibodies, or antigen-binding portions thereof, are provided.

Description

[0001] Related Applications

[0002] This application claims the benefit of priority under 35 USC §119 to U.S. Provisional Application No. 62 / 656,352, filed on April 11, 2018, the entire contents of which are incorporated herein by reference. Background Art

[0003] Yellow fever is an acute viral hemorrhagic disease caused by yellow fever virus (Yellow Fever Virus, YFV). In humans, the main carrier of YFV transmission is the mosquito Aedes aegypti (Aedes aegypti), which transmits several other viruses, including dengue and Zika viruses. YFV is prevalent in tropical and subtropical regions of Africa and Central and South America. Infected individuals have a variety of symptoms ranging from asymptomatic infection to acute viral hemorrhagic disease (10% to 15%), with a mortality rate of 50%. Since the 1930s, live attenuated vaccines have been available on the market. However, global supply shortages have hindered efforts to prevent and control YFV outbreaks. The latest signs of this can be seen in Brazil, where YFV has infected more than 700 people and claimed more than 200 lives since the outbreak began in Brazil in 2017. Currently, there is no YFV treatment available for treating people who are not vaccinated or in which the vaccine does not provide protection. Therefore, there is a demand for YFV treatment. Summary of the invention

[0004] The present disclosure is based at least in part on the discovery of antibodies that specifically bind to YFV and / or neutralize the virus. For example, in some embodiments, antibodies or their antigen-binding portions can prevent YFV from infecting cells. In one embodiment, an antibody or its antigen-binding portion of any one of the compositions or methods provided herein specifically binds to engage the key epitope residues (N106, K93, and K104) that are most exposed to the solvent on the E protein and accessible to the antibody. In one embodiment, an antibody or its antigen-binding portion of any one of the compositions or methods provided herein has energetically favorable complementary positions (CDR) interactions around these key epitope residues and / or is characterized by a threshold minimum binding energy.

[0005] Therefore, one aspect of the present disclosure provides an antibody or antigen binding portion thereof that specifically binds to an epitope of E protein or envelope protein domain II (E-DII) of yellow fever virus. In one embodiment of any one of the compositions or methods provided herein, the E protein or E-DII epitope comprises asparagine at position 106, lysine at position 93, and lysine at position 104 of the E protein of yellow fever virus.

[0006] In one embodiment of any of the methods or compositions provided, the antibody or antigen binding portion thereof comprises three CDRs of a heavy chain variable region (VH). In one embodiment of any of the methods or compositions provided, the three CDRs are those found in any of the VH sequences set forth herein (e.g., in Table 1 (e.g., SEQ ID. NO: 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 one embodiment of any of the methods or compositions provided, the antibody or antigen binding portion thereof comprises three CDRs of a light chain variable region (VL). In one embodiment of any of the methods or compositions provided, the three CDRs are those found in any one of the VL sequences set forth herein, e.g., 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 one embodiment of any of the methods or compositions provided, the antibody or antigen binding portion comprises three CDRs of any one of the VH sequences shown herein (e.g., in Table 1 (e.g., SEQ ID. NO: 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 any one of the VL sequences shown herein (e.g., in Table 2 (SEQ ID. NO: 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 one embodiment of any of the methods or compositions provided, the antibody or antigen binding portion thereof comprises three CDRs of a VH sequence and three CDRs of a VL sequence of any specific combination of a VH sequence and a VL sequence as shown in Table 3. Thus, in one embodiment of any of the methods or compositions provided herein, the antigen binding portion thereof is an antigen binding portion of such an antibody.

[0007] In one aspect, provided herein are nucleic acids encoding the three CDRs of any one of the VH sequences provided herein (eg, provided directly above) and / or the three CDRs of any one of the VL sequences provided herein (eg, provided directly above).

[0008] In one embodiment of any of the methods or compositions provided, the antibody, or antigen binding portion thereof, comprises any of the VH amino acid sequences provided herein, e.g., any of those shown below: SEQ ID. NO: 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 one embodiment of any of the methods or compositions provided, the antibody, or antigen binding portion thereof, comprises any of the VL amino acid sequences provided herein, e.g., any of those shown below: SEQ ID. NO: 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 one embodiment of any of the provided methods or compositions, the antibody, or antigen binding portion thereof, comprises any one of the VH amino acid sequences provided herein, such as any one of the following: SEQ ID. NO: 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 any one of the VL amino acid sequences provided herein, such as any one of the following: SEQ ID. NO: 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 ID. NO: 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 one embodiment of any of the provided methods or compositions, the antibody, or antigen binding portion thereof, comprises any of the specific combinations of combinations of VH amino acid sequences and VL amino acid sequences provided herein, e.g., as shown in Table 3.

[0009] In one aspect, provided herein are nucleic acids encoding any one of the VH sequences provided herein (eg, provided directly above) and / or any one of the VL sequences provided herein (eg, provided directly above).

[0010] In one embodiment of any of the methods or compositions provided herein, the antibody, or antigen binding portion thereof, comprises three CDRs that are at least 90%, 95%, 96%, 97%, 98%, or 99% identical to three CDRs of any one of the VH sequences shown herein, e.g., in Table 1 (e.g., SEQ ID. NO: 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 one embodiment of any of the methods or compositions provided, the antibody, or antigen binding portion thereof, comprises three CDRs that are at least 90%, 95%, 96%, 97%, 98%, or 99% identical to three CDRs of any one of the VL sequences shown herein, e.g., 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 one embodiment of any of the methods or compositions provided, the antibody or antigen binding portion comprises three CDRs that are at least 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of the VH sequences set forth herein (e.g., in Table 1 (e.g., SEQ ID. NO: 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 any one of the VL sequences set forth herein (e.g., in Table 2 (SEQ ID. NO: 1, ID. NO: 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)) have three CDRs that are at least 90%, 95%, 96%, 97%, 98% or 99% identical. In one embodiment of any of the methods or compositions provided, the antibody, or antigen binding portion thereof, comprises three CDRs that are at least 90%, 95%, 96%, 97%, 98%, or 99% identical to a VH sequence in any specific combination of VH and VL sequences provided herein (e.g., as shown in Table 3) and three CDRs that are at least 90%, 95%, 96%, 97%, 98%, or 99% identical to a VL sequence.Thus, in one embodiment of any of the methods or compositions provided herein, the antigen binding portion thereof is an antigen binding portion of such an antibody.

[0011] In one aspect, provided herein are nucleic acids encoding the three CDRs of any one of the VH sequences provided herein (eg, provided directly above) and / or the three CDRs of any one of the VL sequences provided herein (eg, provided directly above).

[0012] In one embodiment of any of the methods or compositions provided, the antibody, or antigen binding portion thereof, comprises a VH amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of the VH amino acid sequences provided herein (e.g., 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 one embodiment of any of the methods or compositions provided, the antibody, or antigen binding portion thereof, comprises a VL sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any of the VL amino acid sequences provided herein (e.g., any of those shown in 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 one embodiment of any of the provided methods or compositions, the antibody, or antigen binding portion thereof, comprises a VH amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of the VH amino acid sequences provided herein (e.g., any one of the following: SEQ ID. NO: 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 a VL amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of the VL amino acid sequences provided herein (e.g., any one of the following: SEQ ID. NO: 1, ID. NO: 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) have a VL sequence that is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical.In one embodiment of any of the methods or compositions provided, the antibody, or antigen binding portion thereof, comprises a VH sequence and a VL sequence that are each independently at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any specific combination of combinations of VH amino acid sequences and VL amino acid sequences provided herein (as shown in Table 3).

[0013] In one aspect, provided herein are nucleic acids encoding a VH sequence as provided directly above and / or a VL sequence as provided directly above.

[0014] In one embodiment of any of the methods or compositions provided herein, the antigen binding portion thereof is an antigen binding portion of any of the antibodies provided herein.

[0015] Any of the antibodies described herein may be full-length antibodies. In one embodiment of any of the methods or compositions provided herein, the antibody or its antigen-binding portion may be human, humanized or chimeric. In one embodiment of any of the methods or compositions provided herein, the antibody or its antigen-binding portion may be a single-chain antibody. Any of the antibodies described herein may be monoclonal or polyclonal. These two terms do not limit the source of the antibody or its preparation method. In one embodiment of any of the methods or compositions provided herein, the antibody or its antigen-binding portion may be a monoclonal antibody or its antigen-binding portion. In addition, in one embodiment of any of the methods or compositions provided herein, its antigen-binding portion may be a scFv, a F(ab')2 fragment, a dAb fragment, a Fab fragment, a Fab' fragment, an Fv or a disulfide-bonded Fv fragment. The antibody or its antigen-binding portion may be a single domain antibody, a bispecific antibody, a multispecific antibody, a bispecific antibody or a dual-specific antibody. In one embodiment of any of the methods or compositions provided herein, the antibody or its antigen-binding portion may be a separated antibody or its antigen-binding portion.

[0016] Also disclosed is a method for treating yellow fever or a disease or condition associated with yellow fever virus by administering a therapeutically effective amount of one or more antibodies or antigen binding portions thereof that specifically bind to YFV to a subject in need of such treatment. In one embodiment of any of the methods provided herein, the subject is not vaccinated against YFV or wherein YFV vaccination does not provide adequate protection. In one embodiment of any of the methods or compositions provided herein, the antibody or its antigen binding portion is any one or more of the antibodies or their antigen binding portions described herein. In one embodiment of any of the methods or compositions provided herein, the antibody or its antigen binding portion specifically binds to the E protein or E-DII epitope of YFV. In one embodiment of any of the methods or compositions provided herein, the antibody or its antigen binding portion specifically binds to the 93rd, 104th, and 106th residues of the E-DII epitope of the E protein or the yellow fever virus E protein. In one embodiment of any of the methods or compositions provided herein, the amount of the antibody or its antigen binding portion effectively reduces one or more yellow fever symptoms in the subject. Any of the anti-YFV antibodies, or antigen-binding portions thereof, can be systemically administered in any of the methods provided herein, such as by an enteral or parenteral route.

[0017] The subject treated with any of the methods described herein can be a patient (e.g., a human patient) suffering from or suspected of suffering from yellow fever or a disease or condition associated with yellow fever virus. In one embodiment of any of the methods provided herein, the subject is a human patient suffering from or suspected of suffering from an acute viral hemorrhagic disease.

[0018] In some aspects, also provided herein are (a) a pharmaceutical composition for treating yellow fever or a disease or condition associated with 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 above antibodies or antigen-binding portions thereof in a medicament and / or in the preparation of a medicament for treating yellow fever or a disease or condition associated with yellow fever virus in a subject.

[0019] In some aspects, methods for producing antibodies or antigen-binding portions thereof, nucleic acids encoding any of the antibodies or antigen-binding portions thereof, vectors that may contain any one or more of the nucleic acids provided herein, and related host cells are also provided herein.

[0020] In one aspect, the method for producing an antibody or an antigen-binding portion thereof is any of the methods described herein. In one embodiment, the method includes considering the structural regions proximal to the different domains present on the viral assembly (e.g., the interchain interface near the icosahedral axis of symmetry) and selecting promising Fv scaffolds. Conventional epitope prediction methods use domain structure to predict epitope surface regions embedded within domain regions. However, neutralizing flavivirus antibodies can recognize quaternary epitope surfaces (spanning two or more E protein chains). Therefore, traditional methods do not contain valuable information. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 An example of an in vivo study design of the efficacy of engineered mAbs against YF-17D-204 in an infection mouse model is shown. The efficacy of the designed mAbs was tested in a lethal model of yellow fever infection in AG129 mice. The protective efficacy of the mAbs was tested either prophylactically or as a treatment.

[0022] Figures 2A to 2B The in vivo efficacy of the designed mAbs against yellow fever virus is shown. Figure 2A Shown are survival curves of YF-17D infected AG129 mice treated with mAb at a dose of 10 mg / kg as prevention (-1) or as treatment (+1 and +1, +4). Figure 2B The blood viral titers of yellow fever virus in treated animals compared to the control group at days 4 and 6 after infection are shown. Administration of mAb resulted in complete protection ( Figure 2A Administration of mAb also resulted in a greater than 2Log10 reduction in viremia on days 4 and 6 after infection ( Figure 2B ). DETAILED DESCRIPTION

[0023] The following description is intended only to illustrate multiple embodiments of the present invention. Therefore, some specific embodiments 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, without departing from the scope of the present invention, multiple changes or equivalents may be made.

[0024] Aspects of the present disclosure relate to antibodies, antigen-binding portions thereof, and pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors, and host cells for preparing such antibodies and fragments. Antibodies and / or antigen-binding portions thereof that specifically bind to yellow fever virus (YFV) and / or neutralize the virus are provided herein. In some embodiments, the antibody or its antigen-binding portion binds to the E-DII epitope of the yellow fever virus and prevents the virus from infecting cells. In some embodiments, the antibody or its antigen-binding portion binds to the E-DII epitope comprising asparagine at position 106, lysine at position 93, and / or lysine at position 104 of the E protein of the yellow fever virus. In some embodiments, antibodies and antigen-binding portions as provided herein are used to treat or prevent yellow fever virus infection in a subject.

[0025] As used herein, antibody (which can be used interchangeably in the plural) broadly refers to an immunoglobulin (Ig) molecule or any functional mutant, variant or derivative thereof. It is expected that its functional mutant, variant and derivative and antigen binding portion retain the basic epitope binding characteristics of the Ig molecule.

[0026] Antibodies are able to specifically bind to a target through at least one antigen recognition site located in the variable region of an immunoglobulin molecule. Generally speaking, a complete 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 3). Each light chain contains a light chain variable region (V L) and constant region (CL). The VH and VL regions can be further subdivided into hypervariable regions known as complementarity determining regions (CDRs), interspersed with more conservative regions known as framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, which are arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Full-length antibodies can be antibodies of any class, such as IgD, IgE, IgG, IgA or IgM (or their subclasses), and the antibody does not need to be of any particular class. Immunoglobulins can be divided into different classes according to the antibody amino acid sequence of the constant domain of its heavy chain. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these can also be 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 α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0027] The term "antigen binding portion" refers to a portion or region of a complete or full-length antibody molecule that can specifically bind to a target. Preferably, the antigen binding portion provided herein retains the ability to specifically bind to YFV. The antigen binding portion may include a heavy chain variable region (VH), a light chain variable region (VL), or both. Each VH and VL typically includes three complementary determining regions CDR1, CDR2, and CDR3.

[0028] Some examples of antigen binding portions include, but are not limited to: (1) Fab fragments, which can be monovalent fragments having a VL-CL chain and a VH-CH chain; (2) F(ab')2 fragments, which can be bivalent fragments having two Fab fragments connected by a disulfide bridge at the hinge region, i.e., a dimer of Fab; (3) Fv fragments having the VL and VH domains of a single arm of an antibody; (4) single-chain Fv (scFv), which can be a single polypeptide chain composed of a VH domain and a VL domain via a peptide linker; (5) (scFv)2, which can contain two VH domains connected by a peptide linker and two VL domains associated with the two VH domains via a disulfide bridge; (6) Fd fragments consisting of VH and CHI domains; (7) dAb fragments, which contain a single variable domain; and (8) isolated complementarity determining regions (CDRs).

[0029] In addition, although the two domains of the Fv fragment, VL and VH, can be encoded by different genes, they can be connected using recombinant methods through a synthetic linker that enables them to be made into a single protein chain, wherein the VL and VH regions pair to form a monovalent molecule (called 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 bispecific antibodies, are also encompassed. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but a linker is used that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and form 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). Diabodies are also encompassed by the term "antigen-binding portion".

[0030] The term "human antibody" refers to an antibody having a variable region and a constant region that substantially corresponds to or is derived from an antibody obtained from a human subject, for example, an antibody encoded by a human germline immunoglobulin sequence or a variant thereof. The human antibody described herein may include one or more amino acid residues not encoded by human germline immunoglobulin sequences (for example, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). Such mutations may be present in one or more CDRs, particularly CDR3, or in one or more framework regions. In some embodiments, human antibodies may have at least one, two, three, four, five or more positions that are not replaced by amino acid residues encoded by human germline immunoglobulin sequences. However, as used herein, the term "human antibody" is not intended to include antibodies wherein the CDR sequences derived from the germline of another mammalian species (such as a mouse) have been grafted onto human framework sequences.

[0031] As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, generated or isolated by recombinant means, such as 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 (see, e.g., Taylor, LD, et al. (1992) Nucl. Acids Res.20:6287-6295; Kellermann SA., and Green LL (2002) Current Opinion in Biotechnology 13:593-597; Little M. et al (2000) Immunology Today 21:364-370) or by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions and constant regions as defined above. However, in certain embodiments, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, when in vivo somatic cell mutagenesis is performed, an animal transgenic for human Ig sequences is used), and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies may be sequences that, although derived from and related to human germline VH and VL sequences, may not naturally exist in the human antibody germline reservoir in vivo.

[0032] 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 comprises asparagine 106, lysine 93, and lysine 104 of the E protein of yellow fever virus.

[0033] The protein sequences of various VH and VL regions are shown in Tables 1 and 2, respectively.

[0034] Table 1. Protein sequences of variable heavy chains

[0035]

[0036]

[0037]

[0038] Table 2. Protein sequences of variable light chains

[0039]

[0040]

[0041]

[0042] "Isolated" material means that it has been altered from its natural state by the hand of man. If an "isolated" material occurs in nature, it has been altered or removed from its original environment, or both. For example, a polypeptide naturally present in a living subject is not "isolated", but if the polypeptide has been substantially separated from the coexisting materials of its natural state and / or is present in a substantially pure state, then the polypeptide is isolated.

[0043] The term "specific binding" refers to a non-random binding reaction between two molecules, such as the combination of an antibody or its antigen binding portion with an antigen epitope. An antibody or its antigen binding portion that "specifically binds" to a target or epitope is a term well known in the art, and the method for determining such specific binding is also well known in the art. If a molecule reacts or associates with a specific target antigen or epitope more frequently, more rapidly, with a longer duration and / or with greater affinity than the reaction or association with an alternative target position / epitope, the molecule is referred to as showing "specific binding". If an antibody or its antigen binding portion binds with greater affinity, avidity, easier and / or longer duration than it binds to other substances, the antibody or its antigen binding portion "specifically binds" to the target antigen. In certain embodiments, when an antibody preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules, it is referred to as specifically binding to an antigen.

[0044] An "epitope" is a region of an antigen that is bound by an antibody. The term encompasses any polypeptide determinant capable of specific binding to an immunoglobulin. In certain embodiments, epitope determinants comprise chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and in certain embodiments, may have specific three-dimensional structural characteristics and / or specific charge characteristics.

[0045] As used herein, the term "neutralization" refers to the activity of a target protein (e.g., yellow fever virus E protein), such as neutralization of a biological activity. In one embodiment, a neutralizing antibody binds to the E-DII epitope of the yellow fever virus E protein and results in inhibition of the biological activity of the yellow fever virus and / or prevents the virus from infecting cells.

[0046] The subject can be a human, but also includes other mammals, particularly those that can be used as laboratory models for human disease, e.g., mice, rats, rabbits, dogs, etc.

[0047] The term "treatment" refers to an act, application or treatment in which direct or indirect medical assistance is given to an object (including a human) for the purpose of improving the object's condition. In particular, in some embodiments, the term refers to reducing the incidence of disease, or alleviating one or more symptoms, eliminating relapses, preventing relapses, preventing the incidence of disease, improving one or more symptoms and / or improving prognosis, or a combination thereof. The skilled person will understand that treatment does not necessarily result in the complete absence or removal of symptoms. For example, for yellow fever virus, "treatment" may refer to reducing the severity or duration of acute viral hemorrhagic disease caused by yellow fever.

[0048] As used herein, an "effective amount" or "effective dose" or "therapeutically effective amount" in connection with the administration of a medicament refers to an amount of a drug or medicament (e.g., an antibody or antigen-binding fragment or portion thereof, or a composition comprising the same) that results in an expected pharmacological outcome, or the effect of treating, curing, preventing or ameliorating a disease or disease symptom, disorder or side effect, or reduces the rate of development of a disease or disorder or any symptom thereof, compared to a corresponding subject not receiving such an amount. The effective amount or effective dose of a medicament may vary depending on the specific active ingredient used, the mode of administration, and / or the age, size, and condition of the subject to be treated.

[0049] The present disclosure also provides a composition (e.g., a pharmaceutical composition) comprising an antibody or an 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 asparagine 106, lysine 93, and lysine 104 of the yellow fever virus E protein. The composition can be prepared from any of the antibodies or antigen binding portions described herein. The antibodies or antigen binding portions thereof described herein, as well as encoding nucleic acids or nucleic acid sets, vectors comprising these, or host cells comprising the vectors can be mixed with a pharmaceutically acceptable carrier (excipient), for example, to form a pharmaceutical composition for 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.

[0050] The pharmaceutical composition may contain a pharmaceutically acceptable carrier, excipient or stabilizer in the form of a lyophilized preparation or an aqueous solution. (Remington: The Science and Practice of Pharmacy 20 th Ed. (2000) Lippincott Williams and Wilkins, Ed. KE Hoover). Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and may include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium 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 (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; 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 nonionic surfactants, such as TWEEN®. TM (Polysorbate), PLURONICS TM (poloxamers) or polyethylene glycol (PEG).

[0051] The phrase "pharmaceutically acceptable" used in conjunction with the compositions of the present disclosure refers to the fact that the molecular entities and other ingredients of such compositions are physiologically tolerable and generally do not produce adverse reactions when administered to a subject. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of a federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopoeia for use in mammals, and more particularly for humans. "Acceptable" means that the carrier is compatible with the active ingredients (e.g., nucleic acids, vectors, cells, or therapeutic antibodies) of the composition and does not negatively affect the subject to which the composition is administered. Any pharmaceutical composition used in the methods of the present invention may include a pharmaceutically acceptable carrier, excipient, or stabilizer in the form of a lyophilized formulation or an aqueous solution.

[0052] 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 nonionic surfactants. See, e.g., Remington: The Science and Practice of Pharmacy 20 th Ed. (2000) Lippincott Williams and Wilkins, Ed. KE Hoover.

[0053] Pharmaceutical compositions may be presented in unit dosage form and may be prepared by any suitable methods, many of which are well known.Such methods may include the step of bringing into association the anti-YFV antibody with the carrier which constitutes one or more accessory ingredients.

[0054] The compositions provided herein can be sterile aqueous preparations, which are preferably isotonic with the blood of the recipient in some embodiments. The aqueous preparations can be formulated using suitable dispersants or wetting agents and suspending agents according to known methods. Sterile aqueous preparations can also be sterile injectable solutions or suspensions in nontoxic parenterally acceptable diluents or solvents. Acceptable carriers and solvents that can be used are water, Ringer's solution, and isotonic sodium chloride solution.

[0055] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA fragments can be linked. In addition, a vector can be capable of directing the expression of a gene to which it is operably linked. Such vectors are referred to herein as "recombinant expression vectors" or "expression vectors."

[0056] 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 specific subject cell, but also to the progeny of such a cell. Because certain modifications may occur in subsequent progeny due to mutations or environmental influences, such progeny may not actually be the same as the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0057] Example

[0058] Using structure-guided analysis of YFV envelope protein epitopes, anti-YFV monoclonal antibodies were designed focusing on the envelope domain II fusion loop. Mutationally restricted epitopes on the surface of yellow fever virus were identified. Suitable antibody scaffolds that satisfied epitope-paratope constraints were then identified and antibodies targeting and neutralizing the virus were engineered.

[0059] Anti-YFV antibodies were cloned, expressed and purified using known methods, and screened for expression and biophysical properties. In silico designed antibodies were cloned into mammalian expression vectors and purified for further analysis. Expression and multiple analytical parameters indicating purity and stability were evaluated. The efficacy of antibodies against yellow fever virus was evaluated using in vitro and in vivo infection models.

[0060] The in vitro neutralization potency of the engineered antibodies was evaluated against the YF17D-204 vaccine strain of yellow fever virus using the plaque reduction neutralization test (PRNT). For selected antibodies, additional evaluation of potency was performed using a mouse model of YFV infection. Rational design of anti-YF mAbs

[0061] Promising anti-YFV monoclonal antibodies must neutralize a wide range of strains and target regions associated with potent protection. Epitopes play a key role in the efficacy of therapeutic antibodies. In YFV infection, the envelope (E) protein is the main target of neutralizing antibodies. In order to define promising epitopes, a structure-guided analysis was performed on the entire YFV assembly to determine spatially aggregated, solvent-accessible and sequence-conserved residues. This was accomplished by mapping the sequence conservation score (calculated based on the alignment of the YFV envelope protein sequence) onto a homology model of the entire YFV E protein assembly. Residues with a sequence conservation greater than 70% and a solvent-accessible surface area > 40% are considered to be putative epitope residues. According to the analysis, the region near the hydrophobic fusion loop of domain II (E-DII) showed high accessibility and conservation in YFV strains.

[0062] To engineer antibodies against the E-DII epitope, a structure-guided search was performed for Fv scaffolds that recognized highly homologous epitope surfaces. Promising scaffolds were docked to the epitope (using the software ZRANK) and ranked based on shape complementarity (>0.6), buried surface area (1,000 Sq.A°), and potential for contact with key E-DII epitope residues (N106, K93, and K104 of the E protein; numbering corresponds to the primary sequence of the 17DYF vaccine strain). The CDR loops of the top-ranked scaffolds were then redesigned by Rosetta Antibody Design to make optimal contacts with the epitope. The engineered antibodies were screened for in vitro neutralization against YFV.

[0063] 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.

[0064] Table 3. YF antibody combination

[0065]

[0066] Illustrative examples of expression levels and biophysical properties

[0067] A subset of YFV antibodies was purified from a large-scale transfection of Expi293 cells. Proteins were purified from cell culture supernatants using HiTrap protein A columns. After dialysis, protein concentrations were estimated by nanodrop using theoretical extinction coefficients. The yields of various antibodies are summarized in Table 4.

[0068] Table 4. Yield of YF mAbs expressed in Expi293 cells

[0069] mAbs Express mg / L 1 34.4 2 171 3 65.8

[0070] 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. Illustrative examples of such analytical biophysical parameters are summarized in Table 5 below.

[0071] Table 5. Purity and stability of selected YF antibodies

[0072]

[0073]

[0074] Additional features of the glycoforms of various antibodies were determined by procainamide label-assisted LC-FLD-MS. Illustrative examples of observed glycosylation profiles are as follows (Table 6).

[0075] Table 6. Glycosylation profile

[0076]

[0077] Illustrative example of neutralization efficacy of engineered antibodies against YF-17D

[0078] Plaque reduction neutralization test (PRNT) was performed to determine the neutralization efficacy of YFV antibodies against YFV (17D-204). Vero cells were infected with the following viruses: virus alone, no virus, or virus pre-incubated with YFV antibodies at various dilutions. 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 the 50% effective concentration (EC50) value was calculated by nonlinear regression using a variable slope (Table 6). The neutralization potency of the antibody is proportional to the reduction of plaques, and the potency is described as the concentration at which 50% of the virus particles are neutralized. The antibody is highly potent in neutralizing the virus and illustrative examples are shown in Table 7 below.

[0079] Table 7. Summary of EC50 values ​​of YF mAbs

[0080] mAbs EC50(μg / ml) STDEV 1 0.012 0.006 2 0.007 0.002 3 0.011 0.010

[0081] Illustrative Examples of In Vivo Efficacy

[0082] Figure 1 An illustrative example of the in vivo efficacy of engineered antibodies against YF-17D-204 in a mouse infection model is provided. The efficacy of the designed antibodies was tested in a lethal model of yellow fever infection in AG129 mice. Figure 1 The protective efficacy of the antibodies is tested prophylactically or as a treatment as shown.

[0083] As an illustrative example shown in FIG2 , administration of the antibody resulted in complete protection compared to the control group ( Figure 2A Administration of the antibody also resulted in a greater than 2 Log reduction in viremia on days 4 and 6 after infection. 10 ( Figure 2B ).

Claims

1. An antibody or an antigen-binding portion thereof, wherein the antibody or the antigen-binding portion thereof specifically binds to the E-DII epitope of yellow fever virus, wherein the antibody or antigen-binding portion thereof comprises a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3 determined according to Kabat numbering of a heavy chain variable region (VH) as shown in SEQ ID.NO:4; and The antibody or antigen-binding portion thereof comprises the light chain CDR1, light chain CDR2 and light chain CDR3 of the light chain variable region (VL) shown in SEQ ID. NO: 38, as determined according to Kabat numbering.

2. The antibody or antigen binding portion thereof of claim 1, wherein the antibody or antigen binding portion thereof comprises a VH, wherein the VH comprises the amino acid sequence of SEQ ID. NO:

4.

3. The antibody or antigen binding portion thereof of claim 1, wherein the antibody or antigen binding portion thereof comprises a VL, wherein the VL comprises the amino acid sequence of SEQ ID. NO:

38.

4. The antibody or antigen binding portion thereof of any one of claims 1 to 3, wherein the antibody or antigen binding portion thereof is a human antibody or antigen binding portion thereof, a chimeric antibody or antigen binding portion thereof, or a humanized antibody or antigen binding portion thereof.

5. The antibody or antigen binding portion thereof of any one of claims 1 to 3, wherein the antibody or antigen binding portion thereof is a single chain antibody or antigen binding portion thereof, a F(ab')2 fragment, a Fab fragment or a Fv fragment.

6. The antibody or antigen-binding portion thereof of any one of claims 1-3, wherein the antibody or antigen-binding portion thereof is an isolated antibody or antigen-binding portion thereof.

7. The antibody or antigen binding portion thereof of any one of claims 1-3, wherein the antibody or antigen binding portion thereof is a monoclonal antibody or antigen binding portion thereof.

8. A composition comprising the antibody or antigen-binding portion thereof of any one of claims 1 to 7 and a carrier.

9. The composition of claim 8, wherein the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

10. Use of the antibody or antigen binding portion thereof of any one of claims 1 to 7 in the preparation of a medicament for treating yellow fever or a disease or condition associated with yellow fever virus.

11. A nucleic acid comprising a nucleotide sequence encoding VH and VL of the antibody or antigen binding portion thereof according to any one of claims 1 to 7.

12. A vector comprising the nucleic acid according to claim 11.

13. The vector of claim 12, wherein the vector is an expression vector.

14. A host cell comprising the nucleic acid of claim 11 or the vector of claim 12 or 13.

15. A method for producing an antibody or antigen-binding portion thereof that binds to yellow fever virus E protein, comprising: The host cell of claim 14 is cultured under conditions that allow expression of the antibody.

16. The method of claim 15, further comprising harvesting the antibody or antigen-binding portion thereof.

17. The method of claim 15 or 16, wherein the antibody or antigen binding portion thereof specifically binds to the E-DII epitope of the Yellow Fever Virus E protein.

18. The method of claim 17, wherein the E-DII epitope comprises residues 93, 104, and 106 of the Yellow Fever Virus E protein.

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