Anti-respiratory syncytial virus antibodies and methods for their production and use

Abstract

Anti-RSV antibodies having neutralizing activity against RSV subtype A and RSV subtype B are provided, as well as methods for their identification, isolation, and production, and methods for their preparation and use. [Solution] The antibodies and their antigen-binding fragments of the present invention are more effective in neutralizing RSV than known antibodies, and therefore can be used to provide higher levels of protection against RSV infection and more effectively treat and / or ameliorate symptoms associated with RSV infection using lower doses of the antibodies, antibody fragments, or pharmaceutical compositions of the present invention.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 411,508, filed October 21, 2016, the entire contents of which are incorporated by reference herein.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on October 20, 2017, is stored under the name "2009186_0218_SL.TXT" and is 1,019,600 bytes in size.

[0003] FIELD OF THEINVENTION In particular, the invention relates to anti-respiratory syncytial virus (RSV) antibodies and functional fragments thereof, nucleic acid sequences encoding these antibodies, and methods and reagents for their preparation and use. [Background technology]

[0004] All references cited herein include, but are not limited to, patents, patent applications, non-patent literature, and publications, all of which are expressly incorporated by reference in their entirety, the contents of which are hereby expressly incorporated by reference herein for all purposes.

[0005] Respiratory syncytial virus (RSV) is associated with significant morbidity and mortality in young children and the elderly, is the leading cause of hospitalization of infants in the United States, and is responsible for an estimated 64 million infections and 160,000 deaths worldwide each year. However, despite decades of research, the development of a safe and effective vaccine or therapeutic and / or prophylactic antibodies against RSV has not yet been achieved, highlighting the need for new strategies to induce or provide a protective immune response. (1-3). Indeed, to date, there is no approved RSV vaccine, and passive prevention using the monoclonal antibody palivizumab (marketed as Synagis®) is limited to high-risk infants, in part because of its limited efficacy.

[0006] Certain populations of children are at risk for developing RSV infection and include premature infants (Hall et al., 1979, New Engl. J. Med. 300:393-396), children with congenital anomalies of the airways, children with bronchopulmonary dysplasia (Groothuis et al., 1988, Pediatrics 82:199-203), children with congenital heart disease (MacDonald et al., New Engl. J. Med. 307:397-400), and children with congenital or acquired immunodeficiencies (Ogra et al., 1988, Pediatr. Infect. Dis. J. 7:246-249; and Pohl et al., 1992, J. Infect. Dis. 165:166-169), and children with cystic fibrosis (Abman et al., 1993, J. Infect. Dis. 165:166-169). al., 1988, J. Pediatr. 1 13:826-830).

[0007] RSV also infects the adult population, where it primarily causes upper respiratory tract disease, but also in older patients (Evans, A.S., eds., 1989, Viral Infections of Humans. Epidemiology and Control, 3 rded., Plenum Medical Book, New York, pages 525-544), as well as immunosuppressed adults, especially bone marrow transplant patients (Hertz et al., 1989, Medicine 68:269-281), may be at increased risk for serious infections and pneumonia. Other at-risk patients include those with congestive heart failure and those with chronic obstructive pulmonary disease (i.e., COPD). Outbreaks have been reported among nursing home residents and institutionalized young adults (Falsey, AR, 1991, Infect. Control Hosp. Epidemiol. 12:602-608; and Garvie et al., 1980, Br. Med. J. 281:1253-1254).

[0008] Treatment options for established RSV disease are limited, but more severe forms of lower respiratory tract disease often require significant supportive care, including administration of humidified oxygen and respiratory support (Fields et al., eds, 1990, Fields Virology, 2001). nd ed., Vol. 1, Raven Press, New York, pages 1045-1072).

[0009] Like other pneumoviruses, RSV expresses two major surface glycoproteins, the fusion protein (F) and the attachment protein (G). Both have been shown to induce protective neutralizing antibody responses, but F is genetically less variable than G, is absolutely required for infection, and is the target of the majority of neutralizing activity in human sera (4-8). RSV F is also the target of the monoclonal antibody palivizumab, which is used to passively protect high-risk infants from severe disease (9). As a result, the RSV F protein is considered a very attractive target for vaccines and antibody-based therapeutics.

[0010] The mature RSV F glycoprotein initially exists in a metastable prefusion conformation (preF) before undergoing a conformational change that inserts the hydrophobic fusion peptide into the host cell membrane (10). Subsequent refolding of F into a stable elongated postfusion conformation (postF) (11, 12) results in fusion of the viral membrane with the host cell membrane. Due to its inherent instability, the preF protein is prone to premature induction of postF both in solution and on the viral surface (13). Recently, stabilization of preF has been achieved by protein engineering (14, 15), and it has been shown in animal models that stabilized preF induces higher titers of neutralizing antibodies than postF (15).

[0011] Despite the importance of neutralizing antibodies in protecting against severe RSV disease, our understanding of the human antibody response to RSV has been limited to studies of human sera and a few RSV-specific human monoclonal antibodies (16-19). The epitopes recognized by these human antibodies, as well as by some murine antibodies, define at least four "antigenic sites" on RSV F (1, 10, 16, 18-20) (see also Table 1, for example). Three of these sites, I, II, and IV, are present in both preF and postF, whereas antigenic site φ is present exclusively in preF. Additional preF-specific epitopes have been defined by antibodies MPE8 (17) and AM14 (21). Serological mapping studies have shown that site φ-directed antibodies are responsible for the majority of neutralizing antibody responses in most individuals (8), but additional antibody specificities that contribute to serum neutralizing activity remain to be determined. In addition, it was previously unknown whether specific antibody sequence features are required to recognize specific neutralization sites, as has been observed for other viral targets.(22-25) Thus, understanding the relationship between neutralizing potency and epitope specificity would be advantageous in the selection and / or design of vaccine antigens, as well as therapeutic and / or prophylactic antibodies, that induce potent neutralizing responses against RSV.

[0012] Treatment options for established RSV disease are limited, but more severe forms of lower respiratory tract disease often require significant supportive care, including administration of humidified oxygen and respiratory support (Fields et al., eds, 1990, Fields Virology, 2001). nd ed., Vol. 1, Raven Press, New York, pages 1045-1072).

[0013] Ribavirin, the only drug approved for the treatment of infection, has been shown to be effective in treating pneumonia and bronchiolitis associated with RSV infection and has been shown to modify the course of severe RSV disease in immunocompetent children (Smith et al., 1991, New Engl. J. Med. 325:24-29). The use of ribavirin is limited due to potential risks to pregnant women who may be exposed to aerosolized drug during administration in the hospital environment.

[0014] Similarly, a vaccine would be useful, but to date no commercially available vaccine has been developed. Several vaccine candidates have been abandoned and others are in development (Murphy et al., 1994, Virus Res. 32:13-36). Vaccine development is problematic, particularly as the peak incidence of lower respiratory tract disease is seen at 2-5 months of age, necessitating immunization in the early neonatal period. However, the immune response of neonates at this time is known to be immature. In addition, infants at this time still have high titers of maternally acquired RSV antibodies, which may reduce the immunogenicity of the vaccine (Murphy et al., 1988, J. Virol. 62:3907-3910; and Murphy et al., 1991, Vaccine 9:185-189).

[0015] Currently, the only approved approach to prevent RSV disease is passive immunization. For example, palivizumab (SYNAGIS®), a humanized antibody specific for an epitope of the F protein, is approved for intramuscular administration to pediatric patients at a recommended monthly dose of 15 mg / kg body weight throughout the RSV season (November-April in the Northern Hemisphere) to prevent severe lower respiratory tract disease caused by RSV. SYNAGIS® is a composite of human (95%) and murine (5%) antibody sequences (Johnson et al., (1997), J. Infect. Diseases 176:1215-1224, and U.S. Patent No. 5,824,307).

[0016] SYNAGIS® has been used successfully to prevent RSV infection in pediatric patients, but multiple intramuscular doses of 15 mg / kg SYNAGIS® are required to achieve prophylactic efficacy. The need to administer multiple intramuscular doses of antibody requires multiple visits to the clinic, which is not only inconvenient for the patient but also introduces the potential for dosing errors. Efforts to improve the therapeutic profile of anti-RSV-F antibodies have led to the identification and development of Motavizumab, also known as NUMAX™. However, clinical trials have revealed severe hypersensitivity reactions in a certain number of patients who received Motavizumab. Since then, no further development of this humanized anti-RSV-F antibody has been attempted. Other antibodies against RSV-F protein include US6656467, US5824307, US7786273, US7670600, US7083784, US6818216, US7700735, US7553489, US7323172, US7229619, US7425618, US7740851, US7658921, US7704505, US7635568, US6855493, US6565849, US7582297, US7208162, US7700720, US641 3771, US5811524, US6537809, US5762905, US7070786, US7364742, US7879329, US7488477, US7867497, US5534411, US6835372, US7482024, US7691603, US8562996, US8568726, US9447173, US20100015596, WO2009088159A1, and WO2014159822. To date, only SYNAGIS® has received regulatory approval for use in preventing RSV infection. [Prior art documents] [Patent documents]

[0017] [Patent Document 1] U.S. Pat. No. 5,824,307 [Patent Document 2] U.S. Patent No. 6,656,467 [Patent Document 3] U.S. Patent No. 5,824,307 [Patent Document 4] U.S. Patent No. 7,786,273 [Patent Document 5] U.S. Patent No. 7,670,600 [Patent Document 6] U.S. Patent No. 7,083,784 [Patent Document 7] U.S. Patent No. 6,818,216 [Patent Document 8] U.S. Patent No. 7,700,735 [Patent Document 9] U.S. Patent No. 7,553,489 [Patent Document 10] U.S. Patent No. 7,323,172 [Patent Document 11] U.S. Patent No. 7,229,619 [Patent Document 12] U.S. Patent No. 7,425,618 [Patent Document 13] U.S. Patent No. 7,740,851 [Patent Document 14] U.S. Patent No. 7,658,921 [Patent Document 15] U.S. Patent No. 7,704,505 [Patent Document 16] U.S. Patent No. 7,635,568 [Patent Document 17] U.S. Patent No. 6,855,493 [Patent Document 18] U.S. Patent No. 6,565,849 [Patent Document 19] U.S. Patent No. 7,582,297 [Patent Document 20] U.S. Patent No. 7,208,162 [Patent Document 21] U.S. Patent No. 7,700,720 [Patent Document 22] U.S. Patent No. 6,413,771 [Patent Document 23] U.S. Pat. No. 5,811,524 [Patent Document 24] U.S. Patent No. 6,537,809 [Patent Document 25] U.S. Patent No. 5,762,905 [Patent Document 26] U.S. Patent No. 7,070,786 [Patent Document 27] U.S. Patent No. 7,364,742 [Patent Document 28] U.S. Patent No. 7,879,329 [Patent Document 29] U.S. Patent No. 7,488,477 [Patent Document 30] U.S. Patent No. 7,867,497 [Patent Document 31] U.S. Pat. No. 5,534,411 [Patent Document 32] U.S. Patent No. 6,835,372 [Patent Document 33] U.S. Patent No. 7,482,024 [Patent Document 34] U.S. Patent No. 7,691,603 [Patent Document 35] U.S. Pat. No. 8,562,996 [Patent Document 36] U.S. Pat. No. 8,568,726 [Patent Document 37] U.S. Patent No. 9,447,173 [Patent Document 38] US Patent Application Publication No. 2010 / 0015596 [Patent Document 39] International Publication No. 2009 / 088159 [Patent Document 40] International Publication No. 2014 / 159822 [Non-patent literature]

[0018] [Non-Patent Document 1] Hall et al.,1979,New Engl.J.Med.300:393-396 [Non-Patent Document 2] Groothuis et al., 1988, Pediatrics 82:199-203 [Non-Patent Document 3] MacDonald et al., New Engl. J.Med.307:397-400 [Non-Patent Document 4] Ogra et al., 1988, Pediatr. Infect. Dis. J. 7:246 - 249 [Non - Patent Document 5] Pohl et al., 1992, J. Infect. Dis. 165:166 - 169 [Non - Patent Document 6] Abman et al., 1988, J. Pediatr. 113:826 - 830 [Non - Patent Document 7] Evans, A.S., eds., 1989, Viral Infections of Humans. Epidemiology and Control, 3rd ed., Plenum Medical Book, New York, pages 525 - 544 [Non - Patent Document 8] Hertz et al., 1989, Medicine 68:269 - 281 [Non - Patent Document 9] Falsey, A.R., 1991, Infect. Control Hosp. Epidemiol. 12:602 - 608 [Non - Patent Document 10] Garvie et al., 1980, Br. Med. J. 281:1253 - 1254 [Non - Patent Document 11] Fields et al., eds, 1990, Fields Virology, 2nd ed., Vol.1, Raven Press, New York, pages 1045 - 1072 [Non - Patent Document 12] Smith et al., 1991, New Engl. J. Med. 325:24 - 29 [Non - Patent Document 13] Murphy et al., 1994, Virus Res. 32:13 - 36 [Non - Patent Document 14] Murphy et al., 1988, J. Virol. 62:3907 - 3910 [Non - Patent Document 15] Murphy et al.,1991,Vaccine9:185-189 [Non-Patent Document 16] Johnson et al.,(1997),J.Infect.Diseases 176:1215-1224 Summary of the Invention [Means for solving the problem]

[0019] There remains a need for highly specific, high affinity, and highly potent neutralizing anti-RSV antibodies and antigen-binding fragments thereof that neutralize at least one, but preferably both, of RSV virus strains of subtype A and subtype B and preferentially recognize the PreF rather than the PostF conformation of the F protein. There also remains a need for anti-RSV and anti-HMPV cross-neutralizing antibodies and antigen-binding fragments thereof.

[0020] The present applicants have discovered, isolated and characterized an extensive panel of RSV F-specific monoclonal antibodies derived from, inter alia, memory B cells of healthy adult human donors, and have used these antibodies to comprehensively map the antigenic topology of RSV F. Advantageously, the majority of the RSV F-specific human antibody repertoire is composed of antibodies with very strong specificity for the PreF conformation of the F protein (compared to the PostF form), many, if not most, of which have demonstrated significant neutralizing potency against one or both of RSV subtype A and RSV subtype B strains in neutralization assays. Indeed, many of these antibodies have demonstrated several times, about 5-100 times or more, greater neutralizing potency than conventional anti-RSV therapeutic antibodies such as D25 and publisumab, and therefore serve as attractive therapeutic and / or prophylactic candidates for treating and / or preventing RSV infection and disease.

[0021] The most potent antibodies are known to target two distinct antigenic sites located near the apex of the preF trimer, providing strong support for the development of therapeutic and / or prophylactic antibodies that target these antigenic sites, as well as preF-based vaccine candidates that preserve these antigenic sites. Furthermore, the neutralizing antibodies described and disclosed herein provide new opportunities for the prevention of severe RSV disease using passive immunological prophylaxis.

[0022] Given the role that the F protein plays in virus-cell fusion and cell-to-cell spread, the antibodies described herein provide a method of inhibiting that process and may therefore be used to prevent infection in patients who have been exposed to or are at risk for RSV infection, or to treat and / or ameliorate one or more symptoms associated with RSV infection in patients who have been exposed to or are at risk for RSV infection or have an infection with RSV. The antibodies and pharmaceutical compositions described herein may also be used to prevent or treat RSV infection in patients who may experience more severe forms of RSV infection due to underlying or pre-existing medical conditions. Patients who may benefit from treatment with the antibodies and / or pharmaceutical compositions of the invention may be premature infants, full-term infants born during RSV season (generally late fall (November) through early spring (April)) who are at risk due to congenital heart disease or other pre-existing or underlying medical conditions such as chronic lung disease, children aged 1 year or older with or without an underlying medical condition, institutionalized or hospitalized patients, or elderly (over 65 years of age) with or without an underlying medical condition such as congestive heart failure (CHF) or chronic obstructive pulmonary disease (COPD). Patients who may benefit from such treatment may suffer from medical conditions resulting from a damaged pulmonary, cardiovascular, neuromuscular, or immune system. For example, the patient may suffer from an airway abnormality or insufficiency, chronic lung disease, chronic or congenital heart disease, neuromuscular disease that interferes with handling of respiratory secretions, or the patient may be immunosuppressed due to severe combined immunodeficiency or severe acquired immunodeficiency disease, or from an infectious or cancerous condition that otherwise results in immunosuppression, or the patient may be immunosuppressed due to treatment with immunosuppressive drugs (e.g., any drug used to treat transplant patients) or radiation therapy. Patients who may benefit from the antibodies and / or pharmaceutical compositions of the invention may be patients suffering from chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), bronchopulmonary dysplasia, congestive heart failure (CHF), or congenital heart disease.

[0023] Since the antibodies and antigen-binding fragments thereof of the present invention are more effective in neutralizing RSV than known antibodies, lower doses of the antibodies or antibody fragments or pharmaceutical compositions of the present invention can be used to achieve higher levels of protection against infection with RSV and more effective treatment and / or amelioration of symptoms associated with RSV infection. Thus, lower doses of antibodies or fragments thereof and / or pharmaceutical compositions that immunospecifically bind to RSV-F antigens are less likely or less likely to cause severe adverse events. Similarly, the use of more effective neutralizing antibodies may reduce the need for more frequent administration of the antibodies or antibody fragments or pharmaceutical compositions than previously believed necessary to prevent infection or neutralize the virus or treat or ameliorate one or more symptoms associated with RSV infection. Symptoms of RSV infection may include a bluish skin color due to lack of oxygen (hypoxia), dyspnea (tightness or shortness of breath), cough, croupy cough ("seal" cough), fever, nasal flaring, nasal congestion (stuffy nose), apnea, loss of appetite, dehydration, anorexia, changes in mental status, or wheezing.

[0024] Such antibodies or pharmaceutical compositions may be useful when administered prophylactically (prior to exposure to and infection with the virus) to reduce the severity or duration of primary infection with RSV or improve at least one symptom associated with the infection. The antibodies or pharmaceutical compositions may be used alone or in combination with a second agent useful in treating RSV infection. In certain embodiments, the antibodies or pharmaceutical compositions may be administered therapeutically (after exposure to and infection with the virus) to reduce the severity or duration of primary infection or improve at least one symptom associated with the infection, either alone or in combination with a second agent. In certain embodiments, the antibodies or pharmaceutical compositions may be used prophylactically as a stand-alone therapy to protect patients at risk of RSV infection, such as those described above. Any of these patient populations may benefit from treatment with the antibodies or pharmaceutical compositions of the present invention, either alone or in combination with a second agent, such as an antiviral therapy such as ribavirin or other antiviral vaccines.

[0025] The antibodies of the present invention may be full length (e.g., IgG1 or IgG4 antibodies) or may comprise only the antigen-binding portion (e.g., Fab, F(ab')2, or scFv fragments) and may be modified to affect functionality, e.g., to eliminate residual effector functions (Reddy et al., (2000), J. Immunol. 164:1925-1933).

[0026] Thus, in certain embodiments, an isolated antibody or antigen-binding fragment thereof that specifically binds to a respiratory syncytial virus (RSV) F protein (F) is provided, wherein at least one, at least two, at least three, at least four, at least five, or at least six of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences of the antibody or antigen-binding fragment thereof are selected from antibody numbers 232 to 372 disclosed in Table 6, and ... at least 70% identical, at least 75% identical, 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 100% identical, and / or any combination thereof to at least one, at least two, at least three, at least four, at least five, or at least six of the amino acid sequences of CDRL1, CDRL2, and / or CDRL3. and the antibody or antigen-binding fragment thereof also has the following characteristics: a) the antibody or antigen-binding fragment thereof cross-competes with the antibody or antigen-binding fragment thereof for binding to RSV-F; b) the antibody or antigen-binding fragment thereof exhibits better binding affinity for the PreF form of RSV-F compared to the PostF form; c) the antibody or antigen-binding fragment thereof exhibits a clean or low polyreactivity profile; d) the antibody or antigen-binding fragment thereof exhibits e) the antibody or antigen-binding fragment thereof exhibits antigenic site specificity for RSV-F at site Φ, site I, site II, site III, site IV, or site V; f) the antibody or antigen-binding fragment thereof exhibits antigenic site specificity for RSV-F site Φ, site V, or site III compared to RSV-F site I, site II, or site IV; g) at least a portion of the epitope with which the antibody or antigen-binding fragment thereof interacts ish) the antibody, or antigen-binding fragment thereof, has an in vitro neutralizing activity (IC, 50 i) the binding affinity and / or epitope specificity of the antibody or antigen-binding fragment thereof to any one of the RSV-F variants designated 1, 2, 3, 4, 5, 6, 7, 8, 9, and DG in Table 7A is reduced or eliminated compared to the binding affinity and / or epitope specificity of the antibody or antigen-binding fragment thereof to RSV-F or RSV-F DS-Cav1; j) the antibody or antigen-binding fragment thereof exhibits a cross-neutralizing potency (IC) against human metapneumovirus (HMPV). 50 k) the antibody or antigen-binding fragment thereof does not comprise D25, MPE8, palivizumab, or motavizumab; or l) the antibody or antigen-binding fragment thereof exhibits a neutralizing potency (IC) that is at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 50-fold, at least about 55-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, greater than about 100-fold, and any number between the aforementioned values. 50 )

[0027] In other specific embodiments, the isolated antibody or antigen-binding fragment thereof comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or at least twelve of the above features a)-l).

[0028] In certain other embodiments, the isolated antibody or antigen-binding fragment thereof comprises: a) the CDRH3 amino acid sequence of any one of the antibodies designated as Antibody No. 232 through Antibody No. 372 disclosed in Table 6; b) the CDRH2 amino acid sequence of any one of the antibodies designated as Antibody No. 232 through Antibody No. 372 disclosed in Table 6; c) the CDRH1 amino acid sequence of any one of the antibodies designated as Antibody No. 232 through Antibody No. 372 disclosed in Table 6; d) the CDRL3 amino acid sequence of any one of the antibodies designated as Antibody No. 232 through Antibody No. 372 disclosed in Table 6; e) the CDRL2 amino acid sequence of any one of the antibodies designated as Antibody No. 232 through Antibody No. 372 disclosed in Table 6; f) the CDRL1 amino acid sequence of any one of the antibodies designated as Antibody No. 232 through Antibody No. 372 disclosed in Table 6; or g) any combination of two or more of a), b), c), d), e), and f).

[0029] In certain other embodiments, the isolated antibody or antigen-binding fragment thereof comprises: a) the heavy chain (HC) amino acid sequence of any one of the antibodies designated Antibody No. 232 to Antibody No. 372 disclosed in Table 6; and / or b) the light chain (LC) amino acid sequence of any one of the antibodies designated Antibody No. 232 to Antibody No. 372 disclosed in Table 6.

[0030] In certain other embodiments, the isolated antibody, or antigen-binding fragment thereof, is selected from the group consisting of antibodies that are at least 70% identical, at least 75% identical, 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, and / or any percentage therebetween, to any one of the antibodies designated Antibody No. 232 through Antibody No. 372 disclosed in Table 6.

[0031] In certain other embodiments, the isolated antibody, or antigen-binding fragment thereof, is selected from the group consisting of the antibodies designated Antibody No. 232 through Antibody No. 372 disclosed in Table 6.

[0032] In other embodiments, isolated nucleic acid sequences encoding antibodies, their light and / or heavy chains, antigen-binding fragments thereof, or light and / or heavy chains encoding antigen-binding fragments of any of the other embodiments disclosed herein are provided.

[0033] In other embodiments, there is provided an expression vector comprising the isolated nucleic acid sequence of any of the other embodiments disclosed herein.

[0034] In other embodiments, the present invention provides a host cell that is transfected, transformed or transduced with a nucleic acid sequence or expression vector of any of the other embodiments disclosed herein.

[0035] In other embodiments, there are provided pharmaceutical compositions comprising one or more of the isolated antibodies or antigen-binding fragments thereof of other embodiments disclosed herein and a pharma- ceutical acceptable carrier and / or excipient.

[0036] In other embodiments, pharmaceutical compositions are provided that include a nucleic acid sequence of the invention, e.g., one or more nucleic acid sequences encoding at least one or both of the light or heavy chains of an antibody of other embodiments disclosed herein, or one or more expression vectors of other embodiments disclosed herein, and a pharma- ceutical acceptable carrier and / or excipient.

[0037] In other embodiments, there is provided a transgenic organism comprising a nucleic acid sequence of any of the other embodiments disclosed herein, or an expression vector of any of the other embodiments disclosed herein.

[0038] In other embodiments, methods are provided for treating or preventing a respiratory syncytial virus (RSV) infection or at least one symptom associated with a RSV infection, comprising administering to a patient in need thereof or suspected of being in need thereof a) one or more antibodies or antigen-binding fragments thereof of other embodiments disclosed herein, b) a nucleic acid sequence of other embodiments disclosed herein, an expression vector of other embodiments disclosed herein, a host cell of other embodiments disclosed herein, or e) a pharmaceutical composition of other embodiments disclosed herein to treat or prevent a RSV infection or treat, alleviate, or reduce the severity of at least one symptom associated with a RSV infection.

[0039] In another embodiment, a method of treating or preventing either a respiratory syncytial virus (RSV) infection and / or a human metapneumovirus (HMPV) infection, or at least one symptom associated with the RSV infection or the HMPV infection is provided, comprising administering to a patient in need thereof or suspected of being in need thereof a) one or more antibodies or antigen-binding fragments thereof of other embodiments disclosed herein, b) a nucleic acid sequence of other embodiments disclosed herein, c) an expression vector of other embodiments disclosed herein, d) a host cell of other embodiments disclosed herein, or e) a pharmaceutical composition of other embodiments disclosed herein to treat or prevent the RSV infection or treat, alleviate, or reduce the severity of at least one symptom associated with the RSV infection. In another embodiment, the one or more antibodies or antigen-binding fragments thereof of a) are selected from the group consisting of the antibodies designated antibody number 340 in Table 6.

[0040] In another embodiment, there is provided the method of the other embodiments, further comprising administering to the patient a second therapeutic agent.

[0041] In other embodiments, methods of other embodiments are provided, wherein the second therapeutic agent is selected from the group consisting of an antiviral drug, a vaccine specific for RSV, a vaccine specific for influenza virus, or a vaccine specific for metapneumovirus (MPV), an siRNA specific for a RSV antigen or a metapneumovirus (MPV) antigen, a secondary antibody specific for a RSV antigen or a metapneumovirus (MPV) antigen, an anti-IL-4R antibody, an antibody specific for an influenza virus antigen, an anti-RSV-G antibody, and an NSAID.

[0042] In certain embodiments, there are provided pharmaceutical compositions comprising one or more nucleic acid sequences encoding at least one of the light or heavy chains of any of the isolated antibodies or antigen-binding fragments thereof, or any other embodiment of the antibodies disclosed herein, or antigen-binding fragments thereof, and a pharma- ceutical acceptable carrier and / or excipient.

[0043] In certain embodiments, pharmaceutical compositions of other embodiments are provided for use in the prevention of a respiratory syncytial virus (RSV) infection in a patient in need thereof or suspected of being in need thereof, or in the treatment of a patient suffering from a RSV infection, or in the amelioration of at least one symptom or complication associated with said infection, where said use results in either the prevention of said infection, or the prevention, amelioration, or reduction in the severity and / or duration of at least one symptom or complication associated with said infection.

[0044] In certain embodiments, the pharmaceutical compositions of other embodiments are provided for use in a patient in need thereof, or suspected of being in need thereof, in the treatment or prevention of either a respiratory syncytial virus (RSV) infection and / or a human metapneumovirus (HMPV) infection, or at least one symptom associated with said RSV infection and / or said HMPV infection, where said use results in either prevention of said infection, or prevention, amelioration, or reduction in severity and / or duration of at least one symptom or complication associated with said infection.

[0045] In certain other embodiments, there is provided the use of the pharmaceutical composition of other embodiments in the manufacture of a medicament for preventing a respiratory syncytial virus (RSV) infection or treating a patient suffering from a RSV infection, or ameliorating at least one symptom or complication associated with said infection, in a patient in need thereof, where said use results in either the prevention of said infection or the prevention, amelioration, or reduction in severity and / or duration of at least one symptom or complication associated with said infection.

[0046] In certain other embodiments, there is provided the use of the pharmaceutical composition of other embodiments in the manufacture of a medicament for the prevention of either a respiratory syncytial virus (RSV) infection and / or a human metapneumovirus (HMPV) infection, or at least one symptom associated with said RSV infection and / or said HMPV infection, in a patient in need thereof or suspected of being in need thereof, whereby said use results in either the prevention of said infection, or the prevention, amelioration, or reduction in severity and / or duration of at least one symptom or complication associated with said infection. The present invention provides, for example, the following items. (Item 1) 1. An isolated antibody or antigen-binding fragment thereof that specifically binds to a respiratory syncytial virus (RSV) F protein (F), wherein at least one of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences of the antibody or antigen-binding fragment thereof is at least 70% identical, at least 75% identical, 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, and / or all percentages therebetween, to at least one of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3 amino acid sequences disclosed in Table 6 of an antibody selected from antibody numbers 232 to 372 disclosed in Table 6, and wherein the antibody or antigen-binding fragment thereof has any of the following characteristics: a) the antibody or antigen-binding fragment thereof cross-competes with the antibody or antigen-binding fragment thereof for binding to RSV-F; b) the antibody or antigen-binding fragment thereof exhibits better binding affinity for the PreF form of RSV-F compared to the PostF form; c) the antibody or antigen-binding fragment thereof exhibits a clean or low polyreactivity profile; d) the antibody or antigen-binding fragment thereof exhibits neutralizing activity against RSV subtype A and RSV subtype B in vitro; e) the antibody or antigen-binding fragment thereof exhibits antigenic site specificity for RSV-F at site Φ, site I, site II, site III, site IV, or site V; f) the antibody or antigen-binding fragment thereof exhibits antigenic site specificity for RSV-F site Φ, site V, or site III compared to RSV-F site I, site II, or site IV; g) at least a portion of the epitope with which the antibody or antigen-binding fragment thereof interacts includes the α3 helix and β3 / β4 hairpin of PreF; h) the antibody, or antigen-binding fragment thereof, has an in vitro neutralizing potency (IC) of between about 0.5 micrograms / milliliter (ug / ml) and about 5 ug / ml, between about 0.05 μg / ml and about 0.5 μg / ml, or less than about 0.05 mg / ml 50 ), i) the binding affinity and / or epitope specificity of the antibody or antigen-binding fragment thereof to any one of the RSV-F variants designated 1, 2, 3, 4, 5, 6, 7, 8, 9, and DG in Table 7A is reduced or eliminated compared to the binding affinity and / or epitope specificity of the antibody or antigen-binding fragment thereof to RSV-F or RSV-F DS-Cav1; j) the antibody or antigen-binding fragment thereof has a cross-neutralizing activity (IC 50 ), k) the antibody or antigen-binding fragment thereof does not comprise D25, MPE8, palivizumab, motavizumab, or AM-14; l) The antibody or antigen-binding fragment thereof has a neutralizing potency (IC) that is at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 50-fold, at least about 55-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, greater than about 100-fold, and any number between the aforementioned values. 50 The antibody or antigen-binding fragment thereof, having one or more of the above characteristics: (Item 2) 2. The isolated antibody or antigen-binding fragment thereof according to item 1, wherein the antibody or antigen-binding fragment thereof comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or at least twelve of features a) to l). (Item 3) The antibody, or antigen-binding fragment thereof, a) the CDRH3 amino acid sequence of any one of the antibodies designated as Antibody No. 232 to Antibody No. 372 in Table 6; b) the CDRH2 amino acid sequence of any one of the antibodies designated as Antibody No. 232 to Antibody No. 372 in Table 6; c) the CDRH1 amino acid sequence of any one of the antibodies designated as Antibody No. 232 to Antibody No. 372 in Table 6; d) the CDRL3 amino acid sequence of any one of the antibodies designated as Antibody No. 232 to Antibody No. 372 in Table 6; e) the CDRL2 amino acid sequence of any one of the antibodies designated Antibody No. 232 to Antibody No. 372 in Table 6; f) the CDRL1 amino acid sequence of any one of the antibodies designated Antibody No. 232 to Antibody No. 372 in Table 6; or g) any combination of two or more of a), b), c), d), e), and f); 3. The isolated antibody or antigen-binding fragment thereof according to item 1 or 2, comprising: (Item 4) The antibody, or antigen-binding fragment thereof, a) any one of the heavy chain (HC) amino acid sequences of the antibodies designated as Antibody No. 232 to Antibody No. 372 in Table 6, and / or b) the light chain (LC) amino acid sequence of any one of the antibodies designated as Antibody No. 232 to Antibody No. 372 in Table 6; 4. The isolated antibody or antigen-binding fragment thereof according to any one of items 1 to 3, comprising: (Item 5) 5. The isolated antibody or antigen-binding fragment thereof according to any one of items 1 to 4, wherein the antibody is selected from the group consisting of antibodies that are at least 70% identical, at least 75% identical, 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, and / or any percentage therebetween, to any one of the antibodies designated as Antibody No. 232 to Antibody No. 372 disclosed in Table 6. (Item 6) 6. The isolated antibody or antigen-binding fragment thereof according to any one of items 1 to 5, wherein the antibody is selected from the group consisting of the antibodies designated as antibody No. 232 to antibody No. 372 in Table 6. (Item 7) 7. An isolated nucleic acid sequence encoding the antibody or antigen-binding fragment thereof according to any one of items 1 to 6. (Item 8) 8. An expression vector comprising the isolated nucleic acid sequence according to item 7. (Item 9) A host cell transfected, transformed or transduced with the nucleic acid sequence of item 7 or the expression vector of item 8. (Item 10) 7. A pharmaceutical composition comprising one or more of the isolated antibodies or antigen-binding fragments thereof according to any one of items 1 to 6, and a pharma- ceutical acceptable carrier and / or excipient. (Item 11) 9. A pharmaceutical composition comprising one or more of the nucleic acid sequences according to item 7 or one or more of the expression vectors according to item 8, together with a pharma- ceutical acceptable carrier and / or excipient. (Item 12) A transgenic organism comprising the nucleic acid sequence according to item 7 or the expression vector according to item 8. (Item 13) 1. A method for treating or preventing a respiratory syncytial virus (RSV) infection or at least one symptom associated with a RSV infection, comprising administering to a patient in need thereof or suspected of being in need thereof: a) one or more antibodies or antigen-binding fragments thereof according to any one of items 1 to 6; b) the nucleic acid sequence according to item 7, c) an expression vector according to item 8, d) a host cell according to item 9, or e) The pharmaceutical composition according to item 10 or 11, to treat or prevent a RSV infection, or to treat, alleviate, or reduce the severity of at least one symptom associated with a RSV infection. (Item 14) 1. A method of treating or preventing either a respiratory syncytial virus (RSV) infection or a human metapneumovirus (HMPV) infection, or at least one symptom associated with a RSV infection or an HMPV infection, comprising administering to a patient in need thereof or suspected of being in need thereof: a) one or more antibodies or antigen-binding fragments thereof according to any one of items 1 to 6; b) the nucleic acid sequence according to item 7, c) an expression vector according to item 8, d) a host cell according to item 9, or e) The pharmaceutical composition according to item 10 or 11, to treat or prevent a RSV infection, or to treat, alleviate, or reduce the severity of at least one symptom associated with a RSV infection. (Item 15) 15. The method of claim 14, wherein the one or more antibodies or antigen-binding fragments thereof comprise antibody number 340. (Item 16) 16. The method according to any one of items 13 to 15, further comprising administering to the patient a second therapeutic agent. (Item 17) 17. The method of claim 16, wherein the second therapeutic agent is selected from the group consisting of an antiviral drug, a vaccine specific for RSV, a vaccine specific for influenza virus, or a vaccine specific for metapneumovirus (MPV), an siRNA specific for a RSV antigen or a metapneumovirus (MPV) antigen, a secondary antibody specific for a RSV antigen or a metapneumovirus (MPV) antigen, an anti-IL-4R antibody, an antibody specific for an influenza virus antigen, an anti-RSV-G antibody, and an NSAID. (Item 18) 8. A pharmaceutical composition comprising one or more of the isolated antibodies or antigen-binding fragments thereof according to any one of items 1 to 7, and a pharma- ceutical acceptable carrier and / or excipient. (Item 19) 19. The pharmaceutical composition according to item 18 for use in the prevention of a respiratory syncytial virus (RSV) infection in a patient in need thereof or suspected of being in need thereof or in the treatment of a patient suffering from a RSV infection or in the amelioration of at least one symptom or complication associated with said infection, wherein said use results in either the prevention of said infection or the prevention, amelioration or reduction in the severity and / or duration of at least one symptom or complication associated with said infection. (Item 20) 19. The pharmaceutical composition according to item 18, for use in a patient in need thereof or suspected of being in need thereof, in the treatment or prevention of a respiratory syncytial virus (RSV) infection or a human metapneumovirus (HMPV) infection or at least one symptom associated with said RSV infection or said HMPV infection, wherein said use results in either prevention of said infection or prevention, amelioration or reduction in severity and / or duration of at least one symptom or complication associated with said infection. (Item 21) 19. Use of the pharmaceutical composition according to item 18 in the manufacture of a medicament for preventing a respiratory syncytial virus (RSV) infection or for treating a patient suffering from a RSV infection or for ameliorating at least one symptom or complication associated with said infection in a patient in need thereof, said use resulting in either the prevention of said infection or the prevention, amelioration or reduction in severity and / or duration of at least one symptom or complication associated with said infection. (Item 22) 19. Use of the pharmaceutical composition according to item 18 in the manufacture of a medicament for the prevention of a respiratory syncytial virus (RSV) infection or a human metapneumovirus (HMPV) infection or at least one symptom associated with said RSV infection or said HMPV infection in a patient in need thereof or in a patient suspected of being in need thereof, wherein said use results in either the prevention of said infection or the prevention, amelioration or reduction in the severity and / or duration of at least one symptom or complication associated with said infection. [Brief description of the drawings]

[0047] [Figure 1-1] 1A-1F illustrate the anti-RSV repertoire cloning and sequence analysis of the identified and isolated antibodies. FIG. 1A: RSV F-specific B cell sorting. FACS plots show RSV F reactivity of IgG+ and IgA+ B cells from healthy adult donors. B cells in quadrant 2 (Q2) were single-cell sorted. FIG. 1B: Isotype analysis. Index sort plots show the percentage of RSV F-specific B cells expressing IgG or IgA. FIG. 1C: Clonal lineage analysis. Each slice represents a clonal lineage, and the size of the slice is proportional to the number of clones within the lineage. The total number of clones is displayed in the center of the circle. Clonal lineages were assigned based on the following criteria: 1) matching variable and binding gene segments, 2) identical CDR3 loop length, and 3) greater than 80% homology in the CDR3 nucleotide sequence. FIG. 1D: VH repertoire analysis. VH germline genes were considered enriched in the RSV repertoire if a given gene was found to be enriched more than three-fold over the non-RSV-specific repertoire (33). Figure 1E: Length distribution of CDRH3. Figure 1F: Somatic hypermutation in VH (excluding CDRH3). The red line indicates the average number of nucleotide substitutions. Each clonal lineage is represented only once in Figures 1D and 1E. Data for non-RSV-reactive IgG were obtained from published sequences obtained by high-throughput sequencing of rearranged antibody variable gene repertoires from healthy individuals (33). [Figure 1-2] Same as above. [Diagram 2]Figures 2A-2D illustrate the conformational state of RSV F in the repertoire and the similar antibody selection observed for subtypes. Figure 2A: IgG affinity for preF and postF is plotted as shown. Figure 2B: Percentage of antibodies in the donor repertoire that recognize both conformations of F (green) or bind only preF (blue) or postF (orange). Figure 2C: Percentage of antibodies in the donor repertoire that specifically bind subtype A (green), subtype B (blue), or both subtypes A and B (red). NB, non-binder. IgG KD was calculated for antibodies with a BLI response >0.1 nm. Antibodies with a BLI response <0.05 nm are designated NB. Figure 2D: Polyreactivity analysis of anti-RSV antibodies. Polyreactivity of the isolated anti-RSV F antibodies was measured using previously reported assays (42, 43). Three panels of control antibodies, 138 antibodies undergoing clinical trials, 39 antibodies approved for clinical use, and a group of 14 broadly neutralizing HIV antibodies were included for comparison. [Figure 3-1] Figures 3A-3G show the mapping and specificity of anti-RSV antibodies to antigenic sites spanning the surface of PreF and PostF. Figure 3A: Previously determined structure of preF with one protomer shown as a ribbon and a rainbow of six antigenic sites colored red to purple. Figure 3B: The percentage of antibodies targeting each antigenic site is shown. Figure 3C: The percentage of preF-specific antibodies targeting each antigenic site. Figure 3D: Apparent antibody binding affinity to subtype A PreF antigenic site. Figure 3E: Apparent binding affinity to subtype A postF antigenic site. Figure 3F: Apparent antibody binding affinity to subtype B PreF antigenic site. Figure 3G: Apparent binding affinity to subtype B postF. Only antibodies with apparent binding affinity >2 nM were included in this analysis, since antibodies with smaller affinities could not be reliably mapped. The red line indicates the median, and the gray dotted line is 2 nM. NB, non-binders. [Figure 3-2] Same as above. [Figure 4-1]Figures 4A-4G illustrate the correlation between neutralizing potency of anti-RSV antibodies and potency against RSV subtypes A and B, respectively, and Pref vs. PostF specificity. Figure 4A: Neutralizing IC50 for antibodies isolated from donor repertoires. The legend on the right color-codes data points based on neutralizing potency. The red and blue dotted lines represent Motavizumab and D25 IC50s, respectively. Figure 4B: Percentage of neutralizing antibodies in donor repertoires against RSV subtype A or subtype B, stratified by potency as indicated in the legend on the right part of the figure. Figure 4C: Percentage of antibodies in donor repertoires that neutralize both RSV subtypes A and B (red), or that neutralize only RSV subtype A (green), or that neutralize only subtype B (blue). Figure 4D: Apparent binding affinity for subtype A, preF, and postF plotted for each antibody (IgG KD was calculated for antibodies with a BLI response >0.1 nm. Antibodies with a BLI response <0.05 nm were labeled NB). Figure 4E: Neutralization IC50 plotted for RSV subtype A PreF-specific, postF-specific, and cross-reactive antibodies. (Red and blue dotted lines represent motavizumab and D25 IC50, respectively. Red bars represent median values. NB, non-binders; NN, non-neutralizing). Figure 4F: Apparent antibody binding affinity for subtype B, preF, and postF. Figure 4G: IC50 plotted for RSV subtype B preF-specific, postF-specific, and cross-reactive antibodies. (Black bars represent median values. NB, non-binders; NN, non-neutralizing). [Figure 4-2] Same as above. [Diagram 5]Figures 5A-5C show that the most potent neutralizing antibodies bind preF with high affinity and recognize antigenic sites φ and V. Figure 5A: Apparent preF KD plotted against neutralization IC50 and colored according to antigenic sites indicated in the legend on the right side of Figure 5C. Figure 5B: Apparent postF KD plotted against neutralization IC50 and colored as in Figure 5A. Figure 5C: Antibodies grouped according to neutralization potency and colored by antigenic site as in the legend on the right side. NB, non-binder; NN, non-neutralizing. IgG KD was calculated for antibodies with BLI responses >0.1 nm. Antibodies with BLI responses <0.05 nm were labeled NB. Statistical significance was determined using unpaired two-tailed t-tests. Pearson's correlation coefficient r was calculated using Prism software version 7.0. Antibodies that failed to bind or neutralize were excluded from statistical analysis due to the inability to calculate the midpoint concentration accurately. [Figure 6] Figures 6A-C show the properties and purification of the pre- and postF selection probes. Figure 6A: Schematic diagram of the fluorescent prefusion RSV F probe shows one PE-conjugated streptavidin molecule bound to four avi-tagged trimeric prefusion F molecules. Figure 6B: Coomassie-stained SDS-PAGE gel demonstrating the isolation of RSV F with one AviTag per trimer using sequential Ni-NTA and Strep-Tactin purification as described in the methods. Figure 6C: Fluorescence size-exclusion chromatography (FSEC) trace of the tetramer probe on a Superose 6 column. The position of the molecular weight standards is indicated by arrows. [Figure 7]Figures 7A-7C show the generation and validation of preF patch panel mutants. Figure 7A: Panel of RSV F mutants used for epitope mapping. Figure 7B: Prefusion RSV F shown as a molecular surface with one promoter colored white. Nine mutants, each containing a patch of mutations, are uniquely colored according to the table in Figure 7A. Figure 7C: Binding of each IgG to fluorescently labeled beads bound to each mutant listed in Figure 7A was measured using a PE-conjugated anti-human Fc antibody on a FLEXMAP 3D flow cytometer (Luminex). The respective reduction in binding of D25 and Motavizumab to patches 1 and 5 is consistent with their structurally defined epitopes (10, 11). AM14 binding was reduced to both patch 3 and patch 9 due to its unique promoter-spanning epitope (21). This distinct binding profile was used to aid in the classification of other possible quaternary specific antibodies in the panel. [Figure 8] Figure 8 shows that antigenic site V is located between the epitopes recognized by D25, MPE8 and Motavizumab. Pre-fusion F is shown with one promoter represented as an image colored according to the location of the antigenic site and the other two protomers colored in gray. D25 and Motavizumab Fab are shown in blue and pink, respectively. The MPE8 binding site is circled in black. Antigenic site V is located between the binding sites of D25 and MPE8 within one protomer, illustrating the competition between site V-directed antibodies and these controls. Competition with Motavizumab can occur across two adjacent protomers (left side) or within one protomer (right side), depending on the angle of approach of these site V-specific antibodies. [Figure 9] Figure 9 shows the percentage of anti-RSV antibodies demonstrating neutralizing activity exhibited by preF-specific antibodies, postF-specific antibodies, and cross-reactive antibodies. Antibodies were stratified according to neutralizing potency, and the percentage of antibodies in each group that were preF-specific (pink), postF-specific (white), or cross-reactive (orange) antibodies was plotted against subtype A (left panel) and subtype B (right panel). [Figure 10] Figures 10A-10C show the relationship between subtype B neutralization and antigenic site specificity for anti-RSV antibodies. Figure 10A: Subtype B preF affinity plotted against neutralization IC50 for all antibodies and colored according to antigenic site according to the color scheme shown on the right side of Figure 10C. Figure 10B: postF affinity plotted against IC50 and colored as in Figure 10A. Figure 10C: Antibodies with preF affinity greater than 2 nM, classified according to neutralizing potency and colored according to antigenic site (right side). [Figure 11] Figure 11 shows in vitro neutralization of RSV A2 for specific anti-RSV antibodies. Inhibition of RSV replication was measured in an ELISA-based neutralization assay using Hep-2 cells. Cells, monoclonal antibodies, and virus were co-incubated at 37°C for 4 days, followed by quantification of viral proteins in infected cells using polyclonal anti-RSV antibodies. Percent inhibition was calculated compared to control cells infected with virus in the absence of neutralizing antibodies. Data are expressed as the half-maximal inhibitory concentration (IC50) that results in a 50% reduction in viral replication and represent the mean + / - SEM of two independent experiments. An isotype-matched control monoclonal antibody (*) was used in each experiment and showed no virus neutralization. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. As used herein, the term "about" when used in reference to a specific recited numerical value means that the value may vary by 1% or less from the recited value. For example, the expression "about 100" as used herein includes 99 and 101, and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0049] definition The "respiratory syncytial virus-F protein", also known as "RSV-F" or "RSV F", is a type I transmembrane surface protein that has an N-terminal cleaved signal peptide and a membrane anchor near the C-terminus (Collins, PL et al., (1984), PNAS (USA) 81:7683-7687). The RSV-F protein is synthesized as an inactive 67 KDa precursor designated F0 (Calder, L Jet et al., (1984), PNAS (USA) 81:7683-7687). al., Virology (2000), 277, 122-131). The F0 protein is proteolytically activated at two sites in the Golgi complex by furin-like proteases to generate two disulfide-linked polypeptides, F2 and F1, from the N- and C-termini, respectively. A 27-amino acid peptide, also known as "pep27", is released. There are furin cleavage sites (FCS) on both sides of pep27 (Collins, PL, Mottet, G. (1991), J. Gen. Virol., 72: 3095-3101; Sugrue, RJ, et al., (2001), J. Gen. Virol., 82, 1375-1386). The F2 subunit consists of heptad repeat C (HRC), while the F1 subunit contains a fusion polypeptide (FP), heptad repeat A (HRA), domain I, domain II, heptad repeat B (HRB), transmembrane (TM) and cytoplasmic domain (CP) (see Sun, Z. et al., Virues (2013), 5:21 1-225). The RSV-F protein plays a role in the fusion of virus particles to the cell membrane and is expressed on the surface of infected cells, thus playing a role in viral cell-cell transmission and syncytium formation. The amino acid sequence of the RSV-F protein is provided in GenBank under the accession number AAX23994.

[0050] A stabilized mutant of the PreF trimer conformation of RSV-F, also known as "RSV-DS-Cav1" or "DS-Cav1", is described, inter alia, in Stewart-Jones et al., PLos One, Vol. 10(6)):e0128779 and WO 20 No. 11 / 050168 and were used to identify, isolate and characterize the antibodies disclosed herein.

[0051] As used herein, the term "laboratory strain" refers to an RSV strain (subtype A or B) that has been passaged extensively in in vitro cell culture. "Laboratory strains" can acquire adaptive mutations that can affect their biological properties. As used herein, "clinical strain" refers to an RSV isolate (subtype A or B) obtained from an infected individual and isolated and grown in tissue culture with few passages.

[0052] The term "effective dose 99" or "ED 99 " refers to the dose of an agent that produces the desired effect of reducing virus-forming plaques by 99% compared to an isotype (negative) control. 99 refers to the dose of anti-RSV-F antibody that neutralizes viral infection in vivo (e.g., reduces viral load by 99%), as described in Example 5.

[0053] The term “IC 50 " refers to the "half maximal inhibitory concentration" which assesses the effectiveness of a compound (e.g., an anti-RSV-F antibody) inhibitor on biological or biochemical utility. This quantitative measure indicates the amount of a particular inhibitor required to inhibit a given biological process by half. In certain embodiments, the RSV virus neutralizing potency of the anti-RSV and / or anti-RSV / anti-HMPV cross-neutralizing antibodies disclosed herein is measured using the neutralizing IC 50 Display as a value.

[0054] "Palivizumab", also known as "SYNAGIS®", is a humanized anti-RSV-F antibody having heavy and light chain variable domains with the amino acid sequences set forth in US 7,635,568 and US 5,824,307. This antibody, which immunospecifically binds to the RSV-F protein, is currently FDA approved for passive immunoprophylaxis of severe RSV disease in high-risk children and is administered intramuscularly at a recommended monthly dose of 15 mg / kg body weight throughout the RSV season (November to April in the Northern Hemisphere). SYNAGIS® is composed of 95% human and 5% murine antibody sequences. Johnson et al., (1997), J. Infect. Diseases See also 176:1215-1224.

[0055] "Motavizumab", also known as "NUMAX™", is a RSV-F specific humanized monoclonal antibody that exhibits enhanced potency derived from in vitro affinity maturation of the heavy and light chain complementarity determining regions of palivizumab. For reference, the amino acid sequence of the NUMAX™ antibody is disclosed in U.S. Patent Application Publication No. 2003 / 0091584, U.S. Patent No. 6,818,216, and Wu et al., (2005) J. Mol. Bio. 350(1): 126-144, and Wu, et al., (2007) J. Mol. Biol. 368: 652-665. The heavy chain of the antibody is also set forth herein as SEQ ID NO: 359, and the light chain of the antibody is set forth herein as SEQ ID NO: 360.

[0056] As used herein, the terms "treat," "treatment," and "treating" refer to the inhibition or amelioration of the progression, severity, and / or duration of upper and / or lower respiratory tract RSV infection and / or human metapneumovirus (HMPV), otitis media, or symptoms or respiratory conditions associated therewith (such as asthma, wheezing, or a combination thereof) as a result of administration of one or more therapeutic agents (including, but not limited to, administration of one or more prophylactic or therapeutic agents). In certain embodiments, these terms refer to the inhibition or inhibition of replication of RSV and / or HMPV, the inhibition or inhibition of spread of RSV and / or HMPV to other tissues or subjects (e.g., transmission to the lower respiratory tract), the inhibition or inhibition of infection of cells by RSV and / or HMPV, or the amelioration of one or more symptoms associated with upper and / or lower respiratory tract RSV infection or otitis media.

[0057] As used herein, the terms "prevent", "preventing" and "prevention" refer to preventing or inhibiting the progression or onset of an upper and / or lower respiratory tract RSV and / or HMPV infection, otitis media or associated respiratory condition in a subject, preventing or inhibiting the progression of an upper respiratory tract RSV and / or HMPV infection to a lower respiratory tract RSV and / or HMPV infection, otitis media or associated respiratory condition due to administration of a therapeutic agent (e.g., a prophylactic or therapeutic agent), preventing the symptoms of an upper and / or lower respiratory tract RSV and / or HMPV infection, otitis media or associated respiratory condition, or administration of a combination of therapeutic agents (e.g., a prophylactic or therapeutic agent). As used herein, the terms "ameliorate" and "ameliorate" refer to a reduction or decrease in the severity of a condition or any of its symptoms.

[0058] As used herein, the term "antibody" is intended to refer to an immunoglobulin molecule comprising four polypeptide chains, two heavy (H) chains and two light (L) chains, inter-connected by disulfide bonds (i.e., an "intact antibody molecule"), as well as multimers thereof (e.g., IgM), or antigen-binding fragments thereof. Each heavy chain (HC) contains a heavy chain variable region ("HCVR" or "V H ") and the heavy chain constant region (domain C H 1. C H 2 and C H Each light chain (LC) consists of a light chain variable region ("LCVR" or "V L ") and the light chain constant region (C L ) and V H and V L The regions can be subdivided into hypervariable regions, also known as complementarity determining regions (CDRs), interspersed with more conserved regions, also known as framework regions (FRs). H and V L consists of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the invention, the FRs of an antibody (or an antigen-binding fragment thereof) may be identical to human germline sequences or may be naturally occurring or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs. Thus, the CDRs in the heavy chain are designated "CHRH1", "CDRH2" and "CDRH3", respectively, and the CDRs in the light chain are designated "CDRL1", "CDRL2" and "CDRL3", respectively.

[0059] Substitution of one or more CDR residues or replacement of one or more CDRs is also possible. Antibodies that can omit one or two CDRs for binding are described in the scientific literature. Padlan et al. (1995 FASEB J.9:133-139) analyzed the contact areas between antibodies and their antigens based on published crystal structures and concluded that only about one-fifth to one-third of the CDR residues are actually in contact with the antigen. Padlan also found a number of antibodies in which one or two CDRs have no amino acids that contact the antigen (Vajdos et al., 2002 J. See also Mol Biol 320:415-428).

[0060] CDR residues that are not in contact with the antigen can be identified based on previous studies, by molecular modeling, and / or empirically from regions of the Kabat CDRs that are outside the Chothia CDRs (e.g., residues H60-H65 of CDRH2 are often not required). When a CDR or a residue(s) thereof is omitted, it is typically substituted with an amino acid that occupies the corresponding position in other human antibody sequences, or the consensus of such sequences. The position of substitution within the CDR, and the substituting amino acid, can also be selected empirically.

[0061] The fully human monoclonal antibodies disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The present invention includes antibodies and antigen-binding fragments thereof derived from any amino acid sequence disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue(s) in the germline sequence from which the antibody is derived, or to the corresponding residue(s) in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are collectively referred to herein as "germline mutations"). Starting from the heavy and light chain variable region sequences disclosed herein, one of skill in the art can readily generate numerous antibodies and antigen-binding fragments containing one or more individual germline mutations, or combinations thereof. In certain embodiments, V H Domain and / or V LAll framework and / or CDR residues within the domains are mutated back to the residue found in the germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only the mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more framework and / or CDR residue(s) are mutated to the corresponding residue(s) in a different germline sequence (i.e., a germline sequence that differs from the germline sequence from which the antibody was derived). Additionally, the antibodies of the invention can include any combination of two or more germline mutations within the framework and / or CDR regions, e.g., certain individual residues are mutated to the corresponding residue in a particular germline sequence, while certain other residues that differ from the original germline sequence are either maintained or mutated to the corresponding residue in a different germline sequence. Once the mutations are complete, antibodies and antigen-binding fragments containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonist or agonist biological properties (as the case may be), reduced immunogenicity, etc. The present invention includes antibodies and antigen-binding fragments obtained by this general method.

[0062] The present invention also includes complete monoclonal antibodies that include any variant of the CDR amino acid sequences disclosed herein that have one or more conservative substitutions, for example, the present invention includes antibodies that have CDR amino acid sequences that have, for example, 10 or less, 8 or less, 6 or less, 4 or less, etc., conservative amino acid substitutions compared to any CDR amino acid sequence disclosed herein.

[0063] The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human monoclonal antibodies of the invention may, for example, include amino acid residues not encoded by human germline immunoglobulin sequences in the CDRs, particularly CDR3 (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

[0064] However, the term "human antibody" as used herein does not contemplate a monoclonal antibody in which the CDR sequences are derived from the germline of another mammalian species (e.g., a mouse) and are grafted onto human FR sequences.

[0065] The term "humanized antibody" refers to a human antibody in which one or more CDRs of the antibody have been replaced with one or more corresponding CDRs from a non-human (e.g., mouse, rat, rabbit, primate) antibody. A humanized antibody may also contain certain non-CDR sequences or residues derived from such a non-human antibody, as well as one or more non-human CDR sequences. Such antibodies may also be referred to as "chimeric antibodies."

[0066] The term "recombinant" generally refers to any protein, polypeptide produced by genetic engineering methods, or cells expressing a gene of interest. The term "recombinant" as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. Proteins used in the immunogenic compositions of the invention may be isolated from natural sources or may be produced by genetic engineering methods.

[0067] The antibodies of the invention may in some embodiments be recombinant human antibodies. As used herein, the term "recombinant human antibody" is intended to include all antibodies, including human or humanized antibodies, prepared, expressed, created or isolated by recombinant means, e.g., antibodies expressed using a recombinant expression vector transfected into a host cell (discussed further below), antibodies isolated from a recombinant combinatorial human antibody library (discussed further below), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, created or isolated by any other means involving splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, in the case of animals transgenic for human Ig sequences, in vivo somatic mutagenesis) and the V H Area and V L The amino acid sequence of the region is human germline V H and V L Although derived and related to the sequence, it may not naturally exist within the human antibody germline repertoire in vivo.

[0068] The terms "specifically bind" or "specifically bind to" and the like mean that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding is at least about 1×10 -6 can be characterized by an equilibrium dissociation constant less than or equal to M (e.g., a small K Drepresents tight binding). Methods for determining whether two molecules specifically bind are well known in the art, such as equilibrium dialysis, surface plasmon resonance, and the like. As described herein, antibodies that specifically bind to, for example, RSV-F are identified by surface plasmon resonance, e.g., BIACORE™, biolayer interferometry, using, for example, a ForteBio Octet HTX instrument (Pall Life Sciences). Furthermore, multispecific antibodies that bind to the RSV-F protein and one or more additional antigens, such as an antigen expressed by HMPV, or bispecific antibodies that bind to two different regions of RSV-F are still considered herein to be "specifically binding" antibodies. In certain embodiments, the antibodies disclosed herein have a specific binding capacity of about 1×10 -6 M, about 1 x 10 -7 M, about 1 x 10 -8 M, about 1 x 10 -9 M, about 1 x 10 -10 M, about 1 x 10 -6 M and approx. 1×10 -7 Between M and approx. 1 x 10 -7 M and approx. 1×10 -8 Between M and approx. 1 x 10 -8 M and approx. 1×10 -9 Between M and approx. 1×10 -9 M and approx. 1×10 -10 This indicates the equilibrium dissociation constant (i.e., specificity) between M and

[0069] The term "high affinity" antibody refers to a monoclonal antibody that has a binding affinity for RSV-F and / or HMPV, the affinity being at least 10 as measured by surface plasmon resonance, e.g., Biolayer Interferometry using a BIACORE™, e.g., a ForteBio Octet HTX instrument (Pall Life Sciences), or solution affinity ELISA. -9 M, more preferably 10 -10 M, more preferably 10 -11 M, more preferably 10 -12 K of M D It is shown as:

[0070] The term "slow off rate," "Koff," or "kd" refers to an antibody that dissociates from RSV-F at a slow off rate of 1×10 or less as determined by surface plasmon resonance, e.g., a BIACORE™ or ForteBio Octet HTX instrument (Pall Life Sciences). -3 s ”1 Less than 1×10 -4 s ”1 It has the following rate constant:

[0071] As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, include naturally occurring, enzymatically-obtainable, synthetic, or genetically engineered polypeptides or glycoproteins that specifically bind to an antigen to form a complex. In certain embodiments, the term "antigen-binding portion" of an antibody or "antibody fragment" as used herein refers to one or more fragments of an antibody that retain the ability to bind to RSV-F and / or HMPV.

[0072] Antibody fragments may include Fab fragments, F(ab')2 fragments, Fv fragments, dAb fragments, fragments containing the CDRs, or isolated CDRs. Antigen-binding fragments of antibodies may be derived from intact antibody molecules using any suitable standard technique, such as, for example, proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable domains and (optional) constant domains. Such DNA is known and / or readily available, for example, commercially, from DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA may be sequenced and then manipulated, chemically or using molecular biology techniques, for example, to place one or more variable and / or constant domains in the appropriate positions, or to introduce codons, generate cysteine ​​residues, modify, add, or delete amino acids, etc.

[0073] Examples of antigen-binding fragments include, but are not limited to, (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single chain Fv (scFv) molecules, (vi) dAb fragments, and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable regions of an antibody (e.g., isolated complementarity determining regions (CDRs) such as CDR3 peptides) or constrained FR3-CDR3-FR4 peptides. As used herein, the phrase "antigen-binding fragment" also includes domain-specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other engineered molecules such as shark variable IgNAR domains.

[0074] An antigen-binding fragment of an antibody generally comprises at least one variable domain, which may be of any size or amino acid composition, and generally comprises at least one CDR adjacent to, or in frame with, one or more framework sequences. L Domain associated with V H In an antigen-binding fragment having a domain, the V H Domain and V L The domains can be positioned relative to each other in any suitable configuration. For example, the variable region can be a dimer, and the V H -V H , V H -V L or V L -V L Alternatively, the antigen-binding fragment of the antibody may comprise a monomeric V H or V L It may include a domain.

[0075] In certain embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody of the invention include: (i) a V H -C H 1, (ii) V H -C H 2. (iii) V H -C H 3. (iv) V H -C h 1-C h 2. (v) V H -C h 1-C h 2-C h 3. (vi) V H -C H 2-C H 3. (vii) V H -C L , (viii) V L -C H 1, (ix) V L -C H 2. (x)V L -C H 3. (xi) V L -C H 1-C H 2. (xii) V L -C H 1-C H 2-C H 3. (xiii) V L -C H 2-C H 3, and (xiv) V L -C LIn any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains can be directly linked to each other or linked by either a full hinge, partial hinge, or linker region. The hinge region can consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids, which provides a flexible or semi-flexible link between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the invention are in non-covalent association with each other and / or with one or more monomeric V H or V L The variable domain may comprise a homodimer or a heterodimer (or other multimer) of any of the variable domain configurations listed above, and a constant domain configuration having domains (e.g., held together by disulfide bond(s)).

[0076] As with intact antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies generally contain at least two different variable domains, each capable of specifically binding to a separate antigen or to a different epitope of the same antigen. Any of the multispecific antibody formats, including the exemplary bispecific antibody formats disclosed herein, can be made available for use with antigen-binding fragments of antibodies of the present invention using routine techniques available in the art.

[0077] In certain embodiments, the antibodies or antibody fragments of the invention may be conjugated to a therapeutic moiety ("immunoconjugate"), such as an antibiotic, a second anti-RSV-F antibody, an anti-HMPV antibody, a vaccine or a toxoid, or any other therapeutic moiety useful in the treatment of RSV infection and / or HMPV infection.

[0078] As used herein, an "isolated antibody" is intended to refer to an antibody or fragment thereof that is substantially free of other antibodies (Abs) having different antigenic specificities (e.g., an isolated antibody that specifically binds to RSV-F and / or HMPV) and is substantially free of Abs that specifically bind to antigens other than RSV-F and / or HMPV.

[0079] As used herein, a "blocking antibody" or "neutralizing antibody" (or an "antibody that neutralizes RSV-F and / or HMPV activity") is intended to refer to an antibody whose binding to a RSV-F or HMPV antigen results in inhibition of at least one biological activity of RSV-F and / or HMPV as disclosed herein. For example, an antibody of the invention may be useful in blocking RSV and / or HMPV fusion to a host cell, or in preventing syncytium formation, or in preventing a primary disease caused by RSV and / or HMPV. Alternatively, an antibody of the invention may demonstrate the ability to ameliorate at least one symptom of RSV infection and / or HMPV infection. This inhibition of RSV-F and / or HMPV biological activity can be assessed by measuring one or more indicators of RSV-F and / or HMPV biological activity by one or more of a number of standard in vitro assays (such as the neutralization assays described herein) or in vivo assays known in the art (e.g., animal models in which protection is examined by inoculation with RSV and / or HMPV and subsequent administration of one or more antibodies, as described herein).

[0080] As used herein, the term "surface resonance plasma" refers to an optical phenomenon that allows for the analysis of real-time biomolecular interactions by detecting changes in protein concentration within a biosensor matrix, for example, using the BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ).

[0081] As used herein, the term "K D" is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.

[0082] The term "epitope" refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule known as the paratope. A single antigen may have multiple epitopes. Thus, different antibodies may bind to different regions on the antigen and have different biological effects. The term "epitope" also refers to a site on an antigen to which B cells and / or T cells respond. It also refers to the region of an antigen that is bound by an antibody. Epitopes can be defined structurally or functionally. Functional epitopes are generally a subset of structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes can also be conformational, i.e., composed of nonlinear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups and, in certain embodiments, may have specific three dimensional structural and / or specific charge characteristics.

[0083] The term "substantial identity" or "substantially identical," when referring to a nucleic acid or a fragment thereof, indicates that there is at least about 90% of the nucleotide bases, and more preferably at least about 95%, 96%, 97%, 98% or 99% nucleotide sequence identity, as measured by any known algorithm of sequence identity, such as FASTA, BLAST or GAP, described below, when optimally aligned with the other nucleic acid, with appropriate nucleotide insertions or deletions. Thus, nucleic acid sequences exhibiting a particular percentage of "identity" share that percentage of identity and / or are "identical" to one another. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in some cases, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0084] In certain embodiments, the disclosed antibody nucleic acid sequences are, e.g., at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to other sequences, and / or all percentages in between, and / or share such percentages of identity with each other (or with specific subsets of the antibody sequences disclosed herein).

[0085] The term "substantial identity" or "substantially identical," as applied to polypeptides, means that two peptide sequences share at least 90% sequence identity, and even more preferably at least 95%, 98% or 99% sequence identity, when optimally aligned, such as with the programs GAP or BESTFIT using default gap weights. Accordingly, amino acid sequences that exhibit a particular percentage of "identity" share that percentage of identity and / or are "identical" to one another for that percentage. Accordingly, amino acid sequences that exhibit a certain percentage of "identity" share that percentage of identity and / or are "identical" to one another for that percentage.

[0086] In certain embodiments, the disclosed antibody amino acid sequences are, e.g., at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, and / or all percentages in between, to other sequences, and / or share such percentages of identity with each other (or with specific subsets of the antibody sequences disclosed herein).

[0087] Preferably, non-identical residue positions differ by conservative amino acid substitutions. A "conservative amino acid substitution" refers to the replacement of an amino acid residue with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially change the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art. (See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331). Examples of groups of amino acids having side chains with similar chemical properties are: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine, 2) aliphatic-hydroxyl side chains: serine and threonine, 3) amide-containing side chains: asparagine and glutamine, 4) aromatic side chains, phenylalanine, tyrosine, and tryptophan, 5) basic side chains: lysine, arginine, and histidine, 6) acidic side chains: aspartic acid and glutamic acid, and 7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443 45. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0088] Polypeptide sequence similarity is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software includes programs such as GAP and BESTFIT that can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species, or between wild-type proteins and their muteins. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignment and percent sequence identity of the regions of most overlap between the query sequence and the search sequence (Pearson (2000) supra). Another preferred algorithm for comparing the sequences of the present invention to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. (See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403 410 and (1997) Nucleic Acids Res. 25:3389 402).

[0089] In certain embodiments, the antibodies or antibody fragments for use in the methods of the invention may be monospecific, bispecific, or multispecific. Multispecific antibodies may be specific for different epitopes of one target polypeptide or may contain antigen binding domains specific for epitopes of two or more target polypeptides. An exemplary bispecific antibody format that can be used in the context of the present invention comprises a first immunoglobulin (Ig)C H 3 domain and a second Ig C H The first and second Ig C HThe three domains differ from each other by at least one amino acid, and the at least one amino acid difference inhibits binding of the bispecific antibody to Protein A compared to a bispecific antibody lacking the amino acid difference. H The IgC3 domain is bound to Protein A and the second ... H The second C3 domain contains a mutation that reduces or eliminates Protein A binding, such as the H95R modification (according to the IMGT exon numbering system, H435R in the EU numbering system). H 3 may further comprise a Y96F modification (IMGT, Y436F in EU). H Further modifications that may be recognized in 3 include D16E, L18M, N44S, K52N, V57M, and V82I for IgG1 monoclonal antibodies (IMGT, D356E, L358M, N384S, K392N, V397M, and V422I for EU), and N44S, K52N, and V82I for IgG2 monoclonal antibodies ( For IgG4 monoclonal antibodies, the residues are: N384S, K392N, and V422I (by IMGT, EU: N384S, K392N, and V422I) and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT, EU: Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I). Variations on the bispecific antibody formats described above are considered to be within the scope of the present invention.

[0090] The phrase "therapeutically effective amount" means an amount that is administered to produce the desired effect. The exact amount will depend on the purpose of the treatment and can be ascertained by one skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0091] An "immunogenic composition" refers to a composition comprising an antigen / immunogen, e.g., a microorganism, such as a virus or bacteria, or a component thereof, a protein, a polypeptide, a fragment of a protein or polypeptide, an inactivated whole cell, a subunit or an attenuated virus, or a polysaccharide, or a combination thereof, which is administered to stimulate the humoral and / or cellular immune system of the recipient against one or more of said antigens / immunogens present in the immunogenic composition. The immunogenic compositions of the invention can be used to treat humans susceptible to, or suffering from or suspected of being susceptible to, RSV infection and / or HMPV infection by means of administering the immunogenic composition via a systemic route. These administrations can include injections, patches, or application of other transdermal delivery devices via intramuscular (im), intradermal (id), intranasal or inhalation routes, or subcutaneous (sc) routes. In certain embodiments, the immunogenic compositions may be used in the manufacture of vaccines that can be used to passively protect or treat mammals, or to induce polyclonal or monoclonal antibodies.

[0092] The terms "vaccine" or "vaccine composition," used interchangeably, refer to a composition that contains at least one immunogenic composition that induces an immune response in an animal.

[0093] In certain embodiments, the protein of interest comprises an antigen. The terms "antigen," "immunogen," "antigenic," "immunogenic," "antigenically active," and "immunologically active," with respect to a molecule, refer to any substance capable of inducing a specific humoral and / or cellular immune response. In certain embodiments, an antigen comprises an epitope, as defined above.

[0094] As used herein, an "immunologically protective amount" refers to an amount of antigen that is effective to induce an immunogenic response in a recipient and is sufficient to prevent or ameliorate signs or symptoms of a disease, including adverse health effects or complications thereof. Either or both humoral or cellular immunity can be induced. The immunogenic response of the animal to the composition can be assessed, for example, indirectly through measurement of antibody titers, lymphocyte proliferation assays, or directly through monitoring of signs and symptoms after inoculation with a microorganism. The protective immunity conferred by the immunogenic composition or vaccine can be assessed, for example, by measuring the reduction in shedding of the inoculated organism, reduction in clinical signs, such as mortality, morbidity, body temperature, and general condition, health, and performance of the subject. The immune response can include, but is not limited to, induction of cellular and / or humoral immunity. The amount of the composition or vaccine that is therapeutically effective can vary depending on the particular organism used or the condition of the animal being treated or vaccinated.

[0095] As used herein, an "immune response" or "immunological response" in a subject refers to the development of a humoral immune response, a cellular immune response, or a humoral and cellular immune response to an antigen / immunogen. A "humoral immune response" refers to one that is mediated, at least in part, by antibodies. A "cellular immune response" refers to one that is mediated by T lymphocytes or other white blood cells, or both, and includes the production of cytokines, chemokines, and similar molecules produced by activated T cells, white blood cells, or both. Immune responses can be determined using standard immunoassays and neutralization assays known in the art.

[0096] As used herein, "immunogenicity" refers to the ability of a protein or polypeptide to elicit a specific immune response against an identified disease-causing bacterium or virus.

[0097] Unless otherwise indicated, the term "antibody" as used herein shall be understood to include antibody molecules comprising two heavy immunoglobulin chains and two light immunoglobulin chains (i.e., "intact antibody molecules"), as well as antigen-binding fragments thereof. As used herein, "antigen-binding portions" of antibodies, "antigen-binding fragments" of antibodies, and the like, include naturally occurring, enzymatically obtained, synthetic, or genetically engineered polypeptides or glycoproteins that specifically bind to an antigen to form a complex.

[0098] Preparation of human antibodies As disclosed herein, anti-RSV and / or anti-RSV / anti-HMPF cross-neutralizing antibodies can be obtained using B cell selection techniques available to those skilled in the art, and for example, as described below in the Examples. Methods for producing human antibodies in transgenic mice are also known in the art and can be used to induce antibodies according to the present disclosure. Any such known methods can be used in the context of the present invention to generate human antibodies that specifically bind to RSV-F (see, for example, U.S. Patent No. 6,596,541).

[0099] In a specific embodiment, the antibodies of the invention have a binding affinity of about 1.0×10 when measured binding to antigen immobilized in either a solid or liquid phase. -7 M ~ approx. 1.0×10 -12 Affinity in the range of M (K D In certain embodiments, the antibodies of the invention have a binding affinity of about 1×10 -7 M ~ approx. 6×10 -10 Affinity in the range of M (K D In certain embodiments, the antibodies of the invention have a binding affinity of about 1×10 -7 M ~ approx. 9×10 -10 Affinity in the range of M (K D ).

[0100] The anti-RSV-F and / or anti-HMPV antibodies and antibody fragments disclosed herein include proteins having amino acid sequences that differ from those of the described antibodies, but retain the ability to bind to RSV-F. Such mutant antibodies and antibody fragments contain one or more amino acid additions, deletions, or substitutions when compared to the parent sequence, but exhibit essentially the same biological activity as the described antibodies. Similarly, DNA sequences encoding the antibodies of the invention include sequences that contain one or more nucleotide additions, deletions, or substitutions when compared to the disclosed sequences, but encode antibodies or antibody fragments that are essentially biologically equivalent to the antibodies or antibody fragments of the invention.

[0101] Two antigen-binding proteins or antibodies are considered to be bioequivalent if they are pharmaceutical equivalents or alternatives that do not show significant differences in the rate and extent of absorption when administered at the same molar dose, either in single or multiple doses, under comparable experimental conditions. Some antibodies are considered equivalents or alternatives when the extent of absorption is comparable but the rate of absorption is not, and may still be considered bioequivalent because such differences in absorption rates are intentional, reflected in the labeling, are not essential, for example, to achieving effective body drug concentrations in chronic use, and are not considered medically significant in the particular pharmaceutical product studied.

[0102] In one embodiment, two antigen binding proteins are bioequivalent if there are no clinically significant differences in their safety, purity, and potency.

[0103] In an embodiment, two antigen binding proteins are bioequivalent if a patient can switch one or more times between the reference product and the biological product without increasing the risk of side effects, such as clinically significant changes in immunogenicity, or can switch one or more times between the reference product and the biological product without a decrease in efficacy compared to continued treatment without such switching.

[0104] In certain embodiments, two antigen binding proteins are biologically equivalent if they both act by a common mechanism or mechanisms of action for one or more conditions of use, to the extent that such mechanisms are known.

[0105] Bioequivalence may be demonstrated by in vivo and / or in vitro methods, including, for example, (a) in vivo studies in humans or other mammals that measure the concentration of the antibody or its metabolites as a function of time in blood, plasma, serum, or other body fluids, (b) in vitro studies that correlate with and are reasonably predictive of human in vivo bioavailability data, (c) in vivo studies in humans or other mammals that measure the relevant acute pharmacological effects of the antibody (or its target) as a function of time, and (d) well-controlled clinical trials that establish the safety, efficacy, or bioavailability or bioequivalence of the antibody.

[0106] Biologically equivalent variants of the antibodies of the invention can be constructed, for example, by making various substitutions of residues or sequences or by deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity can be deleted or replaced with other amino acids to prevent the formation of unnecessary or incorrect intramolecular disulfide bridges during renaturation. Additionally, biologically similar antibodies can include antibody variants that contain amino acid changes that modify the glycosylation characteristics of the antibody, for example, mutations that eliminate or eliminate glycosylation.

[0107] Biological and biophysical characteristics of antibodies In certain embodiments, the antibodies and antigen-binding fragments thereof of the present invention specifically bind to respiratory syncytial virus (RSV) F protein (F), and at least one of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3 amino acid sequences of such antibodies or antigen-binding fragments thereof is at least 70% identical, at least 75% identical, 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, 100% identical, and / or all percentages therebetween, to at least one of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3 amino acid sequences disclosed in Table 6 of an antibody selected from antibody numbers 232 to 372 disclosed in Table 6. In certain embodiments, such antibodies also have at least one, two, three, four, five, six, seven, eight, nine, ten or more of the features disclosed in the following eleven paragraphs.

[0108] Without wishing to be bound by any theory, it is believed that the antibodies and antigen-binding fragments thereof may function by binding to RSV-F, preferably in the PreF conformation, and thereby act to block fusion of the viral membrane with the host cell membrane. The antibodies of the present invention may also function by binding to RSV-F, and thereby block viral transmission between cells and block syncytium formation associated with RSV infection of cells. Advantageously, both RSV subtype A and RSV subtype B are effectively blocked or neutralized by most of the anti-RSV antibodies disclosed herein.

[0109] In certain embodiments, the antibodies and antigen-binding fragments thereof of the present invention exhibit superior binding affinity for PreF-type RSV-F compared to PostF-type RSV-F.

[0110] In other specific embodiments, the antibodies and antigen-binding fragments thereof of the present invention advantageously exhibit a clean or low polyreactivity profile (see, e.g., WO2014 / 179363 and Xu et al., Protein Eng Des Sel, Oct;26(10)663-70) and are therefore particularly suitable for development as safe, effective and viable therapeutic and / or prophylactic anti-RSV and / or HMPV treatments.

[0111] In certain embodiments, the antibodies and antigen-binding fragments thereof, without wishing to be bound by any theory, may function to block or inhibit RSV fusion to the cell membrane, for example, by binding to any one or more of antigenic sites Φ, I, II, III, IV, or site V of the PreF conformation of the F protein. In certain embodiments, the antibodies of the present invention exhibit antigenic site specificity for PreF site Φ, site V, or site III compared to RSV-F site I, site II, or site IV.

[0112] In certain embodiments, at least a portion of the epitopes with which the antibodies and antigen-binding fragments of the present invention interact include the α3 helix and a portion of the β3 / β4 hairpin of PreF. In certain embodiments, substantially all of the epitopes of such antibodies include the α3 helix and the β3 / β4 hairpin of PreF. In yet further embodiments, the antibodies of the present invention cross-compete with antibodies that recognize a portion or substantially all of the α3 helix and the β3 / β4 hairpin of PreF.

[0113] In certain embodiments, the antibodies and antigen-binding fragments thereof of the present invention have an in vitro neutralization potency (IC) of between about 0.5 micrograms per milliliter (ug / ml) and about 5 ug / ml, between about 0.05 ug / ml and about 0.5 ug / ml, or less than about 0.05 mg / ml. 50 ) is shown.

[0114] In certain embodiments, the binding affinity and / or epitope specificity of the antibodies and antigen-binding fragments thereof for any of the RSV-F variants designated as 1, 2, 3, 4, 5, 6, 7, 8, 9, and DG in FIG. 7A is reduced or eliminated compared to the binding affinity and / or epitope specificity of the antibodies or antigen-binding fragments thereof for RSV-F or RSV-F DS-Cav1.

[0115] In certain embodiments, the antibodies and antigen-binding fragments thereof of the present invention have cross-neutralizing potency (IC) against human metapneumovirus (HMPV) and RSV. 50In certain such embodiments, the antibodies and antigen-binding fragments thereof of the present invention have at least one of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3 amino acid sequences of such antibodies that are at least 70% identical, at least 75% identical, 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical, and / or all percentages in between, to at least one of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3 amino acid sequences disclosed in Table 6 of antibody No. 340 disclosed in Table 6.

[0116] In certain embodiments, the antibodies and antigen-binding fragments thereof do not comprise D25, MPE8, palivizumab, motavizumab, or AM-14. In certain embodiments, the antibodies and antigen-binding fragments thereof do not comprise D25, MPE8, palivizumab, or motavizumab. In certain embodiments, the antibodies and antigen-binding fragments thereof do not comprise MPE8, palivizumab, or motavizumab. In certain embodiments, the antibodies and antigen-binding fragments thereof do not comprise D25, palivizumab, or motavizumab. In certain embodiments, the antibodies and antigen-binding fragments thereof do not comprise D25. In certain embodiments, the antibodies and antigen-binding fragments thereof do not comprise MPE8. In certain embodiments, the antibodies and antigen-binding fragments thereof do not comprise palivizumab. In certain embodiments, the antibodies and antigen-binding fragments thereof of the invention do not comprise motavizumab.

[0117] In certain embodiments, the antibodies and antigen-binding fragments thereof of the present invention comprise one or more of D25, MPE8, palivizumab, motavizumab, and / or AM-14.

[0118] In certain embodiments, the antibodies and antigen-binding fragments thereof of the present invention have a neutralizing potency (IC) that is at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 50-fold, at least about 55-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, greater than about 100-fold, and any number between the aforementioned values, greater than D25 and / or palivizumab. 50 ) is shown.

[0119] In certain embodiments, the antibodies and antigen-binding fragments thereof of the present invention comprise the CDRH3 amino acid sequence of any one of the antibodies designated Antibody No. 232 to Antibody No. 372 disclosed in Table 6.

[0120] In certain embodiments, the antibodies and antigen-binding fragments thereof of the present invention comprise the CDRH2 amino acid sequence of any one of the antibodies designated Antibody No. 232 to Antibody No. 372 disclosed in Table 6.

[0121] In certain embodiments, the antibodies and antigen-binding fragments thereof of the present invention comprise the CDRH1 amino acid sequence of any one of the antibodies designated Antibody No. 232 to Antibody No. 372 disclosed in Table 6.

[0122] In certain embodiments, the antibodies and antigen-binding fragments thereof of the present invention comprise the CDRL3 amino acid sequence of any one of the antibodies designated Antibody No. 232 to Antibody No. 372 disclosed in Table 6.

[0123] In certain embodiments, the antibodies and antigen-binding fragments thereof of the present invention comprise the CDRL2 amino acid sequence of any one of the antibodies designated Antibody No. 232 to Antibody No. 372 disclosed in Table 6.

[0124] In certain embodiments, the antibodies and antigen-binding fragments thereof of the present invention comprise the CDRL1 amino acid sequence of any one of the antibodies designated Antibody No. 232 to Antibody No. 372 disclosed in Table 6.

[0125] In certain embodiments, the antibodies and antigen-binding fragments thereof of the present invention comprise any combination of two, three, four, five, or six of the features disclosed in the immediately preceding six paragraphs.

[0126] In certain embodiments, the antibody and antigen-binding fragment thereof of the present invention comprises the heavy chain (HC) amino acid sequence of any one of the antibodies designated as Antibody No. 232 to Antibody No. 372 disclosed in Table 6. In certain embodiments, the antibody and antigen-binding fragment thereof of the present invention comprises the light chain (LC) amino acid sequence of any one of the antibodies designated as Antibody No. 232 to Antibody No. 372 disclosed in Table 6. In certain embodiments, the antibody and antigen-binding fragment thereof of the present invention comprises the heavy chain (HC) amino acid sequence and light chain (LC) amino acid sequence of any one of the antibodies designated as Antibody No. 232 to Antibody No. 372 disclosed in Table 6.

[0127] In certain embodiments, the antibodies and antigen-binding fragments of the present invention are at least 70% identical, at least 75% identical, 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical, and / or all percentages in between, to any one of the antibodies designated Antibody No. 232 to Antibody No. 372 disclosed in Table 6.

[0128] In a specific embodiment, the antibody and antigen-binding fragment thereof of the present invention are selected from the group consisting of the antibodies designated Antibody No. 232 to Antibody No. 372 disclosed in Table 6, respectively.

[0129] In certain embodiments, an isolated nucleic acid sequence encoding an antibody or antigen-binding fragment thereof that specifically binds to a respiratory syncytial virus (RSV) F protein and antigen-binding fragments thereof is provided, wherein at least one of the amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3 of the antibody or antigen-binding fragment thereof is selected from antibody numbers 232 to 372 disclosed in Table 6. 1, CDRL2, and / or CDRL3, to at least one of the amino acid sequences of CDRL1, CDRL2, and / or CDRL3, which are at least 70% identical, at least 75% identical, 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical, and / or all percentages therebetween. In certain embodiments, such nucleic acid sequences are selected from those nucleic acid sequences disclosed in Table 6, and their complements.

[0130] In certain embodiments, isolated nucleic acid sequences encoding the antibodies and antigen-binding fragments thereof of the invention are provided, including sequences encoding the CDRH3 amino acid sequences of the antibodies designated Antibody No. 232 to Antibody No. 372 disclosed in Table 6. In certain embodiments, such nucleic acid sequences are selected from those nucleic acid sequences disclosed in Table 6 and their complements.

[0131] In certain embodiments, isolated nucleic acid sequences encoding the antibodies and antigen-binding fragments thereof of the invention are provided, including sequences encoding the CDRH2 amino acid sequences of the antibodies designated Antibody No. 232 to Antibody No. 372 disclosed in Table 6. In certain embodiments, such nucleic acid sequences are selected from those nucleic acid sequences disclosed in Table 6 and their complements.

[0132] In certain embodiments, isolated nucleic acid sequences encoding the antibodies and antigen-binding fragments thereof of the invention are provided, including sequences encoding the CDRH1 amino acid sequences of the antibodies designated Antibody No. 232 to Antibody No. 372 disclosed in Table 6. In certain embodiments, such nucleic acid sequences are selected from those nucleic acid sequences disclosed in Table 6 and their complements.

[0133] In certain embodiments, isolated nucleic acid sequences encoding the antibodies and antigen-binding fragments thereof of the invention are provided, including sequences encoding the CDRL3 amino acid sequences of the antibodies designated Antibody No. 232 to Antibody No. 372 disclosed in Table 6. In certain embodiments, such nucleic acid sequences are selected from those nucleic acid sequences disclosed in Table 6 and their complements.

[0134] In certain embodiments, isolated nucleic acid sequences encoding the antibodies and antigen-binding fragments thereof of the invention are provided, including sequences encoding the CDRL2 amino acid sequences of the antibodies designated Antibody No. 232 to Antibody No. 372 disclosed in Table 6. In certain embodiments, such nucleic acid sequences are selected from those nucleic acid sequences disclosed in Table 6 and their complements.

[0135] In certain embodiments, isolated nucleic acid sequences encoding the antibodies and antigen-binding fragments thereof of the invention are provided, including sequences encoding the CDRL1 amino acid sequences of the antibodies designated Antibody No. 232 to Antibody No. 372 disclosed in Table 6. In certain embodiments, such nucleic acid sequences are selected from those nucleic acid sequences disclosed in Table 6 and their complements.

[0136] In certain embodiments, isolated nucleic acid sequences encoding the antibodies and antigen-binding fragments thereof of the invention are provided, including sequences encoding the heavy chain (HC) amino acid sequences of the antibodies designated Antibody No. 232 to Antibody No. 372 disclosed in Table 6. In certain embodiments, such nucleic acid sequences are selected from those nucleic acid sequences disclosed in Table 6 and their complements.

[0137] In certain embodiments, isolated nucleic acid sequences encoding the antibodies and antigen-binding fragments thereof of the invention are provided, including sequences encoding the light chain (LC) amino acid sequences of the antibodies designated Antibody No. 232 to Antibody No. 372 disclosed in Table 6. In certain embodiments, such nucleic acid sequences are selected from those nucleic acid sequences disclosed in Table 6 and their complements.

[0138] In certain embodiments, isolated nucleic acid sequences encoding the antibodies and antigen-binding fragments thereof of the invention are provided, wherein such nucleic acid sequences are selected from the group consisting of sequences that are at least 70% identical, at least 75% identical, 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 100% identical, and / or all percentages in between, to any one of the nucleic acid sequences disclosed in Table 6 and their complements, respectively.

[0139] In certain embodiments, there are provided expression vectors comprising the isolated nucleic acid sequences disclosed herein, and throughout, and particularly in the immediately preceding ten paragraphs.

[0140] In certain embodiments, there is provided a host cell transfected, transformed or transduced with the nucleic acid sequence disclosed immediately above and / or the expression vector.

[0141] Epitope mapping and related techniquesAs described above and in the Examples below, applicants have characterized the epitope specificity, bin assignments, and antigenic site assignments of the antibodies and antigen-binding fragments thereof of the present invention. In addition to methods for such characterization, a variety of other techniques are available to those skilled in the art that can be used to perform such characterizations or to determine whether an antibody "interacts with one or more amino acids" in a polypeptide or protein. Exemplary techniques include routine cross-blocking assays, such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb, NY). Other methods include alanine scanning mutation analysis, peptide blot analysis (Reineke (2004) Methods Mol Biol 248:443-63), peptide cleavage analysis, crystallographic studies, and NMR analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be used (Tomer (2000) Protein Science 9:487-496). Another method that can be used to identify amino acids in a polypeptide with which an antibody interacts is hydrogen / deuterium exchange detected by mass spectrometry. In general terms, hydrogen / deuterium exchange involves deuterium labeling of a protein of interest followed by binding of an antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water and exchangeable protons in amino acids protected by the antibody complex undergo back exchange from deuterium to hydrogen at a slower rate than exchangeable protons in amino acids that are not part of the interface. As a result, amino acids that form part of the protein / antibody interface are able to retain deuterium and therefore exhibit a relatively large mass compared to amino acids that are not included in the interface. After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry, thereby revealing deuterium-labeled residues that correspond to the specific amino acids with which the antibody interacts.See, e.g., Ehring (1999) Analytical Biochemistry 267(2):252-259, Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0142] Those skilled in the art will appreciate that epitopes may be formed from both contiguous or non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids generally are retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding generally are lost upon treatment with denaturing solvents. An epitope generally comprises at least 3, usually at least 5 or 8-10 amino acids in a unique spatial conformation.

[0143] Modification-assisted profiling (MAP), also known as antigen structure-based antibody profiling (ASAP), is a method for classifying numerous monoclonal antibodies (mAbs) against the same antigen according to the similarity of the binding profile of each antibody to chemically or enzymatically modified antigen surfaces (US Publication No. 2004 / 0101920). Each category may reflect a unique epitope that is either distinct or partially overlapping with the epitopes represented by the other categories. This technique allows for rapid filtering of genetically identical antibodies so that characterization can be focused on genetically distinct antibodies. When applied to hybridoma screening, MAP can facilitate the identification of rare hybridoma clones that produce monoclonal antibodies with desired characteristics. Using MAP, the antibodies of the present invention can be classified into groups of antibodies that bind to different epitopes.

[0144] In certain embodiments, the antibodies and / or antigen-binding fragments thereof interact with amino acid sequences that include amino acid residues that are modified in one or more of the F protein patch mutants disclosed in the Examples, e.g., and shown in Figure 7A and designated RSV F variants 1, 2, 3, 4, 5, 6, 7, 8, 9, and DG. In certain embodiments, such antibodies and antigen-binding fragments thereof interact with amino acid sequences that include amino acid residues modified in RSV F variant 2. In certain embodiments, the antibodies and / or antigen-binding fragments thereof interact with amino acid residues that extend beyond the region(s) identified above by about 5-10 amino acid residues, or about 10-15 amino acid residues, or about 15-20 amino acid residues toward either the amino or carboxy terminus of the RSV-F protein.

[0145] In certain embodiments, the antibodies of the invention do not bind to the same epitope on the RSV-F protein as palivizumab, motavizumab, MPE8, or AM-14.

[0146] It is understood that a person skilled in the art can easily determine whether an antibody binds to the same epitope as a reference anti-RSV-F antibody or competes for binding with it by using routine methods available in the art. For example, to determine whether a test antibody binds to the same epitope as a reference RSV-F antibody of the present invention, the reference antibody is bound to a RSV-F protein or peptide under saturating conditions. The ability of the test antibody to bind to a RSV-F molecule is then evaluated. If the test antibody can bind to RSV-F after saturation binding with the reference anti-RSV-F antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-RSV-F antibody. Conversely, if the test antibody cannot bind to a RSV-F molecule after saturation binding with the reference anti-RSV-F antibody, the test antibody may bind to the same epitope as the epitope bound by the reference anti-RSV-F antibody of the present invention.

[0147] To determine whether an antibody competes for binding with a reference anti-RSV-F antibody, the binding methodology described above is performed in two steps. In the first step, the reference antibody is bound to a RSV-F molecule under saturating conditions, and then the binding of the test antibody to the RSV-F molecule is evaluated. In the second step, the test antibody is bound to a RSV-F molecule under saturating conditions, and then the binding of the reference antibody to the RSV-F molecule is evaluated. If in both steps only the first (saturating) antibody can bind to the RSV-F molecule, it is concluded that the test antibody and the reference antibody compete for binding to RSV-F. Those skilled in the art will understand that an antibody that competes for binding with a reference antibody does not necessarily bind to the same epitope as the reference antibody, but may sterically block the binding of the reference antibody by binding an overlapping or adjacent epitope.

[0148] Two antibodies bind to the same or overlapping epitopes if they each competitively inhibit (block) the binding of the other to the antigen. That is, one antibody inhibits at least 50%, but preferably 75%, 90%, or even 99%, of the other antibody, as measured in a competitive binding assay, by 1-, 5-, 10-, 20-, or more than 100-fold (see, e.g., Junghans et al., Cancer Res. (1990) 50:1495-1502). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that inhibit or eliminate binding of one antibody inhibit or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that inhibit or eliminate binding of one antibody inhibit or eliminate binding of the other antibody.

[0149] Additional routine experiments (e.g., peptide mutagenesis and binding analysis) can then be performed to confirm whether the observed lack of binding of the test antibody is indeed due to binding to the same epitope as the reference antibody, or whether steric blocking (or another phenomenon) is responsible for the observed lack of binding. This type of experiment can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

[0150] immunoconjugate The present invention includes a human RSV-F monoclonal antibody conjugated to a therapeutic moiety ("immunoconjugate"), such as an agent that can reduce the severity of a primary infection with RSV and / or HMPV or ameliorate at least one of the symptoms associated with RSV infection and / or HMPV infection, such as cough, fever, pneumonia, or the severity thereof. Such an agent can be a second, different antibody against RSV-F and / or HMPV, or a vaccine. The type of therapeutic moiety that can be conjugated to the anti-RSV-F antibody and / or anti-HMPV antibody takes into account the condition to be treated and the desired therapeutic effect to be achieved. Alternatively, if the desired therapeutic effect is to treat sequelae or symptoms associated with RSV and / or HMPV infection, or any other condition resulting from infection, such as, but not limited to, pneumonia, it may be advantageous to appropriately conjugate an agent to treat the sequelae or symptoms of the condition or to reduce any side effects of the antibody of the present invention. Examples of suitable agents for forming immunoconjugates are known in the art, see, for example, WO05 / 103081.

[0151] multispecific antibodies The antibodies of the present invention can be monospecific, bispecific, or multispecific. A multispecific antibody can be specific for different epitopes of one target polypeptide, or can contain antigen-binding domains specific for two or more target polypeptides. See, for example, Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. The antibodies of the present invention can be linked or co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or a fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent bonding, etc.) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific antibody or a multispecific antibody having a second binding specificity.

[0152] An exemplary bispecific antibody format that can be used in accordance with the present invention comprises a first immunoglobulin (Ig) C H 3 domain and second Ig C H 3 domain, and the first and second Ig C H The three domains differ in at least one amino acid, and the at least one amino acid difference inhibits binding of the bispecific antibody to Protein A compared to a bispecific antibody without the amino acid difference. H The IgC3 domain binds to Protein A and H The second C3 domain contains a mutation that reduces or eliminates Protein A binding, such as the H95R modification (according to IMGT exon numbering, H435R in EU numbering). H 3 may further comprise a Y96F modification (IMGT, Y436F in EU). H3Further modifications that may be found in the IgG1 antibodies include D16E, L18M, N44S, K52N, V57M, and V82I (IMGT, EU: D356E, L358M, N384S, K392N, V397M, and V422I), IgG2 antibodies include N44S, K52N, and V82I (IMGT, EU: N384S, K392N, and V422I), and IgG4 antibodies include Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (IMGT, EU: Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I). Variations on the bispecific antibody formats described above are within the scope of the present invention.

[0153] Therapeutic Administration and Formulations The present invention provides therapeutic compositions (including pharmaceutical compositions) comprising the anti-RSV-F and / or HMPV antibodies of the present invention, or antigen-binding fragments thereof. The therapeutic compositions of the present invention are administered with suitable carriers, excipients, and other agents that, when incorporated into the formulation, provide improved mobility, delivery, tolerance, and the like. Numerous suitable formulations can be found in the formulary known to any pharmacist, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (such as LIPOFECTIN™), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998) J Pharm Sci. See also Technol 52:238-31 1.

[0154] The dose of each antibody of the present invention may vary depending on the age and physique of the subject to be administered, the target disease, pathological condition, administration route, and the like. When using the antibodies of the present invention to treat a patient's RSV infection and / or HMPV infection, or to treat one or more symptoms associated with RSV infection and / or HMPV infection, such as cough or pneumonia associated with a patient's RSV infection and / or HMPV infection, or to reduce the severity of the disease, it is usually advantageous to administer each antibody of the present invention intravenously or subcutaneously in a single dose of about 0.01 to about 30 mg / kg body weight, more preferably about 0.1 to about 20 mg / kg body weight, or about 0.1 to about 15 mg / kg body weight, or about 0.02 to about 7 mg / kg body weight, about 0.03 to about 5 mg / kg body weight, or about 0.05 to about 3 mg / kg body weight, or about 1 mg / kg body weight, or about 3.0 mg / kg body weight, or about 10 mg / kg body weight, or about 20 mg / kg body weight. Multiple doses may be administered as necessary. The frequency and duration of the treatment can be adjusted depending on the severity of the condition. In certain embodiments, the antibodies or antigen-binding fragments of the invention can be administered as an initial dose of at least about 0.1 mg to about 800 mg, about 1 mg to about 600 mg, about 5 mg to about 300 mg, or about 10 mg to about 150 mg, up to about 100 mg, or up to about 50 mg. In certain embodiments, the initial dose can be followed by administration of the antibodies or antigen-binding fragments thereof with a second or multiple subsequent doses that can be approximately equal to or less than the initial dose, separated by at least 1 to 3 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 12 weeks, or at least 14 weeks.

[0155] A variety of delivery systems are known and can be used to administer the pharmaceutical compositions of the present invention, including liposomes, microparticles, microcapsules, encapsulation in recombinant cells capable of expressing the mutant virus, and receptor-mediated endocytosis (e.g., Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions may be administered by any suitable route, including, for example, infusion or bolus injection, absorption through epithelial or mucosal linings (e.g., oral mucosa, nasal mucosa, rectal and intestinal mucosa, etc.), and may be administered with other biologically active agents. Administration may be systemic or local. It can be delivered as an aerosolized formulation (see U.S. Publication Nos. 2011 / 0311515 and 2012 / 0128669). Delivery of agents useful for treating respiratory diseases by inhalation has become increasingly popular (AJ Bitonti and JADumont, (2006), Adv. Drug Deliv. Rev, 58:1 106-1118). In addition to being effective in treating localized pulmonary disease, such delivery mechanisms may also be useful for systemic delivery of antibodies (see Maillet et al. (2008), Pharmaceutical Research, Vol. 25, No. 6, 2008).

[0156] Pharmaceutical compositions can also be delivered in vesicles, in particular liposomes (see, eg, Langer (1990) Science 249:1527-1533).

[0157] In certain circumstances, the pharmaceutical composition can be delivered in a controlled release system. In certain embodiments, a pump can be used. In other embodiments, a polymeric material can be used. In yet other embodiments, the controlled release system can be placed close to the target of the composition, thus requiring only a fraction of the systemic dose.

[0158] The injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injections, drip infusions, and the like. These injectable preparations may be prepared by known methods. For example, the injectable preparations may be prepared by dissolving, suspending, or emulsifying the above-mentioned antibody or a salt thereof in a sterile aqueous medium or oily medium that has been conventionally used for injections. Examples of the aqueous medium for injection include physiological saline, isotonic solutions containing glucose, and other adjuvants, which may be used in combination with appropriate solubilizers such as alcohol (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 moles) adduct of hydrogenated castor oil)]. Examples of the oily medium include sesame oil and soybean oil, which may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The injection preparation thus prepared is preferably filled into a suitable ampule.

[0159] The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. In addition, for subcutaneous delivery, a pen delivery device can be easily used to deliver the pharmaceutical composition of the present invention. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge containing the pharmaceutical composition. Once all the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. The disposable pen delivery device does not include a replaceable cartridge. Rather, the disposable pen delivery device is pre-filled with the pharmaceutical composition that is held in a reservoir within the device. Once the pharmaceutical composition in the reservoir is depleted, the entire device is discarded.

[0160] Numerous reusable pen and autoinjector delivery devices are suitable for subcutaneous delivery of the pharmaceutical compositions of the present invention, including, by way of example only, the AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), the DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland), the HUMALOG MIX 75 / 25™ pen, the HUMALOG™ pen, the HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), the NOVOPEN™ I, II, and III (Novo Nordisk, Copenhagen, Denmark), the NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), the BD™ pen (Becton Dickinson, Franklin Lakes, NJ), the OPTIPEN™, and the OPTIPEN™, to name just a few. Examples of disposable pen delivery devices that can be used for subcutaneous delivery of the pharmaceutical compositions of the present invention include, but are not necessarily limited to, the SOLOSTAR pen (Sanofi-Aventis), FLEXPEN (Novo Nordisk), and KWIKPEN (Eli Lilly), the SURECLICK autoinjector (Amgen, Thousand Oaks, CA), PENLET (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and the HUMIRA pen (Abbott Labs, Abbott Park, IL), to name just a few.

[0161] Advantageously, the pharmaceutical composition for oral or parenteral use described above is prepared in a dosage form with a unit dose suitable for the dosage of the active ingredient. Such dosage forms in unit doses include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The content of the antibody described above is generally about 5 to about 500 mg per dosage form per unit dose, particularly in the form of an injection, and for other dosage forms, it is preferable that the antibody described above is contained in an amount of about 5 to about 100 mg and about 10 to about 250 mg.

[0162] Dosage regimen In certain embodiments, multiple doses of antibodies against RSV-F and / or HMPV or pharmaceutical compositions comprising or encoding these antibodies can be administered to a subject over a period of time. The method of this aspect of the invention includes sequentially administering multiple doses of antibodies against RSV-F and / or HMPV to a subject, or sequentially administering multiple doses of a pharmaceutical composition comprising or encoding an antibody or antigen-binding fragment thereof of the invention to a subject. As used herein, "sequentially administering" refers to administering doses of antibodies against RSV-F and / or HMPV or pharmaceutical compositions to a subject at different time points, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The invention includes methods comprising sequentially administering to the patient a single initial dose of an antibody to RSV-F and / or HMPV or a composition comprising or encoding such an antibody, followed by one or more secondary doses of an antibody to RSV-F and / or HMPV or a composition comprising or encoding such an antibody, and, optionally, followed by one or more tertiary doses of an antibody to RSV-F and / or HMPV or a composition comprising or encoding such an antibody.

[0163] The terms "primary dose," "secondary dose," and "tertiary dose" refer to the temporal order of administration of the antibodies or compositions of the invention against RSV-F and / or HMPV. Thus, a "primary dose" is a dose administered at the beginning of a treatment regimen (also known as a "baseline dose"), a "secondary dose" is a dose administered after the primary dose, and a "tertiary dose" is a dose administered after the secondary dose. The primary, secondary, and tertiary doses may all contain the same amount of antibodies against RSV-F and / or HMPV, but generally may differ from one another in terms of frequency of administration. However, in certain embodiments, the amount of antibodies against RSV-F and / or HMPV contained in the primary, secondary, and / or tertiary doses may vary from one another over the course of treatment (e.g., increased or decreased as needed). In certain embodiments, two or more (e.g., two, three, four, or five) doses are administered at the beginning of the treatment regimen as a "loading dose," followed by subsequent doses (e.g., "maintenance doses") administered less frequently.

[0164] In an exemplary embodiment of the invention, each secondary dose and / or tertiary dose is administered 1 to 26 (e.g., 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, or more) weeks after the most recent dose. As used herein, the phrase "most recent dose" refers to the dose of antibody against RSV-F and / or HMPV that a patient receives prior to administering the exact next dose in a series of multiple doses, without any intervening doses.

[0165] The method of this aspect of the invention can include administering any number of secondary and / or tertiary doses of antibodies against RSV-F and / or HMPV to the patient. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., two, three, four, five, six, seven, eight, or more) secondary doses are administered to the patient. Similarly, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., two, three, four, five, six, seven, eight, or more) tertiary doses are administered to the patient.

[0166] In embodiments involving multiple secondary doses, each secondary dose may be administered with the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1-2 weeks after the most recent dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered with the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2-4 weeks after the most recent dose. Alternatively, the frequency with which the secondary doses and / or tertiary doses are administered to the patient may vary over the course of the treatment regimen. The frequency of administration may also be adjusted by the physician over the course of treatment depending on the needs of the individual patient after clinical testing.

[0167] Thus, in certain embodiments, pharmaceutical compositions are provided that include one or more of the antibodies or antigen-binding fragments of the invention disclosed throughout this specification and a pharma- ceutical acceptable carrier and / or one or more excipients. In other particular embodiments, pharmaceutical compositions are provided that include one or more nucleic acid sequences encoding one or more of the antibodies or antigen-binding fragments of the invention, or one or more expression vectors carrying such nucleic acid sequences, and a pharma- ceutical acceptable carrier and / or one or more excipients.

[0168] Therapeutic uses of antibodies and pharmaceutical compositions Due to the binding and interaction with the RSV fusion protein (RSV-F), without wishing to be bound by any theory, it is believed that the antibodies and antigen-binding fragments thereof of the present invention are useful in preventing fusion of the virus with the host cell membrane, preventing the spread of the virus between cells, and inhibiting syncytium formation. In addition, the applicant herein demonstrates that a subset of the anti-RSV antibodies and antigen-binding fragments thereof surprisingly exhibit cross-neutralizing ability against HMPV, and thus the antibodies and antigen-binding fragments thereof, when administered prophylactically, are advantageous in preventing infection of subjects with RSV and / or HMPV. Alternatively, the antibodies of the present invention may be useful in improving at least one symptom associated with infection, such as cough, fever, pneumonia, or reducing the severity, duration, and / or frequency of infection. The antibodies of the present invention are also contemplated for prophylactic use in patients at risk of developing or acquiring RSV and / or HMPV infection. These patients include premature infants, full-term infants born during RSV season (late fall to early spring) and / or HMPV season, elderly patients (e.g., those 65 years of age or older), or patients with immunodeficiency due to illness, or patients treated with immunosuppressant therapeutic drugs, or patients who may have underlying conditions that predispose to RSV infection (e.g., cystic fibrosis patients, patients with congestive heart failure or other cardiac disease, patients with airway disorders, COPD patients) and / or HMPV infection. The antibodies of the invention may be used alone or in combination with a second or third agent to treat RSV and / or HMPV infection or to reduce at least one symptom or complication associated with, associated with, or caused by the RSV and / or HMPV infection, such as fever, cough, bronchiolitis, or pneumonia. Such second or third agents may be delivered simultaneously with the antibody of the invention, or they may be administered separately, before or after the antibody of the invention.The second or third agent may be an antiviral agent such as ribavirin, an NSAID to reduce fever or pain, or other agent, a different antibody that is another second antibody that specifically binds to RSV-F, an agent (e.g., an antibody) that binds to another RSV antigen such as RSV-G, a vaccine against RSV, an antigen such as an siRNA specific for a RSV antigen.

[0169] In yet a further embodiment of the invention, the antibodies of the invention are used for the preparation of a pharmaceutical composition for treating a patient suffering from RSV and / or HMPV infection. In yet another embodiment of the invention, the antibodies of the invention are used for the preparation of a pharmaceutical composition for reducing the severity of a primary infection with RSV and / or HMPV, or for shortening the duration of infection, or for reducing at least one symptom associated with RSV and / or HMPV infection. In a further embodiment of the invention, the antibodies of the invention are used in adjunctive therapy with any other agent useful for treating RSV and / or HMPV infection, such as antiviral agents, toxoids, vaccines, a second RSV-F antibody, or any other antibody specific for a RSV antigen, such as an RSV-G antibody, or any other palliative therapy known to those skilled in the art.

[0170] Thus, in certain embodiments, there is provided a method of treating or preventing a respiratory syncytial virus (RSV) infection or at least one symptom associated with a RSV infection, comprising administering to a patient in need thereof or suspected of being in need thereof one or more of the antibodies of the invention or antigen-binding fragments thereof (or pharmaceutical compositions of the invention) disclosed herein and throughout, such as one or more of the anti-RSV antibodies disclosed in Table 6, thereby treating or preventing the RSV infection or treating, alleviating, or reducing the severity of at least one symptom associated with a RSV infection.

[0171] In certain other embodiments, a method of treating or preventing a respiratory syncytial virus (RSV) infection or at least one symptom associated with a RSV infection is provided, comprising administering to a patient in need thereof or suspected of being in need thereof a nucleic acid sequence encoding one or more of the antibodies of the invention or antigen-binding fragments thereof, such as a nucleic acid sequence disclosed in Table 6, or a complement thereof, thereby treating or preventing the RSV infection or treating, alleviating, or reducing the severity of at least one symptom associated with a RSV infection.

[0172] In a further embodiment, a method is provided for treating or preventing a respiratory syncytial virus (RSV) infection or at least one symptom associated with a RSV infection, comprising administering to a patient in need thereof or suspected of being in need thereof a host cell having a nucleic acid sequence or an expression vector comprising such a nucleic acid sequence, wherein such nucleic acid sequence is selected from the group consisting of the sequences disclosed in Table 6 and complements thereof, thereby treating or preventing the RSV infection or treating, alleviating, or reducing the severity of at least one symptom associated with a RSV infection.

[0173] In a further embodiment, a method of treating or preventing a respiratory syncytial virus (RSV) infection or at least one symptom associated with a RSV infection is provided, comprising administering to a patient in need thereof or suspected of being in need thereof a pharmaceutical composition comprising one or more host cells having one or more of the antibodies or antigen-binding fragments thereof disclosed in Table 6, one or more nucleic acid sequences, or an expression vector comprising the nucleic acid sequences, wherein the nucleic acid sequences are selected from the group consisting of the sequences disclosed in Table 6 and complements thereof, and a pharma- ceutical composition comprising one or more nucleic acid sequences, wherein the nucleic acid sequences are selected from the group consisting of the sequences disclosed in Table 6 and complements thereof, and a pharma- ceutical composition comprising one or more pharma-ceutical carriers and / or one or more excipients, thereby treating or preventing the RSV infection or treating, alleviating, or reducing the severity of at least one symptom associated with a RSV infection.

[0174] In certain embodiments, there is provided a method of treating or preventing either a respiratory syncytial virus (RSV) infection or a human metapneumovirus (HMPV) infection, or at least one symptom associated with the RSV infection or the HMPV infection, comprising administering to a patient in need thereof or suspected of being in need thereof an antibody of the invention disclosed herein and throughout, or an antigen-binding fragment thereof, such as one or more of the anti-RSV antibodies disclosed in Table 6, thereby treating or preventing the RSV infection or treating, alleviating, or reducing the severity of at least one symptom associated with a RSV infection. In certain embodiments, the one or more antibodies or antigen-binding fragments thereof comprise antibody number 340 disclosed in Table 6.

[0175] In certain other embodiments, there is provided a method of treating or preventing either a respiratory syncytial virus (RSV) infection or a human metapneumovirus (HMPV) infection, or at least one symptom associated with the RSV infection or the HMPV infection, comprising administering to a patient in need thereof or suspected of being in need thereof a nucleic acid sequence encoding one or more of the antibodies of the invention or antigen-binding fragments thereof, such as the nucleic acid sequences disclosed in Table 6 and their complements, thereby treating or preventing the RSV infection or treating, alleviating, or reducing the severity of at least one symptom associated with the RSV infection. In certain embodiments, the one or more antibodies or antigen-binding fragments thereof comprise antibody number 340 disclosed in Table 6.

[0176] In a further embodiment, there is provided a method of treating or preventing either a respiratory syncytial virus (RSV) infection or a human metapneumovirus (HMPV) infection, or at least one symptom associated with the RSV infection or the HMPV infection, comprising administering to a patient in need thereof or suspected of being in need thereof a host cell having a nucleic acid sequence or an expression vector comprising such a nucleic acid sequence, the nucleic acid sequence being selected from the group consisting of the sequences disclosed in Table 6 and complements thereof, thereby treating or preventing the RSV infection or treating, alleviating, or reducing the severity of at least one symptom associated with a RSV infection. In a specific embodiment, the one or more antibodies or antigen-binding fragments thereof comprise antibody number 340 disclosed in Table 6.

[0177] In a further embodiment, there is provided a method of treating or preventing either a respiratory syncytial virus (RSV) infection or a human metapneumovirus (HMPV) infection, or at least one symptom associated with said RSV infection or said HMPV infection, comprising administering to a patient in need thereof, or suspected of being in need thereof, one or more of the antibodies of the invention or antigen-binding fragments thereof, one or more nucleic acid sequences thereof, or an expression vector comprising said nucleic acid sequences, wherein said nucleic acid sequences are selected from the group consisting of: and complements thereof, or administering a pharmaceutical composition comprising one or more host cells having one or more nucleic acid sequences selected from the group consisting of the sequences disclosed in Table 6 and complements thereof, and a pharma- ceutical composition comprising any of the sequences disclosed in Table 6 and a pharma- ceutical composition comprising one or more pharma-ceutical carriers and / or one or more excipients, thereby treating or preventing the RSV infection, or treating, alleviating, or reducing the severity of at least one symptom associated with a RSV infection. In certain embodiments, the one or more antibodies or antigen-binding fragments thereof comprise antibody No. 340 disclosed in Table 6.

[0178] Combination therapy As noted above, in certain embodiments, the methods disclosed herein include administering to a subject one or more additional therapeutic agents in combination with antibodies to RSV-F and / or HMPV. As used herein, the phrase "in combination with" means that the additional therapeutic agent is administered before, after, or simultaneously with a pharmaceutical composition comprising the anti-RSV-F antibody. The term "in combination with" also includes sequential or simultaneous administration of the anti-RSV-F antibody and a second therapeutic agent.

[0179] For example, when administered "before" a pharmaceutical composition comprising an anti-RSV-F antibody, the additional therapeutic agent may be administered about 72 hours, about 60 hours, about 48 hours, about 36 hours, about 24 hours, about 12 hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours, about 2 hours, about 1 hour, about 30 minutes, about 15 minutes, or about 10 minutes before administration of the pharmaceutical composition comprising the anti-RSV-F antibody. When administered "after" a pharmaceutical composition comprising the anti-RSV-F antibody, the additional therapeutic agent may be administered about 10 minutes, about 15 minutes, about 30 minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, or about 72 hours after administration of the pharmaceutical composition comprising the anti-RSV-F antibody. Administration "concurrently" or together with a pharmaceutical composition comprising the anti-RSV-F antibody means that the additional therapeutic agent is administered to the subject within less than 5 minutes (before, after, or simultaneously) with administration of a pharmaceutical composition comprising the anti-RSV-F antibody in a separate dosage form, or is administered to the subject as a single combined dosage formulation containing both the additional therapeutic agent and the anti-RSV-F antibody.

[0180] The combination therapy can include an anti-RSV-F antibody of the invention and any additional therapeutic agent with which the antibody of the invention, or a biologically active fragment of the antibody of the invention, can be advantageously combined.

[0181] For example, a second or third therapeutic agent can be used to help reduce the viral load in the lungs, such as an antiviral agent, e.g., ribavirin, and these antibodies can be used in combination with other therapies, such as toxoids, vaccines specific for RSV, secondary antibodies specific for RSV-F, or antibodies specific for other RSV antigens, such as RSV-G.

[0182] Diagnostic Uses of Antibodies The anti-RSV antibodies and antigen-binding fragments thereof of the present invention may also be used to detect and / or measure RSV and / or HMPV in a sample, for example, for diagnostic purposes. It is contemplated that confirmation of an infection believed to be due to RSV and / or HMPV may be performed by measuring the presence of the virus through the use of one or more of the antibodies of the present invention. An example of a diagnostic assay for RSV and / or HMPV may, for example, include contacting a sample obtained from a patient with an anti-RSV-F and / or HMPV antibody of the present invention, which is labeled with a detectable label or reporter molecule or used as a capture ligand to selectively isolate viruses containing the F protein from the patient sample. Alternatively, unlabeled anti-RSV-F and / or HMPV antibodies may be used for diagnostic purposes in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule may be 3 H, 14 C. 32 P, 35 S, or 125 The immunoconjugate may be a radioisotope such as I, a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine, or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, luciferase, etc. Specific examples of assays that can be used to detect or measure F protein and / or RSV, including HMPV, in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).

[0183] Samples that can be used in the RSV and / or HMPV diagnostic assays of the present invention include any tissue or body fluid sample that contains a detectable amount of RSV-F protein and / or HMPV or a fragment thereof and that can be obtained from a patient under normal or pathological conditions. In general, the level of RSV-F and / or HMPV in a particular sample obtained from a healthy patient (e.g., a patient not suffering from a disease or condition associated with the presence of RSV-F and / or HMPV) is measured to first establish a baseline or standard level of F protein from RSV and / or HMPV. This baseline level of RSV-F and / or HMPV can then be compared to the level of RSV-F and / or HMPV measured in a sample obtained from an individual suspected of RSV and / or HMPV infection or a condition associated with such infection. EXAMPLES

[0184] The applicant isolated and characterized 133 RSV F-specific monoclonal antibodies from memory B cells of a healthy adult donor, and used these antibodies to comprehensively profile the human antibody response to the RSV fusion protein (F) and comprehensively map the antigenic topology of RSV F. The antibody response to RSV F was found to consist of a wide variety of clones targeting several antigenic sites. Nearly half of the most potent antibodies targeted a vulnerable, previously undefined site near the apex of the prefusion (preF) conformation of RSV F, strongly supporting the development of RSV antibodies targeting this region and providing a vaccine candidate that preserves the membrane-distal hemisphere of the preF protein. In addition, this class of antibodies exhibits convergent sequence characteristics, thus providing a future means for rapid detection of these types of antibodies in human samples. Many of the preF-specific surface-bound antibodies from this donor were more than 100-fold more potent than palivizumab, and some antibodies cross-neutralized human metapneumovirus (HMPV). Collectively, these results have implications for the design and evaluation of RSV vaccine and antibody-based therapeutic candidates, and provide new options for passive prevention.

[0185] Large-scale isolation of RSV F-specific monoclonal antibodies from healthy human adult donors To comprehensively profile the human antibody response to RSV F, we isolated and characterized 133 monoclonal antibodies from memory B cells of a healthy adult donor ("Donor 006") who had no recorded history of RSV infection, although healthy adults are expected to experience multiple RSV infections throughout their lifetime (26).

[0186] The magnitude of the memory B cell response to RSV F in this donor was assessed by staining peripheral B cells with a mixture of fluorescently labeled pre- and post-fusion RSV F selection probes (Fig. 6A-6B) (11, 15). Both proteins were double labeled to eliminate background from nonspecific fluorochrome binding (27). Flow cytometry analysis revealed that class-switched (IgG + and IgA + ) showed that 0.04-0.18% of peripheral B cells were specific for RSV F (Fig. 1A and B), which is significantly lower than the percentage of RSV F-specific cells observed after experimental RSV infection and suggests that this donor likely had not been recently exposed to RSV (28). Notably, index sorting showed that 17-38% of circulating RSV F-specific B cells expressed IgA, indicating that IgA memory B cells against RSV F are present in the peripheral blood (Fig. 1B).

[0187] Approximately 200 RSV F-specific B cells were single-cell sorted from the donor samples, and antibody variable heavy (VH) and variable light (VL) chain genes were rescued by single-cell PCR (29). 133 cognate heavy and light chain pairs were subsequently cloned and expressed as full-length IgG in engineered Saccharomyces cerevisiae strains for further characterization (30). Preliminary binding studies showed that approximately 80% of the antibodies cloned from RSV F glycoprotein (F)-specific B cells bound to the recombinant RSV F protein.

[0188] Sequence analysis of the RSV F-specific antibody repertoire Sequence analysis of the isolated monoclonal antibodies revealed that the RSV-F-specific repertoire was highly diverse, containing more than 70 unique lineages (Fig. 1C and Table 2). This result is in stark contrast to the relatively partial repertoires observed in HIV-infected patients (37) or healthy donors after influenza vaccination (32). Compared to non-RSV-reactive antibodies (33), the RSV F-specific repertoire was generally skewed toward specific VH germline genes (VH1-18, VH1-2, VH1-69, VH2-70, VH4-304, and VH5-51) (Fig. 1D and Table 2) and toward longer heavy chain third complementarity determining region (CDRH3) lengths (generally about 14-18 amino acids in length, Fig. 1E and Table 2). Interestingly, a bias towards VH1-69 has also been observed in the anti-HIV-1, anti-influenza, and anti-HCV repertoires (34-36), and a recent study reported a substantial increase in the relative usage of VH1-18, VH1-2, and VH1-69 during acute dengue infection (37). It is therefore possible that these specific germline gene segments may have unique properties that facilitate the recognition of viral envelope proteins.

[0189] The average levels of somatic hypermutation (SHM) generally ranged from 16 to 30 nucleotide substitutions per VH gene (excluding CDRH3) (Fig. 1F and Table 2), which is comparable to the average levels of SHM observed in the anti-influenza antibody repertoire (32, 38) and is consistent with the recurrent nature of RSV infection (26). Interestingly, some antibodies contained more than 40 VH gene nucleotide substitutions, suggesting that multiple RSV infections can generate antibodies with very high levels of somatic hypermutation (SHM).

[0190] The majority of antibodies bind only to preF We then used biolayer interferometry to characterize the fusion-dependent markers of Cav1 and Cav2 in pre-fusion (DS-Cav1) or post-fusion (F The apparent binding affinity of the IgG to the furin-cleaved RSV F extracellular domain stabilized in a ΔFP (11, 15) conformation was measured. A relatively large proportion of antibodies (36-67%) bound only to preF (Fig. 2A and B; Table 3). The remaining majority of antibodies bound both preF and postF, and only 5-7% of antibodies showed only postF specificity (Fig. 2A and B; Table 3). The low prevalence of postF-specific antibodies in these donor repertoires is consistent with the observation that less than 10% of anti-RSV F serum binding activity specifically targets postF (8). Interestingly, however, the majority of cross-reactive antibodies bound with high apparent affinity to postF (Fig. 2A; Table 3), suggesting that these antibodies were likely elicited by postF and / or had matured in vivo in affinity to postF. Therefore, the significantly higher ratio of preF- vs. postF-specific antibodies is likely due to the higher immunogenicity of the unique surface of preF compared to postF, rather than to the increased abundance of preF in vivo. Finally, as expected based on the relatively high degree of sequence conservation between RSV subtypes, most antibodies showed binding reactivity to F proteins from both subtypes A and B (Fig. 2C; Table 3).

[0191] Because certain antiviral antibody specificities have been associated with polyreactivity and autoreactivity (39-41), we also tested RSV antibodies for polyreactivity using the high-throughput assay described above that correlates with downstream behaviors such as serum clearance (42, 43). 177 clinical antibodies and several broadly neutralizing HIV antibodies were also included for comparison. Interestingly, in contrast to the numerous broadly neutralizing HIV antibodies described above, the majority of RSV F-specific antibodies lacked significant polyreactivity in this assay (Figure 2D).

[0192] RSV F-specific antibodies target six major antigenic sites To map the antigen specificity of the RSV F-specific antibodies, we first performed competitive binding experiments using the yeast-based assay described above (44). First, these antibodies were tested for competition with the three preF-specific antibodies described above (10, 17, 21), D25, AM14, and MPE8, and with motavizumab, an affinity variant of palivizumab that binds to both preF and postF (10, 11, 45). The non-competing antibodies were then tested for competition with a site IV-directed monoclonal antibody (101F) (46), a site I-directed antibody (site I Ab), and two high affinity antibodies (High Affinity Ab 1 and High Affinity Ab 2, respectively), which did not compete strongly with each other or with any of the control antibodies. Based on the results of this competition assay, each antibody was assigned a bin (see, e.g., Table 4).

[0193] To confirm and improve the resolution of our epitope assignment, we used a Luminex-based assay to measure the binding of each antibody to a panel of preF mutants. Each mutant contained 2-4 mutations clustered together to form a patch on the surface of preF. A total of 9 patches were generated that uniformly covered the surface of preF (Figure 7A-7C). Deglycosylated preF was also included to identify antibodies targeting glycan-dependent epitopes. Binding of each antibody to the 10 preF mutants was compared to that of wild-type preF and used to assign patches (see, e.g., Table 4). The previously characterized antibodies D25, AM14, and motavizumab were used to validate the assay (see, e.g., Figure 7C and Table 4). The combined bin and patch data were then used to assign each antibody to a single antigenic site (Figure 3A-3G) defined based on previously determined F protein structure, resistance mutations, and secondary structure elements. Overall, these data indicate that the majority of isolated antibodies target the six dominant antigenic sites (φ, I, II, III, IV, and V) of prefusion RSV F. Interestingly, only a small proportion of the isolated antibodies had a binding profile similar to that of AM14, suggesting that antibodies targeting this quaternary epitope are not commonly elicited during natural infection. None of the antibodies were sensitive to F deglycosylation, demonstrating that glycan-dependent antibodies are also rarely elicited by natural RSV infection.

[0194] Analysis of the preF and postF binding activity of antibodies targeting each antigenic site (see, e.g., Figures 3C-G; Table 4) revealed that three sites (φ, III, and V) were found primarily in preF. Antibodies targeting sites φ and site III have been reported previously (10, 17), and these sites are located on the top and side of the preF spike, respectively. Approximately 18% of the antibodies from this donor recognized site φ, and approximately 20% recognized site III. A relatively large portion of the antibodies from this donor (approximately 26%) recognized a third preF-specific site, which has not been previously reported and is therefore designated herein as region site V (see, e.g., Figures 3C-G; Table 4). The majority of site V antibodies competed with D25, MPE8, and motavizumab, which was unexpected given the distance between the epitopes recognized by these three antibodies. The patch mutant analysis revealed that these antibodies interact with the α3 helix and β3 / β4 hairpin of preF. This region is located between the epitopes recognized by D25, MPE8, and motavizumab, explaining the unusual competition profile observed for this class of antibodies (see, e.g., FIG. 8). In addition to the three major preF-specific sites, a large number of antibodies that recognize antigenic site IV are preF-specific, likely due to contact with β22, which rapidly rearranges from preF to postF during translocation. Collectively, the epitope mapping data indicate that the majority of isolated antibodies target six dominant antigenic sites, about half of which are exclusively expressed in preF.

[0195] Substantially potent neutralizing antibodies target preF-specific epitopes These antibodies were then tested for neutralizing activity against RSV subtypes A and B using the high-throughput neutralization assay described above (15). More than 70% of the isolated antibodies demonstrated neutralizing activity, and approximately 35%-40% demonstrated strong neutralizing potency (IC 50≦0.05 μg / ml) (see, e.g., Figures 4A and 4B; Table 3). Notably, several clonally unrelated antibodies were more than 5.0-fold more potent than D25 and more than 100-fold more potent than palivizumab (see, e.g., Figure 4A; Table 3). Interestingly, there was no correlation between neutralizing potency and the level of SHM, suggesting that broad-spectrum SHM is not required for potent neutralization of RSV. Consistent with the binding cross-reactivity data, the majority of neutralizing antibodies showed activity against both subtypes A and B (Figures 4A-C; Table 3).

[0196] We next examined the relationship between preF and postF binding affinity and neutralization potency, which clearly demonstrated that the majority of highly potent antibodies bound preferentially or exclusively to preF (e.g., Figures 4D-G; see Table 3). Quantifying this difference revealed that highly potent antibodies (IC 50 <0.05 μg / ml) were specific for preF (e.g., see FIG. 9 ; Table 3 ), and the median IC 50 was 8-fold lower than the median for preF- and postF-cross-binding antibodies and 80-fold lower than the median for antibodies specifically recognizing postF (see, e.g., FIG. 4E; Table 3). Importantly, there was a positive correlation between preF binding and neutralization (P<0.001, r=0.24), and the apparent preF K D S is generally a neutralizing IC 50 (e.g., see Fig. 5A; Table 3). In contrast, there was no correlation between neutralizing potency and postF affinity (P = 0.44, r = -0.07) (e.g., see Fig. 5B; Table 3). This result is consistent with the occupancy model of antibody-mediated neutralization (47) and suggests that DS-Cav1 is a faithful antigen mimic of the native preF trimer. Notably, with an IC of less than 100 pM 50 Very few antibodies were neutralizing at , which is consistent with the upper limit of affinity maturation previously proposed ( 48 , 49 ).

[0197] Next, the relationship between neutralizing potency and antigenic site was analyzed. For example, the results summarized in Figure 5C, Table 3, and Table 4 showed that more than 60% of the highly potent neutralizing antibodies targeted antigenic sites φ and V, two of the three prefusion F-specific sites. In contrast, antibodies targeting sites III and IV showed a broad range of neutralizing potency, and antibodies targeting sites I and II were generally moderately neutralizing to non-neutralizing. Similar results were obtained using the binding affinity and neutralizing potency measured for subtype B (see, e.g., Figures 10A-C; Tables 3 and 4). Interestingly, a subset of site IV-specific antibodies neutralized with a substantially lower potency than expected based on preF binding affinity (see, e.g., Figure 5A; Table 3). This result may suggest that certain epitopes within site IV are less exposed with respect to the native envelope spikes expressed on the crowded surface of virions than recombinant preF.

[0198] Some antibodies cross-neutralize RSV and HMPV Given that RSV and human metapneumovirus (HMPV) F proteins share 33% amino acid identity and that certain RSV F-specific antibodies cross-neutralize HMPV (17, 50), antibodies from this donor were tested for neutralizing activity against HMPV. Of 133 antibodies tested, one neutralized HMPV (see, e.g., Table 5). Sequence analysis revealed that this antibody represents the VH1-46 germline gene and contains a significant degree of somatic hypermutation (see, e.g., Table 2 and Sequence Listing). This cross-neutralizing antibody bound both preF and PostF and competed with MPE8 (see, e.g., Table 5), consistent with previous studies showing that MPE8 cross-neutralizes four pneumoviruses, including RSF and HMPV (17). The results suggest that, in particular, highly conserved epitopes are relatively immunogenic in the context of natural RSV and / or HMPV infection.

[0199] Affinity maturation of RSV F-specific antibodies Some embodiments relate to affinity matured antibodies of any of the antibodies listed in Table 6 (each of which is understood to be a "parent" antibody for generating affinity matured variants). Affinity matured antibodies may be generated by mutagenesis of any one or more CDRs of the parent antibody. In certain embodiments, the invention provides affinity matured variants that include one or more point mutations, e.g., 0, 1, 2 or 3 point mutations, in the CDR sequences of any antibody listed in Table 6 or each of the antibodies that include the six CDR sequences of any antibody listed in Table 6. Affinity matured variants can be generated by any mutagenesis method using standard mutagenesis techniques to optimize binding characteristics, e.g., to increase the affinity of binding or increase the Kon or reduce the Koff, and have a K of at least 2-fold, 5-fold, 1 log, or 2 logs, or 3 logs, compared to the parent antibody. D Such affinity matured antibodies can be characterized by differences in their binding specificity and, for example, optimized affinity for binding to a target epitope as the parent antibody.

[0200] Selected anti-RSV antibodies were identified for affinity maturation. The oligos arrayed contained CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 sequences diversified via NNK diversity. These NNK oligos were incorporated into the parental HC or LC via DNA shuffling as previously reported (Stemmer WP et al., DNA shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution. Proc Natl Acad Sci USA. 1994 Oct 25;91(22):10747-51). Libraries were then generated by transforming VH and VL PCR products into yeast already containing either the parental light or heavy chain plasmids. Diversified libraries were selected using flow cytometry. For each FACS round, the libraries were affinity-pressurized with decreasing amounts of antigen, and clones with improved binding affinity were selected and expanded. Once improved binding populations were observed by flow cytometry (typically after two rounds of selection), single yeast clones were chosen for sequencing and characterization (Table 6).

[0201] Specific embodiments relate to affinity matured variants of antibody 267 in Table 6. In particular, each of antibody numbers 365-372 is an affinity matured variant of antibody number 267 in Table 6.

[0202] Antibody production and purification of affinity matured antibodies Yeast clones were grown to saturation and then induced for 48 hours at 30°C with shaking. After induction, yeast cells were pelleted and the supernatant was collected for purification. IgG was purified using a Protein A column and eluted with acetic acid, pH 2.0. Fab fragments were generated by papain digestion and purified with KappaSelect (GE Healthcare LifeSciences).

[0203] RSV in vitro neutralization in an ELISA-based microneutralization assay In vitro RSV neutralization was tested in an ELISA-based microneutralization assay using RSV-A strain A2 (ATCC, VR1540P). Virus (final multiplicity of infection of approximately 0.25) was added to 96-well plates containing serially diluted monoclonal antibodies in serum-free MEM and preincubated for 30 min at 4°C. Freshly trypsinized Hep-2 cells (1.5 x 10E 4 100 μl / well) were added to each well containing MEM supplemented with 5% FCS. After 4 days of incubation at 37° C. and 5% CO2, the medium was aspirated and the cells were washed twice with 200 μl PBS / well, air-dried and fixed with 100 μl acetone (80%). Quantification of expressed viral proteins by ELISA was used to measure RSV replication. For this purpose, the fixed cells were washed twice with PBS-0.1% Tween-20, blocked with PBS containing 1% skim milk for 1 h at room temperature and then stained with a polyclonal goat anti-RSV antibody preparation (BioRad, #7950-0004) in blocking buffer for 1 h at room temperature. Donkey anti-goat IgG HRP conjugate was used as detection reagent and 1-step-Ultra TMB (Thermo Fisher Scientific, #34209) was used as substrate. The percent inhibition of viral replication was calculated compared to control cells infected with virus in the absence of neutralizing antibodies. Isotype-matched control monoclonal antibodies were used in all experiments. Monoclonal antibody potency was calculated as the half-maximal inhibitory concentration (IC 50 The results are shown in FIG. 11 and show that in this setting, all monoclonal antibodies showed a wide range of IC 50 The values ​​demonstrate that RSV-A2 was neutralized.

[0204] Consideration A detailed understanding of the human antibody response to RSV infection will aid in the development and evaluation of RSV vaccines and therapeutic and / or prophylactic antibody candidates for the treatment and / or prevention of RSV infection. Previous studies have loosely mapped the epitopes targeted by RSV-specific neutralizing antibodies in human sera (4, 8), but the specificity and functional properties of antibodies induced by natural RSV infection remain largely undefined. As disclosed herein, using preF and postF stabilizing proteins (11, 15), a high-throughput antibody isolation platform, and a structure-guided collection of prefusion F mutants, human memory B cell responses to RSV F in naturally infected adult donors were clonally analyzed and highly potent and selective RSV neutralizing, as well as highly potent anti-RSV / anti-HMPV cross-selective and cross-neutralizing, were isolated and characterized.

[0205] In the analyzed repertoires, the ratio of preF-specific antibodies to antibodies recognizing both preF and postF was slightly greater than 1:1 (see, e.g., Fig. 2B). These values ​​are slightly smaller than those reported for human sera, indicating that approximately 70% of anti-F serum binding was specific for preF (8). This discrepancy could be the result of differences between the levels of individual antibodies contained in the sera, differences in the B cell phenotype obtained for a particular specificity, or differences between donors. Despite these small differences, the results of both studies suggest that preF-specific epitopes and epitopes shared by pre and postF are immunogenic during natural RSV infection, whereas the unique surface of postF is significantly less immunogenic.

[0206] The repertoire analysis disclosed herein showed that the majority of RSV F-specific antibodies target six dominant antigenic sites on pre-fusion RSV F: φ, I, II, III, IV, and V. These sites were defined based on previously determined structures, epitope binning / competition assays of the preF protein, resistance mutations, and secondary structure elements. The nomenclature for describing RSV F antigenic sites has evolved over time (6, 51-57), and previous mapping efforts were based on the post-fusion conformation of F and did not include surfaces that are exclusively present in preF. The crystal structure of preF has provided important information about the structure and function of F, as well as new reagents for mapping antibody binding sites on surfaces unique to preF and surfaces shared with postF. To a first approximation, each antibody can be primarily assigned to one of these sites. However, it is possible that antibody epitopes cover the entire surface of F and that there are antibodies that bind to two or more adjacent antigenic sites within a protomer, as well as quaternary antibodies that bind across the protomer.

[0207] Importantly, the results disclosed herein show that the most potently neutralizing antibodies target antigenic sites φ and V, both of which are located near the apex of the preF trimer. These findings are consistent with results from human serum mapping that determined that the majority of neutralizing activity can be removed by preincubation with preF (4, 8) and that preF-specific sites other than site φ account for a significant proportion of preF-specific neutralizing antibodies (8). Although antigenic site φ has been shown to be the target of potently neutralizing antibodies (8, 10), the interaction of antibodies with site V is less understood. Interestingly, most site V-directed antibodies have been found to share several converging sequence features, suggesting that these types of antibodies may be rapidly detected in human samples using high-throughput sequencing techniques (58). Applicants anticipate that this finding will be particularly advantageous in profiling antibody responses to RSV vaccine candidates that aim to preserve the apex of the preF trimer.

[0208] The broad panel of antibodies described herein offers new opportunities for passive prevention as well as treatment of RSV infection. Many of these antibodies neutralize RSV more potently than D25, and they serve as the basis for MEDI8897, a monoclonal antibody currently in clinical trials for the prevention of RSV in at-risk childhood adolescents (59). In addition, we have demonstrated that a subset of these antibodies cross-neutralize HMPV.

[0209] The development of an effective RSV vaccine presents several unique challenges, and the selection of an optimal vaccination strategy is of paramount importance. The detailed analysis of human antibody responses to natural RSV infection presented herein provides insight for the development of such a vaccine. Importantly, these results suggest that immunization of pre-immunized donors with preF immunogens would promote neutralizing responses, whereas the use of postF immunogens is likely to expand B cell clones with moderate or weak neutralizing activity. Similarly, immunization of RSV naive infants with preF immunogens is expected to activate naive B cells that target epitopes associated with substantially stronger neutralizing activity compared to postF immunogens. In addition, an ideal RSV vaccine would preserve antigenic sites φ and V, as these are targeted by the most potent antibodies elicited in response to natural RSV infection.

[0210] Thus, disclosed herein are highly selective, potent, and highly cross-neutralizing anti-RSV and anti-HMPV antibodies and vaccine candidates for the treatment and / or prevention of RSV and / or HMPV infection. Moreover, the reagents disclosed herein provide a useful set of tools for evaluation in clinical trials, which is important in selecting the optimal RSV vaccination or antibody-based treatment strategy from the many currently under investigation (60). [Table 1] [Table 2-1] [Table 2-2] [Table 2-3]

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

Table 2-9

Table 2-10

Table 2-11

Table 2-12

Table 2-13

Table 2-14

Table 2-15

Table 2-16

Table 2-17

Table 3-1

Table 3-2

Table 3-3

Table 3-4

Table 3-5

Table 3-6

Table 3-7

Table 3-8

Table 3-9

Table 3-10

Table 4-1

Table 4-2

Table 4-3

Table 4-4

[0211] Materials and Methods Study design To profile antibody responses to RSV F, peripheral blood mononuclear cells were obtained from adult donors approximately 20-35 years of age, and monoclonal antibodies from RSV F-reactive B cells were isolated. These antibodies were characterized by sequencing, binding, epitope mapping, and neutralization assays. All samples for this study were collected with informed consent from volunteers. The study was not blinded or randomized. At least two independent experiments were performed for each assay.

[0212] Generation of RSV F selection probe The soluble pre-fusion and post-fusion probes were based on the RSV F ΔFP and DS-Cav1 constructs that we had previously crystallized and determined to be in pre-fusion and post-fusion conformations, respectively (11, 15). To increase the binding avidity of the probes and to uniformly orient the RSV F protein, the trimeric RSV F protein was bound to tetrameric streptavidin via biotinylation of the C-terminal AviTag. For each probe, both the C-terminal His-Avi tagged version and the C-terminal StrepTagII version were co-transfected into FreeStyle 293-F cells. First, the secreted proteins were purified with Ni-NTA resin to remove trimers lacking the His-Avi tag. The eluate from the Ni-NTA purification was then purified with Strep-Tactin resin. Due to the low binding of a single StrepTagII to the Strep-Tactin resin, an additional wash step alone was able to remove the StrepTagged trimers. This resulted in the purification of trimers containing two StrepTagII tagged monomers, i.e., only one His-Avi tagged monomer. This purification scheme resulted in a single AviTag per trimer, which greatly reduced the aggregation or "daisy chaining" that occurs when trimeric proteins containing three AviTags are incubated with tetrameric streptavidin. RSV F trimers were biotinylated using biotin ligase BirA according to the manufacturer's instructions (Avidity, LLC). Biotinylated proteins were separated from excess biotin by size exclusion chromatography on a Superdex 200 column (GE Healthcare). Quantitation of the number of biotin moieties per RSV F trimer was performed using Quantitative Analysis. * The procedure was carried out using the Tag Biotin Kit according to the manufacturer's instructions (Vector Laboratories).

[0213] Single B cell sorting Peripheral blood mononuclear cells were stained with anti-human IgG (BV605), IgA (FITC), CD27 (BV421), CD8 (PerCP-Cy5.5), CD14 (PerCP-Cy5.5), CD19 (PECy7), CD20 (PECy7), and a mixture of dual-labeled DS-Cav1 and F ΔFP tetramers (50 nM each). Dual-labeled RSV F tetramers were generated by incubating individual AviTagged RSV F proteins with premium grade phycoerythrin-conjugated streptavidin (Molecular Probes) or premium grade allophycocyanin-conjugated streptavidin at a molar ratio of 4:1 for at least 20 minutes on ice. Tetramers were prepared fresh for each experiment. Single cells were sorted on a BD fluorescence-activated cell sorter Aria II into 96-well PCR plates (BioRad) containing 20 μL / well of lysis buffer [5 μL of 5× first strand cDNA buffer (Invitrogen), 0.25 μL RNaseOUT (Invitrogen), 1.25 μL dithiothreitol (Invitrogen), 0.625 μL NP-40 (New England Biolabs), and 12.6 μL dH20]. The plates were immediately frozen on dry ice and then stored at −80°C.

[0214] Amplification and cloning of antibody variable genes Single B cell PCR was performed as previously described (22). Briefly, IgH, Igλ, and Igκ variable genes were amplified by RT-PCR and nested PCR reactions using a cocktail of IgG- and IgA-specific primers (22). Primers used in the second round of PCR contained 40 base pairs of 5′ and 3′ homology to the cleaved expression vector, allowing cloning by homologous recombination into Saccharomyces cerevisiae (40). PCR products were cloned into S. cerevisiae using the lithium acetate method for chemical conversion (41). Each transformation reaction contained 20 μL of unpurified heavy and light chain PCR products and 200 ng of cleaved heavy and light chain plasmids. Following transformation, individual yeast colonies were picked for sequencing and characterization.

[0215] Expression and purification of IgG and Fab fragments Anti-RSV F IgG was expressed in S. cerevisiae cultures grown in 24-well plates as previously described (23). Fab fragments used in the competition assay were generated by digesting IgG with papain for 2 h at 30°C. Digestion was stopped by adding iodoacetamide, and the Fab and Fc mixture was passed through Protein A agarose to remove Fc fragments and undigested IgG. The Protein A resin flow-through was then passed over CaptureSelect™ IgG-CH1 affinity resin (ThermoFischer Scientific) and eluted with 200 mM acetic acid / 50 mM NaCl pH 3.5 into 1 / 8 volume of 2 M Hepes pH 8.0. The Fab fragments were then buffer exchanged into PBS pH 7.0.

[0216] Biolayer Interferometry Binding Analysis Binding of IgG to DS-Cav1 and FΔFP was determined by BLI measurements using a ForteBio Octet HTX instrument (Pall Life Science). DFor screening, IgG was immobilized on an AHQ sensor (Pall Life Sciences) and exposed to 100 nM antigen in PBS containing 0.1% BSA (PBSF) for the association step, followed by a dissociation step with PBSF buffer. Data were analyzed using ForteBio Data Analysis Software 7. To calculate the association and dissociation rates, the data was fitted to a 1:1 binding model and the K D Let ratio k d / k a The calculation was carried out using

[0217] Antibody competition assay Antibody competition assays were performed as previously described (23). Antibody competition was measured by the ability of a control anti-RSV F Fab to inhibit the binding of yeast surface-expressed anti-RSV F IgG to either DS-Cav1 or FΔ FP. 50 nM biotinylated DS-Cav1 or FΔ FP was preincubated with 1 μM competitor Fab for 30 min at room temperature and then added to the suspension of yeast expressing anti-RSV F IgG. Unbound antigen was removed by washing with PBS containing 0.1% BSA (PBSF). After washing, a 1:500 dilution of streptavidin-Alexa Fluor 1000 Fab was used to bind the yeast surface-expressed anti-RSV F IgG. Bound antigen was detected using 633 and analyzed by flow cytometry using a FACSCanto II (BD Biosciences). The level of competition was assessed by measuring the fold reduction in antigen binding in the presence of competitor Fab compared to antigen-only control. An antibody was considered a competitor if there was a greater than 5-fold reduction in binding in the presence of control Fab compared to antigen-only control.

[0218] Expression, purification, and biotinylation of preF patch mutants A panel of nine patches with 2-4 mutations uniformly covering the surface of the preF molecule was designed based on the structure of prefusion RSV F (10). For known antigenic sites, including those recognized by motavizumab, 101F, D25, AM14, and MPE8, the patches contained residues associated with viral escape or known to be important for antibody binding. When possible, highly conserved residues across 184 subtype A, subtype B, and bovine RSV F sequences were avoided to minimize the possibility of perturbing the protein structure. The mutations present in each patch variant are shown in FIG. 7A. Mutations for each patch variant were cloned into the prefusion stabilized RSV F (DS-Cav1) construct using a C-terminal AviTag for site-specific biotinylation. Proteins were secreted from FreeStyle 293-F cells, purified with Ni-NTA resin, and biotinylated using biotin ligase BirA according to the manufacturer's instructions (Avidity, LLC). Biotinylated proteins were separated from excess biotin by size exclusion chromatography on a Superdex 200 column (GE Healthcare). A deglycosylated version of DS-Cav1 was generated by expressing DS-Cav1 in the presence of 1 μM kifunensine, followed by digestion with 10% (wt / wt) EndoH prior to biotinylation.

[0219] Luminex assay for binding of patch mutants Binding of isolated antibodies to the patch variants was determined using a high-throughput Luminex assay. Each biotinylated variant and DS-Cav1 control were coupled to avidin-coated MagPlex beads (Bio-Rad), with each bead given a bead identification number reflecting a unique ratio of red and infrared dyes embedded within the bead. The coupled beads were then mixed with 6-fold serial dilutions of each antibody ranging from 400 nM to 1.4 pM in a 384-well plate. The beads were washed using a magnetic microplate washer (BioTek) before incubation with a PE-conjugated mouse anti-human IgG Fc secondary antibody (Southern Biotech). The beads were sorted and PE binding was measured using a FLEXMAP 3D flow cytometer (Luminex).

[0220] RSV neutralization assay Virus stocks were prepared and maintained as previously described (61). Recombinant mKate-RSV expressing the prototype subtype A (strain A2) and subtype B (18537) F genes and Katushka fluorescent protein were constructed as described by Hotard et al. (62). HEp-2 cells were maintained in Eagle's minimum essential medium containing 10% fetal bovine serum supplemented with glutamine, penicillin, and streptomycin. Antibody neutralization was measured by a fluorescent plate reader neutralization assay (15). 2.4 × 10 430 μL of the medium containing HEp-2 cells were seeded into a 384-well black optical bottom plate (Nunc, Thermo Scientific). IgG samples were serially diluted 4-fold from 1:10 to 1:163840 and an equal volume of recombinant mKate-RSV A2 was added. The samples were mixed and incubated at 37°C for 1 hour. After incubation, 50 μL of the sample and virus mixture was added to the cells in the 384-well plate and incubated at 37°C for 22-24 hours. The assay plate was then measured for fluorescence intensity at Ex 588 nm and Em 635 nm in a microplate reader (SpectraMax Paradigm, molecular devices). The IC of neutralization for each sample was calculated. 50 was calculated by curve fitting using Prism (GraphPad Software Inc.).

[0221] Human metapneumovirus neutralization assay A given amount of GFP-expressing hMPV recombinant virus (NL / 1 / 00, A1 subline, Bernard Antibodies were mixed with serially diluted monoclonal antibodies (gifts from Dette van den Hoogen and Ron Fouchier, Rotterdam, the Netherlands) and then added to growing cultures of 118 Vero cells in 96-well plates containing Dulbecco's Modified Eagle medium supplemented with 10% fetal bovine serum. After 36 h, medium was removed, PBS was added, and the amount of GFP per well was measured using a Tecan microplate reader M200. Fluorescence values ​​were expressed as a percentage of the no-antibody virus control.

[0222] Polyreactivity assay Antibody polyreactivity was assessed using a previously described high-throughput assay (43) measuring binding to solubilized CHO cell membrane preparations (SMPs). Briefly, 2 million IgG-displaying yeast cells were transferred to a 96-well assay plate, pelleted, and the supernatant removed. The pellet was resuspended in 50 μl of 1:10 diluted stock b-SMP and incubated on ice for 20 min. Cells were then washed twice with ice-cold PBSF, and the cell pellet was resuspended in 50 μl of secondary labeling mixture (Extravidin-R-PE, anti-human LC-FITC, and propidium iodide). This mixture was incubated on ice for 20 min, followed by two washes with ice-cold PBSF. Cells were then resuspended in 100 μl of ice-cold PBSF, and the plate was run on a FACSCanto II (BD Biosciences) using the HTS sample injector. Flow cytometry data were analyzed for mean fluorescence intensity in the R-PE channel and normalized to appropriate controls to assess non-specific binding.

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[0286] An abbreviated sequence listing is shown below in Table 6. This abbreviated sequence listing shows the following 16 sequence elements contained in each of the 133 antibodies identified as described above and designated antibody numbers (Ab#) 232 to 364, in the following order: Heavy chain variable region ("HC") nucleic acid sequence Heavy chain variable region ("HC") amino acid sequence Heavy chain variable region CDR H1 ("H1") amino acid sequence Heavy chain variable region CDR H1 ("H1") nucleic acid sequence Heavy chain variable region CDR H2 ("H2") amino acid sequence Heavy chain variable region CDR H2 ("H2") nucleic acid sequence Heavy chain variable region CDR H3 ("H3") amino acid sequence · Heavy chain variable region CDR H3 (“H3”) nucleic acid sequence Light chain variable region ("LC") nucleic acid sequence Light chain variable region ("LC") amino acid sequence Light chain variable region CDR L1 ("L1") amino acid sequence Light chain variable region CDR L1 ("L1") nucleic acid sequence Light chain variable region CDR L2 ("L2") amino acid sequence Light chain variable region CDR L2 ("L2") nucleic acid sequence Light chain variable region CDR L3 ("L3") amino acid sequence Light chain variable region CDR L3 ("L3") nucleic acid sequence

[0287] The abbreviated sequence listing of antibodies 365 to 372 shows the following 10 sequence elements contained in each of the eight antibodies identified as above and represented by antibody numbers (Ab#) 365 to 372, in the following order: Heavy chain variable region ("HC") nucleic acid sequence Heavy chain variable region ("HC") amino acid sequence Heavy chain variable region CDR H1 ("H1") amino acid sequence Heavy chain variable region CDR H2 ("H2") amino acid sequence Heavy chain variable region CDR H3 ("H3") amino acid sequence Light chain variable region ("LC") nucleic acid sequence Light chain variable region ("LC") amino acid sequence Light chain variable region CDR L1 ("L1") amino acid sequence Light chain variable region CDR L2 ("L2") amino acid sequence Light chain variable region CDR L3 ("L3") amino acid sequence [Table 6-1]

Table 6-2

Table 6-3

Table 6-4

Table 6-5

Table 6-6

Table 6-7

Table 6-8

Table 6-9

Table 6-10

Table 6-11

Table 6-12

Table 6-13

Table 6-14

Table 6-15

Table 6-16

Table 6-17

Table 6-18

Table 6-19

Table 6-20

Table 6-21

Table 6-22

Table 6-23

Table 6-24

Table 6-25

Table 6-26

Table 6-27

Table 6-28

Table 6-29

Table 6-30

Table 6-31

Table 6-32

Table 6-33

Table 6-34

Table 6-35

Table 6-36

Table 6-37

Table 6-38

Table 6-39

Table 6-40

Table 6-41

Table 6-42

Table 6-43

Table 6-44

Table 6-45

Table 6-46

Table 6-47

Table 6-48

Table 6-49

Table 6-50

Table 6-51

Table 6-52

Table 6-53

Table 6-54

Table 6-55

Table 6-56

Table 6-57

Table 6-58

Table 6-59

Table 6-60

Table 6-61

Table 6-62

Table 6-63

Table 6-64

Table 6-65

Table 6-66

Table 6-67

Table 6-68

Table 6-69

Table 6-70

Table 6-71

Table 6-72

Table 6-73

Table 6-74

Table 6-75

Table 6-76

Table 6-77

Table 6-78

Table 6-79

Table 6-80

Table 6-81

Table 6-82

Table 6-83

Table 6-84

Table 6-85

Table 6-86

Table 6-87

Table 6-88

Table 6-89

Table 6-90

Table 6-91

Table 6-92

Table 6-93

Table 6-94

Table 6-95

Table 6-96

Table 6-97

Table 6-98

Table 6-99

Table 6-100

Table 6-101

Table 6-102

Table 6-103

Table 6-104

Table 6-105

Table 6-106

Table 6-107

Table 6-108

Table 6-109

Table 6-110

Table 6-111

Table 6-112

Table 6-113

Table 6-114

Table 6-115

Table 6-116

Table 6-117

Table 6-118

Table 6-119

Table 6-120

Table 6-121

Table 6-122

Table 6-123

Table 6-124

Table 6-125

Table 6-126

Table 6-127

Table 6-128

Table 6-129

Table 6-130

Table 6-131

Table 6-132

Table 6-133

Table 6-134

Table 6-135

Table 6-136

Table 6-137

Table 6-138

Table 6-139

Table 6-140

Table 6-141

Table 6-142

Table 6-143

Table 6-144

Table 6-145

Table 6-146

Table 6-147

Table 6-148

Table 6-149

Table 6-150

Table 6-151

Table 6-152

Table 6-153

[0288] Additional Embodiments EMBODIMENT 1 1. An isolated antibody or antigen-binding fragment thereof that specifically binds to a respiratory syncytial virus (RSV) F protein (F), wherein at least one of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences of the antibody or antigen-binding fragment thereof is at least 70% identical, at least 75% identical, 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, and / or all percentages therebetween to at least one of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3 amino acid sequences disclosed in Table 6 of an antibody selected from antibody numbers 232 to 372 disclosed in Table 6, and wherein the antibody or antigen-binding fragment thereof has the following characteristics: a) the antibody or antigen-binding fragment thereof cross-competes with the antibody or antigen-binding fragment thereof for binding to RSV-F; b) the antibody, or antigen-binding fragment thereof, exhibits better binding affinity for the PreF form of RSV-F compared to the PostF form; c) the antibody or antigen-binding fragment thereof exhibits a clean or low polyreactivity profile; d) the antibody or antigen-binding fragment thereof exhibits neutralizing activity against RSV subtype A and RSV subtype B in vitro; e) the antibody or antigen-binding fragment thereof exhibits antigenic site specificity for RSV-F at site Φ, site I, site II, site III, site IV, or site V; f) the antibody or antigen-binding fragment thereof exhibits antigenic site specificity for RSV-F site Φ, site V, or site III compared to RSV-F site I, site II, or site IV; g) at least a portion of the epitope with which the antibody or antigen-binding fragment thereof interacts includes the α3 helix and β3 / β4 hairpin of PreF; h) The antibody or antigen-binding fragment thereof has an in vitro neutralizing activity (IC) of between about 0.5 micrograms per milliliter (ug / ml) and about 5 ug / ml, between about 0.05 ug / ml and about 0.5 ug / ml, or less than about 0.05 mg / ml. 50 ), i) the binding affinity and / or epitope specificity of the antibody or antigen-binding fragment thereof to any one of the RSV-F variants designated 1, 2, 3, 4, 5, 6, 7, 8, 9, and DG in FIG. 7A is reduced or eliminated compared to the binding affinity and / or epitope specificity of the antibody or antigen-binding fragment thereof to RSV-F or RSV-F DS-Cav1; j) The antibody or antigen-binding fragment thereof has a cross-neutralizing activity (IC 50 ), k) the antibody or antigen-binding fragment thereof does not comprise D25, MPE8, palivizumab, motavizumab, or AM-14; l) The antibody or antigen-binding fragment thereof has a neutralizing potency (IC) that is at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 50-fold, at least about 55-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, greater than about 100-fold, and any number between the aforementioned values. 50 The antibody or antigen-binding fragment thereof has one or more of the following characteristics:

[0289] EMBODIMENT 2 2. The isolated antibody or antigen-binding fragment thereof of embodiment 1, wherein the antibody or antigen-binding fragment thereof comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or at least twelve of features a)-l).

[0290] EMBODIMENT 3 The antibody or antigen-binding fragment thereof is a) the CDRH3 amino acid sequence of any one of the antibodies designated as Antibody No. 232 to Antibody No. 372 in Table 6; b) the CDRH2 amino acid sequence of any one of the antibodies designated as Antibody No. 232 to Antibody No. 372 in Table 6; c) the CDRH1 amino acid sequence of any one of the antibodies designated as Antibody No. 232 to Antibody No. 372 in Table 6; d) the CDRL3 amino acid sequence of any one of the antibodies designated as Antibody No. 232 to Antibody No. 372 in Table 6; e) the CDRL2 amino acid sequence of any one of the antibodies designated Antibody No. 232 to Antibody No. 372 in Table 6; f) the CDRL1 amino acid sequence of any one of the antibodies designated Antibody No. 232 to Antibody No. 372 in Table 6; or g) any combination of two or more of a), b), c), d), e), and f); 3. The isolated antibody or antigen-binding fragment thereof of embodiment 1 or 2, comprising:

[0291] EMBODIMENT 4 The antibody or antigen-binding fragment thereof is a) any one of the heavy chain (HC) amino acid sequences of the antibodies designated as Antibody No. 232 to Antibody No. 372 in Table 6, and / or b) the light chain (LC) amino acid sequence of any one of the antibodies designated as Antibody No. 232 to Antibody No. 372 in Table 6; 4. The isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 3, comprising:

[0292] EMBODIMENT 5 The isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4, wherein the antibody is selected from the group consisting of antibodies that are at least 70% identical, at least 75% identical, 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, and / or any percentage therebetween, to any one of the antibodies designated Antibody No. 232 to Antibody No. 372 disclosed in Table 6.

[0293] EMBODIMENT 6 The isolated antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 5, wherein the antibody is selected from the group consisting of the antibodies designated as Antibody No. 232 to Antibody No. 372 in Table 6.

[0294] EMBODIMENT 7 An isolated nucleic acid sequence encoding the antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 6.

[0295] EMBODIMENT 8 An expression vector comprising the isolated nucleic acid sequence of embodiment 7.

[0296] EMBODIMENT 9 A host cell transfected, transformed or transduced with a nucleic acid sequence according to embodiment 7 or an expression vector according to embodiment 8.

[0297] EMBODIMENT 10 A pharmaceutical composition comprising one or more of the isolated antibodies or antigen-binding fragments thereof according to any one of embodiments 1 to 6, and a pharma- ceutical acceptable carrier and / or excipient.

[0298] EMBODIMENT 11 A pharmaceutical composition comprising one or more of the nucleic acid sequences according to embodiment 7 or one or more of the expression vectors according to embodiment 8, together with a pharma- ceutically acceptable carrier and / or excipient.

[0299] EMBODIMENT 12 A transgenic organism comprising a nucleic acid sequence according to embodiment 7 or an expression vector according to embodiment 8.

[0300] EMBODIMENT 13 1. A method for treating or preventing a respiratory syncytial virus (RSV) infection or at least one symptom associated with a RSV infection, comprising administering to a patient in need thereof or suspected of being in need thereof: a) one or more antibodies or antigen-binding fragments thereof according to any one of embodiments 1 to 6; b) a nucleic acid sequence as defined in embodiment 7, c) an expression vector as defined in embodiment 8; d) a host cell according to embodiment 9, or e) a pharmaceutical composition according to embodiment 10 or embodiment 11, to treat or prevent a RSV infection, or to treat, alleviate, or reduce the severity of at least one symptom associated with a RSV infection.

[0301] EMBODIMENT 14 1. A method of treating or preventing either a respiratory syncytial virus (RSV) infection or a human metapneumovirus (HMPV) infection, or at least one symptom associated with a RSV infection or an HMPV infection, comprising administering to a patient in need thereof or suspected of being in need thereof: a) one or more antibodies or antigen-binding fragments thereof according to any one of embodiments 1 to 6; b) a nucleic acid sequence as defined in embodiment 7, c) an expression vector as defined in embodiment 8; d) a host cell according to embodiment 9, or e) a pharmaceutical composition according to embodiment 10 or embodiment 11, to treat or prevent a RSV infection, or to treat, alleviate, or reduce the severity of at least one symptom associated with a RSV infection.

[0302] EMBODIMENT 15 The method of embodiment 14, wherein the one or more antibodies or antigen-binding fragments thereof comprise antibody number 340.

[0303] EMBODIMENT 16 The method of any one of embodiments 13-15, wherein the method further comprises administering a second therapeutic agent to the patient.

[0304] EMBODIMENT 17 The method of embodiment 16, wherein the second therapeutic agent is selected from the group consisting of an antiviral drug, a vaccine specific for RSV, a vaccine specific for influenza virus, or a vaccine specific for metapneumovirus (MPV), an siRNA specific for a RSV antigen or a metapneumovirus (MPV) antigen, a secondary antibody specific for a RSV antigen or a metapneumovirus (MPV) antigen, an anti-IL-4R antibody, an antibody specific for an influenza virus antigen, an anti-RSV-G antibody, and an NSAID.

[0305] EMBODIMENT 18 A pharmaceutical composition comprising one or more of the isolated antibodies or antigen-binding fragments thereof according to any one of embodiments 1 to 7, and a pharma- ceutical acceptable carrier and / or excipient.

[0306] EMBODIMENT 19 19. The pharmaceutical composition of embodiment 18 for use in the prevention of a respiratory syncytial virus (RSV) infection in a patient in need thereof or suspected of being in need thereof, or for use in the treatment of a patient suffering from a RSV infection, or for the amelioration of the treatment of at least one symptom or complication associated with said infection, wherein said use results in either the prevention of said infection, or the prevention, amelioration, or reduction in the severity and / or duration of at least one symptom or complication associated with said infection.

[0307] EMBODIMENT 20 The pharmaceutical composition of embodiment 18 for use in a patient in need thereof or suspected of being in need thereof in the treatment or prevention of a respiratory syncytial virus (RSV) infection or a human metapneumovirus (HMPV) infection or at least one symptom associated with said RSV infection or said HMPV infection, wherein said use results in either prevention of said infection or prevention, amelioration, or reduction in severity and / or duration of at least one symptom or complication associated with said infection.

[0308] EMBODIMENT 21 Use of the pharmaceutical composition of embodiment 18 in the manufacture of a medicament for preventing a respiratory syncytial virus (RSV) infection or treating a patient suffering from a RSV infection or ameliorating at least one symptom or complication associated with said infection in a patient in need thereof, which results in either preventing said infection or preventing, ameliorating, or reducing the severity and / or duration of at least one symptom or complication associated with said infection.

[0309] EMBODIMENT 22 Use of the pharmaceutical composition of embodiment 18 in the manufacture of a medicament for the prevention of either a respiratory syncytial virus (RSV) infection or a human metapneumovirus (HMPV) infection or at least one symptom associated with said RSV infection or said HMPV infection in a patient in need thereof or suspected of being in need thereof, wherein said use results in either the prevention of said infection or the prevention, amelioration, or reduction in the severity and / or duration of at least one symptom or complication associated with said infection.

Claims

1. 1. An isolated antibody or antigen-binding fragment thereof that specifically binds to a respiratory syncytial virus (RSV) F protein (F), comprising: (A) a heavy chain variable region comprising CDRH1, CDRH2, and CDRH3; and (B) A light chain variable region comprising CDRL1, CDRL2, and CDRL3. Including, a) (i) said CDRH1 comprises the amino acid sequence of SEQ ID NO:563, said CDRH2 comprises the amino acid sequence of SEQ ID NO:565, and said CDRH3 comprises the amino acid sequence of SEQ ID NO:567; (ii) said CDRL1 comprises the amino acid sequence of SEQ ID NO:571, said CDRL2 comprises the amino acid sequence of SEQ ID NO:573, and said CDRL3 comprises the amino acid sequence of SEQ ID NO:575; b) (i) said CDRH1 comprises the amino acid sequence of SEQ ID NO: 2147, said CDRH2 comprises the amino acid sequence of SEQ ID NO: 2148, and said CDRH3 comprises the amino acid sequence of SEQ ID NO: 2149; (ii) said CDRL1 comprises the amino acid sequence of SEQ ID NO:2152, said CDRL2 comprises the amino acid sequence of SEQ ID NO:2153, and said CDRL3 comprises the amino acid sequence of SEQ ID NO:2154; c) (i) said CDRH1 comprises the amino acid sequence of SEQ ID NO: 2157, said CDRH2 comprises the amino acid sequence of SEQ ID NO: 2158, and said CDRH3 comprises the amino acid sequence of SEQ ID NO: 2159; (ii) said CDRL1 comprises the amino acid sequence of SEQ ID NO:2162, said CDRL2 comprises the amino acid sequence of SEQ ID NO:2163, and said CDRL3 comprises the amino acid sequence of SEQ ID NO:2164; d) (i) said CDRH1 comprises the amino acid sequence of SEQ ID NO: 2167, said CDRH2 comprises the amino acid sequence of SEQ ID NO: 2168, and said CDRH3 comprises the amino acid sequence of SEQ ID NO: 2169; (ii) said CDRL1 comprises the amino acid sequence of SEQ ID NO:2172, said CDRL2 comprises the amino acid sequence of SEQ ID NO:2173, and said CDRL3 comprises the amino acid sequence of SEQ ID NO:2174; e) (i) said CDRH1 comprises the amino acid sequence of SEQ ID NO: 2177, said CDRH2 comprises the amino acid sequence of SEQ ID NO: 2178, and said CDRH3 comprises the amino acid sequence of SEQ ID NO: 2179; (ii) said CDRL1 comprises the amino acid sequence of SEQ ID NO:2182, said CDRL2 comprises the amino acid sequence of SEQ ID NO:2183, and said CDRL3 comprises the amino acid sequence of SEQ ID NO:2184; f) (i) said CDRH1 comprises the amino acid sequence of SEQ ID NO: 2187, said CDRH2 comprises the amino acid sequence of SEQ ID NO: 2188, and said CDRH3 comprises the amino acid sequence of SEQ ID NO: 2189; (ii) said CDRL1 comprises the amino acid sequence of SEQ ID NO:2192, said CDRL2 comprises the amino acid sequence of SEQ ID NO:2193, and said CDRL3 comprises the amino acid sequence of SEQ ID NO:2194; g) (i) said CDRH1 comprises the amino acid sequence of SEQ ID NO: 2197, said CDRH2 comprises the amino acid sequence of SEQ ID NO: 2198, and said CDRH3 comprises the amino acid sequence of SEQ ID NO: 2199; (ii) said CDRL1 comprises the amino acid sequence of SEQ ID NO:2202, said CDRL2 comprises the amino acid sequence of SEQ ID NO:2203, and said CDRL3 comprises the amino acid sequence of SEQ ID NO:2204; (i) said CDRH1 comprises the amino acid sequence of SEQ ID NO: 2207, said CDRH2 comprises the amino acid sequence of SEQ ID NO: 2208, and said CDRH3 comprises the amino acid sequence of SEQ ID NO: 2209; (ii) said CDRL1 comprises the amino acid sequence of SEQ ID NO:2212, said CDRL2 comprises the amino acid sequence of SEQ ID NO:2213, and said CDRL3 comprises the amino acid sequence of SEQ ID NO:2214; or i) said CDRH1 comprises the amino acid sequence of SEQ ID NO:2217, said CDRH2 comprises the amino acid sequence of SEQ ID NO:2218, and said CDRH3 comprises the amino acid sequence of SEQ ID NO:2219; (ii) said CDRL1 comprises the amino acid sequence of SEQ ID NO: 2222, said CDRL2 comprises the amino acid sequence of SEQ ID NO: 2223, and said CDRL3 comprises the amino acid sequence of SEQ ID NO: 2224; The isolated antibody or antigen-binding fragment thereof.

2. In a), (i) the heavy chain variable region is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:562; (ii) the light chain variable region is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:570; In b), (i) the heavy chain variable region is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2146; (ii) the light chain variable region is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2151; In c), (i) the heavy chain variable region is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2156; (ii) the light chain variable region is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2161; d), (i) the heavy chain variable region is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2166; (ii) the light chain variable region is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2171; e), (i) the heavy chain variable region is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2176; (ii) the light chain variable region is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2181; f), (i) the heavy chain variable region is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2186; (ii) the light chain variable region is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2191; In g), (i) the heavy chain variable region is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2196; (ii) the light chain variable region is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2201; h), (i) the heavy chain variable region is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2206; (ii) the light chain variable region is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2211; or In i), (i) the heavy chain variable region is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2216; (ii) the light chain variable region is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2221; 2. An isolated antibody or antigen-binding fragment thereof according to claim 1.

3. the heavy chain variable region and the light chain variable region a) SEQ ID NO: 562 and SEQ ID NO: 570, respectively; b) SEQ ID NO: 2146 and SEQ ID NO: 2151, respectively; c) SEQ ID NO: 2156 and SEQ ID NO: 2161, respectively; d) SEQ ID NO: 2166 and SEQ ID NO: 2171, respectively; e) SEQ ID NO: 2176 and SEQ ID NO: 2181, respectively; f) SEQ ID NO: 2186 and SEQ ID NO: 2191, respectively; g) SEQ ID NO: 2196 and SEQ ID NO: 2201, respectively; h) SEQ ID NO: 2206 and SEQ ID NO: 2211, respectively; or i) SEQ ID NO: 2216 and SEQ ID NO: 2221, respectively 2. The isolated antibody or antigen-binding fragment thereof of claim 1, comprising the amino acid sequence of:

4. (A) a heavy chain comprising the heavy chain variable region and a heavy chain constant region; and (B) a light chain comprising the light chain variable region and a light chain constant region. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 3, comprising:

5. 5. The isolated antibody or antigen-binding fragment thereof of claim 4, comprising an immunoglobulin molecule composed of two of said heavy chains and two of said light chains; or a multimer of said immunoglobulin molecule.

6. 6. The isolated antibody or antigen-binding fragment thereof of claim 4 or 5, which is or comprises an IgG1 or IgG4 antibody.

7. Fab fragment, F(ab') ２ 4. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 3, comprising one or more of the following fragments: an Fv fragment, a single chain Fv (scFv) molecule, a dAb fragment, a chimeric antibody, a diabody, a triabody, a tetrabody, a minibody, and / or a small modular immunopharmaceutical (SMIP).

8. An isolated nucleic acid encoding the isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

9. An expression vector comprising the isolated nucleic acid of claim 8.

10. 10. A host cell transfected, transformed or transduced with the isolated nucleic acid of claim 8 or the expression vector of claim 9.

11. A pharmaceutical composition comprising one or more of the isolated antibodies or antigen-binding fragments thereof according to any one of claims 1 to 7, and a pharma- ceutically acceptable carrier and / or excipient.

12. 10. A pharmaceutical composition comprising one or more of the isolated nucleic acids of claim 8 or one or more of the expression vectors of claim 9 and a pharma- ceutically acceptable carrier and / or excipient.

13. A transgenic organism comprising the isolated nucleic acid of claim 8 or the expression vector of claim 9, wherein the transgenic organism is not human.

14. For treating or preventing a respiratory syncytial virus (RSV) infection or at least one symptom associated with a RSV infection. a) one or more isolated antibodies or antigen-binding fragments thereof according to any one of claims 1 to 7; b) an isolated nucleic acid according to claim 8; c) an expression vector according to claim 9, or d) The host cell according to claim 10.

1. A composition comprising: a) a therapeutically effective amount of at least one of the following: a) a therapeutically effective amount of at least one of the following: bluish skin color, hypoxia, dyspnea, shortness of breath, cough, croupy cough, fever, nasal flaring, nasal congestion, apnea, loss of appetite, dehydration, anorexia, altered mental status, wheezing, bronchiolitis, and pneumonia;

15. 15. The method of claim 14, wherein a second therapeutic agent is administered to the patient.

16. 16. The composition of claim 15, wherein the second therapeutic agent is selected from the group consisting of an antiviral drug, an RSV-specific vaccine, an influenza virus-specific vaccine, an siRNA specific for an RSV antigen, a secondary antibody specific for an RSV antigen, an anti-IL-4R antibody, an antibody specific for an influenza virus antigen, an anti-RSV-G antibody, and an NSAID.

17. 12. The pharmaceutical composition of claim 11 for use in the prevention of a respiratory syncytial virus (RSV) infection in a patient in need thereof or suspected of being in need thereof, or in the treatment of a patient suffering from a RSV infection, or in the amelioration of at least one symptom or complication associated with a RSV infection, wherein said use results in either the prevention of the RSV infection or the prevention, amelioration, or reduction in the severity and / or duration of the at least one symptom or complication associated with the RSV infection, wherein the at least one symptom or complication associated with the RSV infection comprises one or more of the following: bluish skin color, hypoxia, dyspnea, shortness of breath, shortness of breath, cough, croupy cough, fever, nasal flaring, nasal congestion, apnea, loss of appetite, dehydration, anorexia, altered mental status, wheezing, bronchiolitis, and pneumonia.

18. 12. Use of the pharmaceutical composition of claim 11 in the manufacture of a medicament for preventing a respiratory syncytial virus (RSV) infection or treating a patient suffering from a RSV infection or ameliorating at least one symptom or complication associated with a RSV infection in a patient in need thereof, resulting in either the prevention of the RSV infection or the prevention, amelioration, or reduction in severity and / or duration of the at least one symptom or complication associated with the RSV infection, wherein the at least one symptom or complication associated with the RSV infection comprises one or more of the following: bluish skin color, hypoxia, dyspnea, shortness of breath, shortness of breath, cough, croupy cough, fever, nasal flaring, nasal congestion, apnea, loss of appetite, dehydration, anorexia, altered mental status, wheezing, bronchiolitis, and pneumonia.

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