WO2022268120A1

WO2022268120A1 - Anti-rsv antibodies and uses thereof

Info

Publication number: WO2022268120A1
Application number: PCT/CN2022/100436
Authority: WO
Inventors: Jingshu XIE; Yanshuang HUO; Xuefeng JI
Original assignee: Biocytogen Pharmaceuticals (Beijing) Co., Ltd.
Priority date: 2021-06-22
Filing date: 2022-06-22
Publication date: 2022-12-29

Abstract

Provided are antibodies that target RSV-F (Respiratory Syncytial Virus Fusion Protein), antigen-binding fragments, and the uses thereof.

Description

ANTI-RSV ANTIBODIES AND USES THEREOF

CLAIM OF PRIORITY

This application claims the benefit of international Application No. PCT/CN2021/101451, filed on June 22, 2021. The entire contents of the foregoing are incorporated herein by reference.

TECHNICAL FIELD

This disclosure relates to anti-RSV-F antibodies, antigen-binding fragments, and the uses thereof.

BACKGROUND

Respiratory syncytial virus (RSV) , also called human respiratory syncytial virus (hRSV) and human orthopneumovirus, is a very common, contagious virus that causes infections of the respiratory tract. It is a negative-sense, single-stranded RNA virus, and its name is derived from the large syncytia that form when infected cells fuse together. RSV infection can present with a wide variety of signs and symptoms that range from mild upper respiratory tract infections (URTI) to severe and potentially life-threatening lower respiratory tract infections (LRTI) requiring hospitalization and mechanical ventilation. While RSV can cause respiratory tract infections in people of all ages and is among the most common childhood infections, its presentation often varies between age groups and immune status. Reinfection is common throughout life, but infants and the elderly remain at highest risk for symptomatic infection (e.g., bronchiolitis or pneumonia) . Thus, there is a need for additional treatment for RSV positive infection beside supportive care in the form of adequate nutrition and oxygen therapy. Antiviral therapies such as Ribavirin have not been proven to be effective in RSV infection. One monoclonal antibody, Palivizumab (also called

) , is registered for prophylaxis against RSV infection. Palivizumab is a genetically engineered (humanized) monoclonal antibody to the fusion protein of RSV. Additional monoclonal antibody drugs against RSV infection include Nirsevimab (MEDI8897) from Sanofi and MK-1654 from Merck. While Palivizumab has been a very effective prophylactic, alternative antibodies and therapies providing additional coverage against RSV would be advantageous.

It is an object to provide means and methods for counteracting and/or preventing an RSV infection. It is a further object to provide alternative and/or improved antibodies against RSV, or functional equivalents of such antibodies, and to provide stable cells capable of producing antibodies or functional equivalents thereof against RSV.

SUMMARY

This disclosure relates to anti-RSV-F (Respiratory Syncytial Virus Fusion Protein) antibodies, antigen-binding fragment thereof, and the uses thereof. The inventors have succeeded in generating RSV-specific antibodies with improved properties over prior RSV-specific antibodies, including improved protection against RSV A subtypes and RSV B subtypes, improved neutralization, and lower IC50 values. Such antibodies have a particular high or strong affinity for RSV and are therefore particularly suitable for counteracting and/or at least in part preventing an RSV infection and/or adverse effects of an RSV infection.

In one aspect, the disclosure provides an antibody or antigen-binding fragment thereof that binds to Respiratory Syncytial Virus Fusion Protein (RSV-F) comprising: a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising

CDRs

1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR3 amino acid sequence. In some embodiments, the selected

VH CDRs

1, 2, and 3 amino acid sequences and the selected VL CDRs, 1, 2, and 3 amino acid sequences are one of the following:

(1) the selected

VH CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 2, 3, respectively, and the selected

VL CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4, 5, 6, respectively;

(2) the selected

VH CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 44, 3, respectively, and the selected

VL CDRs

(3) the selected

VH CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 45, 3, respectively, and the selected

VL CDRs

(4) the selected

VH CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 46, 3, respectively, and the selected

VL CDRs

(5) the selected

VH CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 47, 3, respectively, and the selected

VL CDRs

(6) the selected

VH CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 7, 8, 9, respectively, and the selected

VL CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 10, 11, 12, respectively;

(7) the selected

VH CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 48, 8, 9, respectively, and the selected

VL CDRs

(8) the selected

VH CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 49, 8, 9, respectively, and the selected

VL CDRs

(9) the selected

VH CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 50, 8, 9, respectively, and the selected

VL CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 10, 11, 12, respectively; and

(10) the selected

VH CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 51, 8, 9, respectively, and the selected

VL CDRs

1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 10, 11, 12, respectively.

In some embodiments, the antibody or antigen-binding fragment specifically binds to RSV-F of an RSV subtype A strain. In some embodiments, the antibody or antigen-binding fragment specifically binds to RSV-F of an RSV subtype B strain.

In some embodiments, the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment is a single-chain variable fragment (scFv) or a multi-specific antibody (e.g., a bispecific antibody) .

In one aspect, the disclosure provides a nucleic acid comprising a polynucleotide encoding a polypeptide comprising:

(1) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 23, 24, 25, or 27 binds to RSV-F;

(2) an immunoglobulin light chain or a fragment thereof comprising a

VL comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively, and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 26 binds to RSV-F;

(3) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising

CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 44 and 3, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 23, 24, 25, or 27 binds to RSV-F;

(4) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising

CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 45 and 3, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 23, 24, 25, or 27 binds to RSV-F;

(5) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising

CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 46 and 3, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 23, 24, 25, or 27 binds to RSV-F;

(6) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising

CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 47 and 3, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 23, 24, 25, or 27 binds to RSV-F;

(7) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising

CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 23, 24, 25, or 27 binds to RSV-F;

(8) an immunoglobulin light chain or a fragment thereof comprising a

VL comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively, and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 26 binds to RSV-F;

(9) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising

CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 48, 8, and 9, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 23, 24, 25, or 27 binds to RSV;

(10) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising

CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 49, 8, and 9, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 23, 24, 25, or 27 binds to RSV;

(11) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising

CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 50, 8, and 9, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 23, 24, 25, or 27 binds to RSV; or

(12) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising

CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 51, 8, and 9, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 23, 24, 25, or 27 binds to RSV.

In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a

VH comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively. In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a

VL comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively. In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a

VH comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 44, and 3, respectively. In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a

VH comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 45, and 3, respectively. In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a

VH comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 46, and 3, respectively. In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a

VH comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 47, and 3, respectively. In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a

VH comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively. In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a

VL comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively. In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a

VH comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 48, 8, and 9, respectively. In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a

VH comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 49, 8, and 9, respectively. In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a

VH comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 50, 8, and 9, respectively. In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a

VH comprising CDRs

1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 51, 8, and 9, respectively.

In some embodiments, the VH when paired with a VL specifically binds to RSV-F, or the VL when paired with a VH specifically binds to RSV-F.

In some embodiments, the immunoglobulin heavy chain or the fragment thereof is a humanized immunoglobulin heavy chain or a fragment thereof, and the immunoglobulin light chain or the fragment thereof is a humanized immunoglobulin light chain or a fragment thereof.

In some embodiments, the nucleic acid encodes a single-chain variable fragment (scFv) or a multi-specific antibody (e.g., a bispecific antibody) .

In some embodiments, the nucleic acid is cDNA.

In one aspect, the disclosure provides a vector comprising one or more of the nucleic acids as described herein.

In one aspect, the disclosure provides a vector comprising two of the nucleic acids as described herein.

In some embodiments, the vector encodes the VL region and the VH region that together bind to RSV-F.

In one aspect, the disclosure provides a pair of vectors, In some embodiments, each vector comprises one of the nucleic acids as described herein. In some embodiments, together the pair of vectors encodes the VL region and the VH region that together bind to RSV-F.

In one aspect, the disclosure provides a cell comprising the vector as described herein. In some embodiments, the cell is a CHO cell. In one aspect, the disclosure provides a cell comprising one or more of the nucleic acids as described herein.

In one aspect, the disclosure provides a cell comprising two of the nucleic acids as described herein. In some embodiments, the two nucleic acids together encode the VL region and the VH region that together bind to RSV-F.

In one aspect, the disclosure provides a method of producing an antibody or an antigen-binding fragment thereof, the method comprising

(a) culturing the cell as described herein under conditions sufficient for the cell to produce the antibody or the antigen-binding fragment; and

(b) collecting the antibody or the antigen-binding fragment produced by the cell.

In one aspect, the disclosure provides an antibody or antigen-binding fragment thereof that binds to RSV-F comprising a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%identical to SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 26, and a light chain variable region (VL) comprising an amino acid sequence that is at least 90%identical to 23, 24, 25, or 27.

In some embodiments, the VH comprises the sequence of SEQ ID NO: 21 and the VL comprises the sequence of SEQ ID NO: 25. In some embodiments, the VH comprises the sequence of SEQ ID NO: 16 and the VL comprises the sequence of SEQ ID NO: 25. In some embodiments, the VH comprises the sequence of SEQ ID NO: 17 and the VL comprises the sequence of SEQ ID NO: 24. In some embodiments, the VH comprises the sequence of SEQ ID NO: 16 and the VL comprises the sequence of SEQ ID NO: 24. In some embodiments, the VH comprises the sequence of SEQ ID NO: 17 and the VL comprises the sequence of SEQ ID NO: 25.

In some embodiments, the antibody or antigen-binding fragment specifically binds to RSV-F of an RSV subtype A strain. In some embodiments, the antibody or antigen-binding fragment specifically binds to RSV-F of an RSV subtype B strain. In some embodiments, the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment is a single-chain variable fragment (scFv) or a multi-specific antibody (e.g., a bispecific antibody) .

In one aspect, the disclosure provides an antibody or antigen-binding fragment thereof comprising the

VH CDRs

1, 2, 3, and the

VL CDRs

1, 2, 3 of the antibody or antigen-binding fragment thereof as described herein.

In one aspect, the disclosure provides an antibody or antigen-binding fragment thereof that cross-competes with the antibody or antigen-binding fragment thereof as described herein.

In one aspect, the disclosure provides an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof as described herein covalently bound to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent.

In one aspect, the disclosure provides a method of treating a subject having a Respiratory Syncytial Virus infection, preventing RSV infection in a subject, reducing the risk of RSV infection in a subject, or ameliorate symptoms of RSV infection in a subject, the method comprising administering a therapeutically effective amount of a composition comprising the antibody or antigen-binding fragment thereof as described herein to the subject.

In one aspect, the disclosure provides a method of neutralizing a Respiratory Syncytial Virus (RSV) , the method comprising contacting the RSV with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof as described herein.

In one aspect, the disclosure provides a method of blocking internalization of a Respiratory Syncytial Virus (RSV) by a cell, the method comprising contacting the RSV with an effective amount of a composition comprising the antibody or antigen-binding fragment thereof as described herein.

In one aspect, the disclosure provides a method of identifying a subject as having a Respiratory Syncytial Virus (RSV) infection, the method comprising detecting a sample collected from the subject as having the RSV by the antibody or antigen-binding fragment thereof as described herein, thereby identifying the subject as having the RSV infection. In some embodiments, the sample is a blood sample, a saliva sample, a stool sample, or a liquid sample from the respiratory tract of the subject.

In one aspect, the disclosure provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as described herein, and a pharmaceutically acceptable carrier.

In one aspect, the disclosure provides a pharmaceutical composition comprising the antibody drug conjugate as described herein, and a pharmaceutically acceptable carrier.

In some embodiments, the antibody is a IgG1 antibody. In some embodiments, the antibody is a human IgG1 antibody. In some embodiments, the IgG1 antibody comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 33, 34, or 35.

In one aspect, the disclosure provides an IgG1 antibody or antigen-binding fragment thereof that binds to Respiratory Syncytial Virus Fusion Protein (RSV-F) comprising: a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising CDRs 1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR3 amino acid sequence, wherein the selected VH CDRs 1, 2, and 3 amino acid sequences and the selected VL CDRs, 1, 2, and 3 amino acid sequences are selected from one of the antibodies as set forth in FIG. 6 and FIG. 7. In some embodiments, the antibody is a human IgG1 antibody. In some embodiments, the human IgG1 antibody comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 33, 34, or 35.

In one aspect, the disclosure provides an IgG1 antibody or antigen-binding fragment thereof that binds to Respiratory Syncytial Virus Fusion Protein (RSV-F) comprising: a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%identical to a selected VH sequence, and a light chain variable region (VL) comprising an amino acid sequence that is at least 90%identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are selected from one of the antibodies as set forth in FIG. 8 and FIG. 9. In some embodiments, the antibody is a human IgG1 antibody. In some embodiments, the human IgG1 antibody comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 33, 34, or 35.

In one aspect, the disclosure provides an antibody or antigen-binding fragment thereof comprising

VH CDRs

1, 2, 3 that are identical to

VH CDRs

1, 2, 3 in SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 26, and

VL CDRs

1, 2, 3 that are identical to

VL CDRs

1, 2, 3 in SEQ ID NO: 23, 24, 25, or 27.

In one aspect, the disclosure is related to a food additive comprising the antibody or antigen-binding fragment thereof, the antibody-drug conjugate, or the pharmaceutical composition described herein. In one aspect, the disclosure is related to a method of preventing or treating a Respiratory Syncytial Virus infection in a subject, the method comprising administering the food additive as described herein to the subject, e.g., through oral administration.

In some embodiments, Kabat numbering is used in the present disclosure. In some embodiments, Chothia numbering is used in the present disclosure.

As used herein, the term “antibody” refers to any antigen-binding molecule that contains at least one (e.g., one, two, three, four, five, or six) complementary determining region (CDR) (e.g., any of the three CDRs from an immunoglobulin light chain or any of the three CDRs from an immunoglobulin heavy chain) and is capable of specifically binding to an epitope. Non-limiting examples of antibodies include: monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g., bi-specific antibodies) , single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, an antibody can contain an Fc region of a human antibody. The term antibody also includes derivatives, e.g., bi-specific antibodies, single-chain antibodies, diabodies, linear antibodies, and multi-specific antibodies formed from antibody fragments.

As used herein, the term “antigen-binding fragment” refers to a portion of a full-length antibody, wherein the portion of the antibody is capable of specifically binding to an antigen. In some embodiments, the antigen-binding fragment contains at least one variable domain (e.g., a variable domain of a heavy chain or a variable domain of light chain) . Non-limiting examples of antibody fragments include, e.g., Fab, Fab’, F (ab’) ₂, and Fv fragments.

As used herein, the term “human antibody” refers to an antibody that is encoded by an endogenous nucleic acid (e.g., rearranged human immunoglobulin heavy or light chain locus) present in a human. In some embodiments, a human antibody is collected from a human or produced in a human cell culture (e.g., human hybridoma cells) . In some embodiments, a human antibody is produced in a non-human cell (e.g., a mouse or hamster cell line) . In some embodiments, a human antibody is produced in a bacterial or yeast cell. In some embodiments, a human antibody is produced in a transgenic non-human animal (e.g., a bovine) containing an unrearranged or rearranged human immunoglobulin locus (e.g., heavy or light chain human immunoglobulin locus) .

As used herein, the term “chimeric antibody” refers to an antibody that contains a sequence present in at least two different antibodies (e.g., antibodies from two different mammalian species such as a human and a mouse antibody) . A non-limiting example of a chimeric antibody is an antibody containing the variable domain sequences (e.g., all or part of a light chain and/or heavy chain variable domain sequence) of a non-human (e.g., mouse) antibody and the constant domains of a human antibody. Additional examples of chimeric antibodies are described herein and are known in the art.

As used herein, the term “humanized antibody” refers to a non-human antibody which contains minimal sequence derived from a non-human (e.g., mouse) immunoglobulin and contains sequences derived from a human immunoglobulin. In non-limiting examples, humanized antibodies are human antibodies (recipient antibody) in which hypervariable (e.g., CDR) region residues of the recipient antibody are replaced by hypervariable (e.g., CDR) region residues from a non-human antibody (e.g., a donor antibody) , e.g., a mouse, rat, or rabbit antibody, having the desired specificity, affinity, and capacity. In some embodiments, the Fv framework residues of the human immunoglobulin are replaced by corresponding non-human (e.g., mouse) immunoglobulin residues. In some embodiments, humanized antibodies may contain residues which are not found in the recipient antibody or in the donor antibody. These modifications can be made to further refine antibody performance. In some embodiments, the humanized antibody contains substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops (CDRs) correspond to those of a non-human (e.g., mouse) immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin. The humanized antibody can also contain at least a portion of an immunoglobulin constant region (Fc) , typically, that of a human immunoglobulin. Humanized antibodies can be produced using molecular biology methods known in the art. Non-limiting examples of methods for generating humanized antibodies are described herein.

As used herein, the term “single-chain antibody” refers to a single polypeptide that contains at least two immunoglobulin variable domains (e.g., a variable domain of a mammalian immunoglobulin heavy chain or light chain) that is capable of specifically binding to an antigen. Non-limiting examples of single-chain antibodies are described herein.

As used herein, the term “bispecific antibody” refers to an antibody that binds to two different epitopes. The epitopes can be on the same antigen or on different antigens.

As used herein, the term “trispecific antibody” refers to an antibody that binds to three different epitopes. The epitopes can be on the same antigen or on different antigens.

As used herein, the term “multispecific antibody” refers to an antibody that binds to two or more different epitopes. The epitopes can be on the same antigen or on different antigens. A multispecific antibody can be e.g., a bispecific antibody or a trispecific antibody. In some embodiments, the multispecific antibody binds to two, three, four, five, or six different epitopes.

As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and describe an animal, human or non-human, to whom treatment according to the methods of the present invention is provided. Veterinary and non-veterinary applications are contemplated by the present invention. Human patients can be adult humans or juvenile humans (e.g., humans below the age of 18 years old) . In some embodiments, the subject is a child (e.g., a child younger than 16, 12, 5, 4, 3, 2, or 1 year of age) . In addition to humans, patients include but are not limited to mice, rats, hamsters, guinea-pigs, rabbits, ferrets, cats, dogs, and primates. Included are, for example, non-human primates (e.g., monkey, chimpanzee, gorilla, and the like) , rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits) , lagomorphs, swine (e.g., pig, miniature pig) , equine, canine, feline, bovine, and other domestic, farm, and zoo animals.

As used herein, when referring to an antibody, the phrases “specifically binding” and “specifically binds” mean that the antibody interacts with its target molecule (e.g., RSV-F) preferably to other molecules, because the interaction is dependent upon the presence of a particular structure (i.e., the antigenic determinant or epitope) on the target molecule; in other words, the reagent is recognizing and binding to molecules that include a specific structure rather than to all molecules in general. An antibody that specifically binds to the target molecule may be referred to as a target-specific antibody. For example, an antibody that specifically binds to an RSV-F molecule may be referred to as an RSV-F-specific antibody or an anti-RSV-F antibody.

As used herein, the terms “polypeptide, ” “peptide, ” and “protein” are used interchangeably to refer to polymers of amino acids of any length of at least two amino acids.

As used herein, the terms “polynucleotide, ” “nucleic acid molecule, ” and “nucleic acid sequence” are used interchangeably herein to refer to polymers of nucleotides of any length of at least two nucleotides, and include, without limitation, DNA, RNA, DNA/RNA hybrids, and modifications thereof.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a flow chart showing the first part of an exemplary protocol of making anti-RSV-F antibodies.

FIG. 2 is a flow chart showing the second part of an exemplary protocol of making anti-RSV-F antibodies.

FIG. 3A are gel images of non-reducing SDS-PAGE. M is a protein marker.

FIG. 3B are gel images of reducing SDS-PAGE. M is a protein marker.

FIG. 3C is a table showing the lane number (ID) of the untreated or treated anti-RSV-F antibodies in FIGS. 3A-3B.

FIG. 4A are gel images of non-reducing SDS-PAGE. M is a protein marker.

FIG. 4B are gel images of reducing SDS-PAGE. M is a protein marker.

FIG. 4C is a table showing the lane number (ID) of the untreated or treated anti-RSV-F antibodies in FIGS. 4A-4B.

FIG. 5A are gel images of non-reducing SDS-PAGE. M is a protein marker.

FIG. 5B are gel images of reducing SDS-PAGE. M is a protein marker.

FIG. 5C is a table showing the lane number (ID) of the untreated or treated anti-RSV-F antibodies in FIGS. 5A-5B.

FIG. 6 lists CDR sequences of anti-RSV-F antibody 04-2H10 and CDR sequences of the humanized antibodies thereof as defined by Kabat numbering.

FIG. 7 lists CDR sequences of anti-RSV-F antibody 04-2H10 and CDR sequences of the humanized antibodies thereof as defined by Chothia numbering.

FIG. 8 lists amino acid sequences of heavy chain variable regions (VHs) and light chain variable regions (VLs) of humanized antibodies based on 2H10.

FIG. 9 lists amino acid sequences of heavy chain variable region (VH) and light chain variable region (VL) of mouse 2H10 antibody.

FIG. 10 lists amino acid sequences of RSV-F proteins.

FIG. 11 lists amino acid sequences of wild-type and mutated human IgG1 heavy chain constant domains.

FIG. 12 lists amino acid sequences disclosed herein.

FIG. 13 shows the sequence alignment ofRSF-S2 (SEQ ID NO: 29) , RSF-LONG (SEQ ID NO: 30) , RSF-B1 (SEQ ID NO: 31) , and RSF-B18537 (SEQ ID NO: 32) .

DETAILED DESCRIPTION

The present disclosure provides examples of antibodies, antigen-binding fragments thereof, that bind to RSV-F (Respiratory Syncytial Virus Fusion) .

Respiratory Syncytial Virus

Respiratory syncytial virus (RSV) is an enveloped RNA virus that is a member of the Pneumoviridae family. Upper respiratory tract infections due to RSV reoccur multiple times throughout life, but rarely lead to severe complications in healthy adults. However, RSV infections in infants, the elderly, and the immunocompromised can lead to bronchiolitis or pneumonia, which may result in hospitalization or even death. These complications are a substantial cause of infant mortality worldwide. Although prophylaxis with the monoclonal antibody palivizumab reduces the risk of hospitalization associated with RSV, it must be delivered intravenously multiple times per RSV season and has modest efficacy, preventing its use in developing regions.

RSV is divided into two antigenic subtypes, A and B, based on the reactivity of the F and G surface proteins to monoclonal antibodies. The subtypes tend to circulate simultaneously within local epidemics, although subtype A tends to be more prevalent. Generally, RSV subtype A (RSVA) is thought to be more virulent than RSV subtype B (RSVB) , with higher viral loads and faster transmission time. 16 RSVA and 22 RSVB clades (or strains) have been identified. Among RSVA, the GA1, GA2, GA5, and GA7 clades predominate; GA7 is found only in the United States. Among RSVB, the BA clade predominates worldwide.

RSV has a negative-sense, single-stranded RNA genome. The genome is linear and approximately 15,000 nucleotides in length. It is non-segmented which means that, unlike influenza, RSV cannot participate in the type of genetic reassortment and antigenic shifts responsible for large pandemics. It has 10 genes encoding for 11 proteins. The gene order is NS1-NS2-N-P-M-SH-G-F-M2-L, with the NS1 and NS2 gene serving as nonstructural promoter genes.

RSV is a medium-sized (～150 nm) enveloped virus. While most particles are spherical, filamentous species have also been identified. The genome rests within a helical nucleocapsid and is surrounded by matrix protein and an envelope containing viral glycoproteins. There are 11 proteins, namely protein G, F, SH, M, N, P, L, M2-1, M2-2, NS-1 and NS-2. The two major glycoproteins on the surface of the RSV virion, the attachment glycoprotein (G) and the fusion (F) glycoprotein, control the initial phases of infection. G targets the ciliated cells of the airways, and F causes the virion membrane to fuse with a target cell membrane. The F protein is the major target for antiviral drug development, and both G and F glycoproteins are the antigens targeted by neutralizing antibodies induced by infection.

RSV is highly contagious and can cause outbreaks from both community and hospital transmission. For each person infected with RSV, it is estimated that an average of 5 to 25 uninfected people will become infected. RSV can spread when an infected person coughs or sneezes, releasing contaminated droplets into the air. Transmission usually occurs when these droplets come into contact with (or inoculate) another person's eyes, nose, or mouth. Once infected, people are usually contagious for 3 to 8 days. In infants and in people with weakened immune systems, however, the virus may continue to spread for up to 4 weeks (even after they are no longer showing symptoms) .

Following inoculation of the nose or eyes, RSV infects ciliated columnar epithelial cells of the upper and lower airway. RSV continues to replicate within these bronchial cells for about 8 days. After the first several days, RSV-infected cells will become more rounded and ultimately slough into the smaller bronchioles of the lower airway. This sloughing mechanism is also thought to be responsible for the spread of virus from the upper to lower respiratory tract. Infection causes generalized inflammation within the lungs, including the migration and infiltration of inflammatory cells (such as monocytes and T-cells) , necrosis of the epithelial cell wall, edema, and increased mucous production. Inflammation and cell damage tends to be patchy rather than diffuse. Together, the sloughed epithelial cells, mucous plugs, and accumulated immune cells cause obstruction of the lower airway.

Surface protein F (fusion protein, RSV-F, or RSF) is a type I transmembrane protein encoded by the F gene. RSV-F is synthesized as a 574 amino acid inactive precursor, F0, decorated with 5 to 6 N-linked glycans, depending on the strain. It is also palmitoylated at a cysteine in its cytoplasmic domain. Three F0 monomers assemble into a trimer and, as the trimer passes through the Golgi, the monomers are activated by a furin-like host protease. The protease cleaves twice, after amino acids 109 and 136, generating three polypeptides. The N-terminal and C-terminal cleavage products are the F2 and F1 subunits (named in order of size) , respectively, and are covalently linked to each other by two disulfide bonds. The intervening 27 amino acid peptide, pep27, contains 2 or 3 N-linked glycans, but dissociates after cleavage. The F2 subunit contains two N-linked glycans, whereas the larger F1 subunit contains a single N-linked site. Unlike the others, this F1 glycan is essential for the protein to cause membrane fusion.

RSV-F is responsible for fusion of viral and host cell membranes, as well as syncytium formation between viral particles. Its sequence is highly conserved between strains. Interestingly, while viral attachment appears to involve both F and G proteins, F fusion occurs independently of G. F protein exists in multiple conformational forms. In the prefusion state (PreF) , the protein exists in a trimeric form and contains the major antigenic site

serves as a primary target of neutralizing antibodies in the body. After binding to its target on the host cell surface (its exact ligand remains unclear) , PreF undergoes a conformational change during which

is lost. This change enables the protein to insert itself into the host cell membrane and leads to fusion of the viral and host cell membranes. A final conformational shift results in a more stable and elongated form of the protein (postfusion, PostF) . Opposite of the RSV G protein, the RSV F protein also binds to and activates toll-like receptor 4 (TLR4) , initiating the innate immune response and signal transduction.

According to the UniProt Database (UniProt ID: O36634; SEQ ID NO: 31) , the RSV-F protein from RSV subtype B strain B1 includes a signal peptide, an extracellular region, a transmembrane region, and a cytoplasmic region. The signal peptide corresponds to amino acids 1-25 of SEQ ID NO: 31. The extracellular region corresponds to amino acids 26-524 of SEQ ID NO: 31. The transmembrane region corresponds to amino acids 525-550 of SEQ ID NO: 31. The cytoplasmic region corresponds to amino acids 551-574 of SEQ ID NO: 31.

A detailed description of RSV-F and its function can be found, e.g., in Gilman, Morgan SA, et al., "Transient opening of trimeric prefusion RSV F proteins. " Nature Communications 10.1 (2019) : 1-13; McLellan, Jason S. et al., "Structure and function of respiratory syncytial virus surface glycoproteins. " Challenges and Opportunities for Respiratory Syncytial Virus Vaccines (2013) : 83-104; McLellan, Jason S., et al., "Structure of respiratory syncytial virus fusion glycoprotein in the postfusion conformation reveals preservation of neutralizing epitopes. " Journal of Virology 85.15 (2011) : 7788; Battles, M.B. et al., "Respiratory syncytial virus entry and how to block it. " Nature Reviews Microbiology 17.4 (2019) : 233-245; each of which is incorporated by reference herein in its entirety.

The present disclosure provides anti-RSV-F antibodies, antigen-binding fragments thereof, and methods of using these anti-RSV-F antibodies and antigen-binding fragments to prevent and/or treat RSV-related diseases (e.g., lung infection) .

Antibodies and Antigen Binding Fragments

The present disclosure provides anti-RSV-F antibodies and antigen-binding fragments thereof that comprise complementary determining regions (CDRs) , heavy chain variable regions, light chain variable regions, heavy chains, or light chains described herein.

In general, antibodies (also called immunoglobulins) are made up of two classes of polypeptide chains, light chains and heavy chains. A non-limiting antibody of the present disclosure can be an intact, four immunoglobulin chain antibody comprising two heavy chains and two light chains. The heavy chain of the antibody can be of any isotype including IgM, IgG, IgE, IgA, or IgD or subclasses including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chain can be a kappa light chain or a lambda light chain. An antibody can comprise two identical copies of a light chain and two identical copies of a heavy chain. The heavy chains, which each contain one variable domain (or variable region, VH) and multiple constant domains (or constant regions) , bind to one another via disulfide bonding within their constant domains to form the “stem” of the antibody. The light chains, which each contain one variable domain (or variable region, VL) and one constant domain (or constant region) , each bind to one heavy chain via disulfide binding. The variable region of each light chain is aligned with the variable region of the heavy chain to which it is bound. The variable regions of both the light chains and heavy chains contain three hypervariable regions sandwiched between more conserved framework regions (FR) .

These hypervariable regions, known as the complementary determining regions (CDRs) , form loops that comprise the principle antigen binding surface of the antibody. The four framework regions largely adopt a beta-sheet conformation and the CDRs form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held in close proximity by the framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding region.

Methods for identifying the CDR regions of an antibody by analyzing the amino acid sequence of the antibody are well known, and a number of definitions of the CDRs are commonly used. The Kabat definition is based on sequence variability, and the Chothia definition is based on the location of the structural loop regions. These methods and definitions are described in, e.g., Martin, "Protein sequence and structure analysis of antibody variable domains, " Antibody engineering, Springer Berlin Heidelberg, 2001.422-439; Abhinandan, et al. "Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains, " Molecular immunology 45.14 (2008) : 3832-3839; Wu, T.T. and Kabat, E. A. (1970) J. Exp. Med. 132: 211-250; Martin et al., Methods Enzymol. 203: 121-53 (1991) ; Morea et al., Biophys Chem. 68 (1-3) : 9-16 (Oct. 1997) ; Morea et al., J Mol Biol. 275 (2) : 269-94 (Jan . 1998) ; Chothia et al., Nature 342 (6252) : 877-83 (Dec. 1989) ; Ponomarenko and Bourne, BMC Structural Biology 7: 64 (2007) ; each of which is incorporated herein by reference in its entirety.

The CDRs are important for recognizing an epitope of an antigen. As used herein, an “epitope” is the smallest portion of a target molecule capable of being specifically bound by the antigen binding domain of an antibody. The minimal size of an epitope may be about three, four, five, six, or seven amino acids, but these amino acids need not be in a consecutive linear sequence of the antigen’s primary structure, as the epitope may depend on an antigen’s three-dimensional configuration based on the antigen’s secondary and tertiary structure.

In some embodiments, the antibody is an intact immunoglobulin molecule (e.g., IgG1, IgG2a, IgG2b, IgG3, IgG4, IgM, IgD, IgE, IgA) . The IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved, differ in their constant region, particularly in their hinges and upper CH2 domains. The sequences and differences of the IgG subclasses are known in the art, and are described, e.g., in Vidarsson, et al, "IgG subclasses and allotypes: from structure to effector functions. " Frontiers in immunology 5 (2014) ; Irani, et al. "Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases. " Molecular immunology 67.2 (2015) : 171-182; Shakib, Farouk, ed. The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016; each of which is incorporated herein by reference in its entirety.

The antibody can also be an immunoglobulin molecule that is derived from any species (e.g., human, rodent, mouse, rat, camelid) . Antibodies disclosed herein also include, but are not limited to, polyclonal, monoclonal, monospecific, polyspecific antibodies, and chimeric antibodies that include an immunoglobulin binding domain fused to another polypeptide. The term “antigen binding domain” or “antigen binding fragment” is a portion of an antibody that retains specific binding activity of the intact antibody, i.e., any portion of an antibody that is capable of specific binding to an epitope on the intact antibody’s target molecule. It includes, e.g., Fab, Fab′, F (ab′) 2, and variants of these fragments. Thus, in some embodiments, an antibody or an antigen binding fragment thereof can be, e.g., a scFv, a Fv, a Fd, a dAb, a bispecific antibody, a bispecific scFv, a diabody, a linear antibody, a single-chain antibody molecule, a multi-specific antibody formed from antibody fragments, and any polypeptide that includes a binding domain which is, or is homologous to, an antibody binding domain. Non-limiting examples of antigen binding domains include, e.g., the heavy chain and/or light chain CDRs of an intact antibody, the heavy and/or light chain variable regions of an intact antibody, full length heavy or light chains of an intact antibody, or an individual CDR from either the heavy chain or the light chain of an intact antibody.

In some embodiments, the antigen binding fragment can form a part of a chimeric antigen receptor (CAR) . In some embodiments, the chimeric antigen receptor are fusions of single-chain variable fragments (scFv) as described herein, fused to CD3-zeta transmembrane-and endodomain. In some embodiments, the chimeric antigen receptor also comprises intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS) . In some embodiments, the chimeric antigen receptor comprises multiple signaling domains, e.g., CD3z-CD28-41BB or CD3z-CD28-OX40, to increase potency. Thus, in one aspect, the disclosure further provides cells (e.g., T cells) that express the chimeric antigen receptors as described herein.

In some embodiments, the scFV has one heavy chain variable domain, and one light chain variable domain. In some embodiments, the scFV has two heavy chain variable domains, and two light chain variable domains. In some embodiments, the scFV has two antigen binding regions, and the two antigen binding regions can bind to the respective target antigens.

Anti-RSV-F Antibodies and Antigen-Binding Fragments

The disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to RSV-F. In some embodiments, the antibodies and antigen-binding fragments described herein are capable of binding to RSV-F and prevent an RSV infection (e.g., bronchiolitis and pneumonia) .

The disclosure provides e.g., mouse anti-RSV-F antibodies 04-2H10 ( “2HI0” ) , the chimeric antibodies thereof, and the humanized antibodies thereof (e.g., some of the antibodies as shown in Table 1) .

The CDR sequences for 04-2H10, and 04-2H10 derived antibodies (e.g., humanized antibodies) include CDRs of the heavy chain variable domain, SEQ ID NOs: 1-3, and CDRs of the light chain variable domain, SEQ ID NOs: 4-6 as defined by Kabat numbering. In some embodiments, the CDR sequences for 04-2H10, and 04-2H10 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 1, 44, and 3; and CDRs of the light chain variable domain, SEQ ID NOs: 4-6, as defined by Kabat numbering. In some embodiments, the CDR sequences for 04-2H10, and 04-2H10 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 1, 45, and 3; and CDRs of the light chain variable domain, SEQ ID NOs: 4-6, as defined by Kabat numbering. In some embodiments, the CDR sequences for 04- 2H10, and 04-2H10 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 1, 46, and 3; and CDRs of the light chain variable domain, SEQ ID NOs: 4-6, as defined by Kabat numbering. In some embodiments, the CDR sequences for 04-2H10, and 04-2H10 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 1, 47, and 3; and CDRs of the light chain variable domain, SEQ ID NOs: 4-6, as defined by Kabat numbering.

The CDRs can also be defined by Chothia system. Under the Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 7-9 and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 10-12. In some embodiments, the CDR sequences for 04-2H10, and 04-2H10 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 48, 8, and 9; and CDRs of the light chain variable domain, SEQ ID NOs: 10-12, as defined by Chothia numbering. In some embodiments, the CDR sequences for 04-2H10, and 04-2H10 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 49, 8, and 9; and CDRs of the light chain variable domain, SEQ ID NOs: 10-12, as defined by Chothia numbering. In some embodiments, the CDR sequences for 04-2H10, and 04-2H10 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 50, 8, and 9; and CDRs of the light chain variable domain, SEQ ID NOs: 10-12, as defined by Chothia numbering. In some embodiments, the CDR sequences for 04-2H10, and 04-2H10 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 51, 8, and 9; and CDRs of the light chain variable domain, SEQ ID NOs: 10-12, as defined by Chothia numbering.

The amino acid sequences for heavy chain variable regions and light variable regions of the humanized antibodies are also provided. As there are different ways to humanize a mouse antibody (e.g., a sequence can be modified with different amino acid substitutions) , the heavy chain and the light chain of an antibody can have more than one version of humanized sequences. The amino acid sequences for the heavy chain variable regions of humanized 2H10 antibody are set forth in SEQ ID NOs: 13-22. The amino acid sequences for the light chain variable regions of humanized 2H10 antibody are set forth in SEQ ID NOs: 23-25. Any of these heavy chain variable region sequences (SEQ ID NOs: 13-22) can be paired with any of these light chain variable region sequences (SEQ ID NOs: 23-25) .

Some chimeric and humanized antibodies based on 04-2H10 ( “2HI0” ) are shown in the table below.

Table 1.

Humanization percentage means the percentage identity of the heavy chain or light chain variable region sequence as compared to human antibody sequences in International

Immunogenetics Information System (IMGT) database. The top hit means that the heavy chain or light chain variable region sequence is closer to a particular species than to other species. For example, top hit to human means that the sequence is closer to human than to other species. Top hit to human and Macaca fascicularis means that the sequence has the same percentage identity to the human sequence and the Macaca fascicularis sequence, and these percentages identities are highest as compared to the sequences of other species. In some embodiments, humanization percentage is greater than 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%. A detailed description regarding how to determine humanization percentage and how to determine top hits is known in the art, and is described, e.g., in Jones, et al., "The INNs and outs of antibody nonproprietary names. " MAbs. Vol. 8. No. 1. Taylor &Francis, 2016, which is incorporated herein by reference in its entirety. A high humanization percentage often has various advantages, e.g., more safe and more effective in humans, more likely to be tolerated by a human subject, and/or less likely to have side effects.

Furthermore, in some embodiments, the antibodies or antigen-binding fragments thereof described herein can also contain one, two, or three heavy chain variable region CDRs selected from the group of SEQ ID NOs: 1-3; SEQ ID NOs: 1, 44, and 3; SEQ ID NOs: 1, 45, and 3; SEQ ID NOs: 1, 46, and 3; SEQ ID NOs: 1, 47, and 3; SEQ ID NOs: 7-9; SEQ ID NOs: 48, 8, and 9; SEQ ID NOs: 49, 8, and 9; SEQ ID NOs: 50, 8, and 9; and SEQ ID NOs: 51, 8, and 9; and/or one, two, or three light chain variable region CDRs selected from the group of SEQ ID NOs: 4-6, and SEQ ID NOs: 10-12.

In some embodiments, the antibodies can have a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH CDR3 amino acid sequence, and a light chain variable region (VL) comprising CDRs 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VL CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VL CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VL CDR3 amino acid sequence. The selected

VH CDRs

1, 2, 3 amino acid sequences and the selected VL CDRs, 1, 2, 3 amino acid sequences are shown in FIG. 6 (Kabat CDR) and FIG. 7 (Chothia CDR) .

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 1 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 2 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 3 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 1 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 44 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 3 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 1 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 45 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 3 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 1 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 46 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 3 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 1 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 47 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 3 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 7 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 8 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 9 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 48 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 8 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 9 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 49 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 8 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 9 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 50 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 8 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 9 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 51 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 8 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 9 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 4 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 5 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 6 with zero, one or two amino acid insertions, deletions, or substitutions.

In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 10 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 11 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 12 with zero, one or two amino acid insertions, deletions, or substitutions.

The insertions, deletions, and substitutions can be within the CDR sequence, or at one or both terminal ends of the CDR sequence.

The disclosure also provides antibodies or antigen-binding fragments thereof that bind to RSV-F. The antibodies or antigen-binding fragments thereof contain a heavy chain variable region (VH) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH sequence, and a light chain variable region (VL) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 26, and the selected VL sequence is SEQ ID NO: 23, 24, 25, or 27.

The disclosure also provides antibodies or antigen-binding fragments thereof that can compete with the antibodies described herein. In some aspects, the antibodies or antigen-binding fragments can bind to the same epitope as the antibodies described herein.

To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes) . The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. For purposes of illustration, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

The disclosure also provides nucleic acid comprising a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or an immunoglobulin light chain. The immunoglobulin heavy chain or immunoglobulin light chain comprises CDRs as shown in FIG. 6 or FIG. 7, or have sequences as shown in FIG. 8 and FIG. 9. When the polypeptides are paired with corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region) , the paired polypeptides bind to RSV-F (e.g., RSV-F from RSV subtype A or subtype B strain) .

The anti-RSV-F antibodies and antigen-binding fragments can also be antibody variants (including derivatives and conjugates) of antibodies or antibody fragments and multi-specific (e.g., bi-specific) antibodies or antibody fragments. Additional antibodies provided herein are polyclonal, monoclonal, multi-specific (multimeric, e.g., bi-specific) , human antibodies, chimeric antibodies (e.g., human-mouse chimera) , single-chain antibodies, intracellularly-made antibodies (i.e., intrabodies) , and antigen-binding fragments thereof. The antibodies or antigen-binding fragments thereof can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) , class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) , or subclass. In some embodiments, the antibody or antigen-binding fragment thereof is an IgG antibody or antigen-binding fragment thereof.

Fragments of antibodies are suitable for use in the methods provided so long as they retain the desired affinity and specificity of the full-length antibody. Thus, a fragment of an antibody that binds to RSV-F will retain an ability to bind to RSV-F. An Fv fragment is an antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight association, which can be covalent in nature, for example in scFv. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs or a subset thereof confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) can have the ability to recognize and bind antigen, although usually at a lower affinity than the entire binding site.

Single-chain Fv or (scFv) antibody fragments comprise the VH and VL domains (or regions) of antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. In some embodiments, the linker connecting scFv VH and VL domains is GGGGSGGGGSGGGGS (SEQ ID NO: 52) .

The Fab fragment contains a variable and constant domain of the light chain and a variable domain and the first constant domain (CH1) of the heavy chain. F (ab') ₂ antibody fragments comprise a pair of Fab fragments which are generally covalently linked near their carboxy termini by hinge cysteines between them. Other chemical couplings of antibody fragments are also known in the art.

Diabodies are small antibody fragments with two antigen-binding sites, which fragments comprise a VH connected to a VL in the same polypeptide chain (VH and VL) . By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.

Linear antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.

Antibodies and antibody fragments of the present disclosure can be modified in the Fc region to provide desired effector functions or serum half-life.

Multimerization of antibodies may be accomplished through natural aggregation of antibodies or through chemical or recombinant linking techniques known in the art. For example, some percentage of purified antibody preparations (e.g., purified IgG1 molecules) spontaneously form protein aggregates containing antibody homodimers and other higher-order antibody multimers.

Alternatively, antibody homodimers may be formed through chemical linkage techniques known in the art. For example, heterobifunctional crosslinking agents including, but not limited to SMCC (succinimidyl 4- (maleimidomethyl) cyclohexane-1-carboxylate) and SATA (N-succinimidyl S-acethylthio-acetate) can be used to form antibody multimers. An exemplary protocol for the formation of antibody homodimers is described in Ghetie et al. (Proc. Natl. Acad. Sci. U.S.A. 94: 7509-7514, 1997) . Antibody homodimers can be converted to Fab’ ₂ homodimers through digestion with pepsin. Another way to form antibody homodimers is through the use of the autophilic T15 peptide described in Zhao et al. (J. Immunol. 25: 396-404, 2002) .

In some embodiments, the multi-specific antibody is a bi-specific antibody. Bi-specific antibodies can be made by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers that are recovered from recombinant cell culture. For example, the interface can contain at least a part of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan) . Compensatory “cavities” of identical or similar size to the large side chain (s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine) . This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers. This method is described, e.g., in WO 96/27011, which is incorporated by reference in its entirety.

Bi-specific antibodies include cross-linked or “heteroconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin and the other to biotin. Heteroconjugate antibodies can also be made using any convenient cross-linking methods. Suitable cross-linking agents and cross-linking techniques are well known in the art and are disclosed in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety.

Methods for generating bi-specific antibodies from antibody fragments are also known in the art. For example, bi-specific antibodies can be prepared using chemical linkage. Brennan et al. (Science 229: 81, 1985) describes a procedure where intact antibodies are proteolytically cleaved to generate F (ab’) ₂ fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab’ fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab’ TNB derivatives is then reconverted to the Fab’ thiol by reduction with mercaptoethylamine, and is mixed with an equimolar amount of another Fab’ TNB derivative to form the bi-specific antibody.

Any of the antibodies or antigen-binding fragments described herein may be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or antigen-binding fragment thereof in a subject or in solution) . Non-limiting examples of stabilizing molecules include: a polymer (e.g., a polyethylene glycol) or a protein (e.g., serum albumin, such as human serum albumin) . The conjugation of a stabilizing molecule can increase the half-life or extend the biological activity of an antibody or an antigen-binding fragment in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in a human) .

In some embodiments, the antibodies or antigen-binding fragments described herein can be conjugated to a therapeutic agent. The antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof can covalently or non-covalently bind to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent (e.g., cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin, maytansinoids such as DM-1 and DM-4, dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and analogs) .

Antibody Characteristics

In some embodiments, the antibodies or antigen-binding fragments thereof described herein can neutralize the RSV by blocking virus internalization (e.g., via membrane fusion) .

In some embodiments, the antibody or antigen-binding fragment thereof described herein recognizes an RSV-F from RSV subtype A strain (e.g., strain RSS-2 or strain Long) . In some embodiments, the antibody or antigen-binding fragment thereof described herein recognizes an RSV-F from RSV subtype B strain (e.g., strain B1 or strain 18537) . In some embodiments, the sequence of RSV-F is set forth in SEQ ID NO: 28, 29, 30, 31, or 32. In some embodiments, the antibody or antigen-binding fragment thereof described herein recognizes a recombinant RSV-F.

In some embodiments, the antibody or antigen-binding fragment thereof described herein recognizes all or a portion of RSV-F (e.g., an extracellular region, a transmembrane region and/or a cytoplasmic region of RSV-F) . In some embodiments, the antibody or antigen-binding fragment thereof described herein recognizes an extracellular and transmembrane regions of RSV-F. In some embodiments, the extracellular and transmembrane regions correspond to amino acids 26-524 of SEQ ID NO: 28, 29, 30, 31, or 32.

In some implementations, the antibody (or antigen-binding fragments thereof) specifically binds to RSV-F (e.g., a recombinant RSV-F) with a dissociation rate (koff) of less than 0.1 s ^-1, less than 0.01 s ^-1, less than 0.001 s ^-1, less than 0.0001 s ^-1, or less than 0.00001 s ^-1. In some embodiments, the dissociation rate (koff) is greater than 0.01 s ^-1, greater than 0.001 s ^-1, greater than 0.0001 s ^-1, greater than 0.00001 s ^-1, or greater than 0.000001 s ^-1.

In some embodiments, kinetic association rates (kon) is greater than 1 x 10 ²/Ms, greater than 1 x 103/Ms, greater than 1 x 10 ⁴/Ms, greater than 1 x 10 ⁵/Ms, or greater than 1 x 10 ⁶/Ms. In some embodiments, kinetic association rates (kon) is less than 1 x 10 ⁵/Ms, less than 1 x 10 ⁶/Ms, or less than 1 x 10 ⁷/Ms.

Affinities can be deduced from the quotient of the kinetic rate constants (KD=koff/kon) . In some embodiments, KD is less than 1 x 10 ^-6 M, less than 1 x 10 ^-7 M, less than 1 x 10 ^-8 M, less than 1 x 10 ^-9 M, or less than 1 x 10 ^-10 M. In some embodiments, KD is greater than 1 x 10 ^-7 M, greater than 1 x 10 ^-8 M, greater than 1 x 10 ^-9 M, greater than 1 x 10 ^-10 M, greater than 1 x 10 ^-11 M, or greater than 1 x 10 ^-12 M.

In some embodiments, binding affinity of the antibody or antigen-binding fragment thereof described herein to RSV-F is determined (e.g., by surface plasma resonance (SPR) ) . In some embodiments, the determined KD is less than or about 5 × 10 ^-8 M, less than or about 2 × 10 ^-8 M, less than or about 1 × 10 ^-8 M, less than or about 5 × 10 ^-9 M, less than or about 2 × 10 ^-9 M , less than or about 1 × 10 ^-9 M, less than or about 5 × 10 ^-10 M, less than or about 2 × 10 ^-10 M, or less than or about 1 × 10 ^-10 M.

General techniques for measuring the affinity of an antibody for an antigen include, e.g., ELISA, RIA, and surface plasmon resonance (SPR) . In some embodiments, the antibody binds to all or a portion of RSF (SEQ ID NO: 28) , RSF-S2 (SEQ ID NO: 29) , RSF-LONG (SEQ ID NO: 30) , RSF-B1 (SEQ ID NO: 31) , and/or RSF-B18537 (SEQ ID NO: 32) . In some embodiments, the antibody does not bind to all or a portion of RSF (SEQ ID NO: 28) , RSF-S2 (SEQ ID NO: 29) , RSF-LONG (SEQ ID NO: 30) , RSF-B1 (SEQ ID NO: 31) , and/or RSF-B18537 (SEQ ID NO: 32) .

In some embodiments, thermal stability of an antibody or antigen-binding fragment thereof is determined. The antibody or antigen-binding fragment can have a Tm (e.g., Tm1 or Tm2) greater than 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, or 95 ℃.

In some embodiments, accelerated stability of an antibody or antigen-binding fragment thereof is determined, e.g., by incubating the antibody or antigen-binding fragment thereof at about 4℃, about 25℃, about 37℃, or about 40℃. In some embodiments, the incubation is performed for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks. In some embodiments, parameters of the antibody or antigen-binding fragment thereof (e.g., appearance, thermal stability, purity, hydrophobicity, charge variants, and/or binding activity) are determined using the methods described herein. In some embodiments, the above parameters are not changed after the incubation. In some embodiments, the above parameters are changed by less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%as compared to those of a control sample that is not incubated.

In some embodiments, apparent hydrophobicity of an antibody or antigen-binding fragment thereof is determined, e.g., by Hydrophobic Interaction Chromatography (HIC) separation. In a HIC separation, the hydrophobic ligands on the stationary phase interact with the hydrophobic regions on the surface of the protein and the retention mechanism is due to adsorption-desorption equilibrium in the presence of salts. In practice, proteins bind to the HIC stationary phase in the presence of high concentration of salt, and are eluted in the order of increasing hydrophobicity by decreasing the salt concentration. In some embodiments, the retention time of the main peak for the antibody or antigen-binding fragment thereof is less than 15, less than 14, less than 13, less than 12, less than 11, or less than 10 minutes. In some embodiments, the retention time of the main peak for the antibody or antigen-binding fragment thereof is less than that of a reference antibody (e.g., MEDI8897-IgG1-RYTE) . As such, the antibody or antigen-binding fragment thereof described herein may have a lower tendency to aggregate and/or precipitate. In some embodiments, the percentage of the area of the main peak to the sum of all peak areas for the antibody or antigen-binding fragment thereof is greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99%.

In some embodiments, the antibody or antigen binding fragment thereof can neutralize RSV (e.g., recombinant RSV virus) . In some embodiments, the IC50 (half maximal inhibitory concentration) of the antibody or antigen binding fragment thereof is determined. IC50 is a quantitative measure that indicates how much of the antibody or antigen-binding fragment thereof is need to achieve a 50%RSV neutralization. In some embodiments, the IC50 is less than 25 ng/ml, less than 24 ng/ml, less than 23 ng/ml, less than 22 ng/ml, less than 21 ng/ml, less than 20 ng/ml, less than 19 ng/ml, less than 18 ng/ml, less than 17 ng/ml, less than 16 ng/ml, less than 15 ng/ml, less than 14 ng/ml, less than 13 ng/ml, less than 12 ng/ml, less than 11 ng/ml, less than 10 ng/ml, less than 9 ng/ml, less than 8 ng/ml, less than 7 ng/ml, less than 6 ng/ml, less than 5 ng/ml, less than 4 ng/ml, less than 3 ng/ml, less than 2 ng/ml, or less than 1 ng/ml. In some embodiments, the IC50 is less than 2.5 ng/ml, less than 2.4 ng/ml, less than 2.3 ng/ml, less than 2.2 ng/ml, less than 2.1 ng/ml, less than 2.0 ng/ml, less than 1.9 ng/ml, less than 1.8 ng/ml, less than 1.7 ng/ml, less than 1.6 ng/ml, less than 1.5 ng/ml, less than 1.4 ng/ml, less than 1.3 ng/ml, less than 1.2 ng/ml, less than 1.1 ng/ml, or less than 1.0 ng/ml.

In some embodiments, the antibody or antigen-binding fragment thereof comprises an Fc region from human IgG1, human IgG2, human IgG3, or human IgG4. In some embodiments, the antibody or antigen-binding fragment thereof comprises a wild-type human IgG1 Fc. In some embodiments, sequence of the wild-type IgG1 heavy chain constant domain is set forth in SEQ ID NO: 34.

In some embodiments, the antibody or antigen-binding fragment thereof comprises a human IgG1 Fc containing one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) mutations.

In some embodiments, the antibody or antigen-binding fragment thereof includes the RYTE mutation. The RYTE mutation includes a lysine (K) residue at position 214, a tyrosine (Y) residue at position 252, a threonine (T) residue at position 254, and a glutamate (E) residue at position 256 of the antibody or antigen-binding fragment thereof, according to EU numbering. Details of this mutation can be found, e.g., in Dall'A cqua, et al. "Properties of human IgG1s engineered for enhanced binding to the neonatal Fc receptor (FcRn) . " Journal of Biological Chemistry 281.33 (2006) : 23514-23524; WO2010068722A1; and US8775090B2; each of which is incorporated herein by reference in the entirety. These mutations can enhance the binding affinity of the antibody or antigen-binding fragment thereof with FcRn under low pH conditions, to prolong its half-life in vivo. In some embodiments, sequence of the IgG1 heavy chain constant domain containing the RYTE mutation is set forth in SEQ ID NO: 35.

In some embodiments, the antibody or antigen-binding fragment thereof includes the EDML mutation. The EDML mutation includes an aspartate (D) residue at position 356, and a leucine (L) residue at position 358 of the antibody or antigen-binding fragment thereof, according to EU numbering. Details of this mutation can be found, e.g., in Vidarsson et al., "IgG subclasses and allotypes: from structure to effector functions. " Frontiers in immunology 5 (2014) : 520, which is incorporated herein by reference in its entirety. In some embodiments, sequence of the IgG1 heavy chain constant domain containing the EDML mutation is set forth in SEQ ID NO: 33.

Methods of Making Anti-RSV-F Antibodies

A full-length RSV-F or an isolated fragment (e.g., a fragment including the extracellular and transmembrane regions) of RSV-F can be used as an immunogen to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. Polyclonal antibodies can be raised in animals by multiple injections (e.g., subcutaneous or intraperitoneal injections) of an antigenic peptide or protein. In some embodiments, the antigenic peptide or protein is injected with at least one adjuvant. In some embodiments, the antigenic peptide or protein can be conjugated to an agent that is immunogenic in the species to be immunized. Animals can be injected with the antigenic peptide or protein more than one time (e.g., twice, three times, or four times) .

The full-length polypeptide or protein can be used or, alternatively, antigenic peptide fragments thereof can be used as immunogens. The antigenic peptide of a protein comprises at least 8 (e.g., at least 10, 15, 20, or 30) amino acid residues of the amino acid sequence of RSV-F and encompasses an epitope of the protein such that an antibody raised against the peptide forms a specific immune complex with the protein. As described above, the full length sequence of RSV-F is known in the art (SEQ ID NOs: 28, 29, 30, 31, and 32) .

An immunogen typically is used to prepare antibodies by immunizing a suitable subject (e.g., human or transgenic animal expressing at least one human immunoglobulin locus) . An appropriate immunogenic preparation can contain, for example, a recombinantly-expressed or a chemically-synthesized polypeptide (e.g., a fragment of RSV-F) . The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or a similar immunostimulatory agent.

Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with an RSV-F polypeptide, or an antigenic peptide thereof (e.g., part of RSV-F) as an immunogen. The antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme-linked immunosorbent assay (ELISA) using the immobilized RSV-F polypeptide or peptide. If desired, the antibody molecules can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A of protein G chromatography to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the specific antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler et al. (Nature 256: 495-497, 1975) , the human B cell hybridoma technique (Kozbor et al., Immunol. Today 4: 72, 1983) , the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985) , or trioma techniques. The technology for producing hybridomas is well known (see, generally, Current Protocols in Immunology, 1994, Coligan et al. (Eds. ) , John Wiley &Sons, Inc., New York, NY) . Hybridoma cells producing a monoclonal antibody are detected by screening the hybridoma culture supernatants for antibodies that bind the polypeptide or epitope of interest, e.g., using a standard ELISA assay.

Variants of the antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding a human, humanized, or chimeric antibody, or antigen-binding fragment thereof described herein, or by peptide synthesis. Such variants include, for example, deletions, insertions, or substitutions of residues within the amino acids sequences that make-up the antigen-binding site of the antibody or an antigen-binding domain. In a population of such variants, some antibodies or antigen-binding fragments will have increased affinity for the target protein, e.g., RSV-F. Any combination of deletions, insertions, and/or combinations can be made to arrive at an antibody or antigen-binding fragment thereof that has increased binding affinity for the target. The amino acid changes introduced into the antibody or antigen-binding fragment can also alter or introduce new post-translational modifications into the antibody or antigen-binding fragment, such as changing (e.g., increasing or decreasing) the number of glycosylation sites, changing the type of glycosylation site (e.g., changing the amino acid sequence such that a different sugar is attached by enzymes present in a cell) , or introducing new glycosylation sites.

Antibodies disclosed herein can be derived from any species of animal, including mammals. Non-limiting examples of native antibodies include antibodies derived from humans, primates, e.g., monkeys and apes, cows, pigs, horses, sheep, camelids (e.g., camels and llamas) , chicken, goats, and rodents (e.g., rats, mice, hamsters and rabbits) , including transgenic rodents genetically engineered to produce human antibodies.

Human and humanized antibodies include antibodies having variable and constant regions derived from (or having the same amino acid sequence as those derived from) human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo) , for example in the CDRs.

A humanized antibody, typically has a human framework (FR) grafted with non-human CDRs. Thus, a humanized antibody has one or more amino acid sequence introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain· Hummization can be essentially performed by e.g., substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. These methods are described in e.g., Jones et al., Nature, 321: 522-525 (1986) ; Riechmann et al., Nature, 332: 323-327 (1988) ; Verhoeyen et al., Science, 239: 1534-1536 (1988) ; each of which is incorporated by reference herein in its entirety. Accordingly, “humanized” antibodies are chimeric antibodies wherein substantially less than an intact human V domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically mouse antibodies in which some CDR residues and some FR residues are substituted by residues from analogous sites in human antibodies.

The choice of human VH and VL domains to be used in making the humanized antibodies is very important for reducing immunogenicity. According to the so-called “best-fit” method, the sequence of the V domain of a mouse antibody is screened against the entire library of known human-domain sequences. The human sequence which is closest to that of the mouse is then accepted as the human FR for the humanized antibody (Sims et al., J. Immunol., 151: 2296 (1993) ; Chothia et al., J. Mol. Biol., 196: 901 (1987) ) .

It is further important that antibodies be humanized with retention of high specificity and affinity for the antigen and other favorable biological properties. To achieve this goal, humanized antibodies can be prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen (s) , is achieved.

Ordinarily, amino acid sequence variants of the human, humanized, or chimeric anti-RSV-F antibody will contain an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%percent identity with a sequence present in the light or heavy chain of the original antibody.

Identity with respect to an original sequence is usually the percentage of amino acid residues present within the candidate sequence that are identical with a sequence present within the human, humanized, or chimeric anti-RSV-F antibody or fragment, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.

Additional modifications to the anti-RSV-F antibodies or antigen-binding fragments can be made. For example, a cysteine residue (s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have any increased half-life in vitro and/or in vivo. Homodimeric antibodies with increased half-life in vitro and/or in vivo can also be prepared using heterobifunctional cross-linkers as described, for example, in Wolffet al. (Cancer Res. 53: 2560-2565, 1993) . Alternatively, an antibody can be engineered which has dual Fc regions (see, for example, Stevenson et al., Anti-Cancer Drug Design 3: 219-230, 1989) .

In some embodiments, a covalent modification can be made to the anti-RSV-F antibody or antigen-binding fragment thereof. These covalent modifications can be made by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage. Other types of covalent modifications of the antibody or antibody fragment are introduced into the molecule by reacting targeted amino acid residues of the antibody or fragment with an organic derivatization agent that is capable of reacting with selected side chains or the N-or C-terminal residues.

In some embodiments, antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from 1%to 80%, from 1%to 65%, from 5%to 65%or from 20%to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues; or position 314 in Kabat numbering) ; however, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. In some embodiments, to reduce glycan heterogeneity, the Fc region of the antibody can be further engineered to replace the Asparagine at position 297 with Alanine (N297A) .

In some aspects, the disclosure also provides the use of the antibodies or antigen fragments thereof described herein for manufacture of a medicament for RSV infection.

Recombinant Vectors

The present disclosure also provides recombinant vectors (e.g., an expression vectors) that include an isolated polynucleotide disclosed herein (e.g., a polynucleotide that encodes a polypeptide disclosed herein) , host cells into which are introduced the recombinant vectors (i.e., such that the host cells contain the polynucleotide and/or a vector comprising the polynucleotide) , and the production of recombinant antibody polypeptides or fragments thereof by recombinant techniques.

As used herein, a “vector” is any construct capable of delivering one or more polynucleotide (s) of interest to a host cell when the vector is introduced to the host cell. An “expression vector” is capable of delivering and expressing the one or more polynucleotide (s) of interest as an encoded polypeptide in a host cell into which the expression vector has been introduced. Thus, in an expression vector, the polynucleotide of interest is positioned for expression in the vector by being operably linked with regulatory elements such as a promoter, enhancer, and/or a poly-A tail, either within the vector or in the genome of the host cell at or near or flanking the integration site of the polynucleotide of interest such that the polynucleotide of interest will be translated in the host cell introduced with the expression vector.

A vector can be introduced into the host cell by methods known in the art, e.g., electroporation, chemical transfection (e.g., DEAE-dextran) , transformation, transfection, and infection and/or transduction (e.g., with recombinant virus) . Thus, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant virus) , naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

In some implementations, a polynucleotide disclosed herein (e.g., a polynucleotide that encodes a polypeptide disclosed herein) is introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, or adenovirus) , which may involve the use of a non-pathogenic (defective) , replication competent virus, or may use a replication defective virus. In the latter case, viral propagation generally will occur only in complementing virus packaging cells. Suitable systems are disclosed, for example, in Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA 86: 317-321; Flexner et al., 1989, Ann. N. Y. Acad Sci. 569: 86-103; Flexner et al., 1990, Vaccine, 8: 17-21; U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner-Biotechniques, 6:616-627, 1988; Rosenfeld et al., 1991, Science, 252: 431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, 91: 215-219; Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci. USA, 90: 11498-11502; Guzman et al., 1993, Circulation, 88: 2838-2848; and Guzman et al., 1993, Cir. Res., 73: 1202-1207. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA may also be “naked, ” as described, for example, in Ulmer et al., 1993, Science, 259: 1745-1749, and Cohen, 1993, Science, 259: 1691-1692. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads that are efficiently transported into the cells.

For expression, the DNA insert comprising an antibody-encoding or polypeptide-encoding polynucleotide disclosed herein can be operatively linked to an appropriate promoter (e.g., a heterologous promoter) , such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters are known to the skilled artisan. The expression constructs can further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs may include a translation initiating at the beginning and a termination codon (UAA, UGA, or UAG) appropriately positioned at the end of the polypeptide to be translated.

As indicated, the expression vectors can include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces, and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, Bowes melanoma, and HK 293 cells; and plant cells. Appropriate culture mediums and conditions for the host cells described herein are known in the art.

Non-limiting vectors for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.

Non-limiting bacterial promoters suitable for use include the E. coli lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters and the trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus (RSV) , and metallothionein promoters, such as the mouse metallothionein-I promoter.

In the yeast Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al. (1989) Current Protocols in Molecular Biology, John Wiley &Sons, New York, N. Y, and Grant et al., Methods Enzymol., 153: 516-544 (1997) .

Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986) , which is incorporated herein by reference in its entirety.

Transcription of DNA encoding an antibody of the present disclosure by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type. Examples of enhancers include the SV40 enhancer, which is located on the late side of the replication origin at base pairs 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the expressed polypeptide. The signals may be endogenous to the polypeptide or they may be heterologous signals.

The polypeptide (e.g., antibody) can be expressed in a modified form, such as a fusion protein (e.g., a GST-fusion) or with a histidine-tag, and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties can be added to the polypeptide to facilitate purification. Such regions can be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.

The disclosure also provides a nucleic acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to any nucleotide sequence as described herein, and an amino acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to any amino acid sequence as described herein.

The disclosure also provides a nucleic acid sequence that has a homology of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%to any nucleotide sequence as described herein, and an amino acid sequence that has a homology of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%to any amino acid sequence as described herein.

In some embodiments, the disclosure relates to nucleotide sequences encoding any peptides that are described herein, or any amino acid sequences that are encoded by any nucleotide sequences as described herein. In some embodiments, the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides. In some embodiments, the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, or 400 amino acid residues.

In some embodiments, the amino acid sequence (i) comprises an amino acid sequence; or (ii) consists of an amino acid sequence, wherein the amino acid sequence is any one of the sequences as described herein.

In some embodiments, the nucleic acid sequence (i) comprises a nucleic acid sequence; or (ii) consists of a nucleic acid sequence, wherein the nucleic acid sequence is any one of the sequences as described herein.

The percentage of residues conserved with similar physicochemical properties (percent homology) , e.g. leucine and isoleucine, can also be used to measure sequence similarity. Families of amino acid residues having similar physicochemical properties have been defined in the art. These families include e.g., amino acids with basic side chains (e.g., lysine, arginine, histidine) , acidic side chains (e.g., aspartic acid, glutamic acid) , uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine) , nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) , beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine) . The homology percentage, in many cases, is higher than the identity percentage.

Methods of Treatment

The antibodies or antibody or antigen-binding fragments thereof of the present disclosure can be used for various therapeutic purposes.

In one aspect, the disclosure provides methods for preventing or treating a subject having a Respiratory Syncytial Virus-related disease (e.g., bronchiolitis, pneumonia, or other lung infections) , methods of neutralizing RSV, methods of promoting virus aggregation, methods of inducing Fc-dependent antiviral functions, methods of blocking internalization of the virus by a cell, methods of identifying a subject having an RSV-related disease (e.g., RSV infection) . In some embodiments, the treatment can halt, slow, retard, or inhibit progression of an RSV-related disease. In some embodiments, the treatment can result in the reduction of in the number, severity, and/or duration of one or more symptoms of the RSV-related disease in a subject.

In one aspect, the disclosure features methods that include administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein to a subject in need thereof (e.g., a subject having, or identified or diagnosed as having, an RSV-related disease) .

In some embodiments, the virus that causing the RSV-related disease is an RSV subtype A strain, an RSV subtype B strain, or other strains of RSV having one or more fusion proteins. In some embodiments, the amino acid sequence of the fusion protein of the RSV described herein comprises a sequence that is at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, or at least or about 98%identical to SEQ ID NO: 28, 29, 30, 31, or 32.

In some embodiments, the compositions and methods disclosed herein can be used for treatment of patients at risk for an RSV-related disease. Patients with RSV-related disease can be identified with various methods known in the art.

As used herein, by an “effective amount” is meant an amount or dosage sufficient to effect beneficial or desired results including halting, slowing, retarding, or inhibiting progression of a disease, e.g., a RSV-related disease. An effective amount will vary depending upon, e.g., an age and a body weight of a subject to which the antibody, antigen binding fragment, antibody-encoding polynucleotide, vector comprising the polynucleotide, and/or compositions thereof is to be administered, a severity of symptoms and a route of administration, and thus administration can be determined on an individual basis.

An effective amount can be administered in one or more administrations. By way of example, an effective amount of an antibody or an antigen binding fragment is an amount sufficient to ameliorate, stop, stabilize, reverse, inhibit, slow and/or delay progression of a RSV-related disease in a patient. As is understood in the art, an effective amount of an antibody or antigen binding fragment may vary, depending on, inter alia, patient history as well as other factors such as the type (and/or dosage) of antibody used.

Effective amounts and schedules for administering the antibodies, antibody-encoding polynucleotides, and/or compositions disclosed herein may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage that must be administered will vary depending on, for example, the mammal that will receive the antibodies, antibody-encoding polynucleotides, and/or compositions disclosed herein, the route of administration, the particular type of antibodies, antibody-encoding polynucleotides, antigen binding fragments, and/or compositions disclosed herein used and other drugs being administered to the mammal.

A typical daily dosage of an effective amount of an antibody is 0.01 mg/kg to 100 mg/kg (mg per kg of patient weight) . In some embodiments, the dosage can be less than 100 mg/kg, 50 mg/kg, 25 mg/kg, 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.5 mg/kg, or 0.1 mg/kg. In some embodiments, the dosage can be greater than 25 mg/kg, 20 mg/kg, 15 mg/kg, 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.5 mg/kg, 0.1 mg/kg, 0.05 mg/kg, or 0.01 mg/kg. In some embodiments, the dosage is about 25 mg/kg, 20 mg/kg, 15 mg/kg, 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.9 mg/kg, 0.8 mg/kg, 0.7 mg/kg, 0.6 mg/kg, 0.5 mg/kg, 0.4 mg/kg, 0.3 mg/kg, 0.2 mg/kg, or 0.1 mg/kg.

In any of the methods described herein, the at least one antibody, antigen-binding fragment thereof, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding fragments, or pharmaceutical compositions described herein) and, optionally, at least one additional therapeutic agent can be administered to the subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day) . In some embodiments, at least two different antibodies and/or antigen-binding fragments are administered in the same composition (e.g., a liquid composition) . In some embodiments, at least one antibody or antigen-binding fragment and at least one additional therapeutic agent are administered in the same composition (e.g., a liquid composition) . In some embodiments, the at least one antibody or antigen-binding fragment and the at least one additional therapeutic agent are administered in two different compositions (e.g., a liquid composition containing at least one antibody or antigen-binding fragment and a solid oral composition containing at least one additional therapeutic agent) . In some embodiments, the at least one additional therapeutic agent is administered as a pill, tablet, or capsule. In some embodiments, the at least one additional therapeutic agent is administered in a sustained-release oral formulation.

In some embodiments, the one or more additional therapeutic agents can be administered to the subject prior to, or after administering the at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) . In some embodiments, the one or more additional therapeutic agents and the at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) are administered to the subject such that there is an overlap in the bioactive period of the one or more additional therapeutic agents and the at least one antibody or antigen-binding fragment (e.g., any of the antibodies or antigen-binding fragments described herein) in the subject.

In some embodiments, the subject can be administered the at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) over an extended period of time (e.g., over a period of at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years) . A skilled medical professional may determine the length of the treatment period using any of the methods described herein for diagnosing or following the effectiveness of treatment (e.g., the observation of at least one symptom of RSV-related diseases) . As described herein, a skilled medical professional can also change the identity and number (e.g., increase or decrease) of antibodies or antigen-binding antibody fragments (and/or one or more additional therapeutic agents) administered to the subject and can also adjust (e.g., increase or decrease) the dosage or frequency of administration of at least one antibody or antigen-binding antibody fragment (and/or one or more additional therapeutic agents) to the subject based on an assessment of the effectiveness of the treatment (e.g., using any of the methods described herein and known in the art) .

In some embodiments, the antibodies or antigen-binding fragments thereof can be used for detecting RSV in a subject (e.g., a human) or diagnosing an RSV-related disease. Methods known in the art can be designed, e.g., ELISA, to produce a diagnostic test kit. In some embodiments, one or more antibodies or antigen-binding fragments comprising any of the heavy chain single variable domains as described herein can be used.

In some embodiments, the antibody or antigen-binding fragment thereof can be delivered to a subject by intranasal administration or intraperitoneal administration. In some embodiments, administration of the antibody (or antigen-binding fragment thereof) decreases the viral titer (e.g., in lungs) in the subject to less than 95%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1%as compared to the viral titer of a subject without the administration.

Administration through respiratory tract

The compositions as described herein can be administered through respiratory tract by various means, for example, nasal administration, nasal instillation, insufflation (e.g., nasal sprays) , inhalation (through nose or mouth) , intrapulmonary administration, intratracheal administration, or any combinations thereof. As used herein, the term “nasal instillation” refers to a procedure that delivers a therapeutic agent directly into the nose and onto the nasal membranes, wherein a portion of the therapeutic agent can pass through tracheas and is delivered into the lung.

Because of the occasionally limited functionally for the lungs that are in need of treatment, a therapeutic agent sometimes cannot be effectively delivered to the target sites in the lungs (e.g., bronchioles or alveoli) through respiratory tract administration. In these cases, an agent that can clear the airways can be administered to the subject first. In some embodiments, these agents can induce dilation of bronchial passages, and/or vasodilation in muscle. Such agents include, but are not limited to, beta2 adrenergic receptor agonists, anticholinergic agents, corticosteroids. In some embodiments, an agent for treating asthma can be used.

Pharmaceutical compositions suitable for administering through respiratory tract can include, e.g., liquid solutions, aqueous solutions (where water soluble) , or dispersions, etc. In some embodiments, these compositions can comprise one or more surfactants.

As used herein, the term “respiratory tract” refers to the air passages from the nose to the pulmonary alveoli, including the nose, throat, pharynx, larynx, trachea, bronchi, and any part of the lungs. In some embodiments, the composition is administered to the lungs or any part of the respiratory system.

In some embodiments, the compositions can be administered a subject by a delivery system that can convert the composition into an aerosol form, e.g., a nebulizer, a vaporizer, a nasal sprayer, an inhaler, a soft mist inhaler, a jet nebulizer, an ultrasonic wave nebulizer, a pressurized metered dose inhaler, a breath activated pressurized metered dose inhaler, or a vibrating mesh device. As used herein, the term “inhaler” refers to a device for administering compositions in the form of a spray or dry powder that is inhaled (breathed in either naturally or mechanically forced in to the lungs) through the nose or mouth. In some embodiments, inhalers include e.g., a passive or active ventilator (mechanical with or without an endotracheal tube) , nebulizer, dry powder inhaler, metered dose inhaler, and pressurized metered dose inhaler. Once the antibodies (or antigen-binding fragments thereof) are deposited or localized near cells, a subset of the antibodies can neutralize the virus as described herein.

In some embodiments, the devices can use air (e.g., oxygen, compressed air) or ultrasonic power to break up solutions and suspensions into small aerosol particles (e.g., droplets) that can be directly inhaled from the mouthpiece of the device. In some embodiments, the devices use a mesh/membrane with laser drilled holes (e.g., from 1000 to 7000 holes) that vibrates at the top of the liquid reservoir, and thereby pressures out a mist of very fine droplets through the holes.

The delivery system can also have a unit dose delivery system. The volume of solution or suspension delivered per dose can be anywhere from about 5 to about 2000 microliters, from about 10 to about 1000 microliters, or from about 50 to about 500 microliters. Delivery systems for these various dosage forms can be dropper bottles, plastic squeeze units, atomizers, nebulizers or pharmaceutical aerosols in either unit dose or multiple dose packages.

In some embodiments, the device is a small, hard bottle to which a metered dose sprayer is attached. The metered dose can be delivered by drawing the composition into a chamber of defined volume, which chamber has an aperture dimensioned to aerosolize and aerosol formulation by forming a spray when a liquid in the chamber is compressed. The chamber is compressed to administer the composition. In certain devices, the chamber is a piston arrangement. Such devices are commercially available.

Alternatively, a squeeze bottle with an aperture or opening dimensioned to aerosolize an aerosol formulation by forming a spray when squeezed can be used. The opening is usually found in the top of the bottle, and the top is generally tapered to partially fit in the nasal passages for efficient administration of the aerosol formulation. Preferably, the nasal inhaler can provide a metered amount of the aerosol formulation, for administration of a measured dose of the therapeutic agent.

In some embodiments, the aerosolization of a liquid formulation for inhalation into the lung involves a propellant. The propellant may be any propellant generally used in the art. Specific non-limiting examples of such useful propellants are a chlorofluorocarbon, a hydrofluorocarbon, a hydrochlorofluorocarbon, or a hydrocarbon, including trifluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1, 1, 1, 2-tetrafluoroethane, or combinations thereof.

Pharmaceutically acceptable diluents in such aerosol formulations include but are not limited to sterile water, saline, buffered saline, dextrose solution, and the like. In certain embodiments, a diluent that may be used in the present invention or the pharmaceutical formulation is phosphate buffered saline or a buffered saline solution generally between the pH 7.0-8.0 range (e.g., pH 7.4) , or water.

The aerosol formulation also may optionally include pharmaceutically acceptable carriers, diluents, solubilizing or emulsifying agents, surfactants and excipients.

The present disclosure further contemplates aerosol formulations comprising the composition as described herein and another therapeutically effective agent.

The total amount of the composition delivered to the subject will depend upon several factors, including the total amount aerosolized, the type of nebulizer, the particle size, subject breathing patterns, severity of lung disease, and concentration in the aerosolized solution, and length of inhalation therapy. The amount of composition measured in the alveoli may be substantially less than what would be expected to be from the amount of composition present in the aerosol, since a large portion of the composition may be exhaled by the subject or trapped on the interior surfaces of the nebulizer apparatus.

Skilled practitioners will be able to readily design effective protocols, particularly if the particle size of the aerosol is optimized. In some instances, it is useful to administer higher doses when treating more severe conditions. If necessary, the treatment can be repeated on an ad hoc basis depending upon the results achieved. If the treatment is repeated, the mammalian host can be monitored to ensure that there is no adverse immune response to the treatment. The frequency of treatments depends upon a number of factors, such as the amount of composition administered per dose, as well as the health and history of the subject.

Pharmaceutical Compositions and Routes of Administration

Also provided herein are pharmaceutical compositions that contain at least one (e.g., one, two, three, or four) of the antibodies or antigen-binding fragments described herein. Two or more (e.g., two, three, or four) of any of the antibodies or antigen-binding fragments described herein can be present in a pharmaceutical composition in any combination. The pharmaceutical compositions may be formulated in any manner known in the art.

Pharmaceutical compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) . The compositions can include a sterile diluent (e.g., sterile water or saline) , a fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvents, antibacterial or antifungal agents, such as benzyl alcohol or methyl parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like, antioxidants, such as ascorbic acid or sodium bisulfite, chelating agents, such as ethylenediaminetetraacetic acid, buffers, such as acetates, citrates, or phosphates, and isotonic agents, such as sugars (e.g., dextrose) , polyalcohols (e.g., mannitol or sorbitol) , or salts (e.g., sodium chloride) , or any combination thereof. Liposomal suspensions can also be used as pharmaceutically acceptable carriers (see, e.g., U.S. Patent No. 4,522,811) . Preparations of the compositions can be formulated and enclosed in ampules, disposable syringes, or multiple dose vials. Where required (as in, for example, injectable formulations) , proper fluidity can be maintained by, for example, the use of a coating, such as lecithin, or a surfactant. Absorption of the antibody or antigen-binding fragment thereof can be prolonged by including an agent that delays absorption (e.g., aluminum monostearate and gelatin) . Alternatively, controlled release can be achieved by implants and microencapsulated delivery systems, which can include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc. ) .

Compositions containing one or more of any of the antibodies or antigen-binding fragments described herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in dosage unit form (i.e., physically discrete units containing a predetermined quantity of active compound for ease of administration and uniformity of dosage) .

Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under Good Manufacturing Practice (GMP) conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., the dosage for a single administration) . Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries. The formulation depends on the route of administration chosen. For injection, antibodies can be formulated in aqueous solutions, preferably in physiologically-compatible buffers to reduce discomfort at the site of injection. The solution can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively antibodies can be in lyophilized form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

Toxicity and therapeutic efficacy of compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys) . One can, for example, determine the LD50 (the dose lethal to 50%of the population) and the ED50 (the dose therapeutically effective in 50%of the population) : the therapeutic index being the ratio of LD50: ED50. Agents that exhibit high therapeutic indices are preferred. Where an agent exhibits an undesirable side effect, care should be taken to minimize potential damage (i.e., reduce unwanted side effects) . Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.

Data obtained from cell culture assays and animal studies can be used in formulating an appropriate dosage of any given agent for use in a subject (e.g., a human) . A therapeutically effective amount of the one or more (e.g., one, two, three, or four) antibodies or antigen-binding fragments thereof (e.g., any of the antibodies or antibody fragments described herein) will be an amount that treats the disease in a subject (e.g., inhibits RSV) in a subject (e.g., a human subject identified as having RSV infection) , or a subject identified as being at risk of developing the disease (e.g., a subject who is previously infected with RSV but now has been cured) , decreases the severity, frequency, and/or duration of one or more symptoms of a disease in a subject (e.g., a human) . The effectiveness and dosing of any of the antibodies or antigen-binding fragments described herein can be determined by a health care professional or veterinary professional using methods known in the art, as well as by the observation of one or more symptoms of disease in a subject (e.g., a human) . Certain factors may influence the dosage and timing required to effectively treat a subject (e.g., the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and the presence of other diseases) .

Exemplary doses include milligram or microgram amounts of any of the antibodies or antigen-binding fragments described herein per kilogram of the subject’s weight (e.g., about 1 μg/kg to about 500 mg/kg; about 100 μg/kg to about 500 mg/kg; about 100 μg/kg to about 50 mg/kg; about 10 μg/kg to about 5 mg/kg; about 10 μg/kg to about 0.5 mg/kg; about 1 μg/kg to about 50 μg/kg; about 500 μg/kg to about 5 mg/kg; or about 500 μg/kg to about 2 mg/kg) . While these doses cover a broad range, one of ordinary skill in the art will understand that therapeutic agents, including antibodies and antigen-binding fragments thereof, vary in their potency, and effective amounts can be determined by methods known in the art. Typically, relatively low doses are administered at first, and the attending health care professional or veterinary professional (in the case of therapeutic application) or a researcher (when still working at the development stage) can subsequently and gradually increase the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, and the half-life of the antibody or antibody fragment in vivo.

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. The disclosure also provides methods of manufacturing the antibodies or antigen binding fragments thereof for various uses as described herein.

In some embodiments, the disclosure is related to a food additive that comprises the antibody or antigen-binding fragment thereof described herein, e.g., to prevent or treat RSV infection. It will be clear to one skilled in the art that although the compositions described herein can be administered orally. In the case that the compositions are given orally, they may be in the form of tablets, capsules, powder, syrups, etc. In some embodiments, the antibody or antigen-binding fragment thereof described herein is delivered with food.

EXAMPLES

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Example 1. Generating Mouse Anti-RSV-F Antibodies

The anti-RSV-F antibodies were collected by the methods as described below.

To generate mouse antibodies against RSV-F protein, 6-8 weeks old female BALB/c mice were immunized with RSV-F protein (RSV-F; SEQ ID NO: 28) . Anti-RSV-F antibodies were collected by the methods as described below and shown in FIG. 1 and FIG. 2.

Immunization of mice

6-8 weeks old female BALB/c mice were immunized with His-tagged RSV-F proteins at 20 μg/mouse at a concentration of 100 μg/ml. The His-tagged RSV-F proteins were emulsified with adjuvant and injected at four positions on the back of the mice. For the first subcutaneous (s.c. ) injection, the diluted antigen was emulsified with Complete Frelmd's Adjuvant (CFA) in equal volume. In the following subcutaneous injections, the protein was emulsified with Incomplete Freund's Adjuvant (IFA) in equal volume. Three days after the third injection or the booster immunization, blood (serum) was collected and analyzed for antibody titer using ELISA.

In another experiment, 6-8 weeks old female BALB/c mice were immunized by injecting the expression plasmid encoding RSV-F protein into the mice. The plasmids encoding the antigen were injected into the tibialis anterior muscle (intramuscular injection; i.m. injection) of the mice by using gene guns at the concentration of 1000 μg/ul at 60 μg per mouse. At least four injections were performed with at least 14 days between two injections. Blood (serum) was collected seven days after the last immunization and the serum was tested for antibody titer by ELISA.

Procedures to enhance immunization were also performed at least fourteen days after the previous immunization (either by injecting the plasmid or by injecting the proteins) . Jurkat cells were intravenously injected into the mice through tail veins. Spleen was then collected four days after the injection.

Fusion of SP2/0 cells and spleen cells

Spleen tissues were grinded. Spleen cells were first selected by CD3e Microbeads and Anti-Mouse IgM Microbeads, and then fused with SP2/0 cells. The cells were then plated in 96-well plates with hypoxanthine-aminopterin-thymidine (HAT) medium.

Primary screening of hybridoma

Primary screening of the hybridoma supernatant in the 96-well plates was performed using Fluorescence-Activated Cell Sorting (FACS) pursuant to standard procedures. Chinese hamster ovary (CHO) cells were added to 96-well plates (2 × 104 cells per well) before the screening. 50 μl of supernatant was used. The antibodies that were used in experiments were

(1) Fluorescein (FITC) -conjugated AffiniPure F (ab) 2 Fragment Goat Anti-Mouse IgG, Fcγ Fragment Specific, and

(2) Alexa

647-conjugated AffiniPure F (ab) ₂ Fragment Goat Anti-Human IgG, Fcγ Fragment Specific.

Sub-cloning

Sub-cloning was performed using

2. In short, the positive wells identified during the primary screening were transferred to semisolid medium, and IgG positive clones were identified and tested. FITC anti-mouse IgG Fc antibody was used.

Ascites fluid antibodies

1 × 10 ⁶ positive hybridoma cells were injected intraperitoneally to

mice (Biocytogen Pharmaceuticals (Beijing) , Beijing, China; Catalog number: B-CM-002) . Monoclonal antibodies were produced by growing hybridoma cells within the peritoneal cavity of the mouse. The hybridoma cells multiplied and produced ascites fluid in the abdomens of the mice. The fluid contained a high concentration of antibody which can be harvested for later use.

Purification of antibodies

Antibodies in ascites fluid were purified using GE AKTA ^TM protein chromatography (GE Healthcare, Chicago, Illinois, United States) . 04-2H10 ( “2H10” ) was produced by the methods described above.

The VH, VL and CDR regions for some of the antibodies were determined. The heavy chain CDR1, CDR2, CDR3, and light chain CDR1, CDR2, and CDR3 amino acid sequences of 2H10 are shown in SEQ ID NOs: 1-6 (Kabat numbering) or SEQ ID NOs: 7-12 (Chothia numbering) .

Example 2. Humanization of mouse antibodies

The starting point for humanization was the mouse antibodies (e.g., 2H10) . The amino acid sequences for the heavy chain variable region and the light chain variable region of these mouse antibodies were determined.

Ten humanized heavy chain variable region variants (SEQ ID NOs: 13-22) and three humanized light chain variable region variants (SEQ ID NOs: 23-25) for 2H10 were constructed, containing different modifications or substitutions.

These humanized heavy chain variable region variants can be combined with any of the light chain variable region variants derived from the same mouse antibody. For example, 2H10-H1 (SEQ ID NO: 13) can be combined with any humanized light chain variable region variant based on the same mouse antibody 04-2H10 (e.g., SEQ ID NO: 23-25) , and the antibody will be labeled accordingly. For example, if 2H10-H1 is combined with 2H10-K3 (SEQ ID NO: 25) , the antibody is labeled as 2H10-H1K3.

Each humanized heavy chain variable region variant can be connected with a human heavy chain constant region to generate a complete humanized antibody heavy chain, and each humanized light chain variable region variant can be connected with a human light chain constant region to generate a complete humanized antibody light chain. Mutations can also be introduced within the constant regions of the antibody. For example, the RYTE mutation (i.e., K214R, M252Y, S254T, and T256E within the heavy chain constant region of wild-type human IgG1) were introduced to enhance the binding affinity of the antibody with FcRn under low pH conditions, to prolong the half-life of the antibody. For instance, 2H10-H1K3-IgG1 is an antibody having the heavy chain variable region 2H10-H1 (SEQ ID NO: 13) and the light chain variable region 2H10-K3 (SEQ ID NO: 25) , each connected with human IgG1 constant domains. 2H10-H1K3-IgG1-RYTE shares the same heavy chain and light chain variable regions, except that the human IgG1 heavy chain constant domain includes the RYTE mutation.

Unless otherwise specified in the following experiments, the antibodies used were all purified by a protein A column connected with the AKTA ^TM chromatography system.

Example 3. In vitro testing of Respiratory Syncytial Virus Fusion Protein (RSV-F) Antibodies

Detection of the binding activities of Anti-RSV-F Antibodies to F protein of RSV Subtype A and Subtype B strains

25 μl CHO-S cells transiently transfected to express the RSV-F protein were added to each well (5 × 104 cells per well) of a multi-well plate. The purified antibodies were titrated to a final concentration of 10 μg/ml, and then added to each well at 25 μl per well. The plate was incubated for 30 minutes at 4 ℃. After being washed with phosphate-buffered saline (PBS) twice, 50 μl of Alexa

647 AffiniPure F (ab') ₂ Fragment Goat Anti-Human IgG, Fcγ fragment specific (or AF647; Jackson Immuno Research, Cat#109-606-170) with 1: 500 dilution was added into each well, and incubated for 30 minutes at 4 ℃, followed by PBS wash.

Vectors encoding the extracellular and transmembrane regions of RSV-F proteins (corresponding to amino acids 26-524 of the full-length RSV-F sequence (SEQ ID NO: 28) ) from 4 common RSV strains were used to transfect CHO cells. Specifically, the RSV strains and the corresponding full-length RSV-F proteins are as follows: (1) human respiratory syncytial virus A (strain RSS-2) , also known as RSV-F S2 (or RSF-S2; SEQ ID NO: 29) ; (2) human respiratory syncytial virus A (strain Long) , also known as RSV-F LONG (or RSF-LONG; SEQ ID NO: 30) ; (3) human respiratory syncytial virus B (strain B1) , also known as RSV-F B1 (or RSF-B1; SEQ ID NO: 31) ; and (4) human respiratory syncytial virus B (strain 18537) , also known as RSV 18537 (B) strain F (or RSF-B18537; SEQ ID NO: 32) . The sequence alignment of RSF-S2, RSF-LONG, RSF-B1, and RSF-B18537 are shown in FIG. 13. As shown in FIG. 13, these RSV-F proteins are not identical. The difference in certain amino acid residues may impact the binding of the antibodies to RSV-F proteins. Thus, it is advantageous if an antibody can bind to RSV-F proteins from different strains with high affinities.

The signals for AF647 were determined by flow cytometry (Model: Thermo Attune ^TM NX) and the detection results are summarized in the table below. Specifically, the percentage of cells having AF647 signals over the total cells are listed. CHO-S-RSF (T) -S2-V5, CHO-S- RSF (T) -LONG-V5, CHO-S-RSF (T) -B1-V5, and CHO-S-RSF (T) -B18537-V5 represent CHO-Scells transfected to express the extracellular and transmembrane regions of RSF-S2, RSF-LONG, RSF-B1, and RSF-B18537, respectively. The expressed RSV-F proteins were labeled with a V5 tag.

Table 2.

Note: (T) : transfected; V5: V5 tag; NC: negative control.

The above results indicate that the antibodies disclosed herein can bind to the RSV-F proteins from different RSV strains. Specifically, 2H10-H1K1-IgG1, 2H10-H1K2-IgG1 and 2H10-H1K3-IgG1 antibodies showed a relatively low binding capacity as compared to others.

The above results for some antibodies were further confirmed in another experiment. Specifically, the purified antibodies were titrated to final concentrations of 10, 1, or 0.1 μg/ml. The detection results are shown in the tables below. When the concentration of the anti-RSV-F antibodies increased, the detected AF647 signal also increased, suggesting that the humanized antibodies can bind to RSV-F.

Table 3.

Table 4.

Here, 2H10-mHvKv-IgG1 is a chimeric anti-RSV-F antibody. It has the heavy chain variable region (SEQ ID NO: 26) and light chain variable region (SEQ ID NO: 27) from the mouse anti-RSV-F antibody 2H10, and human IgG1 antibody constant domains (CL, CH1, CH2, and CH3; SEQ ID NO: 34 for CH1-CH3) . MEDI8897-IgG1-RYTE has the heavy chain variable region (SEQ ID NO: 36) and light chain variable region (SEQ ID NO: 37) from MEDI8897, and human IgG1 antibody constant domains with the RYTE mutation (CL, CH1, CH2, CH3; SEQ ID NO:35 for CH1-CH3) .

Other antibodies disclosed herein, e.g., 2H10-H1K1-IgG1, 2H10-H2K1-IgG1, 2H10-H3K2-IgG1, 2H10-H4K3-IgG1, 2H10-H5K2-IgG1, 2H10-H5K3-IgG1, 2H10-H7K3-IgG1, 2H10-H8K1-IgG1, 2H10-H9K3-IgG1, and 2H10-H10K1-IgG1, are humanized antibodies. Each of them has human IgG1 antibody constant domains (CL, CH1, CH2, CH3; SEQ ID NO: 34 for CH1-CH3) . The numbers immediately after “H” and “K” indicate the humanized heavy chain and light chain variable region variants, respectively. For example, 2H10-H3K2-IgG1 includes the humanized 2H10 heavy chain variable region H3 (SEQ ID NO: 15) and humanized 2H10 light chain variable region K2 (SEQ ID NO: 24) . Similarly, 2H10-H5K3-IgG1 includes humanized 2H10 heavy chain variable region H5 (SEQ ID NO: 17) and humanized 2H10 light chain variable region K3 (SEQ ID NO: 25) .

Detection of the binding activities of Anti-RSV-F Antibodies (with IgG constant domain mutations) to F protein of RSV Subtype A and Subtype B strains

The RSV-F binding activities of the following antibodies: 2H10-mHvKv-IgG1, 2H10-H9K3-IgG1-RYTE, 2H10-H5K2-IgG1-RYTE, Suptavumab-IgG1-EDML, MK1654-IgG1-RYTE, MEDI-493-IgG1, and MEDI8897-IgG1-RYTE were detected using the method described above. CHO-S-RSF (T) represents CHO-Scells transfected to express the full-length protein of RSV-F (SEQ ID NO: 28) with a V5 tag.

Suptavumab-IgG1-EDML has the heavy chain variable region (SEQ ID NO: 38) and light chain variable region (SEQ ID NO: 39) from Suptavumab, and human IgG1 antibody constant domains with the EDML mutation (CL, CH1, CH2, CH3; SEQ ID NO: 33 for CH1-CH3) . The EDML mutation (i.e., E356D and M358L within the heavy chain constant region of wild-type human IgG1) were introduced. MK1654-IgG1-RYTE has the heavy chain variable region (SEQ ID NO: 40) and light chain variable region (SEQ ID NO: 41) from MK1654, and human IgG1 antibody constant domains with the RYTE mutation (CL, CH1, CH2, CH3; SEQ ID NO:35 for CH1-CH3) . MEDI-493-IgG1 has the heavy chain variable region (SEQ ID NO: 42) and light chain variable region (SEQ ID NO: 43) from MEDI-493, and human IgG1 antibody constant domains (CL, CH1, CH2, CH3; SEQ ID NO: 34 for CH1-CH3) .

Table 5. Binding activities of anti-RSV-F antibodies with mutations

Table 6. Binding activities of anti-RSV-F antibodies with mutations

Determination of the binding affinity of Anti-RSV-F Antibodies to RSV-F protein

The binding affinity of the anti-RSV-F antibodies to recombinant RSV-F protein was measured by surface plasmon resonance (SPR) using Biacore ^TM (Biacore, INC, Piscataway N. J. ) 8K biosensor equipped with pre-immobilized Protein A sensor chips. Specifically, purified anti-RSV-F antibodies were injected into the Biacore ^TM 8K biosensor at 10 μL/min for about 50 seconds to achieve a desired protein density (e.g., about 50 response units (RU) ) . A His-tagged human RSV fusion protein (Sino Biological, Cat#11049-V08B) at a concentration of 100 nM was then injected at 30 μL/min for 180 seconds· Dissociation was monitored for 600 seconds. The chip was regenerated after the last injection of each titration with glycine (pH 2.0, 30 μL/min for 30 seconds) .

Kinetic association rates (kon) and dissociation rates (koff) were obtained simultaneously by fitting the data globally to a 1: 1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994. Methods Enzymology 6.99-110) using Biacore ^TM 8K Evaluation Software 3.0. Affinity values were deduced from the quotient of the kinetic rate constants (KD=koff/kon) .

As a person of ordinary skill in the art would understand, the same method with appropriate adjustments for parameters (e.g., antibody concentration) was performed for each tested antibody. The results for the tested antibodies are summarized in the table below.

Table 7.

In a different experiment, anti-RSV-F antibodies at concentrations of 200, 100, 50, 25, 12.5, 6.25, 3.125, or 1.5625 nM were used. The binding affinities of 2H10-H5K2-IgG1-RYTE and 2H10-H9K3-IgG1-RYTE were determined using the above method. The results showed that the affinities of H5K2 and H9K3 did not change significantly after introducing the RYTE mutations.

Table 8.

Example 4. Stability testing of Respiratory Syncytial Virus Fusion Protein (RSV-F) Antibodies

Determination of the thermal stability of Anti-RSV-F Antibodies

Thermofluor assays wereperformed using the Protein Thermal Shift ^TM Dye Kit (Thermo Fisher Scientific) and QuantStudio ^TM 5 Real Time PCR Systems (Thermo Fisher Scientific) . The assays measured the thermal stability using a fluorescent dye that binds to hydrophobic patches exposed as the protein unfolds.

The experiments were performed according to the manufacturer's protocol. In Step 1, samples were heated to 25 ℃ at 1.6℃/second. In Step 2, samples were heated to 99 ℃ at 0.05 ℃/second. The table below summarizes the melting temperature Tm for the tested humanized anti-RSV-F antibodies.

Table 9.

The results showed that both the chimeric antibody 2H10-mHvKv-IgG1 and the humanized antibody 2H10-H9K3-IgG1, 2H10-H4K3-IgG1, 2H10-H5K2-IgG1, and 2H10-H4K2-IgG1 had Tm1 and Tm2 above 70 ℃. Thus, all of the tested antibodies exhibited a good thermal stability.

Determination of accelerated stability of Anti-RSV-F Antibodies

Three humanized anti-RSV-F antibodies 2H10-mHvKv-IgG1, 2H10-H9K3-IgG1, and 2H10-H4K2-IgG1 (stock solution concentration: 10 mg/ml, dissolved in PBS) were diluted to 5 mg/ml using a buffer at pH 6.0 (3 mg/ml histidine, 80 mg/ml sucrose, and 0.2 mg/ml Tween 80) . The diluted antibodies were kept in sealed Eppendorf tubes at 4 ± 3℃ (hereinafter referred to as 4 ℃) for 7 days; or at 40 ± 2 ℃ (hereinafter referred to as 40 ℃) for 7 days, and their thermal stability was evaluated. Specifically, the following tests were performed: (1) observing the solution appearance and presence of visible non-soluble objects; (2) detecting changes of the thermal stability of the antibodies using the Protein Thermal Shift ^TM Dye Kit (indicated as Tm1 (if any) and Tm2) ; (3) detecting the purity changes of antibodies by Size-Exclusion Ultra Performance Liquid Chromatography (SEC-UPLC) (indicated as the percentage of the main peak area to the sum of all peak areas (Purity, %) and the retention time of the main peak (RT, min) ) ; (4) detecting changes in the apparent hydrophobicity of the antibodies using the Hydrophobic Interaction Chromatography-High Performance Liquid Chromatography (HIC-HPLC) method (indicated as the retention time of the main peak (HIC, min) and the percentage of the area of the main peak to the sum of all peak areas (Perc %) ) ; (5) detecting charge variants in the antibodies by the Capillary Isoelectric Focusing (cIEF) method (indicated as the percentages of the main component, acidic component, and alkaline component) ; (6) detecting antibody activity changes by FACS (indicated as the binding of different concentrations of the antibodies to the RSV-F protein) . The standard of the detection items are shown in the table below.

Table 10.

The detection results are as follows:

1) Appearance: After storage at 4 ℃ or 40 ℃ for 7 days, respectively, there was no change of appearance observed for each sample.

2) Thermal stability (Tm value) : After storage at 4 ℃ or 40 ℃ for 7 days, respectively, the thermodynamic stability of antibody molecules in each sample did not change.

3) Purity:

a) SEC-UPLC method: After storage at 4℃ or 40℃ for 7 days, respectively, the molecular structure in each antibody sample was well preserved; the purity did not change; and there were no obvious aggregates or fragments.

b) SDS-PAGE method: After storage at 4℃ or 40℃ for 7 days, respectively, the purity of each sample did not change; and there were no obvious aggregates or fragments (See FIGS. 3A-3C) .

4) Apparent hydrophobicity (HIC-HPLC method) : Under the condition of storage at 4 ℃ for 7 days, the apparent hydrophobicity of the antibody molecules in each sample did not change. Under the condition of storage at 40 ℃ for 7 days, the apparent hydrophobicity of 2H10-H9K3-IgG1 did not change. However, 2H10 -mHvKv-IgG1 and 2H10-H4K2-IgG1 both showed spurious peaks, but the change was within 20%.

5) Charge variant (cIEF method) : After storage at 4℃ or 40℃ for 7 days, respectively, the antibody main peak, and peaks representing the acidic or alkaline component in the 2H10-mHvKv-IgG1 sample did not change significantly. After storage at 4℃ for 7 days, the antibody main peak, and peaks representing the acidic or alkaline component in the 2H10-H4K2-IgG1 and 2H10-H9K3-IgG1 did not change significantly. However, after storage at 40℃ for 7 days, the peak representing the acidic component changed slightly (within 20%) .

6) Antibody activity (FACS method) : After storage at 4℃ or 40℃ for 7 days, respectively, all of the antibody samples (at concentrations of 10 μg/ml, 1μg/ml, and 0.1 μg/ml) can bind to the RSV-F protein expressed on cell surface.

Detailed results are shown in the table below.

Table 11.

Note: ND: not detected.

Specific procedures for each test item are described below.

The thermal stability (e.g., Tm1 and Tm2) of the antibody samples was determined as described above.

In the SEC-UPLC method, the antibody samples were diluted to 1 mg/mL with purified water and an Agilent 1290 chromatograph system (connected with XBridge ^TM Protein BEH SEC column (

Waters Corporation) ) was used. The following parameters were used: mobile phase: 25 mmol/L phosphate buffer (PB) + 300 mmol/L NaCl, pH 6.8; flow rate: 1.8 ml/min; column temperature: 25 ℃; detection wavelength: 280 nm; injection volume: 10 μL; sample tray temperature: about 4℃; and running time: 7 minutes.

In the HIC-HPLC method, an Agilent 1260 chromatograph system (connected with ProPac ^TM HIC-10 column (4.6 x 250 mm, Thermo Scientific) ) was used, and samples were diluted using mobile phase A to 0.5 mg/mL. The following parameters were used: mobile phase A: 1.0 M ammonium sulfate, 20 mM sodium acetate, 10%acetonitrile pH 5.0; mobile phase B: 20 mM sodium acetate, 10%acetonitrile pH 5.0; flow rate: 0.8 ml/min; gradient: 0 min 100%A, 2 min 100%A, 32 min 100%B, 34 min 100%B, 35 min 100%A, and 45 min 100%A; column temperature: 30 ℃; detection wavelength: 280 nm; injection volume: 10 μL; sample tray temperature: about 10 ℃; and running time: 30 minutes.

In the cIEF method, a Maurice cIEF Method Development Kit (Part #PS-MDK01-C, ProteinSimple) was used for sample preparation. Specifically, 8 μL protein sample was mixed with the following reagents in the kit: 1 μL Maurice cIEF pI Marker-4.05, 1 μL Maurice cIEF pI Marker-9.99, 35 μL 1%Methyl Cellulose Solution, 2 μL Maurice cIEF 500 mM Arginine, 4 μL Ampholytes (Pharmalyte pH ranges 3-10) , and water (added to make a final volume of 100 μL) . On the Maurice analyzer (Protein Simple, Santa Clara, CA) , Maurice cIEF Cartridges (PS-MC02-C) were used to generate imaging capillary isoelectric focusing spectra. The sample was focused for a total of 10 minutes. The analysis software installed on the instrument was used to integrate the absorbance of the 280 nm-focused protein.

Non-reducing SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) was performed using a 4-12%acrylamide gel. The protein samples were prepared as follows. First, 2.4 μl of the protein sample was mixed with 6 μl Tris-Glycine SDS Sample Buffer (2 ×) (Thermo, LC2676) and 3.6 μl distilled water. The mixture was then boiled for 2 minutes and instantly centrifuged before loading. Reducing SDS-PAGE was performed using a 4-12%acrylamide gel. The protein samples were prepared as follows. First, the protein samples were diluted to 1 mg/ml. 3 μl of the diluted protein sample was mixed with 3 μl SDS-PAGE Sample Loading Buffer (5X) (Beyotime, P0015L) and 9 μl distilled water. The mixture was then boiled for 2 minutes and instantly centrifuged before loading. 10 μg of each sample was loaded to the gel.

In the FACS method, similar to the binding activity detection method described herein, 25 μl CHO cells transiently transfected to express the RSF-S2 protein (5 × 10 ⁴ cells) were added to each well in a plate. The antibody samples were titrated to final concentrations of 10 μg/ml, 1 μg/ml and 0.1 μg/ml, respectively. The titrated antibodies were added to each well at 25 μl per well at 4 ℃ and incubated for 30 minutes. After being washed with phosphate-buffered saline (PBS) twice, 50 μl of AF647 with 1∶500 dilution was added into each well, and incubated for 30 minutes at 4 ℃, followed by PBS wash.

Determination of accelerated stability of Anti-RSV-F Antibodies (with IgG constant domain mutations)

2H10-H9K3-IgG1-RYTE and 2H10-H5K2-IgG1-RYTE were selected, and stock solutions of the two antibodies were prepared (10 mg/ml antibody, 20 mM histidine, pH 6.0) . Accelerated stability of the antibodies were determined using the methods described herein. The stock solutions were diluted to 5 mg/ml using a buffer at pH 6.0 (3 mg/ml histidine, 80 mg/ml sucrose, and 0.2 mg/ml Tween 80) . Results for SDS-PAGE methods are shown in FIGS. 4A-4C. Detailed results are shown in the table below.

Table 12.

Note: ND: not detected.

The original 10 mg/ml solution was centrifuged in an ultrafiltration tube using the pH 6.0 buffer (3 mg/mL histidine, 80 mg/mL sucrose, 0.2 mg/mL Tween 80) to make a 100 mg/ml (high concentration) solution. Results for SDS-PAGE methods are shown in FIGS. 5A-5C. Detailed results are shown in the table below.

Table 13.

Note: ND: not detected.

According to the above stability test results, multiple 2H10 antibodies (H5K2, H9K3, and H4K2) were determined to be stable in the buffer at different concentrations or subtype mutations. Specifically, after 2H10 antibody preparations were stored at 4℃ or 40℃ for 7 days, the appearance of all samples remained unchanged, and the antibody structure was intact. The antibody content, thermodynamic stability, hydrophobicity, charge variants, and antibody activity did not show any obvious changes.

It is known that apparent hydrophobicity is considered to be the dominant mechanism of protein aggregation tendency, and higher hydrophobicity is usually associated with higher aggregation and precipitation tendency. The apparent hydrophobicity of all 2H10 antibodies is lower than that of MEDI8897-IgG1-RYTE, which indicates that the 2H10 antibodies may have advantages in subsequent drug development.

Example 5. Anti-RSV-F antibody and RSV subtype A virus strain neutralization ability test

Anti-RSV-F antibodies were serially diluted in a 96-well plate. Then, the recombinant RSV virus (150 μL, 320 TCID ₅₀/ml) was added and mixed with the antibodies. Next, 100 μL 293T cells (5 × 10 ⁵ cells/well) were added to the corresponding wells, and the plate was incubated at 37℃, 5%CO ₂ for 48 hours. After the incubation, 100 μl supernatant was discarded by pipetting, and 100 μl luciferase detection reagent was added to each well. The plate was then incubated at room temperature in dark for 2 minutes. Afterwards, 150 μl solution in each well was transferred to a new plate, which was placed in a plate reader to measure chemiluminescence signals. The neutralization inhibition ratio was calculated as follows:

Inhibition ratio = [1- (Ab-CC) / (VC-CC) ] × 100%

wherein Ab is the average signal of the antibody wells; CC is the average signal of the cell control wells (culture medium only) ; VC is the average signal of the virus control wells (culture medium with the recombinant RSV virus added) . According to the inhibition ratio, IC50 can be calculated by the Reed-Muench algorithm. Details of this method can be found, e.g., in Reed, Lowell Jacob, and Hugo Muench. "Asimple method of estimating fifty per cent endpoints. " American Journal of Epidemiology 27.3 (1938) : 493-497, which is incorporated herein by reference in its entirety. The IC50 of anti-RSV-F antibodies were determined and the results are listed in the table below.

Table 14. IC50

Antibody	IC50 (ng/mL)
2H10-mHvKv-IgG1	3.44
2H10-H2K2-IgG1	11.7
2H10-H2K3-IgG1	5.42
2H10-H3K2-IgG1	2.88
2H10-H3K3-IgG1	3.21
2H10-H4K2-IgG1	4.87
2H10-H4K3-IgG1	5.29
2H10-H5K1-IgG1	5.49
2H10-H5K2-IgG1	4.89
2H10-H5K3-IgG1	4.94
2H10-H7K1-IgG1	4.29
2H10-H7K3-IgG1	2.91
2H10-H8K1-IgG1	3.67
2H10-H8K2-IgG1	1.68
2H10-H9K1-IgG1	3.77
2H10-H9K2-IgG1	20.13
2H10-H9K3-IgG1	3.88

The IC50 of Suptavumab-IgG1-EDML, 2H10-H9K3-IgG1-RYTE, 2H10-H9K3-IgG1, 2H10-H5K2-IgG1-RYTE, and MEDI 493 were also determined in a different experiment. The results are listed in the table below. The results showed that the IC50 of H9K3 did not change significantly after the RYTE mutation was introduced. In addition, most of the antibodies exhibited better neutralizing activity than the control drugs.

Table 15. IC50

Antibody	IC50 (ng/mL)
Suptavumab-IgG1-EDML	2.46
2H10-H9K3-IgG1-RYTE	2.03
2H10-H9K3-IgG1	2.51
2H10-H5K2-IgG1-RYTE	1.18
MEDI 493	85.82

Example 6. Epitope correlation analysis of purified anti-RSV-F antibodies

Relative positions of target protein epitope between a pair of purified anti-RSV-F monoclonal antibodies were analyzed through a surface plasmon resonance (SPR) competition experiment. A total of 6 monoclonal antibodies were used to study the binding inhibition (blocking) effect of each antibody on another antibody: 2H10-H5K3-IgG1, 2H10-H4K2-IgG1, 2H10-H5K2-IgG1, 2H10-H9K3-IgG1, 2H10-H4K3-IgG1 and 2H10-mHvKv-IgG1.

HBS-EP+ buffer (10 mM 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES) , 150 mM NaCl, 3 mM ethylenediaminetetraacetic acid (EDTA) and 0.05%P20, pH 7.4) was diluted from HBS-EP+ buffer (10×) as the running buffer throughout the experiment. Anti-His antibodies were fixed on the surface of a Series S sensor Chip CM5 by amino group coupling to generate an anti-His chip (i.e., CM5-Anti-His-Channel 1, 8-Chip) . Then, 1M ethanolamine, pH 8.5 was injected to block the remaining active carboxyl groups on the chip surface, followed by equilibration using the HBS-EP+ buffer for 2 hours. Recombinant RSV-F protein with His-tag (1 μg/ml) were injected into the Biacore ^TM 8K biosensor at 10 μL/min for 70 seconds and captured on the anti-His chip to achieve a desired protein density (i.e., 200 RU) . A pair of antibodies (200 nM each) was continuously injected at 30 μL/min into the chip. The first injected antibody (analyte 1) had a binding time of 180 seconds, and then the second antibody (analyte 2) was injected with a binding time of 180 seconds. After injection of the antibodies in each analysis cycle, the chip was regenerated twice with a glycine buffer (pH 1.7; 30 μL/min for 20 seconds) . Each pair of monoclonal antibodies was subjected to the same experimental steps to obtain the binding inhibition data when each monoclonal antibody was paired with another antibody.

The binding value of each antibody was obtained using Biacore ^TM Insight Evaluation Software. To quantify the interference of one antibody binding to another, a binding ratio was calculated to compare each pair of antibodies. The binding ratio is defined as the binding value of the second antibody (analyte 2) , divided by the binding value of the first antibody (analyte 1) . In summary, after the CDR region is mutated, these antibodies share identical or overlapping epitopes. The analysis results are listed in the table below.

Table 16.

Example 7. In vivo testing method

In a BALB/c mouse model infected with RSV recombinant virus of subtype A, the virus dose and the protective effect of the antibodies can be determined. Specifically, mice are placed into several groups (4 mice per group) . On day 0, the treatment group mice are injected intraperitoneally (i. p. ) with antibodies at a certain dose level (e.g., 2 mg/kg, 4 mg/kg, or 8 mg/kg) , and the PBS control group mice are injected with an equal volume of PBS. On day 1, the mice are anesthetized, and then the mice are infected by nasal drops (60 μL/mouse) of 10 ⁴ TCID ₅₀ RSV recombinant virus of subtype A.

On the 4th day post infection, an in vivo imaging instrument can be used to detect the expression level and tissue distribution of luciferase in each group of mice (e.g., the luminescence of the recombinant virus in the nasal cavity and lungs) . Specifically, the mice can be administered with luciferase substrate D-Luciferin (75 mg/kg of mouse body weight) by intraperitoneal injection. After 10 minutes, the luminescence level in the mouse can be detected using the in vivo imaging instrument. The image luminescence acquisition time is 1 min, and the total body luminescence represents the expression level of the recombinant virus luciferase protein.

The antibodies can also be administered after infection. The recommended dose level for the treatment group mice is 1 mg/kg, 2 mg/kg, 5 mg/kg, 10 mg/kg or 20 mg/kg.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

An antibody or antigen-binding fragment thereof that binds to Respiratory Syncytial Virus Fusion Protein (RSV-F) comprising:

a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR3 amino acid sequence; and

a light chain variable region (VL) comprising CDRs 1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR3 amino acid sequence,

wherein the selected VH CDRs 1, 2, and 3 amino acid sequences and the selected VL CDRs, 1, 2, and 3 amino acid sequences are one of the following:

(1) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 2, 3, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4, 5, 6, respectively;

(2) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 44, 3, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4, 5, 6, respectively;

(3) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 45, 3, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4, 5, 6, respectively;

(4) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 46, 3, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4, 5, 6, respectively;

(5) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 47, 3, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4, 5, 6, respectively;

(6) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 7, 8, 9, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 10, 11, 12, respectively;

(7) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 48, 8, 9, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 10, 11, 12, respectively;

(8) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 49, 8, 9, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 10, 11, 12, respectively;

(9) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 50, 8, 9, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 10, 11, 12, respectively; and

(10) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 51, 8, 9, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 10, 11, 12, respectively.
The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3 respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively.
The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 1, 44, and 3 respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively.
The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 1, 45, and 3 respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively.
The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 1, 46, and 3 respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively.
The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 1, 47, and 3 respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively.
The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively.
The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 48, 8, and 9, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively.
The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 49, 8, and 9, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively.
The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 50, 8, and 9, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively.
The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 51, 8, and 9, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively.
The antibody or antigen-binding fragment thereof of any one of claims 1-11, wherein the antibody or antigen-binding fragment specifically binds to RSV-F of an RSV subtype A strain.
The antibody or antigen-binding fragment thereof of any one of claims 1-11, wherein the antibody or antigen-binding fragment specifically binds to RSV-F of an RSV subtype B strain.
The antibody or antigen-binding fragment thereof of any one of claims 1-13, wherein the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof.
The antibody or antigen-binding fragment thereof of any one of claims 1-14, wherein the antibody or antigen-binding fragment is a single-chain variable fragment (scFv) or a multi-specific antibody (e.g., a bispecific antibody) .
A nucleic acid comprising a polynucleotide encoding a polypeptide comprising:

(1) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 23, 24, 25, or 27 binds to RSV-F;

(2) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively, and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 26 binds to RSV-F;

(3) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 44 and 3, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 23, 24, 25, or 27 binds to RSV-F;

(4) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 45 and 3, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 23, 24, 25, or 27 binds to RSV-F;

(5) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 46 and 3, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 23, 24, 25, or 27 binds to RSV-F;

(6) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 47 and 3, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 23, 24, 25, or 27 binds to RSV-F;

(7) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 23, 24, 25, or 27 binds to RSV-F;

(8) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively, and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 26 binds to RSV-F;

(9) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 48, 8, and 9, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 23, 24, 25, or 27 binds to RSV;

(10) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 49, 8, and 9, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 23, 24, 25, or 27 binds to RSV;

(11) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 50, 8, and 9, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 23, 24, 25, or 27 binds to RSV; or

(12) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 51, 8, and 9, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 23, 24, 25, or 27 binds to RSV.
The nucleic acid of claim 16, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively.
The nucleic acid of claim 16, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively.
The nucleic acid of claim 16, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 44, and 3, respectively.
The nucleic acid of claim 16, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 45, and 3, respectively.
The nucleic acid of claim 16, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 46, and 3, respectively.
The nucleic acid of claim 16, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 47, and 3, respectively.
The nucleic acid of claim 16, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively.
The nucleic acid of claim 16, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively.
The nucleic acid of claim 16, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 48, 8, and 9, respectively.
The nucleic acid of claim 16, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 49, 8, and 9, respectively.
The nucleic acid of claim 16, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 50, 8, and 9, respectively.
The nucleic acid of claim 16, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 51, 8, and 9, respectively.
The nucleic acid of any one of claims 16-28, wherein the VH when paired with a VL specifically binds to RSV-F, or the VL when paired with a VH specifically binds to RSV-F.
The nucleic acid of any one of claims 16-29, wherein the immunoglobulin heavy chain or the fragment thereof is a humanized immunoglobulin heavy chain or a fragment thereof, and the immunoglobulin light chain or the fragment thereof is a humanized immunoglobulin light chain or a fragment thereof.
The nucleic acid of any one of claims 16-30, wherein the nucleic acid encodes a single-chain variable fragment (scFv) or a multi-specific antibody (e.g., a bispecific antibody) .
The nucleic acid of any one of claims 16-31, wherein the nucleic acid is cDNA.
A vector comprising one or more of the nucleic acids of any one of claims 16-32.
A vector comprising two of the nucleic acids of any one of claims 16-32, wherein the vector encodes the VL region and the VH region that together bind to RSV-F.
A pair of vectors, wherein each vector comprises one of the nucleic acids of any one of claims 16-32, wherein together the pair of vectors encodes the VL region and the VH region that together bind to RSV-F.
A cell comprising the vector of claim 33 or 34, or the pair of vectors of claim 35.
The cell of claim 36, wherein the cell is a CHO cell.
A cell comprising one or more of the nucleic acids of any one of claims 16-32.
A cell comprising two of the nucleic acids of any one of claims 16-32.
The cell of claim 39, wherein the two nucleic acids together encode the VL region and the VH region that together bind to RSV-F.
A method of producing an antibody or an antigen-binding fragment thereof, the method comprising

(a) culturing the cell of any one of claims 36-40 under conditions sufficient for the cell to produce the antibody or the antigen-binding fragment; and

(b) collecting the antibody or the antigen-binding fragment produced by the cell.
An antibody or antigen-binding fragment thereof that binds to RSV-F comprising a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%identical to SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 26,, and a light chain variable region (VL) comprising an amino acid sequence that is at least 90%identical to 23, 24, 25, or 27.
The antibody or antigen-binding fragment thereof of claim 42, wherein the VH comprises the sequence of SEQ ID NO: 21 and the VL comprises the sequence of SEQ ID NO: 25.
The antibody or antigen-binding fragment thereof of claim 42, wherein the VH comprises the sequence of SEQ ID NO: 16 and the VL comprises the sequence of SEQ ID NO: 25.
The antibody or antigen-binding fragment thereof of claim 42, wherein the VH comprises the sequence of SEQ ID NO: 17 and the VL comprises the sequence of SEQ ID NO: 24.
The antibody or antigen-binding fragment thereof of claim 42, wherein the VH comprises the sequence of SEQ ID NO: 16 and the VL comprises the sequence of SEQ ID NO: 24.
The antibody or antigen-binding fragment thereof of claim 42, wherein the VH comprises the sequence of SEQ ID NO: 17 and the VL comprises the sequence of SEQ ID NO: 25.
The antibody or antigen-binding fragment thereof of any one of claims 42-47, wherein the antibody or antigen-binding fragment specifically binds to RSV-F of an RSV subtype A strain.
The antibody or antigen-binding fragment thereof of any one of claims 42-47, wherein the antibody or antigen-binding fragment specifically binds to RSV-F of an RSV subtype B strain.
The antibody or antigen-binding fragment thereof of any one of claims 42-49, wherein the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof.
The antibody or antigen-binding fragment thereof of any one of claims 42-50, wherein the antibody or antigen-binding fragment is a single-chain variable fragment (scFv) or a multi-specific antibody (e.g., a bispecific antibody) .
An antibody or antigen-binding fragment thereof comprising the VH CDRs 1, 2, 3, and the VL CDRs 1, 2, 3 of the antibody or antigen-binding fragment thereof of any one of claims 1-15 and 42-51.
An antibody or antigen-binding fragment thereof that cross-competes with the antibody or antigen-binding fragment thereof of any one of claims 1-15 and 42-52.
An antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1-15 and 42-53 covalently bound to a therapeutic agent.
The antibody drug conjugate of claim 54, wherein the therapeutic agent is a cytotoxic or cytostatic agent.
A method of treating a subject having a Respiratory Syncytial Virus infection, preventing RSV infection in a subject, reducing the risk of RSV infection in a subject, or ameliorate symptoms of RSV infection in a subject, the method comprising administering a therapeutically effective amount of a composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-15 and 42-53 to the subject.
A method of neutralizing a Respiratory Syncytial Virus (RSV) , the method comprising contacting the RSV with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof of any one of claims 1-15 and 42-53.
A method of blocking internalization of a Respiratory Syncytial Virus (RSV) by a cell, the method comprising

contacting the RSV with an effective amount of a composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-15 and 42-53.
A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-15 and 42-53, and a pharmaceutically acceptable carrier.
A pharmaceutical composition comprising the antibody drug conjugate of claim 54 or 55, and a pharmaceutically acceptable carrier.
The antibody or antigen-binding fragment thereof of any one of claims 1-15 and 42-53, or the antibody-drug conjugate of claims 54 or 55, wherein the antibody is a IgG1 antibody.
The antibody or antigen-binding fragment thereof of any one of claims 1-15 and 42-53, or the antibody-drug conjugate of claims 54 or 55, wherein the antibody is a human IgG1 antibody.
The antibody or antigen-binding fragment thereof, or the antibody-drag conjugate of claim 62, wherein the IgG1 antibody comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%identical to SEQ ID NO: 33, 34, or 35.
An antibody or antigen-binding fragment thereof comprising VH CDRs 1, 2, 3 that are identical to VH CDRs 1, 2, 3 in SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 26, and VL CDRs 1, 2, 3 that are identical to VL CDRs 1, 2, 3 in SEQ ID NO: 23, 24, 25, or 27.
A food additive comprising the antibody or antigen-binding fragment thereof of any one of claims 1-15, 42-53, and 61-64, the antibody-drug conjugate of claim 54 or 55, or the pharmaceutical composition of claim 59 or 60.
A method of preventing a Respiratory Syncytial Virus infection in a subject, the method comprising administering the food additive of claim 65 to the subject.