EA039682B1 - Antibodies that neutralize rsv, mpv and pvm and uses thereof - Google Patents

Abstract

The invention relates to antibodies and antigen binding fragments thereof that neutralize infection of RSV, MPV and PVM. The invention also relates to a nucleic acid molecule, an expression vector comprising the same, a cell and a pharmaceutical composition for the manufacture of a medicament comprising one of the above mentioned components or two antibodies. Furthermore, the invention relates to the use of antibodies, antibody fragments and nucleic acid molecules in the diagnosis, treatment or attenuation of RSV or MPV infection and RSV and MPV co-infection. The invention also includes a method of the attenuation of RSV and MPV infection or reduction of the risk of infection development.

Description

The invention relates to antibodies or antigen-binding fragments neutralizing RSV, MPV and PVM infection, their production and use. Thus, the invention may be directed to an isolated antibody, eg, a monoclonal antibody, a human antibody, a human monoclonal antibody, a variant antibody, or an antigen-binding fragment that cross-reacts to neutralize RSV, MPV, and PVM infection.

Prior Art

Respiratory syncytial virus (RSV) and metapneumovirus (MPV) and murine pneumonia virus (PVM) are upper respiratory infection viruses belonging to the paramyxovirus family that infect a general target population and represent a major health problem in neonates and immunocompromised patients. .

RSV is the leading cause of acute respiratory disease in children and adults worldwide. Hospitalization is required for 0.5-3.2% of children with RSV infection (Thompson W.W. et al., 2003, JAMA: The Journal of the American Medical Association, 289:179-186) and 5 to 10% of children have chronic severe infection, a factor thought to precipitate airflow obstruction and asthma-like symptoms later in childhood. Immunity to RSV appears to be of short duration, thus reinfections are frequent (Ogra, 2003, Pediatric Respiratory Reviews 5 Suppl A:S119-126).

Human MPV was first isolated in 2001 and is now recognized as the second leading cause of acute respiratory illness in children and adults; it is estimated that it infects more than 50% of children by age two and virtually all children by age five. MPV is responsible for approximately 5 to 15% of cases of respiratory disease in hospitalized young children (Alto, 2004, The Journal of the American Board of Family Practice/American Board of Family Practice, 17:466-469; Williams et al., 2004 , N. Engl. J. Med., 350:443-450). MPV infection represents a significant burden of disease in at-risk preterm infants, chronic lung disease in preterm infants, congestive heart disease, and immunodeficiency (Martino et al., 2005, Biology of Blood and Marrow Transplantation: Journal of the American Society for Blood and Marrow Transplantation, 11:781-796).

Mixed infections of MPV and RSV may be common given their prevalence and overlapping winter epidemics. Although it is still unclear whether a synergistic pathology between the two viruses can occur, in particular, exacerbations leading to severe respiratory disease have been observed in some children with mixed MPV and RSV infection (Greensill, 2003, Emerging Infectious Diseases 9:372).

RSV, which belongs to the Pneumovirus genus of the Pneumoviriniae subfamily, and MPV, which belongs to the Metapneumovirus genus of the Pneumoviriniae subfamily, share some similarities in their genetic structure, despite the fact that MPV does not contain the non-structural NS1 and NS2 genes found in RSV. The envelopes of RSV and MPV contain three virally encoded transmembrane surface glycoproteins: the major attachment protein G, the fusion protein F, and the small hydrophobic protein SH. Although the envelopes of RSV and MPV contain proteins that are functionally similar, however, it should be noted that the F proteins of RSV and MPV share only 33% amino acid sequence identity. In addition, antisera generated against RSV or MPV do not give a neutralizing cross-reaction for both viruses (Wyde et al. cross reaction for both RSV and MPV.

The F-glycoproteins RSV and MPV control viral entry by fusion of the virion envelope and the host cell's plasma membrane. Subsequently, during infection, an F protein expressed on the cell surface can mediate fusion with neighboring cells to form syncytia (Collins et al., 1984 PNAS, 81:7683-7687). In both cases, the N-terminus of the F-subunit, which is obtained by proteolytic cleavage and which contains a hydrophobic amino acid fragment called a fusion peptide, is inserted directly into the target membrane to initiate the fusion. After binding to the target cell and subsequent activation, the metastable F protein undergoes a series of structural rearrangements prior to fusion, which result in the incorporation of the fusion peptide into the target cell membrane, followed by the formation of a stable helical strand that is formed as the viral and cell membranes approach each other. . Such structural changes lead to the formation of a stable F-protein after fusion.

Vaccines against RSV or MPV infection are not currently available. Formalin-inactivated RSV and adjuvanted aluminum (FIRSV) vaccines tested in the 1960s were found to provoke full-blown disease in children after natural RSV infection, leading to severe fever and severe pneumonia, leading to high hospitalization rates. and even some fatal cases (Fulginiti et al., 1969, American Journal of Epidemiology, 89:435-448; Kapikian et al., 1969, American Journal of Epidemiology, 89:405-421; Kim et al., 1969 , American Journal of Epidemiology, 89:422-434). Similarly, formalin-inactivated MPV vaccines demonstrated immune-mediated advanced disease in young adults.

- 1,039,682 cynomolgus monkeys (de Swart et al., 2007, Vaccine, 25:8518-8528). In addition, antiviral therapies such as ribavirin have not been shown to be effective for RSV or MPV infection.

From epidemiological studies as well as from animal studies, evidence has been obtained for the role of serum antibodies in protection against the RSV virus. In children, maternal antibody titers correlate with resistance to serious disease (Glezen et al., 1981, The Journal of Pediatrics, 98:708-715), and in adults, the incidence and severity of lower respiratory tract disease is reduced in the presence of high levels of neutralizing antibodies against Serum RSV (McIntosh et al., 1978, The Journal of Infectious Diseases, 138:24-32). The monoclonal antibody palivizumab (Synagis) has been approved for the prevention of RSV infection in preterm infants. However, palivizumab is not always effective in preventing RSV infection and is not therapeutically effective. In addition, long-term replication in the lung of RSV in the presence of palivizumab in animals is accompanied by the emergence of resistant strains of the virus (Zhao and Sullender, 2005, Journal of Virology, 79:3962-3968). There are currently no monoclonal antibodies to treat or prevent MPV infection.

The lack of a well-functioning animal model for the most severe forms of RSV infection is due to the fact that RSV and MPV are host-specific Pneumovirus pathogens. The development of new drugs for the treatment of RSV and MPV infections has been hampered by the lack of an animal model capable of reproducing all the symptoms and severity of human disease. In fact, RSV and MPV are not natural pathogens of the mouse and only induce a limited, minimally symptomatic, and rapidly reversible primary infection in response to a massive, non-physiological virus inoculum. Mouse pneumonia virus (PVM) is a naturally occurring rodent Pneumovirus pathogen that belongs to the same family, subfamily, and genus (Pneumovirus) of human and bovine RSV. The PVM F protein shares only 40% amino acid identity with the human RSV F protein, but shares the same genetic organization except for the M2-L overlap, which is present in RSV but not in PVM. Infection with the natural pathogen PVM of the mouse reproduces many of the signs and symptoms of the most severe forms of RSV as seen in children. PVM infection is characterized by rapid viral replication accompanied by a massive inflammatory response that leads to respiratory failure and death (Rosemberg and Domachowske, 2008, Immunology Letter, 118:6-12). Thus, PVM infection in mice is considered to be the most relevant animal model of severe human RSV and MPV infections.

The lack of prophylactic treatment for MPV infection and vaccines against RSV and MPV infections, as well as the therapeutic efficacy of palivizumab, points to the need for new prophylactic and therapeutic agents against such important human pathogens. Given the prevalence and likelihood of mixed infection, it is highly desirable to have a single agent that is capable of providing prevention as well as treatment or reduction in RSV and MPV infection, and an animal model in which to test the agent. Thus, there is a need for broad spectrum cross-reactive neutralizing antibodies that protect against a broad spectrum of paramyxoviruses, eg at least RSV and MPV and preferably RSV, MPV and PVM.

The essence of the invention

In a first embodiment, the invention discloses an isolated antibody or antigen-binding fragment thereof that neutralizes RSV (respiratory syncytial virus) infection, wherein the antibody or antigen-binding fragment comprises (i) complementarity determining regions (CDR)1, CDR2, and heavy chain CDR3 represented by in the sequence of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the light chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 respectively; (ii) the heavy chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, SEQ ID NO: 39, and SEQ ID NO: 40, respectively, and the light chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 4, SEQ ID NO: 41 and SEQ ID NO: 42, respectively; (iii) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, SEQ ID NO: 39 and SEQ ID NO: 40, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 4 , SEQ ID NO: 5 and SEQ ID NO: 6, respectively; (iv) the heavy chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the light chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 4, SEQ ID NO: 41 and SEQ ID NO: 42, respectively; (v) the heavy chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively, and the light chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: : 4, SEQ ID NO: 22 and SEQ ID NO: 23, respectively; (vi) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, SEQ ID NO: 39 and SEQ ID NO: 53, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 4 , SEQ ID NO: 41 and SEQ ID NO: 54, respectively; (vii) the heavy chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, SEQ ID NO: 39, and SEQ ID NO: 53, respectively, and the sequence

- 2 039682 light chain CDR1, CDR2 and CDR3 as set forth in SEQ ID NO: 4, SEQ ID NO: 22 and SEQ ID NO: 23, respectively; or (viii) the heavy chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively, and the light chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: : 4, SEQ ID NO: 41 and SEQ ID NO: 54, respectively.

Moreover, the antibody or its antigen-binding fragment can bind the F-protein of RSV before the fusion, but not the F-protein after the fusion. Or not bind to the RSV F protein at a site that overlaps the I antigenic site, the II antigenic site, or the IV antigenic site.

They are also able to neutralize both group A and group B RSV infection or RSV and MPV (metapneumovirus, or RSV, MPV and PVM (murine pneumonia virus) infections).

In addition, the antibody or antigen-binding fragment thereof can bind to each of the conserved regions in the N-terminal region of the RSV F protein containing any of the amino acid sequences of SEQ ID NOs: 64-69.

The concentration of antibody or antigen-binding fragment required to neutralize 50% of RSV is 500 ng/mL or less.

Another embodiment of the invention relates to an antibody or antigen-binding fragment containing (i) a heavy chain variable region at least 80% identical to the amino acid sequence of SEQ ID NO: 13, and a light chain variable region at least 80% amino acid identical. the sequences of SEQ ID NO: 14;

(ii) a heavy chain variable region at least 80% identical to the amino acid sequence of SEQ ID NO: 17 and a light chain variable region at least 80% identical to the amino acid sequence of SEQ ID NO: 14;

(iii) a heavy chain variable region at least 80% identical to the amino acid sequence of SEQ ID NO: 49 and a light chain variable region at least 80% identical to the amino acid sequence of SEQ ID NO: 50;

(iv) a heavy chain variable region at least 80% identical to the amino acid sequence of SEQ ID NO: 49 and a light chain variable region at least 80% identical to the amino acid sequence of SEQ ID NO: 14; (v) a heavy chain variable region at least 80% identical to the amino acid sequence of SEQ ID NO: 17 and a light chain variable region at least 80% identical to the amino acid sequence of SEQ ID NO: 50;

(vi) a heavy chain variable region at least 80% identical to the amino acid sequence of SEQ ID NO: 29 and a light chain variable region at least 80% identical to the amino acid sequence of SEQ ID NO: 30;

(vii) a heavy chain variable region at least 80% identical to the amino acid sequence of SEQ ID NO: 33 and a light chain variable region at least 80% identical to the amino acid sequence of SEQ ID NO: 30;

(viii) a heavy chain variable region at least 80% identical to the amino acid sequence of SEQ ID NO: 59 and a light chain variable region at least 80% identical to the amino acid sequence of SEQ ID NO: 60;

(ix) a heavy chain variable region at least 80% identical to the amino acid sequence of SEQ ID NO: 59 and a light chain variable region at least 80% identical to the amino acid sequence of SEQ ID NO: 30; or (x) a heavy chain variable region at least 80% identical to the amino acid sequence of SEQ ID NO: 33 and a light chain variable region at least 80% identical to the amino acid sequence of SEQ ID NO: 60.

And the antibody or antigen-binding fragment thereof may comprise (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14; (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14; (iii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 50; (iv) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14; (v) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 50; (vi) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 29, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30; (vii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 33, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30; (viii) a heavy chain variable region comprising an amino acid

- 3 039682 the lot sequence of SEQ ID NO: 59, the variable region of the light chain, including the amino acid sequence of SEQ ID NO: 60; (ix) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 59, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30; or (x) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 33 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 60.

The antibody or antigen-binding fragment thereof may be a human antibody, monoclonal antibody, human monoclonal antibody, single chain antibody, Fab, Fab', F(ab') ₂ , Fv or scFv.

They may be intended to treat or attenuate RSV or MPV infection, or both RSV and MPV.

In another aspect, the invention comprises a nucleic acid molecule comprising a polynucleotide encoding an antibody or antibody fragment of the invention. Moreover, the nucleotide sequence can be at least 75% identical to any of the nucleotide sequences of SEQ ID NOs: 7-12, 15, 16, 18, 24 28, 31, 32, 34, 43-48, 51-52, 55- 58 or 61-62.

In yet another aspect, the invention comprises an expression vector containing the nucleic acid molecule of the invention. The invention also includes a cell that expresses an antibody of the invention, or an antigen-binding fragment thereof.

The invention also relates to a pharmaceutical composition for the manufacture of a medicament for the treatment or attenuation of RSV or MPV or both RSV and MPV infections, comprising an antibody of the invention or an antigen-binding fragment thereof, a nucleic acid molecule of the invention, a vector containing a nucleic acid molecule of the invention, a cell, expressing an antibody or antibody fragment of the invention; and a pharmaceutically acceptable diluent or carrier. The invention also contains a pharmaceutical composition for the manufacture of a drug for the treatment or attenuation of RSV or MPV or both RSV and MPV infections, containing the first antibody or antigen-binding fragment and the second antibody or antigen-binding fragment, where the first antibody is an antibody according to the invention, and the second antibody is an antibody, or antigen-binding fragment thereof, that neutralizes infection with RSV or MPV or RSV and MPV, or all three of RSV, MPV and PVM.

The scope of the invention includes the use of an antibody of the invention, or an antigen-binding fragment thereof, in the manufacture of a medicament for the treatment or attenuation of RSV or MPV, or both RSV and MPV infections, or in the diagnosis of RSV or MPV infection. As well as use to monitor the quality of anti-RSV or anti-MPV vaccines.

As well as the use of a nucleic acid molecule in the manufacture of a medicament for the treatment or attenuation of RSV or MPV or both RSV and MPV infections, or in the diagnosis of RSV or MPV infection.

In another aspect, the invention includes a method of attenuating RSV or MPV infection or reducing the risk of developing RSV and MPV infection, comprising administering to an individual in need thereof a therapeutically effective amount of an antibody of the invention, or an antigen-binding fragment thereof.

Description of drawings

In FIG. 1 shows the results of screening of monoclonal antibodies produced by immortalized EBV memory B cells from 7 donors (donors 1-7) for their ability to neutralize RSV or MPV infection in vitro.

In FIG. 2 shows the results of neutralization of RSV and MPV with monoclonal antibodies HMB2430, HMB3210, mAb 234 and palivizumab.

In FIG. 3 shows the binding of RSV F protein or tetanus toxin protein by monoclonal antibodies motavizumab, mAb 234, HMB2430 and HMB3210 as measured by ELISA.

In FIG. 4 shows the binding of labeled monoclonal antibodies to RSV-infected Hep-2 cells in the presence of a large excess of these unlabeled antibodies.

In FIG. 5 shows the binding of monoclonal antibodies HMB2430, HMB3210, mAb 234 and motavizumab to the RSV F protein from lysates of RSV-infected Hep-2 cells under reducing or non-reducing conditions as measured by Western blot analysis.

In FIG. 6 shows binding of monoclonal antibodies HMB3210 and mAb 234 to MPV F protein from lysates of MPV-infected LLC-MK2 cells under reducing or non-reducing conditions as measured by Western blot analysis.

In FIG. 7 shows the results of neutralization of the long RSV strain and the PZ-MARM6 isolate with monoclonal antibodies HMB2430, HMB3210, mAb234 and palivizumab.

In FIG. 8 shows the results of the analysis of HMB3210v2 on SDS-PAGE gel under reducing conditions after incubation in the presence of (+) or absence of (-) N-glycosidase PNGase F. The black box indicates the minor fraction of the light chain of HMB3210, which is glycosylated.

In FIG. 9 shows the results of neutralization of I-PV 03/01-6621 MPV and A2 RSV monoclonal

- 4 039682 antibodies HMB3210v2 and HMB3210v3.

In FIG. 10 shows the results of neutralization of a panel of MPV and RSV strains with monoclonal antibodies HMB3210v2 and HMB3210v3.

In FIG. 11 shows the results of neutralization of I-PV 03/01-6 621 MPV and A2 RSV with variants of the monoclonal antibodies HMB3210 and HMB2430 modified at the level of germline genes.

In FIG. 12 shows the results of a size exclusion chromatography analysis of recombinant RSV F protein after fusion incubated with or without HMB3210 or palivizumab.

In FIG. 13 shows the results of size exclusion chromatography analysis of recombinant RSV F protein before fusion, incubated with or without HMB3210 or palivizumab.

In FIG. 14 shows the binding of HMB3210v3 or palivizumab (PVZ) to RSV F proteins before and after fusion as measured by surface plasmon resonance (SPR).

In FIG. 15 shows virus neutralization and viral spread inhibition by human monoclonal antibodies HMB3210v3 and D25.

In FIG. 16 shows the prophylactic efficacy of HMB3210v3 and palivizumab in RSV or MPV infection.

In FIG. 17 shows the therapeutic efficacy of HMB3210 in STAT1 deficient mice infected with RSV.

In FIG. 18 shows the prophylactic and therapeutic efficacy of HMB3210 in mice infected with a lethal dose of PVM.

In FIG. 19 shows blocking of the rise in lung viral titers in mice treated with HMB3210v3 on days 3, 4, or 5 after lethal PVM infection.

In FIG. 20 shows the prophylactic and therapeutic efficacy of HMB3210v3 variants carrying a wild-type Fc or a LALA mutation in mice infected with a lethal dose of PVM.

In FIG. 21 shows an alignment indicating the high conservation of the YLSALR peptide recognized by HMB3210 in RSV, BRSV, PVM and MPV sequences compared to parainfluenza virus 5 (PIV5).

In FIG. 22 is a pre-fusion model of the RSV F protein showing the location of the YLSALR peptide and the palivizumab proximal region (PVZ), and strip plots showing the rearrangement of the PVZ and HMB3210v3 regions of the RSV F protein in pre- and post-fusion conformations.

In FIG. 23 shows a high degree of conservation of the HMB3210 core epitope in strains 364 RSV, 162 MPV, 8 BRSV, and 5 PVM.

In FIG. 24A, 24B, and 24C show the sequences of various heavy and light chain variants of HMB3210.

In FIG. 25A and 25B show the sequences of different HMB2430 heavy and light chain variants.

Detailed description

The application is based in part on the discovery and isolation of antibodies that cross-neutralize with RSV and MPV or RSV, MPV and PVM, as well as the epitopes to which the antibodies of the invention bind. Such antibodies are required to neutralize RSV and MPV or RSV, MPV and PVM with one or more antibodies. In addition, to reduce the cost of obtaining medicines. For the treatment of RSV and/or MPV infection, cross-neutralizing antibodies are obtained in high titers. In addition, epitopes recognized by such antibodies may be part of a vaccine capable of inducing broad spectrum protection against RSV and MPV.

Although antibodies neutralize RSV, MPV, and PVM, disease treatment, vaccine development, etc., can only refer to RSV and MPV, as these viruses are human pathogens while PVM is a mouse pathogen. In the present description, the terms RSV and MPV and RSV, MPV and PVM are used interchangeably depending on the context.

Thus, in one aspect, the invention relates to an isolated antibody, antibody variants and antigen-binding fragments thereof that neutralize RSV and MPV or RSV, MPV and PVM, wherein the RSV may be human RSV or bovine RSV. The antibodies are described to neutralize human RSV (hRSV) and bovine RSV (bRSV). The MPV may be a human MPV.

The application also discloses an isolated antibody, or an antigen-binding fragment thereof, that neutralizes group A and group B RSV infection or group A and group B MPV infection, or group A and group B RSV infection, and group A and group B MPV.

As stated above, RSV, MPV and PVM share some similarities in their genetic structure. Amino acid sequences of G and F proteins are classified into groups A and B in RSV and MPV; MPV is further divided into 4 subgroups: A1, A2, B1 and B2. PVMs are not divided into groups or subgroups. The F protein of RSV, MPV, or PVM is a type I transmembrane surface protein that contains a signal peptide cleaved from the N-terminus and a membrane anchor.

- 5 039682 near the C-terminus. The F proteins of RSV and MPV are synthesized as inactive F0 precursors, which assemble into homotrimers and are activated by cleavage. The F protein is formed by three domains (DI-DIII), a fusion peptide (FP) and three regions formed by seven repeats (HR-A, -B and -C). The F-glycoproteins of RSV and MPV regulate viral entry through the fusion of the viral envelope and the plasma membrane of the host cell. In both cases, the N-terminus of the F subunit, which is formed by proteolytic cleavage and contains the fusion peptide, is inserted directly into the target membrane to initiate the fusion. After binding to the target cell and subsequent activation, the metastable F-protein undergoes a series of structural rearrangements before fusion, which lead to the incorporation of the fusion peptide into the target cell membrane, followed by the formation of a stable helical strand, which forms as the viral and cell membranes approach . Such structural changes lead to the formation of a stable F-protein after fusion. At a later stage of infection, the F-protein expressed on the cell surface of infected cells can mediate fusion with neighboring uninfected cells and the formation of large syncytia.

The epitopes for palivizumab and motavizumab were mapped to the post-fusion antigenic region II of the RSV F protein (also referred to as region A) formed by residues 255-275. MAB19 and 101F target the post-fusion antigenic region IV of the RSV F protein (also referred to as region C) of RSV, formed by residues 422-438. MAB19 was tested in a clinical trial but failed to demonstrate significant efficacy (Johnson et al., 1999, The Journal of Infectious Diseases, 180:35-40; Meissner et al., 1999, Antimicrobial Agents and Chemotherapy, 43:1183-1188) .

To be effective, antibodies must recognize a pre-fusion F protein whose conformation is appropriate to block viral entry, and preferably not recognize an excess post-fusion F protein that can act as a trap, thus wasting the antibody and reducing its effectiveness. To date, no antibodies have been isolated that recognize the RSV F protein before fusion but do not recognize the RSV F protein after fusion.

According to the application, an isolated antibody or antigen-binding fragment thereof that specifically binds to the RSV F protein before fusion and does not bind to the RSV F protein after fusion. In another embodiment, the isolated antibody, or antigen-binding fragment thereof, specifically binds to the F protein of RSV and MPV prior to fusion and to the non-F protein of RSV and MPV after fusion. They also specifically bind to the F protein of RSV, MPV, and PVM prior to fusion, but do not bind to the F protein of RSV, MPV, and PVM after fusion.

The application discloses antibodies that bind to the F protein of RSV, MPV and PVM. Although there is only approximately 33 and 40% amino acid sequence identity between the F proteins of RSV and MPV or RSV and PVM, respectively, the antibodies of the application recognize a common epitope presented on the F proteins of RSV, MPV and PVM. This epitope is different from all epitopes recognized by currently known antibodies such as palivizumab, motavizumab, mAb 101F, etc. Antibodies according to the application, for example, do not bind to the antigenic site II (recognized by motavizumab and palivizumab), nor to the antigenic site IV (recognized by mAb 101F), nor to the antigenic site I (which binds mAb 131-2A). The epitopes on the RSV F-protein recognized by the antibodies according to the application also differ from the epitopes recognized by mAb D25, an antibody specific only to RSV. In addition, the epitopes on the MPV F protein recognized by the antibodies described in the application differ from the epitopes recognized by mAb 234 (which recognizes an epitope on the MPV F protein that corresponds to the antigenic site II on the RSV F protein). In general, antibodies according to the application recognize a conformational epitope. In one embodiment, the conformational epitope is only presented under non-reducing conditions.

In addition, the conformational epitope may contain disulfide bonds between amino acid residues in the F protein.

As demonstrated herein, antibodies or antigen-binding fragments, according to the application, specifically bind to several different strains of RSV and MPV and neutralize RSV and MPV. In addition, they specifically bind to and cross-neutralize group A and group B RSVs, as well as group A and group B MPVs, including all relevant MPV subgroups (eg, A1, A2, B1 and B2).

The antibody and antigen-binding fragment, according to the application, have a high neutralizing activity. Their concentration required to neutralize 50% of RSV, MPV and PVM may be, for example, about 500 ng/ml or less. For 50% neutralization of RSV, MPV and PVM, may be approximately 500, 450, 400, 350, 300, 250, 200, 175, 150, 125, 100, 90, 80, 70, 60, or approximately 50 ng/mL or less . This means that only low concentrations of antibody are needed for 50% neutralization of RSV, MPV and PVM. Specificity and activity can be measured by standard assays as known to those skilled in the art.

Antibodies according to the application can be human antibodies, monoclonal antibodies, human monoclonal antibodies, recombinant antibodies or purified antibodies. It reveals

- 6 039682 you also fragments of antibodies, in particular fragments that retain the antigen-binding activity of antibodies. Such fragments include, but are not limited to, single chain antibodies, Fab, Fab', F(ab')2, Fv or scFv. Although the description, in some places, may explicitly refer to antigen-binding fragment(s), antibody fragment(s), antibody variant(s) and/or derivative(s), it should be understood that the term antibody or antibody of the invention includes all categories of antibodies, namely antigen-binding fragment(s), fragment(s) of antibodies, variant(s) and derivative(s) of antibodies.

The applicant has determined the sequences of the heavy chains and light chains of several antibodies of the invention, where each sequence contains three CDRs on the heavy chain and three CDRs on the light chain. The amino acid positions of the CDRs are determined according to the IMGT numbering system. The sequences of CDRs, heavy chains, light chains, as well as sequences of nucleic acid molecules encoding CDRs, heavy chains, light chains of the antibodies of the invention are described in the Sequence Listing. The heavy chain CDRs of an antibody are referred to as CDRH1 (or HCDR1), CDRH2 (or HCDR2), and CDRH3 (or HCDR3), respectively. Similarly, the CDRs of antibody light chains are referred to as CDRL1 (or LCDR1), CDRL2 (or LCDR2), and CDRL3 (or LCDR3), respectively. In table. 1 shows the SEQ ID numbers for the amino acid sequences of the six heavy and light chain CDRs, respectively, of the antibodies of the invention.

Table 1. SEQ ID numbers for CDR polypeptides of antibodies that neutralize _________ RSV, MPV, and PVM_________

SEQID NO. for CDR polypeptides CDRH1 CDRH2 CDRH3 CDRL1 CDRL2 CDRL3 option 1 3210 one 2 3 4 5 6 option 2 3210 one 2 3 4 5 6 option 3 3210 one 2 3 4 5 35 option 4 3210 one 39 40 4 41 42 option 5 3210 one 39 40 4 5 6 option 6 3210 one 2 3 4 41 42 option 1 2430 nineteen 20 21 4 22 23 option 2 2430 nineteen 20 21 4 22 23 option 3 2430 one 39 53 4 41 54 option 4 2430 one 39 53 4 22 23 option 5 2430 nineteen 20 21 4 41 54

An antibody or antibody fragment may contain at least one CDR with a sequence that shares at least 95% sequence identity with any one of SEQ ID NOs: 1-6, 19-23, 35, 39-42, or 53-54. The CDRs of antibody 3210 and antibody 2430 variants are shown in FIG. 24 and 25, respectively (CDRs in bold).

Also an antibody or antigen binding fragment comprising a heavy chain containing one or more (i.e. one, two, or all three) heavy chain CDRs from 3210 Option 1, 3210 Option 2, 3210 Option 3, 3210 Option 4, 3210 Option 5, option 2410, option 1 2430, option 2 2430, option 3 2430, option 4 or option 5 2430.

In yet another embodiment, the antibody or antigen-binding fragment comprises a heavy chain CDR1 with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 19; A heavy chain CDR2 with the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 20 or SEQ ID NO: 39 and a heavy chain CDR3 with the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 21, SEQ ID NO: 40 or SEQ ID NO: 53. In certain embodiments, the antibody or antibody fragment as provided herein comprises a heavy chain comprising the amino acid sequence of (i) SEQ ID NO: 1 for CDRH1, SEQ ID NO: 2 for CDRH2, and SEQ ID NO: 3 for CDRH3, (ii) SEQ ID NO: 1 for CDRH1, SEQ ID NO: 39 for CDRH2 and SEQ ID NO: 40 for CDRH3, (iii) SEQ ID NO: 19 for CDRH1, SEQ ID NO: 20 for CDRH2 and SEQ ID NO: 21 for CDRH3 or (iv) SEQ ID NO: 1 for CDRH1, SEQ ID NO: 39 for CDRH2, and SEQ ID NO: 53 for CDRH3.

Also provided is an antibody or antigen-binding fragment comprising a light chain containing one or more (i.e., one, two, or all three) light chain CDRs from option 1 3210, option 2 3210, option 3 3210, option 4 3210, option 5 3210 , variant 6 2430, variant 1 2430, variant 2 2430, variant 3 2430, variant 4 2430, or variant 5 2430. The antibody or antigen binding fragment may comprise a light chain CDR1 with the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 with the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 22 or SEQ ID NO: 41 and a light chain CDR3 with the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 23, SEQ ID NO: 35, SEQ ID NO: 42 or SEQ ID NO : 54. Antibody or antibody fragment as provided herein

- 7 039682 description, contains a light chain containing the amino acid sequence of (i) SEQ ID NO: 4 for CDRL1, SEQ ID NO: 5 for CDRL2 and SEQ ID NO: 6 for CDRL3; (ii) SEQ ID NO: 4 for CDRL1, SEQ ID NO: 5 for CDRL2, and SEQ ID NO: 35 for CDRL3; (iii) SEQ ID NO: 4 for CDRL1, SEQ ID NO: 41 for CDRL2, and SEQ ID NO: 42 for CDRL3; (iv) SEQ ID NO: 4 for CDRL1, SEQ ID N0; 22 for CDRL2 and SEQ ID NO: 23 for CDRL3 or (v) SEQ ID NO: 4 for CDRL1, SEQ ID NO: 41 for CDRL2 and SEQ ID NO: 54 for CDRL3.

And also the antibody according to the application, or its antigen-binding fragment, may contain all the CDRs of option 1 of the antibody 3210 listed in table. 1, and neutralize RSV, MPV and PVM infection. The antibody according to the application or its antigennegative fragment may contain all CDR variant 2 antibodies 3210 listed in table. 1 and neutralizes RSV, MPV and PVM infection. In another embodiment, the antibody or antigen-binding fragment may contain all of the CDRs of variant 3 of the 3210 antibody listed in Table 1. 1 and neutralizes RSV, MPV and PVM infection. In addition, the antibody, according to the application or its antigennegative fragment may contain all CDR variant 4 antibodies 3210 listed in table. 1, and neutralize RSV, MPV and PVM infection. And the antibody, or antigen-binding fragment thereof, may contain all of the CDRs of variant 5 of antibody 3210 listed in Table 1. 1, and neutralize RSV, MPV and PVM infection. Also, the antibody, or antigen-binding fragment thereof, may contain all of the 3210 antibody variant 6 CDRs listed in Table 1. 1, and neutralize RSV, MPV and PVM infection.

The antibody, according to the application, or antigennegative fragment may contain all CDR variant 1 antibodies 2430 listed in table. 1, and neutralize RSV, MPV and PVM infection. The antibody, or antigen-binding fragment thereof, may contain all of the 2430 antibody variant 2 CDRs listed in Table 1. 1, and neutralize RSV, MPV and PVM infection. Also, the antibody or antigen-binding fragment may contain all of the CDRs of variant 3 of the 2430 antibody listed in Table 1. 1, and neutralize RSV, MPV and PVM infection. The antibody, or antigen-binding fragment thereof, may contain all of the 2430 antibody variant 4 CDRs listed in Table 1. 1, and neutralize RSV, MPV and PVM infection. And the antibody of the invention, or antigen-binding fragment thereof, may contain all of the CDRs of variant 5 of antibody 2430 listed in Table 1. 1, and neutralize RSV, MPV and PVM infection.

The amino acid sequence SEQ ID numbers for the heavy chain variable region (VH) and the light chain variable region (VL) of the antibodies of the invention, as well as the SEQ ID numbers for the nucleic acid sequences encoding them, are listed in Table 1. 2.

- 8 039682

Table 2. SEQ ID numbers for amino acid residues and nucleic acid VH and V _L for antibodies ___________ that neutralize RSV, MPV and PVM________________

SEQID NO. for V _H and V _L amino acid and nucleic acid residues chain V _H chain v _L Amino acid V _H Amino acid _vL nucleic acid V _H nucleic acid V _L option 1 3210 VH. one VL thirteen fourteen fifteen sixteen option 2 3210 VH. 2 VL 17 fourteen eighteen sixteen option 3 3210 VH. 2 VL.3 17 37 eighteen 38 option 4 3210 VH. 3 VL. 4 49 fifty 51 52 option 5 3210 VH. 3 VL 49 fourteen 51 sixteen option 6 3210 VH. 2 VL. 4 17 fifty eighteen 52 option 1 2430 VH. one VL 29 thirty 31 32 option 2 2430 VH. 2 VL 33 thirty 34 32 option 3 2430 VH. 3 VL.2 59 60 61 62 option 4 2430 VH. 3 VL 59 thirty 61 32 option 5 2430 VH. 2 VL.2 33 60 34 62

An antibody or antibody fragment according to the application may contain a heavy chain variable region containing an amino acid sequence that is approximately 70, 75, 80, 85, 90, 95, 97, 98, 99, or 100% identical to the sequence described in any SEQ ID NO : 13, 17, 29, 33, 49, or 59. They may also contain a light chain variable region containing an amino acid sequence that is approximately 70, 75, 80, 85, 90, 95, 97, 98, 99, or 100% identical the sequence described in SEQ ID NO: 14, 30, 37, 50, or 60. In addition, the antibody or antibody fragment may contain a heavy chain or light chain variable region containing an amino acid sequence that is approximately 70, 75, 80, 85, 90%, 95%, 97%, 98%, 99% or 100% identical to the sequences shown in FIG. 24 and 25.

The antibody or antigen-binding fragment thereof can neutralize RSV, MPV and PVM infection and contain a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 13 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 14; or a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 13, a light chain variable region containing the amino acid sequence of SEQ ID NO: 37; or a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 13, a light chain variable region containing the amino acid sequence of SEQ ID NO: 50; or a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 17, a light chain variable region containing the amino acid sequence of SEQ ID NO: 14; or a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 17, a light chain variable region containing the amino acid sequence of SEQ ID NO: 37; or a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 49, a light chain variable region containing the amino acid sequence of SEQ ID NO: 50; or a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 49, a light chain variable region containing the amino acid sequence of SEQ ID NO: 14; or a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 49, a light chain variable region containing the amino acid sequence of SEQ ID NO: 37; or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17 and the variable region

- 9 039682 light chain containing the amino acid sequence of SEQ ID NO: 50.

In addition, the antibody or antigen-binding fragment may neutralize RSV, MPV and PVM infection and contain a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 29 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 30; or a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 29 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 60; or a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 33, a light chain variable region containing the amino acid sequence of SEQ ID NO: 30; or a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 59, a light chain variable region containing the amino acid sequence of SEQ ID NO: 60; or a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 59, a light chain variable region containing the amino acid sequence of SEQ ID NO: 30; or a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 33 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 60.

Examples of antibodies include, but are not limited to, HMB3210 variant 1, HMB3210 variant 2, HMB3210 variant 3, variant 4, HMB3210 variant 5 HMB3210, variant 6 HMB3210, variant 1 HMB2430, variant 2 HMB2430, variant 3 HMB2430, variant 4 HMB2430 or variant 5 HMB2430.

An antibody or fragment thereof can bind to the same epitope as an antibody or antigen-binding fragment of the invention, or an antibody that competes for binding to the antibody or antigen-binding fragment.

As can be seen from Table. 1 and 2, the CDRs, heavy chains, and light chains of disclosed antibodies can be interchanged to produce new antibodies that retain their binding and neutralizing abilities. Thus, antibodies can include antibodies and antigennegative fragments containing any combination of the CDRs shown in table. 1, or heavy and light chains shown in table. 2.

Antibodies may also contain hybrid antibody molecules that contain one or more CDRs from an antibody of the invention and one or more CDRs from another antibody in the same epitope. Such hybrid antibodies may contain three CDRs from an antibody of the invention and three CDRs from another antibody for the same epitope. Exemplary hybrid antibodies may comprise (i) three light chain CDRs from an antibody of the invention and three heavy chain CDRs from another antibody for the same epitope, or (ii) three heavy chain CDRs from an antibody of the application and three light chain CDRs from another antibody. .

Sequence variants listed in the application include natural variants resulting from somatic mutation in vivo during an immune response, or in vitro when culturing immortalized B cell clones. Alternatively, variants may arise from degeneracy of the genetic code, or may result from errors in transcription or translation.

Additional antibody sequence variants having improved affinity and/or activity can be generated by methods known in the art. For example, amino acid substitutions can be used to generate antibodies with further improved affinity. Alternatively, codon optimization of the nucleotide sequence can be used to improve translation efficiency in expression systems for antibody production. In addition, polynucleotides containing a sequence optimized for antibody specificity or neutralizing activity by site-directed mutagenesis for any nucleic acid sequences are also disclosed in the application.

An antibody sequence variant may have 70% or more (ie, 75%, 80, 85, 90, 95, 97, 98, 99% or more) amino acid sequence identity with the sequences listed in the application. In some embodiments, such sequence identity is calculated relative to the full length of the reference sequence (ie, the sequence listed in the application). In some additional embodiments, percent identity as defined herein is as determined using BLAST version 2.1.3 using the default settings set by NCBI (the National Center for Biotechnology Information; http://www.ncbi. nlm.nih.gov/) [Blosum matrix 62, gap creation penalty=11 and gap extension penalty=1].

The invention also includes nucleic acid sequences encoding part or all of the light and heavy chains and CDRs of the antibodies of the present invention. Disclosed herein are nucleic acid sequences encoding part or all of the light and heavy chains and CDRs of the antibodies of the invention. In table. 2 shows SEQ ID numbers for nucleic acid sequences encoding heavy chain and light chain variable regions of some exemplary antibodies of the invention. In table. 3 shows the SEQ ID numbers for nucleic acid sequences encoding antibody CDRs. Due to the redundancy of the genetic code, there are variants of these nucleic acid sequences that encode the same amino acid sequences.

Table 3

SEQ ID numbers for CDR polynucleotides of antibodies that neutralize RSV, MPV and PVM

SEQID NO. for CDR polynucleotides CDRH1 CDRH2 CDRH3 CDRL1 CDRL2 CDRL3 option 1 3210 7 eight nine ten eleven 12 option 2 3210 7 eight nine ten eleven 12 option 3 3210 7 eight nine ten eleven 36 option 4 3210 43 44 45 46 47 48 option 5 3210 43 44 45 ten eleven 12 option 6 3210 7 eight nine 46 47 48 option 1 2430 24 25 26 ten 27 28 option 2 2430 24 25 26 ten 27 28 option 3 2430 55 44 56 57 47 58 option 4 2430 55 44 56 ten 27 28 option 5 2430 24 25 26 57 47 58

Nucleic acid sequences may include nucleic acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98 %, or at least 99% identity with the nucleic acid encoding the heavy or light chain of the antibody of the invention. Also, the nucleic acid sequence may comprise a nucleic acid sequence encoding a heavy or light chain CDR of an antibody of the invention. For example, a nucleic acid sequence contains a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identical nucleic acid SEQ ID NO: 7-12, 15, 16, 18, 24-28, 31-32, 34, 36, 38, 43-48, 51-52, 55-58, or 61-62.

In yet another embodiment, nucleic acid sequences may include nucleic acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identity with a nucleic acid encoding a heavy or light chain of an antibody of the invention as shown in FIG. 24 and 25.

In addition, the application describes expression vectors containing the nucleic acid sequence of the invention. Cells transformed with such vectors are also within the scope of the invention. Examples of such cells may include eukaryotic cells such as yeast cells, animal cells, or plant cells. Also, the cells may be mammalian, eg human, CHO, HEK293T, PER.C6, NS0, myeloma or hybridoma cells.

The application discloses monoclonal antibodies that bind to an epitope capable of binding to the antibodies or antigen-binding fragments of the invention.

Monoclonal and recombinant antibodies are particularly useful for identifying and purifying individual polypeptides or other antigens against which they are directed. Antibodies, according to the application, may have the additional useful property that they can be used as reagents in immunoassays, radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA). In such applications, antibodies can be labeled with an analytically detectable reagent such as a radioactive isotope, fluorescent molecule, or enzyme. Antibodies can also be used for molecular identification and characterization (epitope mapping) of antigens.

Antibodies according to the application can be associated with a drug for delivery to the treatment site or associated with a detectable label to facilitate visualization of the site containing cells of interest, cells infected with RSV or MPV or RSV and MPV. Methods for binding antibodies to drugs and detectable labels are well known in the art, as are imaging methods using detectable labels. Labeled antibodies can be used in a wide range of assays using a wide range of labels. Detection of antibody-antigen complex formation between an antibody of the invention and an epitope of interest (epitope or RSV or MPV or both) can be facilitated by attaching a detectable substance to the antibody. Suitable detection methods include the use of labels such as radionuclides, enzymes, coenzymes, fluorescent substances, chemiluminescent substances.

- 11 039682 agents, chromogens, enzyme substrates or cofactors, enzyme inhibitors, prosthetic group complexes, free radicals, particles, dyes, and the like. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent substance is luminol; examples of bioluminescent substances include luciferase, luciferin and aequorin, and examples of a suitable radioactive substance include 125I, 131I, 35S or 3H. Such labeled reagents can be used in a number of well known assays such as radioimmunoassays, enzyme immunoassays such as ELISA, fluorescence immunoassays, and the like. (See, for example, US 3766162, US 3791932, US 3817837 and US 4233402).

An antibody of the invention may be conjugated to a therapeutic molecule such as a cytotoxin, a therapeutic agent, or a radioactive metal ion or radioactive isotope. Examples of radioactive isotopes include, but are not limited to, I-131, I-123, I-125, Y-90, Re-188, Re-186, At-211, Cu-67, Bi-212, Bi-213, Pd-109, Tc-99, In-111 etc. Such antibody conjugates can be used to modify a given biological response; imply that the drug molecule is not limited to classical chemical therapeutic agents. For example, the drug molecule may be a protein or polypeptide having the desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, Pseudomonas aeruginosa exotoxin, or diphtheria toxin.

Methods for conjugating such a therapeutic molecule to antibodies are well known. See, for example, Arnon et al. (1985) Monoclonal Antibodies for Immunotargeting of Drugs in Cancer Therapy in Monoclonal Antibodies and Cancer Therapy, ed. Reisfeld et al. (Alan R. Liss, Inc.), pp. 243-256; ed. Hellstrom et al., (1987) Antibodies for Drug Delivery in Controlled Drug Delivery, ed. Robinson et al. (2d ed; Marcel Dekker, Inc.), pp. 623-653; Thorpe (1985) Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review in Monoclonal Antibodies '84: Biological and Clinical Applications, ed. Pinchera et al., pp. 475-506 (Editrice Kurtis, Milano, Italy, 1985); Analysis, Results and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy in Monoclonal Antibodies for Cancer Detection and Therapy, ed. Baldwin et al. (Academic Press, New York, 1985), pp. 303-316 and Thorpe et al., (1982) Immunol. Rev., 62: 119-158.

Alternatively, an antibody or antibody fragment thereof can be conjugated to a second antibody or antibody fragment thereof to form an antibody heteroconjugate as described in US 4,676,980. Linkers between labels and antibodies of the invention can also be used (eg, US 4,831,175). Antibodies or their antigen-binding fragments can be directly labeled with radioactive iodine, indium, yttrium, or other radioactive species known in the art (eg, US 5,595,721). Treatment may consist of a combination of treatment with conjugated and unconjugated antibodies administered simultaneously or sequentially (eg WO00/52031, WO 00/52473).

The antibodies of the invention can also be attached to a solid support. In addition, the antibodies of the invention, or functional antibody fragments thereof, can be chemically modified by covalent conjugation to a polymer, for example, to increase their circulating half-life. Examples of polymers and methods for attaching them to peptides are provided in US 4,766,106, US 4,179,337, US 4,495,285, and US 4,609,546. In some embodiments, polymers can be selected from polyoxyethylated polyols and polyethylene glycol (PEG). PEG is water soluble at room temperature and has the general formula: R(O--CH2--CH2)nO--R where R can be hydrogen or a protecting group such as an alkyl or alkanolic group. The protecting group may contain from 1 to 8 carbon atoms, for example be methyl. The symbol n is a positive integer and is from 1 to 1000 or from 2 to 500. The average molecular weight of PEG is from 1000 to 40000, according to the application. In an additional embodiment, the molecular weight of PEG is from 2000 to 20000 or from 3000 to 12000. In one embodiment, the PEG may contain at least one hydroxy group, in another embodiment, a terminal hydroxy group and a terminal hydroxy group that is activated to interact with a free amino group on the inhibitor. However, it should be understood that the type and number of reactive groups can be changed to obtain a covalently conjugated PEG/antibody.

Also suitable are water-soluble polyoxyethylene polyols. These include polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol (POG), and the like. In one embodiment, POG is used, without being bound by any theory, due to the fact that the glycerol backbone of polyoxyethylated glycerol is similar to the natural backbone found, for example, in mono-, di-, triglycerides of animals and humans, therefore such a branched chain is not necessarily recognized in the body as a foreign agent. In some embodiments, POG may have a molecular weight in the same range as PEG. Another drug delivery system that can be used to increase circulatory half-life

- 12 039682 is a liposome. Methods for making delivery systems based on liposomes are known to the person skilled in the art. Other drug delivery systems are also known in the art and are described, for example, in Poznansky et al., (1980) and Poznansky (1984).

Antibodies, according to the application, may be in purified form. Typically, the antibody is contained in a composition that is substantially free of other polypeptides, e.g., where other polypeptides comprise less than 90% (by weight), typically less than 60%, and more, typically less than 50% of the composition.

The antibodies of the application may be immunogenic in a non-human (or heterologous) host, such as mice. In particular, the antibodies may contain an idiotope that is immunogenic in a host (not a human). Antibodies claimed for use in humans include antibodies that cannot be readily isolated from hosts such as mice, goats, rabbits, rats, non-primate mammals, etc. and generally cannot be obtained by humanization or from xeno mice .

Antibodies according to the application can be of any isotype (for example, IgA, IgG, IgM, ie heavy chain α, γ or μ), but, as a rule, are IgG. In the IgG isotype, the antibodies may be a subclass of IgG1, IgG2, IgG3, or IgG4.

Antibodies according to the application may contain a light chain to or λ.

Obtaining antibodies

Antibodies of the invention can be obtained by any method known in the art. For example, the basic methodology for obtaining monoclonal antibodies using hybridoma technology is well known (Kohler G. and Milstein C.,. 1975; Kozbar et al., 1983). In one embodiment, the alternative EBV immortalization method described in WO2004/076677 is used.

In the manner described in WO 2004/076677, antibody producing B cells can be transformed with EBV and a polyclonal B cell activator. Additional cell growth and differentiation stimulators can be added to further improve efficiency during the transformation step. Such stimulants may be cytokines such as IL-2 and IL-15. In one aspect, IL-2 is added during the immortalization step to further improve the efficiency of immortalization, but its use is optional. Immortalized B cells obtained by such methods can then be cultured by methods known in the art and isolated from them antibodies.

The method described in WO 2010/046775 can culture plasma cells in limited quantities or as individual plasma cells on microwell culture plates. Antibodies can be isolated from a culture of plasma cells. In addition, RNA can be extracted from the plasma cell culture and PCR can be carried out by methods known in the art. The VH and VL regions of the antibodies can be amplified by reverse transcription PCR, sequenced and cloned into an expression vector which is then transfected into HEK293T cells or other host cells. Cloning the nucleic acid into expression vectors, transfecting host cells, culturing the transfected host cells, and isolating the antibody produced can be carried out by any means known to one of skill in the art.

If desired, antibodies can be further purified using filtration, centrifugation, and various chromatographic techniques such as HPLC or affinity chromatography. Methods for purifying antibodies, such as monoclonal antibodies, including methods for preparing pharmaceutical grade antibodies, are well known in the art.

Antibody fragments of the invention can be obtained from antibodies by methods that include cleavage with enzymes such as pepsin or papain and/or cleavage of disulfide bonds by chemical reduction. In addition, antibody fragments can be obtained by cloning and expression of a portion of the heavy or light chain sequences. Antibody fragments include Fab, Fab', F(ab')2 and Fv fragments. Single chain Fv fragments (scFv) are derived from the heavy and light chains of an antibody of the invention. In addition, monomers and dimers of heavy or light chains, single domain heavy chain antibodies, single domain light chain antibodies, as well as single chain antibodies, for example, single chain Fv, in which the variable domains of heavy and light chains are connected by a peptide linker, can be obtained.

Antibody fragments can provide monovalent or multivalent interactions and can be contained in a number of structures, as described above. For example, scFv molecules can be synthesized to produce a trivalent antibody or a tetravalent antibody. The scFv molecules may contain the domain of the Fc region resulting in divalent mini-antibodies. In addition, the sequences according to the application can be a component of multispecific molecules, in which the sequences are directed to epitopes, and other regions of the molecules are associated with other targets. Illustrative molecules include, but are not limited to, Fab2 bispecific, Fab3 trispecific, scFv bispecific, and bivalent antibodies (Holliger and Hudson, 2005, Nature Biotechnology, 9:11261136).

To obtain DNA sequences encoding antibodies or antibody fragments according to

- 13 039682 of the present invention, standard methods of molecular biology can be used. Desired DNA sequences can be synthesized in whole or in part using oligonucleotide synthesis methods. As appropriate, site-directed mutagenesis and polymerase chain reaction (PCR) methods can be used.

Any suitable host/vector system can be used to express DNA sequences encoding antibody molecules of the present invention or fragments thereof. Bacterial, eg, E. coli, and other microorganism-based systems can be partially used to express antibody fragments such as Fab and F(ab')2 fragments, and in particular Fv fragments and single chain antibody fragments, eg single chain Fv.

Expression systems based on eukaryotic host cells, such as mammals, can be used to produce larger antibody molecules, including full antibody molecules. Suitable mammalian host cells include, but are not limited to, CHO, HEK293T, PER.C6, NS0, myeloma or hybridoma cells.

The present application also describes a method for producing an antibody molecule of the present invention, comprising culturing a host cell containing a vector encoding a nucleic acid of the present invention under conditions suitable for expressing a protein from DNA encoding an antibody molecule of the present invention, and isolating the antibody molecule.

The antibody molecule may contain only a heavy or light chain polypeptide, in which case only the sequence encoding the heavy chain or light chain polypeptide needs to be used for transfection of the host cells. To obtain products containing both heavy and light chains, the cell line can be transfected with two vectors, where the first vector encodes a light chain polypeptide and the second vector encodes a heavy chain polypeptide. In addition, a single vector can be used, where the vector contains sequences encoding light chain and heavy chain polypeptides.

Also, the antibodies of the invention can be produced by (i) expression of the nucleic acid in a host cell, and (ii) isolation of the expressed antibody product. In addition, the method may include (iii) purification of the isolated antibody.

Transformed B cells and cultured plasma cells can be screened for cells producing antibodies of the desired specificity or function.

The screening step can be performed by any immunological assay, eg, ELISA, tissue or cell staining (including transfected cells), neutralization assay, or one of a number of other methods known in the art to identify the desired specificity or function. The assay may be selected based on the simple recognition of one or more antigens, or may be selected based on the desired function, e.g. to select neutralizing antibodies rather than just antigen-binding antibodies, select antibodies that can alter characteristics of target cells such as their signaling cascades, their shape, their rate of growth, their ability to influence other cells, their response to the influence of other cells or other agents, or changing conditions, their status of differentiation, etc.

Individual clones of transformed B cells can then be obtained from a culture of positive transformed B cells. The cloning step to isolate individual clones from a mixture of positive cells can be performed using limiting dilution, micromanipulation, single cell isolation by cell sorting, or other method known in the art.

Nucleic acid from cultured plasma cells can be isolated, cloned and expressed in HEK2 93T cells or other known host cells by methods known in the art.

Immortalized B cell clones or transfected host cells can be used in various ways, for example, as a source of monoclonal antibodies, as a source of nucleic acid (DNA or mRNA) encoding a monoclonal antibody of interest, for research, and so on.

The application describes a composition containing immortalized memory B cells or transfected host cells producing antibodies that neutralize RSV, MPV and PVM infection.

An immortalized B cell clone or cultured plasma cells can also be used as a source of nucleic acid for cloning antibody genes for subsequent recombinant expression. Recombinant expression is more common for pharmaceutical purposes than expression by B cells or hybridomas, for example, for reasons of stability, reproducibility, ease of culture, etc.

Thus, the application discloses a method for producing a recombinant cell, comprising the steps of: (i) obtaining one or more nucleic acids (eg, heavy and/or light chain mRNA) encoding an antibody of interest from a clone of B cells or cultured plasma cells; (ii) inserting the nucleic acid into the expression vector; and (iii) transfecting the vector into a host cell to allow expression of the presenting antibody in that host cell.

- 14 039682

Similarly, the application discloses a method for producing a recombinant cell, comprising the steps of: (i) sequencing a nucleic acid(t) encoding an antibody of interest from a clone of B cells or cultured plasma cells, and (ii) using the sequencing information from step (i) to obtain a nucleic acid acid(s) to be introduced into a host cell to cause the antibody of interest to be expressed in that host cell. The nucleic acid may, but need not, be processed between steps (i) and (ii) to introduce restriction sites, to change codon usage, and/or to optimize transcriptional and/or translational regulatory sequences.

The application can also be referred to a method for obtaining a transfected host cell, comprising the step of transfecting the host cell with one or more nucleic acids that encode an antibody of interest, where the nucleic acids are nucleic acids that were obtained from a clone of immortalized B cells or a cultured plasma cell . Thus, methods of pre-producing a nucleic acid(t) and then using it to transfect a host cell can be done at different times, by different people in different places (eg, different countries).

Such recombinant cells can then be used for expression and culture. They are particularly suitable for the expression of antibodies for large scale pharmaceutical production. They can also be used as an active ingredient in a pharmaceutical composition. Any suitable culture method may be used, including, but not limited to, static culture, spinner culture, ascitic fluid, hollow fiber bioreactor cartridge, modular mini-fermenter, mixing tank, microcarrier culture, ceramic rod irrigation, etc.

Methods for obtaining and sequencing immunoglobulin genes from B cells or plasma cells are well known in the art (eg, see Chapter 4 of Kuby Immunology, 4th edition, 2000).

The transfected host cell may be a eukaryotic cell, including yeast and animal cells, in particular mammalian cells (e.g. CHO cells, NS0 cells, human cells such as PER.C6 or HKB-11 cells, myeloma cells), as well as plant cells. cells.

Preferred expressing hosts can glycosylate the antibody, in particular with carbohydrate structures, which are not themselves immunogenic in humans. In one embodiment, the transfected host cell may grow in a serum-free medium. In another embodiment, the transfected host cell may grow in culture without the presence of animal-derived products. The transfected host cell can also be cultured to form a cell line.

The application discloses a method for producing one or more nucleic acid molecules (eg, heavy and light chain genes) that encode an antibody of interest, comprising the steps of: (i) obtaining an immortalized B cell clone or culturing plasma cells of the invention; (ii) obtaining from a clone of B cells or cultured plasma cells a nucleic acid encoding an antibody of interest. In addition, a method for obtaining a nucleic acid sequence encoding an antibody of interest may include the steps of: (i) obtaining an immortalized B cell clone or culturing plasma cells according to the invention; (ii) sequencing the nucleic acid encoding the antibody of interest from a clone of B cells or cultured plasma cells.

According to the application, a method for obtaining a nucleic acid molecule(s) that encodes an antibody of interest, comprising the step of obtaining a nucleic acid that was obtained from a clone of transformed B cells or cultured plasma cells according to the invention. Thus, methods for first obtaining a clone of B cells or a cultured plasma cell and then obtaining nucleic acid(s) from a clone of B cells or cultured plasma cells can be carried out at different times, they can be carried out by different people in different places (for example, , in different countries).

A method for producing an antibody (for example, for pharmaceutical use) may include the steps of: (i) obtaining and/or sequencing one or more nucleic acids (for example, heavy and light chain genes) from a selected clone of B cells or cultured plasma cells expressing the representative an antibody of interest; (ii) inserting the nucleic acid(s) or using the nucleic acid sequence(s) to obtain an expression vector; (iii) transfecting a host cell that can express the antibody of interest; (iv) culturing or subculturing the transfected host cells under conditions where the antibody of interest is expressed, and optionally (v) purifying the antibody of interest.

Also, the method for producing an antibody may include the steps of culturing or subculturing a population of transfected host cells under conditions where the antibody of interest is expressed, and optionally purifying the antibody of interest, where said population

The transfected host cells were prepared by (i) providing the nucleic acid(t) encoding the selected antibody of interest, which is produced by a clone of B cells or cultured plasma cells obtained as described above, (ii) inserting the nucleic acid( m) into an expression vector, (iii) transfecting the vector into host cells that can express the antibody of interest, and (iv) culturing or subculturing the transfected host cell containing the inserted nucleic acids to produce the antibody of interest. Thus, methods for pre-producing a recombinant host cell and then culturing it to express an antibody can be performed at different times, by different people in different places (eg, different countries).

epitopes

As indicated above, the antibodies according to the application can be used to map the epitopes to which they bind. The inventors have discovered that the described neutralizing antibodies are directed to epitopes occurring in the F protein before the fusion, but not after the fusion. In one embodiment, the antibodies, or antigen-binding fragments thereof, bind to the RSV F protein prior to fusion and do not bind to the RSV F protein after fusion. In another embodiment, the antibodies, or antigen-binding fragments thereof, bind to the F protein of RSV and MPV prior to fusion and do not bind to the F protein of RSV and MPV after fusion. In yet another embodiment, the antibodies or antigen-binding fragments thereof bind to the F protein of RSV, MPV and PVM prior to fusion, but do not bind to the F protein of RSV, MPV and PVM after fusion.

The epitopes to which antibodies bind can be linear (continuous) or conformational (discontinuous). In one embodiment, the antibodies and antibody fragments will bind to a conformational epitope. Alternatively, the conformational epitope will only be presented under non-reducing conditions. Without wishing to be bound by any theory, the conformational epitope to which antibodies bind is due to the presence of disulfide bonds between amino acid residues in the F protein.

In another embodiment, the epitope to which the antibodies bind may be different from antigenic region I, antigenic region II, antigenic region IV, as determined in the post-fusion RSV F protein and the corresponding regions in the MPV F protein. Alternatively, the epitopes may not cross-compete with palivizumab, motavizumab, mAb 101F, mAb 131-2A or mAb D25 for binding to the RSV F protein and do not cross compete with mAb 234 for binding to the MPV F protein.

In another embodiment, the antibody-binding region may contain a polypeptide located at the N-terminus of the RSV F protein, including residues SAVSKGYLSALRTGWYTSVIT (SEQ ID NO: 63). The core portion in this polypeptide is formed by Y(x1)S(x2)LRTGW residues that are highly conserved for RSV, MPV, and PVM, and where the amino acid at position (x1) may be, but is not limited to, L, F, or K, and where the amino acid at position (x2) may be, but is not limited to, A or V. Examples of polypeptide variants that antibodies can bind to include, but are not limited to, YLSALRTGW (SEQ ID NO: 64), YLSVLRTGW (SEQ ID NO : 65), YFSALRTGW (SEQ ID NO: 66), YFSVLRTGW (SEQ ID NO: 67), YKSALRTGW (SEQ ID NO: 68), and YKSVLRTGW (SEQ ID NO: 69).

Polypeptides that can bind to antibodies can find a number of uses. Polypeptides and polypeptide variants thereof, in purified or synthetic form, can be used to enhance immune responses (ie, as a vaccine or to produce antibodies for other applications) or to screen serum for antibodies that immunologically interact with an epitope or its mimotopes. In one embodiment, such polypeptides or polypeptide variants, or an antigen containing such polypeptides or polypeptide variants, can be used as a vaccine to enhance the immune response, which contains the same antibodies as described. Antibodies and antibody fragments can also be used in a method for monitoring the quality of vaccines. In particular, antibodies can be used to verify that an antigen in a vaccine contains the correct immunogenic epitope in the correct conformation. You can also consider using the antibody or its antigen-binding fragment to monitor the quality of a vaccine against RSV or MPV or RSV and MPV, for example, by checking that the antigen of said vaccine contains a specific epitope in the correct conformation.

Polypeptides that bind to the antibodies of the present invention may also be useful for screening for ligands that bind to said polypeptides. Such ligands include, but are not limited to, antibodies, including antibodies from camelids, sharks, and other species, antibody fragments, peptides, phage display technology products, aptamers, adnectins, or fragments of other viral or cellular proteins that can block an epitope and thus prevent infection.

Pharmaceutical compositions

The pharmaceutical composition may contain one or more of the antibodies or antibody fragments of the invention; nucleic acid encoding such antibodies or fragments; vectors encoding nucleic acids or polypeptides recognized by antibodies or antigen-binding

- 16 039682 a general fragment according to the invention. The pharmaceutical composition may also contain a pharmaceutically acceptable carrier or excipient. While the carrier or excipient may facilitate administration, it should not by itself induce the formation of antibodies that are harmful to the individual receiving the composition. It also should not be toxic. Suitable carriers can be large slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive viral particles.

Pharmaceutically acceptable salts may be used, for example inorganic acid salts such as hydrochlorides, hydrobromides, phosphates and sulfates, or organic acid salts such as acetates, propionates, malonates and benzoates.

Pharmaceutically acceptable carriers in therapeutic compositions may additionally contain liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances such as wetting agents or emulsifiers or pH buffering agents may be contained in such compositions. Such carriers make it possible to prepare pharmaceutical compositions in the form of tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions, for ingestion by an individual.

The composition may be presented in several forms of administration, forms including, but not limited to, those forms suitable for parenteral administration, for example, by injection or infusion, for example, by bolus injection or continuous infusion. Where the product is intended for injection or infusion, it may be in the form of a suspension, solution or emulsion in an oily or aqueous vehicle, and it may contain adjuvants such as suspending agents, preservatives, stabilizers and/or dispersing agents. Alternatively, the antibody molecule may be in a dry form that must be reconstituted using a suitable sterile liquid prior to use.

After receiving the composition according to the invention can be administered directly to the individual. In one embodiment, the compositions are adapted for administration to a mammal, such as humans

Pharmaceutical compositions can be administered by any of a number of routes, including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intraperitoneal, intrathecal, intraventricular, transdermal, transdermal, topical, subcutaneous, intranasal, enteral, sublingual, intravaginal, or rectal way. For the introduction of pharmaceutical compositions, according to the application, you can also use needleless syringes. As a rule, therapeutic compositions can be obtained in the form of injectable solutions, as well as in the form of liquid solutions or suspensions. Solid forms suitable for dissolution or suspension in liquid carriers prior to injection may also be prepared.

Direct delivery of the compositions is typically by injection, subcutaneously, intraperitoneally, intravenously or intramuscularly, or into the extracellular space of the tissue. The compositions can also be administered to the lesion. The dosage treatment may be a single dosing regimen or a multiple dosing regimen. Known antibody-based pharmaceuticals provide guidance regarding the frequency of administration, such as whether the pharmaceutical should be delivered daily, weekly, monthly, etc. Frequency and dosage may also depend on the severity of symptoms.

The compositions can be obtained in various forms. For example, the compositions can be prepared as injectable solutions as well as liquid solutions or suspensions. It is also possible to prepare solid forms suitable for dissolution or suspension in liquid vehicles prior to injection (eg, lyophilized formulations such as Synagis™ and Herceptin™ for reconstitution with sterile water containing a preservative). The composition can be prepared for topical administration, for example in the form of an ointment, cream or powder. The composition can be prepared for oral administration, for example, as a tablet or capsule, as a spray, or as a syrup (optionally flavored). The composition can be prepared for pulmonary administration, for example, as an inhalation aerosol, using a fine powder or spray. The composition can be obtained in the form of a suppository or pessary. The composition can be prepared for nasal, ear or ocular administration, for example in the form of drops. The composition may be in the form of a kit designed so that the combined composition is reconstituted immediately prior to administration to the individual. For example, the lyophilized antibody may be provided in kit form with sterile water or sterile buffer.

It should be understood that the active ingredient in the composition may be an antibody molecule, an antibody fragment, or variants and derivatives thereof. It undergoes degradation in the gastrointestinal tract. Thus, if the composition is to be administered using the gastrointestinal tract, the composition must contain an agent that protects the antibody from degradation, but which releases the antibody after absorption from the gastrointestinal tract.

A comprehensive discussion of pharmaceutically acceptable carriers is available in Gennaro (2000) Re

- 17 039682 mington: The Science and Practice of Pharmacy, 20th edition, ISBN: 0683306472.

Pharmaceutical compositions disclosed in the application will typically have a pH of 5.5 to 8.5, in some embodiments, it may be from 6 to 8, and in other embodiments, about 7. The pH can be maintained through the use of a buffer. The composition may be sterile and/or non-pyrogenic. The composition may be isotonic to humans. In one embodiment, the pharmaceutical compositions may be supplied in hermetically sealed containers.

Pharmaceutical compositions may contain an effective amount of one or more antibodies and/or a polypeptide containing an epitope that binds to the antibody i. an amount that is sufficient to treat, ameliorate, ameliorate, or prevent the desired disease or condition, or to produce a detectable therapeutic effect. Therapeutic effects also include the reduction or amelioration of pathogenic activity or physical symptoms. The exact effective amount for any particular individual will depend on their size, weight and health status, the nature and severity of the condition, and the therapeutic agents or combination of therapeutic agents chosen to be administered. The effective amount for a given situation is determined by routine experimentation and is at the discretion of the clinician. For the compositions described, an effective dose will generally be from about 0.01 to about 50 mg/kg, or from about 0.05 to about 10 mg/kg, for the individual to whom it is administered. Known antibody-based pharmaceuticals provide recommendations for such a dose, for example, Herceptin™ is administered by intravenous infusion of a 21 mg/ml solution with an initial loading dose of 4 mg/kg body weight and a weekly maintenance dose of 2 mg/kg body weight; Rituxan™ is administered weekly at 375 mg/m ² etc.

In one embodiment, the compositions may contain more than one (eg, 2, 3, etc.) antibodies to provide an additive or synergistic therapeutic effect. In another embodiment, the composition may contain one or more (eg, 2, 3, etc.) antibodies and one or more (eg, 2, 3, etc.) additional antibodies against RSV, MPV or RSV and MPV. It is also possible to administer antibodies together with antibodies specific for other pathogens, eg influenza A or influenza B virus. Antibodies can be administered in combination/simultaneously or separately from antibodies specific for pathogens other than RSV or MPV.

The pharmaceutical composition may contain two or more antibodies, where the first antibody is an antibody as disclosed herein and the second antibody is specific for RSV, MPV or RSV and MPV or another pathogen that may be present in the individual with mixed infection who is administered pharmaceutical composition.

Examples of antibodies specific to and neutralizing RSV, MPV, and PVM include, but are not limited to, HMB3210 variant 3, HMB3210 variant 1, HMB3210 variant 2, HMB3210 variant 4, HMB3210 variant 5, HMB3210 variant 6, HMB2430 variant 1, option 2 HMB2430, option 3 HMB2430, option 4 HMB2430 or option 5 HMB2430.

The pharmaceutical composition may contain HMB3210 variant 1 or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier. The pharmaceutical composition may also contain HMB3210 variant 2, or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier. And the pharmaceutical composition may contain HMB3210 variant 3, or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier. The pharmaceutical composition may also contain variant 4 of the HMB3210 antibody, or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier. The pharmaceutical composition may contain HMB3210 variant 5 or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier. The pharmaceutical composition may also contain HMB3210 variant 6 or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier.

In yet another embodiment, the pharmaceutical composition may contain version 1 of the HMB2430 antibody, or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition may contain version 2 of the HMB2430 antibody, or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition may contain version 3 of the HMB2430 antibody, or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition may contain version 4 of the HMB2430 antibody, or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition may contain version 5 of the HMB2430 antibody, or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier.

Antibodies can be administered (in combination or separately) in conjunction with other therapeutic agents, for example, chemotherapeutic compounds, radiation therapy, etc. In one embodiment, the therapeutic compounds include antiviral compounds such as Tamiflu™. Such combination treatment provides an additive or synergistic improvement in therapeutic efficacy compared to individual therapeutic agents when used alone.

- 18 039682 introduction. The term synergy is used to describe the combined effect of two or more active agents that is greater than the sum of the individual effects of each respective active agent. Thus, when the combined action of two or more agents results in a synergistic inhibition of an activity or process, it should be understood that the inhibition of the activity or process is greater than the sum of the inhibitory effects of each respective active agent. The term synergistic therapeutic effect refers to the therapeutic effect observed for a combination of two or more therapeutic agents, where the therapeutic effect (as determined by any of a number of parameters) is greater than the sum of the individual therapeutic effects observed for the respective individual therapies.

Antibodies can be administered to individuals who have not previously shown a response, ie. they have been shown to be resistant to treatment for RSV or MPV infection. Such treatment may include prior treatment with an antiviral agent. This may be due, for example, to an infection resistant to an antiviral agent by a strain of RSV, MPV or RSV and MPV.

In one embodiment, the composition may contain antibodies, where the antibodies may comprise at least 50 wt.% (for example, 60, 70, 75, 80, 85, 90, 95, 97, 98, 99% or more) of the total protein in compositions. In such a composition, the antibodies are in a purified form.

The application describes a method for producing a pharmaceutical composition, which includes the steps of (i) obtaining an antibody and (ii) mixing the purified antibody with one or more pharmaceutically acceptable carriers.

In another embodiment, the method for preparing a pharmaceutical composition may include the step of mixing the antibody with one or more pharmaceutically acceptable carriers, where the antibody is a monoclonal antibody that was obtained from a transformed B cell or a cultured plasma cell. Thus, the methods of first obtaining a monoclonal antibody and then obtaining a pharmaceutical preparation can be carried out at different points in time, they can be carried out by different people in different places (for example, in different countries).

As an alternative to an antibody or a B cell, a nucleic acid (typically DNA) encoding a monoclonal antibody of interest (or an active fragment thereof) derived from a B cell or cultured plasma cells can be used for therapeutic purposes. The nucleic acid can be expressed in situ in the individual to provide the desired therapeutic effect. Suitable gene therapy and delivery of nucleic acid vectors are known in the art.

The compositions may be immunogenic compositions, and in some embodiments, may be vaccine compositions comprising an antigen epitope, a recognized antibody, or an antigen-binding fragment thereof. Vaccines can be prophylactic (ie prevent infection) or therapeutic (ie treat or improve infection).

The compositions may contain an antimicrobial agent, particularly when packaged in a multiple dose format. They may contain a detergent such as Tween (polysorbate) such as Tween 80. Detergents are typically present in low levels, eg less than 0.01%. The compositions may also contain sodium salts (eg sodium chloride) to impart tonicity. The concentration of NaCl 10±2 mg/ml is typical.

In addition, the compositions may contain a sugar alcohol (eg, mannitol) or a disaccharide (eg, sucrose or trehalose), for example, about 15-30 mg/ml (for example, 25 mg/ml), in particular if they are to be lyophilized, or if they contain a substance that has been reconstituted from a lyophilized substance. The pH of the lyophilization composition can be adjusted from 5 to 8 or 5.5 to 7 or about 6.1 prior to lyophilization.

The compositions may also contain one or more immunoregulatory agents. In one embodiment, one or more immunoregulatory agents may include an adjuvant.

Epitope-based compositions can elicit a cell-mediated immune response as well as a humoral immune response to effectively combat RSV and MPV infection. Such an immune response can induce long-lasting (eg, neutralizing) antibodies and cell-mediated immunity that can respond rapidly to exposure to RSV or MPV or RSV and MPV.

Types of medical treatment and application

Antibodies and antibody fragments or derivatives and variants thereof can be used to treat an RSV or MPV infection or a mixed RSV and MPV infection, to prevent an RSV or MPV or RSV and MPV infection, or to diagnose an RSV or MPV infection.

Diagnostic methods may include bringing the antibody or antibody fragment into contact with a sample. Such samples may be tissue samples obtained from, for example, nasal passages, paranasal sinuses, salivary glands, lungs, liver, pancreas, kidney, ear, eye, placenta, digestive tract, heart, ovaries, pituitary gland, adrenal glands, thyroid gland, brain or skin. Diagnostic methods may also include detecting

- 19 039682 antigen/antibody complex.

Thus (i) an antibody, antibody fragment or variants and derivatives thereof, (ii) immortalized B cell clones, (iii) epitopes capable of binding to an antibody, or (iv) ligands, preferably an antibody, capable of binding to an epitope that binds with an antibody, for use in therapy.

The application describes a method of treating a subject, comprising administering to the subject an antibody, an antibody fragment or variants and derivatives thereof, or a ligand, preferably an antibody, capable of binding to an epitope that binds to the antibody. In one embodiment, the method results in a reduction in RSV or MPV infection in an individual. In another embodiment, the method prevents, reduces the risk of, or retards the development of an RSV or MPV infection in an individual.

The application can also be referred to the use of (i) an antibody, an antibody fragment or variants and derivatives thereof, (ii) an immortalized B cell clone, (iii) an epitope capable of binding to an antibody, (iv) a ligand, preferably an antibody, that binds to the epitope capable of binding to an antibody, or (v) a pharmaceutical composition for (i) the manufacture of a medicament for treating or attenuating RSV or MPV or RSV and MPV infection (ii) a vaccine or (iii) diagnosing RSV and MPV infection.

The application may relate to a composition for use as a medicament for the prevention or treatment of RSV or MPV infection. It also refers to the use of antibodies and/or a protein containing an epitope to which such an antibody binds in the manufacture of a medicament for the treatment of an individual and/or diagnosis in an individual. It also relates to a method of treating an individual, comprising the step of administering the composition to the individual. In some embodiments, the individual may be a human. One way to test the effectiveness of therapeutic treatment includes monitoring the symptoms of the disease after administration of the composition. Treatment may be a single dosing regimen or a multiple dosing regimen.

In one embodiment, the antibody, antibody fragment, immortalized B cell clone, epitope, or composition can be administered to an individual in need of such treatment. Such an individual includes, but is not limited to, an individual who is particularly at risk or predisposed to RSV or MPV infection, including, for example, an immunocompromised individual. The antibody or antibody fragment can also be used in passive immunization or active vaccination.

Antibodies and their fragments, as described in this application, can also be used in a kit for the diagnosis of RSV or MPV infection. In addition, epitopes capable of binding to an antibody can be used in a kit to monitor the effectiveness of vaccination methods by detecting the presence of protective anti-RSV antibodies or anti-MPV antibodies. Antibodies, antibody fragments or variants and derivatives thereof can also be used in a kit to monitor the production of a vaccine with the desired immunogenicity.

The application may refer to an epitope that specifically binds to an antibody, or antigen-binding fragment thereof, for use in (i) therapy, (ii) in the manufacture of a medicament for treating or attenuating infection with RSV or MPV or RSV and MPV, (iii) as a vaccine or (iv) to screen for ligands capable of neutralizing RSV or MPV or RSV and MPV infection.

The application can be related to a method for producing a pharmaceutical agent, including the step of mixing a monoclonal antibody with one or more pharmaceutically acceptable carriers, where the monoclonal antibody is a monoclonal antibody that was obtained from the transfected host cell. Thus, methods for pre-producing (e.g., expressing and/or purifying) a monoclonal antibody and then mixing it with the pharmaceutical carrier(s) can be done at different times, by different people at different locations (e.g., at different locations). countries).

Starting from a transformed B cell or a cultured plasma cell, various steps of culture, subculture, cloning, subcloning, sequencing, nucleic acid production, etc. can be carried out. with optimization at each step to preserve the antibody expressed by the transformed B cell or cultured plasma cell. In one embodiment, the above methods further include optimization methods (eg, affinity maturation or optimization) applied to antibody-encoding nucleic acids.

In all of these methods, the nucleic acid used in the expression host can be manipulated to insert, remove, or alter specific nucleic acid sequences. Changes resulting from such manipulation include, but are not limited to, changes to introduce restriction sites, to improve codon usage, to add or optimize transcriptional and/or translational regulatory sequences, and so on. You can also change the nucleic acid to change the encoded amino acids. For example, it may be useful to introduce one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) substitutions

- 20 039682 no acids, deletions and/or insertions in the amino acid sequence of an antibody. Such point mutations may modify effector functions, antigen binding affinity, post-translational modifications, immunogenicity, etc., may include amino acids to attach covalent groups (eg, labels), or may introduce labels (eg, for purification). Mutations may be introduced at specific sites, or may be introduced randomly followed by selection (eg, molecular evolution). For example, one or more nucleic acids encoding any of the CDRs, heavy chain variable regions, or light chain variable regions of antibodies can be subjected to random or site mutagenesis. Such changes can lead to an iterative process where the initial changes are maintained and new changes are introduced at other nucleotide positions. In addition, it is possible to combine changes obtained from independent stages. Various properties introduced into the encoded amino acid sequences may include, but are not limited to, increased affinity.

General provisions

As used herein, the terms antigen-binding fragment, fragment, and antibody fragment are used interchangeably to refer to any antibody fragment of the invention that retains the antigen-binding activity of the antibody. Examples of antibody fragments may include, but are not limited to, single chain antibody, Fab, Fab', F(ab') ₂ , Fv or scFv. In addition, the term antibody, as used herein, includes antibodies and antigen-binding fragments thereof.

As used herein, a neutralizing antibody is an antibody that neutralizes, eg, prevents, inhibits, reduces, hinders, or interferes with the ability of a pathogen to initiate and/or maintain an infection in a host. The terms neutralizing antibody and antibody that neutralizes or antibodies that neutralize are used interchangeably herein. Such antibodies can be used alone or in combination as prophylactic or therapeutic agents after appropriate formulation in association with active vaccination, as a diagnostic agent, or as a means of obtaining, as described in the present description.

The term containing includes including as well as consisting, for example, a composition containing X may consist solely of X or may contain some addition, such as X+Y.

The word essentially does not completely exclude, for example, a composition that essentially does not contain Y may contain absolutely no Y. If necessary, the word essentially can be omitted in the definition of the invention.

The term approximately in relation to the numerical value x means, for example, x±5% or x±7%, or x±10%, or x±12%, or x±15%, or x±20%.

It is assumed that the term disease, as used herein, is generally synonymous with, and is used interchangeably with, the terms disorder and condition (as in a medical condition), because they all reflect an abnormal condition of a human or animal body or one of its parts that interfere with normal functioning, as a rule, manifest themselves with distinctive signs and symptoms, and lead to the fact that the duration or quality of life of a person or animal is reduced.

As used herein, reference to treating an individual or patient is intended to include prevention, prophylaxis, mitigation, amelioration, and therapy.

The terms individual or patient are used interchangeably herein to refer to all mammals, including humans. Examples of individuals include humans, cows, dogs, cats, horses, goats, sheep, pigs and rabbits. In one embodiment, the patient is a human.

Examples

Illustrative embodiments of the present invention are provided in the following examples. The following examples are provided by way of illustration only and to assist one skilled in the art in making use of the invention. The examples are not intended to limit the scope of the invention in any way.

Example 1 Isolation and Characterization of Monoclonal Antibodies from Human Memory B Cells Capable of Cross-Neutralizing RSV and MPV

From a cohort of 125 blood donors, the authors selected 7 donors who had high titers of antibodies against RSV and MPV in serum. CD22+ IgG+ B cells were isolated from cryopreserved peripheral blood mononuclear cells (PBMC) and immortalized at 3-5 cells/well using Epstein-Barr virus (EBV) and oligodeoxynucleotide 2006 CpG and irradiated allogeneic PBMC as feeder cells. Culture supernatants were harvested after 14 days and analyzed for the presence of neutralizing antibodies by microneutralization assay based on infection of Hep-2 cells with RSV strain A2 or LLC-MK2 cells with MPV strain A1 I-PV-03/01-6621. Pure supernatants

- 21 039682 were incubated with 50-100 TCID ₅₀ viruses for 1 hour at room temperature before adding Hep-2 or LLC-MK2 target cells, which were incubated for 6 or 8 days, respectively. Viable cells were then detected using a spectrophotometer by adding WST-1 reagent (Roche) to the cultures for 3 to 4 hours.

From three independent experiments, 36 mAbs were isolated that neutralized MPV (FIG. 1, left panel) and from five independent experiments, 136 mAbs were isolated that neutralized RSV (FIG. 1, right panel). A secondary screen was then performed to test whether the isolated mAbs were able to neutralize MPV and RSV. Using this strategy, two mAbs isolated from one of the donors (don. 5) were found to cross-neutralize RSV and MPV: (i) HMB2430, which was initially selected based on RSV neutralization, and (ii) HMB3210, which was initially selected based on MPV neutralization.

The HMB2430 and HMB3210 VH and VL genes were cloned into IgG1 expression vectors and recombinant mAbs were generated by transient transfection of 293 Freestyle (293F) cells.

Supernatants from transfected cells were harvested after 10 days of culture and IgG was affinity purified by protein A chromatography. homology analysis performed using the IMGT database, but distinguished in N regions by the frequency of IGHD use (D3-10*01 and D5-24*01 for HMB2430 and HMB3210, respectively) and by the pattern of somatic mutations, and thus they were not considered clonally related.

The half maximal inhibitory concentration (IC ₅₀ ) of HMB2430 and HMB3210 was determined using the microneutralization assay described above with 100 tissue culture infectious doses 50 (TCID50) of the virus. IC ₅₀ values were calculated by interpolating neutralization curves fitted using 4-parameter non-linear regression with variable slope. The results of the analysis are shown in Fig. 2 and in table. 4.

________________________________________________________________________Table 4

mAb IC50 (ng/ml) Viruses (group) NMV2430 NMV3210 Palivizumab 234 A2 RSV (A) 146 86 524 >20000 MPV I-PV 03/01-6621 (A1) 393 52 >20000 7

Example 2. The degree of reactivity of all groups and subgroups of RSV and MPV

To assess the reactivity spectrum of HMB2430 and HMB3210, purified mAbs were tested by FACS for binding to RSV-infected Hep-2 cells or to MPV-infected LLC-MK2 cells using the following RSV and MPV strains: A2 RSV (A, 1961 Australia; A/A2/61) , RSV Long (A, Maryland US, 1956; A/Long/56), RSV Randall (A, Chicago US, 1958; A/Randall/58), RSV 9320 (B, Massachusetts US, 1977; B/9320/77 ), WV/14617/85 (B, Huntington West Virginia, 1985; B/14617/85), 18537 (B, Washington District of Columbia US, 1962; B/18537/62), MPV I-PV-03/01 -6621 (A1, Pavia IT, 2001; A1/6621/01), MPV I-PV-02/06-8938 (A2, Pavia IT, 2006; A2/8938/06), I-PV-03/04- 4702 (B1, Pavia IT, 2004; B1/4702/04) and I-PV-02/04-3817 (B2, Pavia IT, 2004; B2/3817/04). Three previously described mAbs were tested in parallel: (i) RSV-specific motavizumab; (ii) MPV-specific mAb 234; and (iii) influenza A-specific FO32 (used as a negative control). All mAbs were tested for binding to infected or uninfected cells at 10 μg/ml. HMB2430 and HMB3210 interacted with all 6 tested strains of RSV and all 4 tested strains of MPV characteristic of known groups and subgroups of RSV and MPV (Table 5). In contrast, motavizumab interacted with all 6 RSV strains tested, but did not interact with any of the 4 MPV strains tested. In turn, mAb 234 interacted with all 4 MPV strains tested, but did not interact with any of the 6 RSV strains tested.

- 22 039682

Table 5. Staining of Hep-2 or LLC-MK2 cells infected with different strains of RSV or MPV, respectively, with FO32 (negative control), motavizumab, 234, HMB2430, and

HMB3210 as measured by flow cytometry. Monoclonal antibody (10 µg/ml) Virus FO32 Motavizumab mAb 234 NMV2430 NMV3210 RSV A/A2/61 - + - + + RSV A/Long/56 - + - + + RSV A/Randall/58 - + - + + RSV B/18537/62 - + - + + RSV B/14617/85 - + - + + RSV B/9320/77 - + - + + MPV Al/6621/01 - - + + + MPV A2/8938/06 - - + + + MPV Bl/4702/04 - - + + + MPV B2/3817/04 - - + + + Mock LLC-MK2 - - - - - Mock Hep-2 - - - - -

(-)<5% stained cells (+)>50% stained cells

Example 3 Binding to Recombinant RSV and MPV F Protein by ELISA and Staining of Transfected Cells

To identify the target antigen recognized by HMB2430 and HMB3210 on RSV and MPV, the authors analyzed two mAbs in parallel with motavizumab and mAb 234 for their ability to bind to the homotrimeric soluble RSV F protein (strain A2), which was obtained from transiently transfected 293F cells. . As shown in FIG. 3, HMB2430 and HMB3210 interacted specifically with RSV F protein by ELISA and showed a different binding profile compared to motavizumab. In addition, HM2430 and HMB3210 stained intracellularly with 293F cells transiently transfected with mammalian cell-based expression vectors encoding the full-length F protein from RSV (A2 strain) or MPV (NL/1/99 B1 strain) (Table 6), indicating that HMB2430 and HMB3210 recognize a common epitope presented on the F proteins of RSV and MPV. This finding is particularly surprising given that the F proteins of RSV and MPV share only 33-35% amino acid sequence identity. As expected, palivizumab and motavizumab bound to cells expressing the RSV F protein, but not to cells expressing the MPV F protein (Table 6). In turn, mAb 234 bound to cells expressing the MPV F protein, but not to cells expressing the RSV F protein (Table 6).

Table 6. Staining of non-transfected 293F cells or 293F cells transfected

F-protein of RSV or MPV as measured by flow cytometry

Antibody (10 µg/ml) 293F+RSV F (A/A2/61; A) 293F+MPV F (NL/1/99; B1) Untransfected 293F NMV3210 + + - NMV2410 + + - Palivizumab + - - Motavizumab + - - 234 - + -

(-)<5% stained cells (+)>50% stained cells

Example 4 Epitope Mapping Using Binding Inhibition Assay on RSV Infected Cells

In order to establish the F protein epitope recognized by HMB2430 and HMB3210, we developed a binding inhibition assay using Hep-2 cells infected with the A2 strain of RSV. The following panel of RSV F-protein-specific mAbs were acquired or generated by gene synthesis: (i) motavizumab specific for antigenic site II; (ii) 101F specific for antigenic site IV; (iii) D25 of uncertain specificity; (iv) 131-2a, specific for antigenic site I. The mAb was labeled with biotin and tested for binding to infected Hep-2 cells to determine the optimal concentration of mAb needed to obtain 70-80% of the maximum

- 23 039682 binding. Biotin-labeled mAbs were then used as probes to assess whether their binding (measured using fluorophore-conjugated streptavidin) was inhibited by pre-incubation of RSV A2-infected cells with a 50-fold excess of homologous or heterologous unlabeled mAbs. As expected, biotin labeled HMB2430 binding was blocked by preincubation of cells with unlabeled HMB2430, but it was also partially blocked with unlabeled HMB3210 (FIG. 4). In contrast, binding of all other tested biotin-labeled mAbs was not prevented by pre-incubation with HMB2430 or HMB3210 (FIG. 4). Taken together, these results suggest that HMB2430 and HMB3210 recognize overlapping epitopes on the F protein that are common in RSV and MPV, and that these epitopes differ from epitopes in the antigenic region II of the F protein (recognized by motavizumab and palivizumab) , antigenic site IV (recognized by mAb 101F) and antigenic site I (recognized by mAb 131-2A). In addition, the epitopes recognized by mAb HMB2430 and HMB3210 on the RSV F protein differ from the unknown epitope recognized by mAb D25.

Example 5 Monoclonal Antibody Reactivity to RSV and MPV F Proteins under Reducing and Non-Reducing Conditions

To further confirm the discovery that HMB2430 and HMB3210 recognize RSV and MPV F protein, the authors tested two mAbs for their ability to stain RSV and MPV F proteins in a Western blot. Hep-2 cells were infected with RSV and LLC-MK2 cells were infected with MPV, lysed with mild detergent and run on SDS-PAGE gel under reducing or non-reducing conditions. The proteins were then transferred to a PVDF membrane which was then incubated with HMB2430, HMB3210, motavizumab, or mAb 234. mAb binding was detected using an anti-human HRP conjugated antibody in combination with an ECL Western blot detection reagent. HMB2430 and HMB3210 bind to F-protein derived from RSV-infected cells (FIG. 5) and MPV-infected cells (FIG. 6) under non-reducing conditions. MPV-specific mAb 234 (which recognizes an epitope on the MPV F protein that corresponds to antigenic site II on the RSV F protein) bound to the MPV F protein under non-reductive conditions, but did not bind to the RSV F protein. In contrast, the RSV-specific mAb motavizumab bound to the RSV F protein under both reductive and non-reductive conditions, confirming the recognition of a predominantly linear epitope. These results suggest that, unlike motavizumab and palivizumab, HMB2430 and HMB3210 recognize conformational epitopes that are also due to the presence of disulfide bonds between amino acid residues in RSV and MPV F proteins.

Example 6 Neutralization of all groups and subgroups of RSV and MPV HMB2430 and HMB3210

Purified HMB2430 and HMB3210 mAbs were tested for their ability to neutralize RSV or MPV infection of Hep-2 or LLC-MK2 cells, respectively. The following strains of RSV and MPV were tested: A2 RSV (A, 1961 Australia; A/A2/61), RSV Long (A, Maryland US, 1956; A/Long/56), RSV Randall (A, Chicago US, 1958; A/Randall/58), RSV 9320 (B, Massachusetts US, 1977; B/9320/77), WV/14617/85 (B, Huntington West Virginia, 1985; B/14617/85), 18537 (B, Washington District of Columbia US, 1962; B/18537/62), RSV 9727/2009 (B, Pavia IT, 2009; B/9727/09), RSV 9736/2009 (B, Pavia IT, 2009; B/9736/09 ), RSV 9847/2009 (B, Pavia IT, 2009; B/9847/09), MPV I-PV-03/01-6621 (A1, Pavia IT, 2001; A1/6621/01), MPV I-PV -02/06-8938 (A2, Pavia IT, 2006; A2/8938/06), I-PV-02/06-8908 (A2, Pavia IT, 2006; A2/8908/06), I-PV-02 /06-8909 (A2, Pavia IT, 2006; A2/8909/06), I-PV-03/04-4702 (B1, Pavia IT, 2004; B1/4702/04) and I-PV02/04-3817 (B2, Pavia IT, 2004; B2/3817/04).

In the same experiment, HMB2430 and HMB3210 were compared with palivizumab (RSV specific) and mAb 234 (MPV specific). HMB3210 neutralized all 11 tested strains of RSV and all 6 tested strains of MPV characteristic of known groups and subgroups of RSV and MPV (Table 7). HMB2430 neutralized all 11 RSV strains tested and all MPV A1 and A2 strains tested, but not MPV B1 or B2 strains tested. As expected, palivizumab neutralized all 11 RSV strains tested but none of the 6 MPV strains tested, while mAb 234 neutralized all 6 MPV strains tested but none of the 11 RSV strains tested.

HMB3210 and HMB2430 effectively neutralized all 11 RSV strains tested (mean IC ₅₀ values of 0.070 and 0.133 µg/mL, respectively). These values were on average 5.4 and 2.6 times higher than the IC ₅₀ value of palivizumab (0.284 µg/mL).

HMB3210 effectively neutralized all 6 strains of MPV tested (mean IC ₅₀ 0.113 µg/mL), which was on average 1.7 times lower than the IC ₅₀ value of 234 (0.046 µg/mL).

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Table 7. Neutralization of RSV and MPV strains

1C ₅₀ Neutralization (mcg/ml) Virus palivizumab. mAb 234 HMB2430 HMB3210 RSV A/A2/61 0.617 - 0.350 0.184 RSV A/Long/56 0.599 - 0.361 0.187 RSV A/Randall/58 0.440 - 0.179 0.116 RSV A/9846/09 0.283 - 0.123 0.06 RSV A/9835/09 0.284 - 0.076 0.063 RSV B/18537/62 0.143 - 0.094 0.034 RSV B/14617/85 0.129 - 0.096 0.038 RSV B/9727/09 0.275 - 0.084 0.051 RSV B/9320/77 0.069 - 0.021 0.012 RSV B/9736/09 0.092 - 0.027 0.007 RSV B/9847/09 0.209 - 0.053 0.026 MPV Al/6621/01 - 0.040 0.744 0.071 MPV A2/8938/06 - 0.044 1,049 0.045 MPV A2/8908/06 - 0.057 2.795 0.066 MPV A2/8909/06 - 0.012 0.161 0.007 MPV Bl/4702/04 - 0.019 - 0.029 MPV B2/3817/04 - 0.106 - 0.465

Example 7 No selection of RSV and MPV escaped mutants

HMB3210, palivizumab, and mAb 234 were tested for their ability to select monoclonal antibody resistant mutants of RSV or MPV (MARM) in vitro. Despite repeated attempts, HMB3210 did not select any MARM RSV or MPV when tested against 32***10e6 TCID50 RSV A/Long/58 TCID50 and against 16***10e6 TCID50 MPV A1/6621/01. In contrast, palivizumab selected MARMs at a high frequency (a total of 85 palivizumab-independent MARMs were selected from input data of 16***-10e6 TCID50 RSV A/Long/58, corresponding to a TCID50 frequency of 1 out of 185,000). mAb 234 does not select any MARM MPV under the same experimental conditions. The difficulty in isolating the MARM mAb 234 is consistent with the published data of Ulbrandt et al. (J. General. Virol., 2008), which demonstrated that a high level of virus was necessary to isolate a small number of MARMs. Escaped viruses were mapped for the K242N mutation using the B1 NL/1/99 MPV isolate. Palivizumab-independent MARMs (PZ-MARMs) were harvested and the F-protein of 10 of them were subjected to full sequencing (Table 8). PZ-MARM2, PZ-MARM3, PZ-MARM4, PZMARM5, PZ-MARM6, PZ-MARM8 and PZ-MARM10 had the same two amino acid mutations (P101S/K272T); PZ-MARM1 also contained two amino acid mutations at the same position but with a different amino acid substitution (P101S/K272Q); PZ-MARM7 contained a single amino acid mutation (K272N) again at position 272, finally PZ-MARM9 contained a mutation in common with other PZ-MARMs and a unique mutation at position 262 (P101S/N262Y). Point mutations in this region (nucleotide position 827 and 828) have already been described (Zhao et al., Virology, 2004) and result in two different amino acid substitutions at position 272 (K272Q and K272M). The first mutation (ie K272Q) is also present in PZ-MARM1, described in the present description. Viruses carrying such point mutations were completely resistant to the prophylactic effects of palivizumab in cotton rats (Zhao et al., Virology, 2004), and similar mutations, among others, have been described in RSV-infected immunocompromised cotton rats treated prophylactically with palivizumab.

HMB3210, HMB2430 and palivizumab were then tested for their ability to neutralize PZ-MARM6 infection of Hep-2 cells. Although palivizumab did not neutralize PZ-MARM6, HMB3210 and HMB2430 effectively neutralized this virus to levels comparable to those observed for the corresponding wild-type virus (Fig. 7).

Taken together, these results demonstrate that HMB3210 and HMB2430 do not select MARM RSV or MPV in vitro. These results are consistent with the notion that the target epitopes recognized by HMB3210 and HMB2430 are very conserved, and that mutations that abolish antibody binding are extremely rare or may be associated with loss of viral replication.

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Table 8. MARM RSV Amino Acid Changes Selected by Palivizumab

a.k. position (nucleotide position) 101 (314) 262 (797) 272 (827) 272 (828) 272 (829) RSV A/Long/56 R N To To To PZ-MAARM1 S (S to T) Q (A on WITH) PZ-MAARM2 S (S to T) T (A to C) PZ-MAARM3 S (S to T) T (A an C) PZ-MAARM4 S (S to T) T (A to C) PZ-MARM5 S (S to T) T (A to C) PZ-MARM6 S (S to T) T (A to C) PZ-MAARM7 R N (G to T) PZ-MARM8 S (S to T) T (A to C) PZ-MAARM9 S (S to T) Y (A to T) PZ-MARM10 S (S to T) T (A to C)

Example 8 Removal of glycosylation site in LCDR3 does not affect HMB3210 activity

The light chain variable region of HMB3210 contains an N-glycosylation motif (NxS/T, where x can be any amino acid except proline) in LCDR3 at position 113 (IMGT numbering). The asparagine at position 113 (N113) replaces the serine contained in the germline. The presence of glycosylation motifs in the variable region can have a positive or negative effect on the activity of the antibody and is recognized to be responsible for the heterogeneity of the antibody. The glycan content of the HMB3210 light chain was assessed on SDS-PAGE gel under reducing conditions after incubation in the presence or absence of PNGase F N-glycosidase (FIG. 8). This analysis indicated that a minority of the light chain is actually glycosylated. The N113 residue was then removed and the corresponding germline-encoded serine residue was restored. In parallel, another somatic mutation in the framework region -1 of the light chain (P7T) was also removed to restore the germ-encoded proline residue at position 7 (IMGT numbering in the corresponding IGLV1 gene -40*02). A new HMB3210 variant (referred to as HMB3210v3) was then made, consisting of HMB3210 heavy chain VH.2 (SEQ ID 17) and VL. 3 HMB3210 light chain, and tested its neutralizing activity against RSV strains A2 and MPV strains I-PV 03/01-6621 in parallel with HMB3210v2 (consisting of HMB3210 heavy chain VH.2 (SEQ ID NO: 17) and light chain VL SEQ ID NO : fourteen). This variant (HMB3210v3) showed slightly improved neutralization against the tested strains of RSV and MPV (FIG. 9), thus demonstrating that the removal of the light chain glycosylation site did not affect binding to RSV and MPV target epitopes. The two antibody variants were then tested in parallel against a panel of RVS and MPV viruses and demonstrated to have comparable activities against all tested viruses (FIG. 10). In conclusion, HMB3210v3 is non-glycosylated in the light chain variable region and is generally weakly mutated compared to the heavy and light chain germline genes, containing only 8 somatic amino acid mutations in the heavy chain and 4 in the light chain: S58A (HCDR2), I65S (HCDR2 ), Y66D (HFR3), V71A (HFR3), N85T (HFR3), Y88F (HFR3), V101I (HFR3), Y103F (HFR3), G56D (LCDR2), S65N (LCDR2), G78A (LFR3), and S109R (LCDR3) ) (all positions are indicated according to the IMGT numbering).

Example 9 HMB3210 and HMB2430 cross-reactivity against MPV due to somatic mutations

To understand the role of somatic mutations in the cross-reactivity of HMB2430 and HMB3210 against RSV and MPV, mAb variants were synthesized without modification at the germline level and tested for their ability to neutralize RSV A2 and I-PV strains 03/01-6621 MPV. mAb HMB2430 and HMB3210 without germline modification (HMB2430-GL and HMB3210-GL, respectively)

- 26 039682 but) consisted of VH.3 and VL. 2 for HMB2430-GL and from VH.3 and VL.4 for HMB3210-GL. Both forms of mAb, without modification at the germline level, effectively neutralized RSV to levels comparable to those observed for the original HMB3210 and HMB2430 with somatic mutations (Fig. 11). However, HMB3210-GL and HMB2430-GL were unable to neutralize MPV, thus indicating that somatic mutations are necessary to neutralize MPV. To further understand whether heavy or light chain somatic mutations are responsible for MPV neutralization, we generated antibodies carrying either the heavy or light chain in the germline configuration: HMB2430-VHGL-VLSM, consisting of VH.3 and VL; HMB2430VHSM-VLGL consisting of VH.2 and VL.2; HMB3210-VHGL-VLSM consisting of VH. 3 and VL; HMB3210VHSM-VLGL consisting of VH.2 and VL.4. Deletion of somatic mutations in the HMB3210 heavy chain did not affect the neutralization of RSV or MPV viruses, while deletion of somatic mutations in the HMB2430 heavy chain affected the neutralization of MPV, although retaining the neutralization of RSV. Deletion of somatic mutations in the HMB2430 and HMB3210 light chain abolished MPV neutralization without affecting RSV neutralization (FIG. 11). Taken together, these findings indicate that HMB3210 and HMB2430 were originally selected for RSV and then developed, through the accumulation of somatic mutations, cross-reactivity against MPV. Only 3 somatic mutations in the light chain CDR result in the acquisition of anti-MPV cross-reactivity in an RSV-specific antibody without modification at the germline level. Of note, HMB2430 and HMB3210 (clonal unrelated) contain the same somatic mutation in LCDR3 S to R at position 109.

Example 10 Recognition of HMB3210 F protein conformations before and after fusion

A DNA construct encoding F residues 26-136 and 147-512 of RSV (corresponding to the F ectodomain without the fusion peptide of the A2 strain of RSV) with a C-terminal histidine tag was subjected to codon optimization and synthesized. Recombinant F was expressed using a baculovirus expression vector in Sf21 cells and purified from the supernatant by nickel affinity and size exclusion chromatography (SEC). A similar construct has already been used by other researchers (Swanson et al., PNAS, 2011 and McLellan et al., J. Virol., 2011) to study the crystal structure of the F-protein after fusion. The protein was analyzed under non-reductive conditions on a SDS-PAGE gel and the ®65-70 kDa band was obtained, and then analyzed by SEC on an S200 column F-protein after RSV fusion eluting as a symmetrical peak with an apparent molecular weight of ®150 kDa, corresponding to M _w trimeric F-protein and partially coincides with the elution volume of human IgG1 antibodies. The post-fusion F protein was incubated with HMB3210 or palivizumab and the two mixtures were run through an S200 column. Incubation of the F-protein after fusion with palivizumab shifted the elution peak to a lower elution volume (corresponding to an apparent M _w ® 300 kDa) compared to F-protein alone, indicating that palivizumab binds to the F-protein after fusion, as already described. . It should be noted that post-fusion incubation of HMB3210 with F protein did not result in a shift in elution volume (FIG. 12). The fact that HMB3210 and the F protein elute as independent molecules after fusion indicates that HMB3210, unlike palivizumab, does not bind to the F protein after fusion.

A stabilized pre-fusion form of the full length RSV F protein was then synthesized according to a strategy adapted by Magro et al., PNAS, 2012, by replacing 4 amino acid residues (L481, D489, S509 and D510) with cysteines and by replacing 9 basic amino acid residues (R106, R108, R109, K131, K132, R133, K134, R135 and R136) at two F-protein cleavage sites into N-residues for furin cleavage ablation. The F protein sequence was further modified by inserting a TEV cleavage site after the transmembrane region followed by GFP and a 6-His tag at the C-terminus to facilitate purification. The four cysteines introduced were positioned according to the F structure prior to PIV-5 fusion such that intermonomeric disulfide bonding was possible only when the F protein was in the pre-fusion conformation, but not when refolded into the post-fusion structure, thus providing stabilization of the F protein prior to fusion. Magro et al. described that the pre-fusion stabilized F protein is heterogeneous due to the fact that it also contains a portion of the molecules in which additional cysteine residues are not linked by disulfide bonds. The F protein construct described herein was generated using a baculovirus expression vector in Sf21 cells, solubilized from the cell membrane with a mild detergent, and purified by nickel affinity and size exclusion chromatography. Purified pre-fusion F protein was analyzed by SEC on an S200 column and eluted as a symmetrical peak with an apparent molecular weight of ®150 kDa, which corresponds to the M _w of trimeric F protein and which overlaps with the elution volume of human IgG1. Incubation of the F protein prior to fusion with palivizumab shifted the elution peak to a lower elution volume (corresponding to an apparent M _w ® 300 kDa) compared to F protein alone, and also induced the formation of a high molecular weight complex that eluted in the dead volume of the column, which may be associated with the formation of larger aggregates. A similar elution volume shift was also observed when HMB3210v2 was incubated with the protein until confluence (FIG. 13). These results indicate

- 27 039682 that palivizumab binds to the pre-fusion and post-fusion forms of the F protein, while HMB3210 selectively recognizes the pre-fusion form of the F protein. The two F proteins (forms before and after fusion) were also tested by surface plasmon resonance (SPR).

Palivizumab bound pre-fusion and post-fusion proteins with similar affinities, while HMB3210v3 selectively bound the pre-fusion F protein with high affinity (0.1 nM Kd compared to palivizumab 2 nM Kd) (Figure 14).

Example 11 HMB3210v3 cross-neutralizes two animal paramyxoviruses: bovine respiratory syncytial virus (BRSV) and murine pneumonia virus (PVM)

The reactivity spectrum of HMB3210 was also evaluated on two other animal paramyxoviruses: BRSV and PVM, two viruses that share 81 and 40% amino acid identity in the F protein with RSV, respectively. HMB3210 was tested for its ability to neutralize PVM strain 15 and BRSV strain RB94 and was shown to be effective against these viruses with IC ₅₀ values of 100 and 10 ng/mL, respectively. These results indicate that along with the human paramyxoviruses RSV and MPV, HMB3210 is also effective against two other viruses of the Paramyxoviridae family.

Example 12 Inhibition of the spread of the HMB3210 virus

The authors also measured the ability of HMB3210 and RSV specific antibody D25 to prevent cell-to-cell spread of the virus, which was described as a hallmark of anti-RSV antibodies independent of neutralizing activity. The authors infected Hep-2 cells with RSV A or B strains, added various concentrations of antibodies after 20 hours, and analyzed the formation of syncytia on day 3 to determine the 50% concentration of the antibody that inhibits the spread of the virus, referred to in the present description as IS50. Both antibodies were able to inhibit the spread of the virus, but at higher concentrations. Of note, in this assay, HMB3210 showed an IS50 comparable to that of the more potent neutralizing antibody D25 (FIG. 15).

Example 13 Prophylactic and Therapeutic Efficacy of HMB3210 Against RSV, MPV and PVM

In a mouse model of RSV, HMB3210 was on average five to ten times more effective than palivizumab in reducing lung RSV titers and was effective at concentrations as low as 0.12 mg/kg (Fig. 16a). In a mouse model of MPV, HMB3210 was relatively effective (FIG. 16b). To test the therapeutic potential of HMB3210, we infected RSV STAT1 deficient mice and administered HMB3210 on days 1, 2, or 3 post-infection. Despite the limitations of this model due to weak viral replication, MB3210 demonstrated therapeutic efficacy at all time points and reduced viral and inflammatory cytokine titers in the lung (FIG. 17).

To test HMB3210 in a more relevant animal model of acute lower respiratory tract infection, the authors used its cross-reactivity against the PVM virus, a virus that causes fatal disease in mice after a very small inoculum and reproduces signs of severe RSV and MPV infection in humans. Under prophylactic conditions, HMB3210 completely protected mice from lethality at 0.12 mg/kg and from body weight loss at 0.6 mg/kg (FIG. 18a). In addition, under therapeutic conditions, HMB3210 completely protected against lethality when administered 3 days post-infection at 30 and 5 mg/kg, and provided significant protection when administered on days 4 or 5 at 30 mg/kg (Fig. 18b-d). In this system, ribavirin, which is the only approved standard of care in human therapy as described previously (Bonville et al., 2004, Journal of Virology, 78:7984-7989), was ineffective. Of note, therapeutic delivery of HMB3210 effectively blocked further increases in viral RNA in the lung (Fig. 19). To determine the role of effector mechanisms in vivo, the authors compared HMB3210 IgG1 with a mutant lacking complement and Fc receptor binding (HMB3210-LALA). The two antibodies were compared for in vitro neutralizing activity and were shown to be equivalent. When compared in limited amounts under prophylactic conditions (0.12 mg/kg), HMB3210-LALA showed significantly reduced efficacy (FIG. 20a). In contrast, when used under therapeutic conditions, HMB3210-LALA was as effective as HMB3210 at all doses tested (FIG. 20b). The therapeutic efficacy of HMB3210 in a PVM infection model where ribavirin is ineffective may be due to a combination of factors such as effective neutralizing and spread-inhibiting activity, selective protein recognition prior to fusion, which avoids wasting of the antibody by excess post-fusion proteins acting as decoys, and the inability to select elusive mutants. Surprisingly, no effector functions are required for the therapeutic efficacy of HMB3210 in a PVM mouse model, suggesting that the activity of the antibody in vivo is due predominantly to neutralization of the virus and inhibition of viral spread.

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Example 14 HMB3210 binds to a highly conserved beta chain in the pre-fusion F protein that is not available in the post-fusion F protein

To identify an epitope recognized by HMB3210, the authors screened a library of 7095 structured peptides containing the complete human RSV F protein sequence. The experiments presented in Example 5 demonstrated that HMB3210 interacted in Western blots with RSV and MPV F proteins under non-reductive conditions, confirming that HMB3210 targets an epitope whose conformation is stable in the presence of the anionic detergent SDS (Figures 5 and 6) . The library screening approach resulted in the identification of a putative HMB3210 epitope in the N-terminal region of F2, including residues SAVSKGYLSALRTGWYTSVIT (SEQ ID NO: 63). The core sequence in this region, YLSALRTGW (SEQ ID NO: 64), is highly conserved across RSV, MPV, BRSV and PVM (Fig. 21), where the variants YLSVLRTGW (SEQ ID NO: 65), YFSALRTGW (SEQ ID NO: 66) , YFSVLRTGW (SEQ ID NO: 67), YKSALRTGW (SEQ ID NO: 68), and YKSVLRTGW (SEQ ID NO: 69) are also recognized by HMB3210. This sequence is not exposed on the surface of the RSV F protein after fusion, but in the RSV F protein model prior to fusion, built around the structure of the PIV5 F protein (Yin et al., 2006, Nature, 439:38-44), it is suggested that it is located in the exposed beta chain close to the loop region targeted by palivizumab (Fig. 22). This mapping is consistent with the pre-fusion specificity of HMB3210 for the F protein demonstrated in Example 10. This epitope is similar to, but different from, the epitope recognized by palivizumab and is centered around the YLSVLRTGW sequence, which is highly conserved across 551 virus strains, including the 364 strains of HRSV, 162 HMPV, 8 BRSV and 5 PVM (Fig. 23).

Table of sequences and SEQ ID numbers

SEQID NO Description Subsequence one a.k. CDRH1 GFTFSSYS 2 a.k. CDRH2 ISASSSYS 3 a.k. CDRH3 ARARATGYSSITPYFDI 4 a.k. CDRL1 SSNIGAGYD 5 a.k. CDRL2 DNN 6 a.k. CDRL3 QSYDRNLSGV 7 nucleotides. CDRH1 ggattcaccttcagtagttatagc eight CDRH2 nucleotides attagtgcaagtagcagttacagc nine CDRH3 nucleotides gcgagagctcgggcaactggctacagttccattaccccctactttgacatt ten CDRL1 nucleotides agctccaacatcggggcaggttatgat And CDRL2 nucleotides gataacaac 12 CDRL3 nucleotides cagtcctatgacaggaacctgagtggtgtc thirteen a.k. heavy chain EEQLLESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAP GKGLEWVS SIS AS S S YSD YADS AKGRFTISRDNAKTSLFLQM NSLRAEDTAIYFC ARARATGYS SITPYFDIWGQGTLVTVS S fourteen a.k. light chain QSVVTQTPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLP GTAPKLLIYDNNNRPSGVPDRFSASKSGTSASLAITGLQAEDE ADYYCQSYDRNLSGVFGTGTKVTVL fifteen heavy nucleotides chains gaggaacagctgctagagtctgggggaggcctggtcaagcctggggggtccctgagactctcc tgtgcagcctctggattcaccttcagtagttatagcatgaactgggtccgccaggctccagggaa ggggctggagtgggtctcatccattagtgcaagtagcagttacagcgattacgcagactcagcg aagggccgattcaccatctccagagacaacgccaagacctcactgtttctgcaaatgaacagcct gagagccgaggacacggctatctatttctgtgcgagagctcgggcaactggctacagttccatta ccccctactttgacatttggggccagggaaccctggtcaccgtctcctcag sixteen light nucleotides chains cagtctgtcgtgacgcagacgccctcagtgtctggggccccagggcagagggtcaccatctcct gcactgggagcagctccaacatcggggcaggttatgatgtacactggtaccagcaacttccagg aacagcccccaaactcctcatctatgataacaacaatcgaccctcaggggtcccggaccgattct ctgcctccaagtctggcacctcagcctccctggccatcaccgggctccaggctgaggatgaggc tgattattactgccagtcctatgacaggaacctgagtggtgtcttcggaactgggaccaaggtcac cgtcctag

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17 a. to. heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAP GKGLEWVS SIS AS S S YSD YADS AKGRFTISRDNAKTSLFLQM NSLRAEDTAIYFC ARARATGYS SITPYFDIWGQGTLVTVS S eighteen a. to. heavy chain gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcc tgtgcagcctctggattcaccttcagtagttatagcatgaactgggtccgccaggctccagggaa ggggctggagtgggtctcatccattagtgcaagtagcagttacagcgattacgcagactcagcg aagggccgattcaccatctcc agagac aacgcc aagacctcactgtttctgc aaatgaac agcct gagagccgaggacacggctatctatttctgtgcgagagctcgggcaactggctacagttccatta ccccctactttgacatttggggccagggaaccctggtcaccgtctcctcag nineteen a.k. CDRH1 GFAFTGYG 20 a.k. CDRH2 ITAGSSYI 21 a.k. CDRH3 ARVASPLVRGLHLDY 22 a.k. CDRL2 AND 23 a.k. CDRL3 QSYDRTLSVV 24 CDRH1 nucleotides ggattcgcattcactggttatggt 25 CDRH2 nucleotides atcactgctggaagctcatacatc 26 CDRH3 nucleotides gcgagagttgcgtctcctctggttcggggactccacttagactac 27 CDRL2 nucleotides gctaacgac 28 CDRL3 nucleotides cagtcctatgaccgcaccctgagtgtagtg 29 a.k. heavy chain EVHLVESGGGLVKPGGSLRLSCAASGFAFTGYGLNWVRQVP GKGLEWVSSITAGSSYIDYAESVKGRFTISRDNGKNTLFLQM SDLRADDTAVYYCARVASPLVRGLHLDYWGQGALVTVSS thirty a.k. light chain QSVLTQPPSMSGAPGQRVTISCTGGSSNIGAGYDVQWYQQL PGAAPKLLIYANDNRPSGVPDRFSGSKSGTSGSLVIAGLRAE DEADYYCQSYDRTLSVVFGGGTKLTVL 31 heavy nucleotides chains gaggtgcacctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcc tgtgcagcctctggattcgcattcactggttatggtctaaattgggtccgccaggttccagggaag ggcctggagtgggtttcatccatcactgctggaagctcatacatcgactacgcagagtcagtgaa gggccgattcaccatctccagagacaacggcaagaatacactgttcctgcaaatgagcgacctg agagccgacgacacggctgtctattactgtgcgagagttgcgtctcctctggttcggggactcca cttagactactggggccagggagccctggtcaccgtctcctcag 32 light nucleotides chains cagtctgtgctgacgcagccgccctcaatgtccggggccccagggcagagggtcaccatctcc tgcactgggggcagctccaacatcggggcaggttatgatgtgcagtggtaccagcaacttccag gagcagcccccaaactcctcatctatgctaacgacaatcggccctcaggggtccctgaccgattc tctggctccaagtctggcacctcaggctccctagtcatcgctggcctccgggctgaggatgagg ctgattattactgccagtcctatgaccgcaccctgagtgtagtgttcggcggagggaccaagctg accgtcctgg

- 30 039682

33 a. to. heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFAFTGYGLNWVRQVP GKGLEWVSSITAGSSYIDYAESVKGRFTISRDNGKNTLFLQM SDLRADDTAVYYCARVASPLVRGLHLDYWGQGALVTVSS 34 heavy nucleotides chains gaggtgcagctggtggagtctgggggaggcctggtcaagcctggggggtccctgagactctcc tgtgcagcctctggattcgcattcactggttatggtctaaattgggtccgccaggttccagggaag ggcctggagtgggtttcatccatcactgctggaagctcatacatcgactacgcagagtcagtgaa gggccgattcaccatctccagagacaacggcaagaatacactgttcctgcaaatgagcgacctg agagccgacgacacggctgtctattactgtgcgagagttgcgtctcctctggttcggggactcca cttagactactggggccagggagccctggtcaccgtctcctcag 35 a.k. CDRL3 QSYDRSLSGV 36 CDRL3 nucleotides cagtcctatgacaggagcctgagtggtgtc 37 a.k. light chain QSVVTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLP GTAPKLLIYDNNNRPSGVPDRFSASKSGTSASLAITGLQAEDE ADYYCQSYDRSLSGVFGTGTKVTVL 38 light nucleotides chains cagtctgtcgtgacgcagccgccctcagtgtctggggccccagggcagagggtcaccatctcct gcactgggagcagctccaacatcggggcaggttatgatgtacactggtaccagcaacttccagg aacagcccccaaactcctcatctatgataacaacaatcgaccctcaggggtcccggaccgattct ctgcctccaagtctggcacctcagcctccctggccatcaccgggctccaggctgaggatgaggc tgattattactgccagtcctatgacaggagcctgagtggtgtcttcggaactgggaccaaggtcac cgtcctag 39 a.k. CDRH2 ISSSSSYI 40 a.k. CDRH3 ARARATGYNSITPYFDI 41 a.k. CDRL2 GNS 42 a.k. CDRL3 QSYDSSLSGV 43 CDRH1 nucleotides ggcttcacattcagctcctactct 44 CDRH2 nucleotides atctcaagctcctctagttacatc 45 CDRH3 nucleotides gcccgggctagagcaacaggctataacagcattactccttactttgacatc 46 CDRL1 nucleotides tcatccaacatcggc 47 CDRL2 nucleotides gggaacagc 48 CDRL3 nucleotides cagtcttatgattcttctctgtctggagtc 49 a.k. heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAP GKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQM NSLRAEDTAVYYCARARATGYNSITPYFDIWGQGTLVTVSS fifty a.k. light chain QSVVTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLP GTAPKLLIYGNSNRPSGVPDRFSGSKSGTSASLAITGLQAEDE ADYYCQSYDSSLSGVFGTGTKVTVL

- 31 039682

51 heavy nucleotides chains gaggtgcagctggtggagagcggaggcggactggtcaaacctggcgggtcactgagactgtc atgcgcagcaagcggcttcacattcagctcctactctatgaactgggtgcgacaggctcctggca agggactggagtgggtctctagtatctcaagctcctctagttacatctactatgcagactccgtgaa gggaaggttcaccatctcacgcgataacgccaaaaatagcctgtatctgcagatgaattccctga gagccgaagacaccgctgtctactattgcgcccgggctagagcaacaggctataacagcattac tccttactttgacatctggggacagggcacactggtgaccgtctcctca 52 light nucleotides chains cagtccgtcgtcactcagcctccaagcgtcagcggggcacctgggcagcgggtcacaatctcat gcactgggtcctcatccaacatcggcgctgggtacgacgtgcactggtatcagcagctgcctgg aacagcacctaagctgctgatctacgggaacagcaatcggccatctggagtccccgatagattc agcggatccaaatctggcaccagtgcctcactggctattacagggctgcaggcagaggacgaa gccgattactattgccagtcttatgattcttctctgtctggagtcttcggcaccggcacaaaagtcac cgtcctg 53 a.k. CDRH3 ARVASPMVRGLHFDY 54 a.k. CDRL3 QSYDSSLVV 55 CDRH1 nucleotides ggcttttacctttagctcctactct 56 CDRH3 nucleotides gcccgcgtcgctagccctatggtgcggggggctgcattttgattat 57 CDRL1 nucleotides tcttcaaacatcggcgctgggtacgac 58 CDRL3 nucleotides cagagctacgattcatccctgagcgtggtc 59 a.k. heavy chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAP GKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQM NSLRAEDTAVYYCARVASPMVRGLHFDYWGQGTLVTVSS 60 a.k. light chain QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLP GTAPKLLIYGNSNRPSGVPDRFSGSKSGTSASLAITGLQAEDE ADYYCQSYDSSLSVVFGGGTKLTVL 61 heavy nucleotides chains gaagtgcagctggtggaatctgggggcgggctggtcaaacctggcggaagtctgaggctgtcc tgtgctgctagtggctttacctttagctcctactctatgaactgggtgcgacaggcacctggcaag ggactggagtgggtctctagtatctcaagctcctctagttacatctactatgctgactccgtgaagg gccggttcaccatctcaagagataacgcaaaaaatagcctgtatctgcagatgaattccctgagg gcagaagacacagccgtgtactattgcgcccgcgtcgctagccctatggtgcgggggctgcatt ttgattattggggacagggaactctggtgaccgtctcatcc 62 light nucleotides chains cagagcgtcctgacccagccaccatccgtgagcggcgcacccggccagcgagtgactatttcc tgtaccggcagttcttcaaacatcggcgctgggtacgacgtgcactggtatcagcagctgcctgg aacagcacctaagctgctgatctacgggaacagcaatcggccatctggagtccccgatagattc agcggatccaaatctggcaccagtgcctcactggctattacagggctgcaggcagaggacgaa gccgattactattgccagagctacgattcatccctgagcgtggtcttcggaggcggcacaaaact gactgtcctg 63 a.k. SAVSKGYLSALRTGWYTSVIT 64 a.k. YLSALRTGW 65 a.k. YLSVLRTGW 66 a.k. YFSALRTGW 67 a.k. YFSVLRTGW 68 a.k. YKSALRTGW 69 a.k. YKSVLRTGW

CLAIM

Claims (24)

CLAIM 1. An isolated antibody or antigen-binding fragment thereof that neutralizes RSV (respiratory syncytial virus) infection, wherein the antibody or antigen-binding fragment contains (i) complementarity-determining regions of the heavy chain CDR1, CDR2 and CDR3 shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and the light chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; (ii) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, SEQ ID NO: 39 and SEQ ID NO: 40, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 4 , SEQ ID NO: 41 and SEQ ID NO: 42, respectively; (iii) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, SEQ ID NO: 39 and SEQ ID NO: 40, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 4 , SEQ ID NO: 5 and SEQ ID NO: 6, respectively; (iv) the heavy chain CDR1, CDR2 and CDR3 sequences shown in - 32 039682 SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the light chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 4, SEQ ID NO: 41, and SEQ ID NO: 42, respectively; (v) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 4 , SEQ ID NO: 22 and SEQ ID NO: 23, respectively; (vi) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 1, SEQ ID NO: 39 and SEQ ID NO: 53, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 4 , SEQ ID NO: 41 and SEQ ID NO: 54, respectively; (vii) the heavy chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 1, SEQ ID NO: 39, and SEQ ID NO: 53, respectively, and the light chain CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 4 , SEQ ID NO: 22 and SEQ ID NO: 23, respectively; or (viii) the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 4, SEQ ID NO: 41 and SEQ ID NO: 54, respectively. 2. An antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof binds the RSV F protein prior to fusion, but not the F protein after fusion. 3. An antibody according to claim 1 or 2, or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment neutralizes both group A and group B RSV infection. 4. An antibody according to claim 1 or 2, or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment neutralizes RSV and MPV (metapneumovirus) infections. 5. An antibody according to claim 1 or 2, or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment neutralizes RSV, MPV, and PVM (murine pneumonia virus) infections. 6. An antibody according to claim 1 or 2, or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof does not bind to the RSV F protein at a site that overlaps the I antigenic site, the II antigenic site, or the IV antigenic site. 7. An antibody according to claim 1 or 2, or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof binds to each of the conserved regions in the N-terminal region of the RSV F protein comprising any of the amino acid sequences of SEQ ID NOs: 64-69. 8. An antibody according to claim 1 or 2, or an antigen-binding fragment thereof, wherein the concentration of the antibody or antigen-binding fragment thereof required to neutralize 50% of RSV is 500 ng/mL or less. 9. An antibody according to claim 1 or 2, or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises (i) a heavy chain variable region at least 80% identical to the amino acid sequence of SEQ ID NO: 13, and a light chain variable region, at least 80% identical to the amino acid sequence of SEQ ID NO: 14; (ii) a heavy chain variable region at least 80% identical to the amino acid sequence of SEQ ID NO: 17 and a light chain variable region at least 80% identical to the amino acid sequence of SEQ ID NO: 14; (iii) a heavy chain variable region at least 80% identical to the amino acid sequence of SEQ ID NO: 49 and a light chain variable region at least 80% identical to the amino acid sequence of SEQ ID NO: 50; (iv) a heavy chain variable region at least 80% identical to the amino acid sequence of SEQ ID NO: 49 and a light chain variable region at least 80% identical to the amino acid sequence of SEQ ID NO: 14; (v) a heavy chain variable region at least 80% identical to the amino acid sequence of SEQ ID NO: 17 and a light chain variable region at least 80% identical to the amino acid sequence of SEQ ID NO: 50; (vi) a heavy chain variable region at least 80% identical to the amino acid sequence of SEQ ID NO: 29 and a light chain variable region at least 80% identical to the amino acid sequence of SEQ ID NO: 30; (vii) a heavy chain variable region at least 80% identical to the amino acid sequence of SEQ ID NO: 33 and a light chain variable region at least 80% identical to the amino acid sequence of SEQ ID NO: 30; (viii) a heavy chain variable region at least 80% identical to the amino acid sequence of SEQ ID NO: 59 and a light chain variable region at least 80% identical to the amino acid sequence of SEQ ID NO: 60; (ix) a heavy chain variable region at least 80% identical to the amino acid sequence of SEQ ID NO: 59 and a light chain variable region at least 80% identical to the amino acid sequence of SEQ ID NO: 30; or (x) a heavy chain variable region at least 80% identical to the amino acid sequence of SEQ ID NO: 33 and a light chain variable region at least 80% identical to the amino acid sequence of SEQ ID NO: 60. - 33 039682 10. An antibody according to claim 1 or 2 or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: : fourteen; (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14; (iii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 50; (iv) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14; (v) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 50; (vi) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 29, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30; (vii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 33, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30; (viii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 59, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 60; (ix) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 59, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 30; or (x) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 33 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 60. 11. An antibody according to one of the preceding claims or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is a human antibody, monoclonal antibody, human monoclonal antibody, single chain antibody, Fab, Fab', F(ab')2, Fv or scFv. 12. An antibody according to one of the preceding claims, or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is intended to treat or attenuate infection with RSV or MPV, or both RSV and MPV. 13. Nucleic acid molecule containing a polynucleotide encoding an antibody or antibody fragment according to any one of the preceding paragraphs. 14. The nucleic acid molecule according to claim 13, where the nucleotide sequence is at least 75% identical to any of the nucleotide sequences of SEQ ID NOs: 7-12, 15, 16, 18, 24-28, 31, 32, 34, 43- 48, 51-52, 55-58 or 61-62. 15. An expression vector containing a nucleic acid molecule according to claim 13 or 14. 16. A cell expressing an antibody according to any one of claims 1 to 12 or an antigen-binding fragment thereof, wherein the cell contains an expression vector according to claim 15. 17. A pharmaceutical composition for the manufacture of a medicament for the treatment or attenuation of RSV or MPV or both RSV and MPV infections, containing an antibody according to any one of claims 112 or an antigen-binding fragment thereof, a nucleic acid molecule according to claim 13 or 14, a vector according to claim 15 or a cage according to claim 16, and a pharmaceutically acceptable diluent or carrier. 18. A pharmaceutical composition for the manufacture of a medicament for treating or attenuating RSV or MPV or both RSV and MPV infections, comprising a first antibody or antigen-binding fragment thereof and a second antibody or antigen-binding fragment thereof, wherein the first antibody is an antibody according to any one of claims 1- 12 and the second antibody neutralizes RSV or MPV or both RSV and MPV infections. 19. The use of an antibody according to any one of claims 1 to 12, or an antigen-binding fragment thereof, in the manufacture of a medicament for the treatment or attenuation of RSV or MPV or both RSV and MPV infections. 20. The use of an antibody according to any one of claims 1 to 12 or an antigen-binding fragment thereof in the diagnosis of RSV or MPV infection. 21. The use of a nucleic acid molecule according to claim 13 or 14 in the manufacture of a medicament for the treatment or attenuation of RSV or MPV or both RSV and MPV infections. 22. The use of a nucleic acid molecule according to claim 13 or 14 in the diagnosis of RSV or MPV infection. 23. The use of an antibody according to any one of claims 1 to 12, or an antigen-binding fragment thereof, for monitoring the quality of anti-RSV or anti-MPV vaccines. 24. A method for attenuating RSV or MPV infection or reducing the risk of developing RSV and MPV infection, comprising administering to an individual in need thereof a therapeutically effective amount of an antibody according to any one of claims 1 to 12, or an antigen-binding fragment thereof.