EA039222B1 - Antibody neutralizing human respiratory syncytial virus - Google Patents

Abstract

The invention relates to monoclonal antibodies which have high anti-RSV neutralizing titers. The invention further relates to isolated nucleic acids encoding the antibodies of the invention and host cells transformed therewith. The invention yet further relates to diagnostic, prophylactic and therapeutic methods employing the antibodies and nucleic acids of the invention, particularly as a passive immunotherapy agent in infants and the elderly.

Description

The field of technology to which the invention belongs

The present invention relates to human monoclonal antibodies having high titers in the neutralization of RSV, as well as to the use of these antibodies as a passive immunotherapeutic agent for children and the elderly.

Prerequisites for the invention

Paramyxoviruses are enveloped, negative RNA viruses that are dangerous pathogens of humans and animals. Human respiratory syncytial virus (hRSV, RSV) belongs to the family Paramyxoviridae, subfamily Pneumovirinae. Two subtypes of the virus, type A and type B, have been identified as the leading cause of severe, and sometimes even fatal, respiratory infections in children less than 6 months of age. Adults with underlying diseases such as COPD, asthma, cancer, immunocompromised conditions including HIV infection or a transplant condition are also at risk of developing severe RSV infection. RSV is responsible for 15% of annual hospitalizations of adults over the age of 50 due to acute respiratory infections. In the US, RSV causes more than 100,000 hospital admissions each year and is estimated to cause approximately 160,000 deaths worldwide each year. There is currently no vaccine against RSV, and a trial using formalin-inactivated virus has been associated with increased disease severity in children after RSV infection. Other members of the family, including human metapneumovirus (hMPV) and human parainfluenza virus (hPVV), also cause acute respiratory infections similar to hRSV.

The hRSV genome is a single-stranded, negative-polar RNA molecule, approximately 15 kb in size, that encodes 11 proteins. Two of these proteins are the main surface glycoproteins of the virion. They are:

(i) an attachment protein (G) that mediates the attachment of the virus to cells; and (ii) a fusion protein (F), which stimulates both the fusion of viral and cell membranes during the initial stages of the infection cycle and the fusion of infected cell membranes with those of neighboring cells to form the characteristic syncytium.

The attachment G protein binds to cell surface receptors and interacts with the F protein. This interaction triggers a conformational change in the F-protein that induces membrane fusion, resulting in the release of the viral ribonucleic complex into the cytoplasm of the host cell.

Monoclonal antibodies to F-protein or G-protein have been shown to have a neutralizing effect in vitro and a prophylactic effect in vivo. See, for example, Beeler and Coelingh, 1989, J. Virol. 63:2941-50; Garcia-Barreno et al., 1989, J. Virol. 63:925-32; Taylor et al., 1984, Immunology, 52:137-142; Walsh et al., 1984, Infection and Immunity, 43:756-758 and US Pat. and also by evaluating the binding of antibodies to peptides derived from the RSV F protein. These studies have shown that neutralizing antibodies often target two different linear epitopes. See Graham et al., 2015, Curr. Opin. Immunol. 35:30-38 for a review of antigenic site forms before and after F-protein fusion. Antigenic site II (also called site A) contains residues 255-275 and is the target of palivizumab (Synagis®, AstraZeneca). This epitope was predicted to be conformation dependent, and the structure of the more potent palivizumab derivative complexed with this epitope revealed that the linear epitope adopts a helix-loop-helix conformation. Antigenic site IV (also referred to as site C) contains residues 422-438 and is the target of mAbs 19 and 101F. This epitope is C-terminal to the cysteine-rich region and is part of domain II, which in the homologous F-glycoproteins of paramyxoviruses has an unchanged structure in conformations before and after fusion. Antibodies 5C4, AM22, and D25 allowed demarcation of the epitope designated site 0, which is present only in the pre-F-fusion form, and were 50 times more potent than palivizumab. See McLellan et al., 2013, Science, 340:1113-1117; International Patent Application No. WO 2008/147196 and US Patent No. 8568726. Other anti-hRSV antibodies are described in International Patent Applications Nos. WO 94/06448 and WO 92/04381 and US Patent No. 8221759.

RSV vaccine for active immunization, if available, should not be used to treat newborn infants with immature immune systems or patients whose immune systems are suppressed. For patients requiring prophylactic passive immunotherapy due to a more chronic form of the disease, the current treatment is intermittent intravenous injections of human pooled plasma-derived IgG. With such therapy, due to low titers of neutralizing anti-RSV antibodies, it is necessary to administer a large amount of globulin (for example, 0.75 g per 1 kg of body weight) and constantly require intravenous administration in a clinic or hospital setting that lasts a long time (2-4 hours ), which is performed monthly during periods of high risk (autumn, winter and early spring).

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The essence of the invention

The invention relates to antibodies to the RSV F-protein and their antigen-binding fragments having the structural and functional features described below.

In one embodiment, the invention relates to an antibody, or antigen-binding fragment thereof, that binds to human RSV F protein, which comprises a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3. In specific embodiments, the heavy chain or heavy chain variable region is not contains the amino acid sequence of SEQ ID NO: 9. In one embodiment, the antibody, or antigen-binding fragment thereof, optionally has at least one of the following characteristics: (i) binds to a pre-fusion form of a human RSV F protein with a Kd value of about 1x10' 9 to about 1 x 10' ¹² M as determined by surface plasmon resonance (eg, Biacore) or a similar method (eg, KinExa or OCTET) or (ii) binds to the post-fusion form of the human RSV F protein with a Kd value of about 1 x 10'6 up to about 1x10'9 M when determined by surface plasmon resonance (e.g. Biacor e) or a similar method (eg KinExa or OCTET). In specific embodiments, the heavy chain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 23.

In another embodiment, the invention relates to an antibody, or antigen-binding fragment thereof, that binds to human RSV F protein, which comprises a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 6. In specific embodiments, the light chain or light chain variable region is not contains the amino acid sequence of SEQ ID NO: 8. In specific embodiments, the light chain is not linked to a heavy chain containing the amino acid sequence of SEQ ID NO: 9. In specific embodiments, the light chain is linked to a heavy chain containing the amino acid sequence of SEQ ID NO: 7. In one embodiment, the antibody, or antigen-binding fragment thereof, optionally has at least one of the following characteristics: (i) binds to a pre-fusion form of the human RSV F protein with a Kd value of from about 1x10'9 to about ^1x10'12 M when determined surface plasmon resonance method (for example example, Biacore) or a similar method (e.g., KinExa or OCTET) or (ii) binds to the post-fusion form of the human RSV F protein with a Kd value of about 1x10'9 to about 1x10'11 M as determined by surface plasmon resonance (e.g. , Biacore) or a similar method (eg KinExa or OCTET). In specific embodiments, the light chain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 25.

In another embodiment, the invention provides an antibody, or antigen-binding fragment thereof, that binds to human RSV F protein, which comprises (i) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2; and (iii) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3. In specific embodiments, the heavy chain or heavy chain variable region does not comprise the amino acid sequence of SEQ ID NO: 9. In one embodiment, the antibody or antigen-binding fragment thereof, optionally, has at least one of the following characteristics: (i) binds to the pre-fusion shape of the human RSV F protein with a Kd value of about 1x10'9 to about 1x10' ¹² M as determined by surface plasmon resonance (eg, Biacore) or by a similar method (eg, KinExa or OCTET) or (ii) binds to the post-fusion form of the human RSV F protein with a Kd value of about 1x10'9 to about 1x10'11 M as determined by surface plasmon resonance (eg, Biacore) or equivalent method (for example, KinExa or OCTET). In specific embodiments, the heavy chain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 23.

In one embodiment, the antibody, or antigen-binding fragment thereof, comprises (i) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4; (ii) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and (iii) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 6. In specific embodiments, the light chain or light chain variable region does not comprise the amino acid sequence of SEQ ID NO: 8. In specific embodiments, the light chain is not linked to the heavy chain. a chain containing the amino acid sequence of SEQ ID NO: 9. In specific embodiments, the light chain is linked to a heavy chain containing the amino acid sequence of SEQ ID NO: 7. In one embodiment, the antibody or fragment thereof optionally has at least one of the following characteristics : (i) binds to the pre-fusion shape of the human RSV F protein with a Kd value of about 1x10'9 to about 1x10' ¹² M as determined by surface plasmon resonance (eg, Biacore) or a similar method (eg, KinExa or OCTET) or (ii) binds to the post-fusion form of the human RSV F protein with a Kd value of about 1 x 10' ⁹ to about 1 x 10 ^-11 M as determined by surface plasmon resonance (eg Biacore) or a similar method (eg KinExa or OCTET). In specific embodiments, the light chain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 25.

In one embodiment, the antibody, or antigen-binding fragment thereof, comprises (i) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and (vi) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 6. In specific embodiments, the heavy chain or heavy chain variable region does not comprise the amino acid sequence of SEQ ID NO: 9. In one embodiment, the antibody or antigen-binding fragment thereof, optionally, has at least one of the following characteristics: (i) binds to the pre-fusion shape of the human RSV F protein with a Kd value of from about 1 x 10' ⁹ to about 1 x 10' ¹² M as determined by surface plasmon resonance (eg, Biacore) or by a similar method (eg, KinExa or OCTET) or (ii) binds to the post-fusion form of the human RSV F protein with a Kd value of about ^1x10'9 to about 1x10'11 M as determined by surface plasmon resonance (eg, Biacore) or equivalent method (for example, KinExa or OCTET).

In specific embodiments, the heavy chain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 23 and the light chain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 25.

In another embodiment, the invention provides an antibody, or antigen-binding fragment thereof, that binds to human RSV F protein, which comprises (i) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and (vi) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 6; wherein the antibody or antigen-binding fragment thereof contains a heavy chain variable region having at least 90, 95, 96, 97, 98 or 99% identity with the heavy chain variable region consisting of SEQ ID NO: 7, and the light chain variable region, having at least 90%, 95%, 96%, 97%, 98%, or 99% identity with the light chain variable region consisting of SEQ ID NO: 8. In specific embodiments, the heavy chain or heavy chain variable region does not contain the amino acid sequence of SEQ ID NO: 9. In these aforementioned embodiments, sequence variations occur in the framework regions. In one embodiment, the antibody, or antigen-binding fragment thereof, optionally has at least one of the following characteristics: (i) binds to a pre-fusion form of the human RSV F protein with a Kd value of about 1 x 10' ⁹ to about 1 x 10' ¹² M when determined surface plasmon resonance (eg, Biacore) or a similar method (eg, KinExa or OCTET) or (ii) binds to the post-fusion form of the human RSV F protein with a Kd value of about 1 x 10' ⁹ M to about 1 x 10 ^-1 M when determined by surface plasmon resonance (eg Biacore) or a similar method (eg KinExa or OCTET). In specific embodiments, the heavy chain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 23 and the light chain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 25.

In another embodiment, the invention also provides an antibody or antigen-binding fragment thereof that binds to human RSV, which comprises (i) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and (vi) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 6. In specific embodiments, the heavy chain or heavy chain variable region does not comprise the amino acid sequence of SEQ ID NO: 9. In one embodiment, the antibody or antigen-binding fragment thereof has 1, 2 or 3 amino acid substitutions in heavy chain CDR regions (SEQ ID NOs: 1-3) and/or in light chain CDR regions

- 3 039222 (SEQ ID NO: 4-6). The VH sequence of SEQ ID NO: 7 contains the CDR regions of SEQ ID NO: 1-3; and the VL sequence SEQ ID NO: 8 contains the CDR regions of SEQ ID NO: 4-6. In one embodiment, the antibody, or antigen-binding fragment thereof, optionally has at least one of the following characteristics: (i) binds to a pre-fusion form of the human RSV F protein with a Kd value of about 1 x 10' ⁹ to about 1 x 10' ¹² M when determined by surface plasmon resonance (eg, Biacore) or a similar method (eg, KinExa or OCTET) or (ii) binds to the post-fusion form of the human RSV F protein with a Kd value of about ^1x10'9 to about 1x10'1 M as determined by the method surface plasmon resonance (eg Biacore) or a similar method (eg KinExa or OCTET).

In specific embodiments, the heavy chain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 23 and the light chain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 25.

In one embodiment, the invention relates to an antibody or antigen-binding fragment containing: a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 7, and/or a light chain variable region containing the amino acid sequence of SEQ ID NO: 8, wherein the antibody or its antigen-binding fragment binds to the human RSV F protein. In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain containing, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 23, and a light chain containing, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 25 , wherein the antibody or antigen-binding fragment thereof binds to the human RSV F protein. In one embodiment, the antibody, or antigen-binding fragment thereof, optionally has at least one of the following characteristics: (i) binds to a pre-fusion form of the human RSV F protein with a Kd value of about 1 x 10' ⁹ to about 1 x 10' ¹² M when determined by surface plasmon resonance (eg, Biacore) or a similar method (eg, KinExa or OCTET) or (ii) binds to the post-fusion form of the human RSV F protein with a Kd value of about ^1x10'9 to about 1x10'11 M as determined by the method surface plasmon resonance (eg Biacore) or a similar method (eg KinExa or OCTET).

In another embodiment, the invention provides an antibody, or antigen-binding fragment thereof, that binds to the same human RSV F protein epitope as an antibody comprising the heavy chain of SEQ ID NO: 23 and the light chain of SEQ ID NO: 25, wherein the antibody or its antigen-binding fragment has at least one of the following characteristics: (i) binds to a pre-fusion form of the human RSV F protein with a Kd value of about 1 x 10' ⁹ to about 1 x 10' ¹² M as determined by surface plasmon resonance (e.g., Biacore) or a similar method (eg, KinExa or OCTET) or (ii) binds to the post-fusion form of the human RSV F protein with a Kd value of about 1 x 10' ⁹ to about 1 x 10 ^-11 M as determined by surface plasmon resonance (eg, Biacore) or by a similar method (for example, KinExa or OCTET). In one embodiment, the antibody has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the heavy chain variable region and/or the light chain variable region of SEQ ID NOs: 7 and 8, respectively. In another embodiment, the antibody or antigen-binding fragment thereof has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions in the heavy chain variable region of SEQ ID NO: 7 and/or the light chain variable region of SEQ ID NO: 8. In specific embodiments, the heavy chain or the heavy chain variable region does not contain the amino acid sequence of SEQ ID NO: 9.

In another embodiment, the invention relates to an antibody, or antigen-binding fragment thereof, that cross-blocks the binding (or is a competitor) of an antibody comprising the heavy chain of SEQ ID NO: 23 and the light chain of SEQ ID NO: 25 to human RSV, wherein the antibody or its the antigen-binding fragment has at least one of the following characteristics: (i) binds to a pre-fusion shape of the human RSV F protein with a Kd value of about 1 x 10' ⁹ to about 1 x 10' ¹² M as determined by surface plasmon resonance (eg, Biacore) or by a similar method (eg, KinExa or OCTET) or (ii) binds to the post-fusion form of the human RSV F protein with a Kd value of about ^1x10'9 to about 1x10 ^-11 M as determined by surface plasmon resonance (eg, Biacore) or equivalent method (for example, KinExa or OCTET). In one embodiment, the antibody or antigen-binding fragment thereof has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the heavy chain variable region of SEQ ID NO: 7 or the light chain variable region of SEQ ID NO: 8. In another embodiment, the antibody or antigen-binding fragment thereof has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions in the heavy chain variable region of SEQ ID NO: 7 or the light chain variable region of SEQ ID NO: 8. In another embodiment, the antibody or its antigen-binding fragment has

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1, 2, or 3 amino acid substitutions in heavy chain CDR regions (SEQ ID NOs: 1-3) and/or in regions

Light chain CDRs (SEQ ID NOs: 4-6). In specific embodiments, the heavy chain or heavy chain variable region does not contain the amino acid sequence of SEQ ID NO: 9.

In one embodiment, the invention relates to an isolated antibody or antigen-binding fragment thereof that binds to human RSV F-protein, which contains a heavy chain containing the amino acid sequence of SEQ ID NO: 7 or a variant thereof having up to 30 amino acid substitutions, and/or a light chain. a chain containing the amino acid sequence of SEQ ID NO: 8 having up to 12 amino acid substitutions. In specific embodiments, the heavy chain or heavy chain variable region does not contain the amino acid sequence of SEQ ID NO: 9.

In specific embodiments, the invention provides an isolated antibody, or antigen-binding fragment thereof, that binds to a human RSV F protein, wherein the antibody binds to a human RSV F protein through one or more of the following interactions, or all of the following interactions:

1) the CDR3 loop of the light chain through Phe 91 and Leu 92 residues interacts with the Arg 429 F side chain of the human RSV protein by forming two hydrogen bonds between the oxygen atoms of the carbonyl groups Phe 91 and Leu 92 in the CDR3 loop and the nitrogen atoms of the guanidine groups Arg 429 F - human RSV protein;

2) the CDR2 loop of the light chain through the residues Asp 50 and Glu 55 forms hydrogen bonds with Asn 426 and Lys 445 of the human RSV F protein;

3) the heavy chain CDR3 loop through Tyr 104 and Tyr 110 residues forms a surface for van der Waals interactions with Ile 432 on the human RSV F protein;

4) the CDR3 loop of the heavy chain forms a hydrogen bond through Asn 107 with Lys 433 of the human RSV F protein; And

5) the light chain folds opposite Glu 161 and Ser 182 of the adjacent trimer monomer prior to RSV fusion.

In specific aspects of any of the above embodiments, the antibody or antigen-binding fragment thereof is isolated.

In specific aspects of any of the above embodiments, the antibody, or antigen-binding fragment thereof, is a recombinant antibody.

In specific aspects of any of the above embodiments, the antibody, or antigen-binding fragment thereof, is a full length antibody.

In specific aspects of any of the above embodiments, an antibody or antigen-binding fragment thereof of the invention may comprise a heavy chain variable region consisting of (a) any of the heavy chain variable regions described above and (b) a leader peptide (e.g., the leader peptide of SEQ ID NO: 10). In specific aspects of any of the above embodiments, an antibody or antigen-binding fragment thereof of the invention may comprise a light chain variable region consisting of (a) any of the variable regions described above and (b) a leader peptide (e.g., the leader peptide of SEQ ID NO: 10).

In specific aspects of any of the above embodiments, the antibody or antigen-binding fragment thereof of the invention is an antibody comprising any of the heavy chain variable regions described above and any heavy chain constant domain of a human antibody. In one embodiment, the antibody or antigen-binding fragment of the invention is of the IgG isotype and contains a heavy chain constant domain of a human IgG1, IgG2, IgG3, or IgG4. In one embodiment, an antibody or antigen-binding fragment thereof of the invention comprises a human IgG1 heavy chain constant domain, wherein the IgG1 constant domain is afucosylated.

In specific aspects of any of the above embodiments, an antibody or antigen-binding fragment thereof of the invention may comprise any of the light chain variable regions described above and a human antibody light chain constant domain. In one embodiment, an antibody or antigen-binding fragment thereof of the invention comprises a human antibody kappa light chain constant domain, or a variant thereof, where the variant has up to 20, 10, 5, 3, 2, or 1 amino acid substitutions. In another embodiment, an antibody or antigen-binding fragment thereof of the invention comprises a human lambda antibody light chain constant domain, or a variant thereof, where the variant has up to 20, 10, 5, 3, 2, or 1 amino acid substitutions. In one embodiment, an antibody or antigen-binding fragment thereof of the invention comprises a human antibody kappa light chain constant domain comprising the amino acid sequence of SEQ ID NO: 14.

In one embodiment, an anti-hRSV F protein antibody of the invention has a complete tetrameric structure with two light chains and two heavy chains, with each light chain containing a variable region containing SEQ ID NO: 8 and a human antibody kappa light chain constant domain ( SEQ ID NO: 14); and each heavy chain contains a variable region containing

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SEQ ID NO: 7, and human IgG1 constant domain (SEQ ID NO: 13).

In specific aspects of any of the above embodiments, an anti-hRSV F protein antibody or antigen-binding fragment of the invention may be conjugated to at least one prophylactic or therapeutic agent. In one embodiment, the therapeutic agent is a triple antibody or fragment thereof, an immunomodulator, a hormone, a cytotoxic agent, an enzyme, a radioactive isotope, a second antibody conjugated to at least one immunomodulator, an enzyme, a radiolabel, a hormone, an antisense oligonucleotide, or a cytotoxic agent, or their combination.

The invention also relates to isolated polypeptides containing any amino acid sequence from SEQ ID NO: 1-8, 2 3 or 25 or a fragment of any of these sequences. In specific embodiments, the polypeptides containing the heavy chain amino acid sequences do not contain the amino acid sequence of SEQ ID NO: 9.

The invention also relates to isolated nucleic acids encoding any of the antibodies or antigen-binding fragments to the hRSV F protein of the invention. In one embodiment, the invention relates to selected nucleic acids encoding any of the polypeptides of SEQ ID NO: 1-8, 23 or 25, while these polypeptides may optionally contain a leader sequence. In specific embodiments, the implementation of the polypeptides containing the amino acid sequences of the heavy chain do not contain the amino acid sequence of SEQ ID NO: 9. The invention also relates to expression vectors containing a nucleic acid encoding any of the polypeptides of SEQ ID NO: 1-8, 23 or 25 (when these polypeptides may optionally contain a leader sequence). In specific embodiments, the polypeptides containing the heavy chain amino acid sequences do not contain the amino acid sequence of SEQ ID NO: 9. These isolated nucleic acids and expression vectors containing them can be used to express the antibodies of the invention, or antigen-binding fragments thereof, in recombinant host cells. . Thus, the invention also relates to host cells containing isolated nucleic acids encoding any of the polypeptides of SEQ ID NO: 1-8, 23 or 25 (while these polypeptides may optionally contain a leader sequence). In specific embodiments, the polypeptides containing the heavy chain amino acid sequences do not contain the amino acid sequence of SEQ ID NO: 9. In one embodiment, the host cell is a Chinese hamster ovary cell. In one embodiment, the host cell is a yeast cell, such as a Pichia cell or a Pichia pastoris host cell.

The invention also relates to pharmaceutical compositions containing an antibody or antigen-binding fragment of the invention and a pharmaceutically acceptable carrier or diluent. In one embodiment, the pharmaceutically acceptable carrier or diluent is L-histidine. In one aspect of this embodiment, the antibody or antigen binding fragment is formulated in a solution of 10 mM L-histidine, 7% (w/v) sucrose, and 0.02% (w/v) polysorbate-80, pH 6.0. The antibody or antigen-binding fragment is typically present in such a preparation at a concentration of about 100 mg/ml.

In one embodiment, the present invention relates to compositions containing an antibody or antigen-binding fragment of the invention and containing an additional prophylactic or therapeutic agent. In one embodiment, the additional prophylactic or therapeutic agent is selected from the group consisting of a second anti-hRSV antibody, or an antigen-binding fragment thereof. In one embodiment, the second anti-hRSV antibody or antigen-binding fragment of the invention comprises (i) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and (vi) a light chain variable region CDR3 containing the amino acid sequence of SEQ ID NO: 6. In specific embodiments, the heavy chain or heavy chain variable region does not contain the amino acid sequence of SEQ ID NO: 9.

The invention also relates to a container or injection device containing any of the antibodies or antigen-binding fragments to the hRSV F protein of the invention. In one embodiment, an antibody or antigen-binding fragment to hRSV F protein of the invention comprises (i) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and (vi) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 6. In specific embodiments, the heavy chain or heavy chain variable region does not comprise the amino acid sequence of SEQ ID NO: 9. In specific embodiments, the heavy chain contains, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 23; and the light chain contains, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 25.

The invention also relates to a method for producing an antibody or antigen-binding fragment to the hRSV F-protein of the invention, comprising culturing a host cell containing a polynucleotide encoding the heavy chain and/or light chain of an antibody of the invention (or its antigen-binding fragment), under conditions favorable for polynucleotide expression; and optionally recovering the antibody or antigen-binding fragment from the host cell and/or culture medium. In one embodiment, the heavy chain encoding polynucleotide and the light chain encoding polynucleotide are in the same vector. In another embodiment, the heavy chain encoding polynucleotide and the light chain encoding polynucleotide are in different vectors. In one embodiment, the heavy chain encoding polynucleotide and the light chain encoding polynucleotide encode an antibody or antigen binding fragment comprising (i) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and (vi) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 6. In specific embodiments, the heavy chain or heavy chain variable region does not comprise the amino acid sequence of SEQ ID NO: 9. In specific embodiments, the heavy chain comprises, consists of essentially or consists of the amino acid sequence of SEQ ID NO: 23 and the light chain contains, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 25.

The invention also relates to a method for preventing or treating hRSV infection in a patient, comprising administering to the patient an effective amount of an antibody or antigen-binding fragment against hRSV F protein of the invention, optionally in combination with an additional prophylactic or therapeutic agent or therapeutic procedure. In one embodiment, the patient receiving treatment is a human. In one embodiment, the additional prophylactic or therapeutic agent is selected from the group consisting of a second anti-hRSV antibody or antigen-binding fragment thereof, a nucleic acid encoding an antibody or antigen-binding fragment to the RSV F protein, or an antibody conjugate. In one embodiment, an antibody or antigen-binding fragment to hRSV F protein of the invention comprises (i) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and (vi) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 6. In specific embodiments, the heavy chain or heavy chain variable region does not comprise the amino acid sequence of SEQ ID NO: 9. In specific embodiments, the heavy chain comprises, consists of essentially or consists of the amino acid sequence of SEQ ID NO: 23 and the light chain contains, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 25.

The invention also relates to a method for preventing or treating hRSV infection in a patient, comprising administering to the patient an effective amount of an antibody or antigen-binding fragment against hRSV F protein of the invention, optionally in combination with an additional prophylactic or therapeutic agent or therapeutic procedure. In one embodiment, an antibody or antigen-binding fragment to hRSV F protein of the invention comprises (i) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and (vi) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 6. In specific embodiments, the heavy chain or heavy chain variable region does not comprise the amino acid sequence of SEQ ID NO: 9. In specific embodiments, the heavy chain comprises, consists of essentially or consists of the amino acid sequence of SEQ ID NO: 23 and the light chain contains, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 25.

- 7 039222

The invention also relates to a vaccine or immunogenic composition containing an antibody or antigen-binding fragment of the invention. In one embodiment, an antibody or antigen-binding fragment to hRSV F protein of the invention comprises (i) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and (vi) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 6. In specific embodiments, the heavy chain or heavy chain variable region does not comprise the amino acid sequence of SEQ ID NO: 9. In specific embodiments, the heavy chain comprises, consists of essentially or consists of the amino acid sequence of SEQ ID NO: 23 and the light chain contains, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 25.

In one embodiment, the vaccine or immunogenic composition further comprises an antigen selected from RSV F protein and RSV G protein, as well as fragments thereof.

The invention also relates to a method for detecting the presence of RSV in a sample (by detecting an F-protein or fragment thereof), including contacting the sample with an antibody or antigen-binding fragment of the invention and detecting the presence of a complex between the antibody or fragment and the peptide; while the detection of the complex indicates the presence of the RSV F-protein. In one embodiment, an antibody or antigen-binding fragment of the invention comprises (i) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and (vi) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 6. In specific embodiments, the heavy chain or heavy chain variable region does not comprise the amino acid sequence of SEQ ID NO: 9. In specific embodiments, the heavy chain comprises, consists of essentially or consists of the amino acid sequence of SEQ ID NO: 23 and the light chain contains, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 25.

The invention also relates to a method for increasing the anti-hRSV activity of an antibody to hRSV F-protein, which includes obtaining the original antibody to hRSV F-protein and enhancing the effector function of the original antibody to hRSV F-protein; the activity of the resulting antibody to the hRSV F-protein is increased compared to the activity of the original antibody to the hRSV F-protein. As used herein, the term parent antibody refers to an antibody having a wild-type Fc region and/or wild-type glycosylation (ie, a glycosylation pattern resulting from the expression of a polypeptide in a non-genetically modified mammalian host cell). The effector function of the parent antibody can be enhanced by mutations in its Fc region or by altering its glycosylation pattern, for example by creating an afucosylated antibody (as described in more detail below). In one embodiment, the effector function of the parent anti-hRSV F protein antibody is enhanced by introducing mutations in the Fc region of the parent anti-hRSV F protein antibody. In another embodiment, the effector function of the parent anti-hRSV F protein antibody is enhanced by removing fucose residues from the antibody, or by expressing the antibody in a host cell that is genetically modified to eliminate the activity of an enzyme that adds fucose residues to glycoproteins.

Brief description of the drawings

In FIG. 1A, 1B show binding curves (ELISA analysis) of anti-human RSV antibodies: D25, palivizumab, and RB1 with pre-fusion (A) and post-fusion (B) forms of human RSV F protein.

In FIG. 2A, 2B show neutralization curves for anti-human RSV antibodies for RSV A Long strain (A) and RSV B Washington strain (B).

In FIG. 3A, 3B show epitope mapping for RB1 by alanine-scanning mutagenesis of the F-fusion protein (A) and residues of the mapped epitope on the crystal structure of the pre-fusion F-protein form (B).

In FIG. 4A-4D show the efficacy of RB1 versus D25 in cotton hamster lung in RSV A infection model as a function of antibody concentrations (A) and in RSV B infection model as a function of antibody concentrations (B) or number of virus particles (PFU/g). present in tissues, depending on the dose of antibody in RSV A infection (C) and RSV B infection (D).

In FIG. 5A-5D show the efficacy of RB1 versus D25 in cotton hamster nose in an RSV A infection model as a function of antibody concentrations (A) and in an RSV B infection model as a function of antibody concentrations (B) or number of viral particles (PFU/g). present in tissues,

- 8 039222 depending on the dose of antibodies in RSV A infection (C) and RSV B infection (D).

In FIG. 6 shows the binding curve (ELISA assay) of the anti-human RSV antibody RB1+YTE with

F-protein of human RSV A.

In FIG. 7 shows the pharmacokinetic properties in rhesus monkeys of the RB1-YTE antibody (RB1+YTE) compared to motavizumab having the YTE mutation set.

Detailed description of the invention

Abbreviations.

In the text of the detailed description and examples of the invention, the following abbreviations are used:

ADCC Antibody dependent cellular cytotoxicity

CDC Complement dependent cytotoxicity

CDR Complementarity-determining region in immunoglobulin variable regions, defined using the Rabat numbering system

CHO Chinese hamster ovary

ELISA Enzyme-linked immunosorbent assay FR HRP IC50 IgG Antibody framework region: immunoglobulin variable excluding CDR regions Horseradish peroxidase Concentration resulting in 50% inhibition Immunoglobulin G areas Kabat Alignment and numbering system for immunoglobulin residues, first proposed by Elvin A. Rabat ((1991) Sequences of Proteins of Immunological Interest, 5 ^th Ed., Public Health Service, National Institutes of Health, Bethesda, Md.)

Mab or Mab Monoclonal antibody or Mab

V-region A segment of IgG chains whose sequence varies between antibodies. It continues to residue 109 in the light chain and residue 113 in the heavy chain according to the Rabat system.

VH Immunoglobulin heavy chain variable region

VK Immunoglobulin kappa light chain variable region

Definitions.

To facilitate understanding of the invention, some technical and scientific terms are explained below. Unless specifically indicated otherwise in the present description, all other technical and scientific terms used in the present description have the meaning that is usually given to them by specialists in the field to which this invention pertains.

When used in the present description, including the appended claims, terms in the singular include the corresponding terms in the plural, unless the context clearly indicates otherwise.

The terms administration and exposure when applied to an animal, human, experimental patient, cell, tissue, organ, or biological fluid means bringing an exogenous pharmaceutical, therapeutic, diagnostic agent, or composition into contact with an animal, human, patient, cell, tissue, organ, or biological fluid. The action on the cell includes the creation of contact of the reagent with the cell, as well as the creation of contact of the reagent with the liquid when the liquid is in contact with the cell. The terms administration and exposure also refer to in vitro and ex vivo exposure to, for example, a cell with a reagent, diagnostic, binding compound, or another cell.

RSV disease means any disease caused, directly or indirectly, by respiratory syncytial virus (RSV) infection, as well as diseases or conditions that predispose a patient to RSV infection. Examples of diseases that fall into this category include pneumonia and bronchiolitis. Diseases and conditions in this category (i.e., those that place a patient at risk for severe RSV infection) include cystic fibrosis, congenital heart disease, cancer, age-related immunosuppression, transplantation, and, in

- 9 039222 in general, any condition that causes immunosuppression or a decrease in the function of the immune system, such as a condition after an organ transplant operation or premature birth.

The terms "treat" or "treatment" refer to the administration of a therapeutic agent, such as a composition comprising any of the antibodies or antigen-binding fragments of the present invention, internally or externally to a person or patient who has one or more symptoms of a disease or is suspected of having a disease for which the agent has therapeutic activity. Typically, the agent is administered in an amount effective to relieve one or more symptoms of a disease in a treated patient or group of patients by either causing regression or inhibiting the progression of such symptom(s) to any clinically measurable degree. The amount of a therapeutic agent that is effective in alleviating any particular symptom of a disease may vary depending on such factors as the condition of the disease, the age and body weight of the patient, and the ability of the drug to elicit the desired response in the patient. Whether a symptom has improved can be assessed by measuring any of the clinical indicators commonly used by physicians or other qualified medical personnel to assess the severity or state of progression of a symptom. Treatment with anti-RSV antibodies can also be combined with other interventions (using antibodies, nucleic acids, vaccines, and small molecule compounds) to treat respiratory diseases caused by pathogens.

The terms prevent or prevent means administering a prophylactic agent, such as a composition comprising any of the antibodies or antigen-binding fragments of the present invention, internally or externally to a patient or patient at risk of hRSV infection for which the agent has prophylactic activity. Prevention includes reducing the likelihood or severity of subsequent RSV infection, relieving symptoms associated with lower respiratory tract infection (LRTI) when RSV is infected, and inducing immunity to protect against RSV infection. Typically, the agent is administered in an amount effective to neutralize RSV in the lungs and/or nose to block infection. The amount of a prophylactic agent that is effective in alleviating any particular symptom of the disease may vary depending on such factors as the age and weight of the patient, and the ability of the agent to elicit the desired response in the patient. Whether a symptom of a disease has improved can be assessed by measuring any of the clinical measures typically used by physicians or other qualified medical personnel to assess the severity or state of progression of a symptom, or in some cases based on a reduction in the need for hospitalization.

F protein hRSV.

The human RSV F protein is synthesized as a metastable trimeric precursor (F0) that is proteolytically cleaved into covalently linked F1 and F2 subunits. The atomic structures of the F-protein trimers in the pre-fusion form were determined for representatives of PGV-5 and RSV of the paramyxovirus family. See McLellan et al., 2011, J. Virol. 85:7788-7796 (RSV) and Welch et al., 2012, Proc. Natl. Acad. sci. 109:16672-16677 (PGV). The pre-fusion form of the F protein has a short C-terminal cytoplasmic fragment, one transmembrane domain, a helical stem, and a globular head domain. The atomic structures of the F-proteins VBN, hPGV-3, and RSV in the post-fusion form showed that a large refolding event occurs to convert the pre-fusion form to the post-fusion form of the F protein, which rearranges part of the globular head domain to form a six-helix bundle. These structures, along with data on peptide inhibition, provided a model of F-mediated membrane fusion in which F1/F2 rearrangement occurs upon activation, with the introduction of a hydrophobic fusion peptide from the N-terminus of F1 into the target cell membrane, leading to the formation of a pre-hairpin intermediate structure. This relatively elongated structure binds the virus to the cell membrane and breaks down to form a stable six-stranded bundle structure upon fusion. The transition from the pre-fusion metastable form to the pre-hairpin intermediate structure and then to the post-fusion conformation occurs in the direction of decreasing free energy, with the post-fusion form being the most stable state, and the energy released during F-protein refolding is expended on membrane fusion .

The term hRSV F protein includes the human RSV F protein as well as fragments thereof, such as its mature fragment without the signal peptide. In one embodiment, the amino acid sequence of the human RSV F protein is the amino acid sequence listed in Genbank under accession number AAR14266 (hRSV B strain 9320).

Anti-hRSV antibodies and their antigen-binding fragments.

The present invention relates to antibodies or antigen-binding fragments thereof that bind human RSV F protein, preferably from both A and B strains of RSV, that bind both the pre-F protein fusion form and the post-F protein fusion form, and variants the use of such antibodies or fragments. In some embodiments, antibodies to the RSV F protein are isolated. The antibodies described herein bind to an epitope at site IV of the F protein. In any of the specific embodiments of the invention described herein, the heavy chain or variable region of the heavy chain does not contain the amino acid sequence of SEQ ID NO: 9 and/or the light chain or variable region of the light chain does not contain the amino acid sequence of SEQ ID NO: 8. in specific embodiments, the heavy chain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 23 and the light chain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 25.

In preferred embodiments, the RSV F protein antibodies are fully human. A major advantage of the monoclonal antibodies of the invention results from the fact that they contain human antibody CDR3 sequences and, in some embodiments, may be fully human monoclonal antibodies. Thus, in vivo use of fully human monoclonal antibodies of the invention for immunoprophylaxis and immunotherapy of RSV disease greatly reduces the problem of host immune response to passively administered antibodies. Such a problem usually occurs when using prior art monoclonal antibodies of xenogeneic or chimeric origin. A second important aspect of this advantage is the potential safety of these human monoclonal antibodies for gene therapy applications in which the expression of xenogeneic or chimeric proteins containing non-human sequences cannot be stopped.

As used herein, the term antibody or antigen-binding fragment thereof against the RSV F protein means an antibody or antigen-binding fragment thereof that specifically binds to the human RSV F protein. An antibody or antigen-binding fragment thereof that specifically binds to human RSV is an antibody or antigen-binding fragment thereof that binds to the pre-fusion or post-fusion form of the human RSV F protein with a Kd value of about 1 nM or higher affinity (e.g., 1 nM - 2 pM, 1 nM, 100, 10 or 2 pM), but does not bind to other proteins lacking RSV F-protein sequences. In one embodiment, an antibody of the invention that specifically binds to human RSV F protein also cross-reacts with bovine RSV F protein. As used herein, the term cross-reactivity refers to the ability of an antibody to interact with a homologous protein from other species. Whether an antibody binds specifically to the human RSV F protein can be determined using any assay known in the art. Examples of assays known in the art for determining binding affinity include surface plasmon resonance (eg Biacore) or a similar method (eg KinExa or OCTET).

The present invention includes antibodies to hRSV F protein and methods of using the same. As used herein, the term antibody refers to any form of antibody that has the desired biological activity. Thus, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies containing two light chains and two heavy chains), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, and chimeric antibodies.

The present invention includes antigen-binding fragments to hRSV F-protein and methods of using them. When used in the present description, unless otherwise indicated, the terms antibody fragment or antigen-binding fragment means antigen-binding fragments of antibodies, i.e. antibody fragments that retain the ability to specifically bind to an antigen that binds a full-length antibody, such as fragments that retain one or more CDR regions. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments; diabody; linear antibodies; single chain antibody molecules, eg scFv; as well as multispecific antibodies formed from antibody fragments.

The present invention includes Fab-fragments to the F protein of RSV and methods of their use. The Fab fragment consists of one light chain, as well as CH1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. The Fab fragment may be a cleavage product of an antibody with papain.

The present invention includes antibodies to the F-protein of RSV and antigen-binding fragments containing the Fc region, as well as methods of their use. The Fc region contains two heavy chain fragments containing the CH1 and CH2 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and hydrophobic interactions of the CH3 domains.

The present invention includes Fab'-fragments to the F protein of RSV and methods of their use. A Fab' fragment contains one light chain and a portion or fragment of one heavy chain containing a VH domain and a CH1 domain, as well as a region between the CH1 and CH2 domains so that an interchain disulfide bond can be formed between the two heavy chains of the two Fab' fragments with the formation of the F(ab') ₂ molecule.

- 11 039222

The present invention includes F(ab') ₂ fragments to the RSV F protein and methods of using them. The F(ab') ₂ fragment contains two light chains and two heavy chains containing part of the constant region between the CH1 and CH2 domains so that an interchain disulfide bond is formed between the two heavy chains. Thus, the F(ab') ₂ fragment consists of two Fab' fragments that are held together by a disulfide bond between the two heavy chains. The F(ab') ₂ fragment may be the product of cleavage of an antibody with pepsin.

The present invention includes Fv-fragments to the F-protein of RSV and methods of their use. The Fv region contains both heavy and light chain variable regions, but no constant regions.

The present invention includes scFv fragments to the RSV F protein and methods for their use. The term single-chain Fv, or scFv, antibodies means antibody fragments containing VH and VL domains of the antibody, and these domains are on the same polypeptide chain. Typically, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which allows the scFv to form the structure required for antigen binding. For a review on scFv, see Pluckthun (1994), The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, p. 269-315. See also International Patent Application Publication WO 88/01649 and US Patent Nos. 4,946,778 and 5,260,203.

The present invention includes RSV F-protein domain antibodies and methods of using the same. A domain antibody is an immunologically functional fragment of an immunoglobulin containing only a heavy chain variable region or a light chain variable region. In some cases, two or more VH regions are covalently linked by a peptide linker to form a divalent domain antibody. The two VH regions of a bivalent domain antibody may target the same or different antigens.

The present invention includes divalent antibodies to the RSV F protein and methods of using the same. A bivalent antibody contains two antigen-binding sites. In some cases, the two binding sites have the same antigenic specificity. However, bivalent antibodies can be bispecific (see below).

The present invention includes diabodies to the RSV F protein and methods for their use. As used herein, the term diabody refers to small antibody fragments with two antigen-binding sites, the fragments containing a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL- vh). As a result of using a linker that is too short to allow pairing between two domains on the same strand, the domains are forced to pair with complementary domains on the other strand to form two antigen binding sites. Diabodies are described in more detail, for example, in EP 404097, WO 93/11161 and Holliger et al. (1993), Proc. Natl. Acad. sci. USA 90:6444-6448. For a review of recombinant antibody variants, see in general Holliger and Hudson (2005), Nat. Biotechnol. 23:1126-1136.

Typically, an antibody or antigen-binding fragment of the invention that is modified in some way retains at least 10% of its binding activity (as compared to the parent antibody), on a molar basis. Preferably, the antibody or antigen-binding fragment of the invention retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the binding affinity for the RSV F protein of that of the parent antibody. It is also contemplated that an antibody or antigen-binding fragment of the invention may have conservative or non-conservative amino acid substitutions (such variants are referred to as conservative variants or functionally conservative variants of an antibody) that do not significantly alter its biological activity.

The present invention includes isolated antibodies to the hRSV F protein and antigen-binding fragments thereof, as well as methods for their use. The isolated antibodies or antigen-binding fragments thereof are at least partially free of other biological molecules from the cells or cell cultures in which they are produced. Such biological molecules include nucleic acids, proteins, lipids, carbohydrates, or other material such as cell debris and growth media. In addition, the isolated antibody or antigen-binding fragment may be at least partially free of components of the expression system, such as biological molecules from its host cell or growth medium. As a rule, the term isolated should not mean the complete absence of such biological molecules or the absence of water, buffers or salts or components of a pharmaceutical preparation containing antibodies or fragments.

The present invention includes monoclonal antibodies to the hRSV F protein and antigen-binding fragments thereof, as well as monoclonal antibody compositions containing several isolated monoclonal antibodies. As used herein, the term monoclonal antibody refers to a population of substantially homogeneous antibodies, ie. the antibody molecules that make up the population are identical in amino acid sequence, with the exception of possible natural mutations, which may be present in small quantities. On the contrary, ordinary

- 12 039222 (polyclonal) preparations of antibodies, as a rule, contain many different antibodies having different amino acid sequences in their variable domains, in particular their CDR regions, which are often specific for different epitopes. The term "monoclonal" indicates the nature of the antibody as being derived from a substantially homogeneous population of antibodies, and should not imply the need to produce the antibody in a particular manner. For example, monoclonal antibodies used in accordance with the present invention can be obtained by recombinant DNA methods (see, for example, US patent No. 4816567).

As a rule, the structural unit of the main (or full-length) antibodies is a tetramer. Each tetramer contains two identical pairs of polypeptide chains, each pair containing one light (about 25 kDa) and one heavy (about 50-70 kDa) chain. The amino-terminal portion of each chain contains a variable region, or domain, of about 100-110 or more amino acids, which plays a major role in antigen recognition. The carboxy-terminal portion of the heavy chain may contain a constant region, or domain, which plays a major role in effector function. Typically, human antibody light chains are classified as kappa and lambda light chains. In addition, human antibody heavy chains are generally classified as mu, delta, gamma, alpha, or epsilon chains, and define the isotype of the antibody as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by a J region of about 12 or more amino acids, with the heavy chain also containing a D region of about 10 more amino acids. See mainly Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ^Ed ., Raven Press, NY (1989)). In the context of an antibody or antigen-binding fragment thereof, the terms domain and region may be used interchangeably when appropriate.

The variable regions of each light/heavy chain pair form the antibody binding site. Thus, typically, an intact antibody has two binding sites. With the exception of bifunctional or bispecific antibodies, the two binding sites are generally the same.

Typically, the heavy and light chain variable domains contain three hypervariable regions, also referred to as complementarity determining regions (CDRs), located among the relatively conserved framework regions (FRs). The CDRs are typically aligned with the framework regions, which allows for the binding of a particular epitope. Typically, from the N-terminus to the C-terminus, the variable domains of both the light and heavy chains contain FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Generally, amino acids are assigned to each domain according to the definitions given in Sequences of Proteins of Immunological Interest, Rabat, et al.; National Institutes of Health, Bethesda, Md.; 5th Ed ^.; NIH Publ. no. 91-3242 (1991); Rabat, 1978, Adv. Prot. Chem. 32:175; Rabat, et al., 1977, J. Biol. Chem. 252:6609-6616; Chothia et al., 1987, J. Mol. Biol. 196:901-917 or Chothia et al., 1989, Nature 342:878-883.

As used herein, the term hypervariable region refers to the amino acid residues of an antibody or antigen-binding fragment thereof that are responsible for antigen binding. The hypervariable region contains amino acid residues from the complementarity determining region or CDR (ie CDRL1, CDRL2 and CDRL3 in the light chain variable domain and CDRH1, CDRH2 and CDRH3 in the heavy chain variable domain). See Rabat et al. (1991), Sequences of Proteins of Immunological Interest, 5th ^Ed ., Public Health Service, National Institutes of Health, Bethesda, Md. (determination of antibody CDR regions based on sequence); see also Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917 (determination of antibody CDR regions based on structure). As used herein, the term framework or FR residues refers to variable domain residues other than the hypervariable region residues defined herein as CDR residues.

The terms isolated nucleic acid molecule or isolated polynucleotide means a DNA or RNA polynucleotide of genomic, mRNA, cDNA or synthetic origin, or some combination thereof, that is not associated with a full or partial polynucleotide, in which the isolated polynucleotide occurs naturally or is associated with a polynucleotide, with which it is not associated in nature. For the purposes of this invention, it should be understood that the term nucleic acid molecule containing a particular nucleotide sequence does not encompass intact chromosomes. Isolated nucleic acid molecules containing specific nucleotide sequences may contain, in addition to these sequences, coding sequences for up to ten or even up to twenty or more other proteins or parts or fragments thereof, or may contain operably linked regulatory sequences that control the expression of the coding region of these nucleotide sequences, and/or may contain vector sequences.

The term control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences that are suitable for prokaryotes, for example, include a promoter sequence, optionally an operator, and a ribosome binding site. Promoters, polyadenylation signals, and enhancers are known to be used in eukaryotic cells.

- 13 039222 sulfur.

A nucleic acid, or polynucleotide, is operably linked when it is operably linked to another nucleotide sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that is involved in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is located to facilitate translation. Typically, but not always, the term operably linked means that the linked DNA sequences are contiguous and, in the case of a secretory leader, are contiguous in read phase as well. However, enhancers need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites are not present, synthetic oligonucleotide adapters or linkers are used in accordance with common practice.

As used herein, the terms cell, cell line, and cell culture are used interchangeably, and all such definitions include cell progeny. Thus, the terms transformants and transformed cells include the primary specified cell and cultures derived from it, without regard to the number of passages. It should also be understood that not all offspring will have exactly identical DNA due to intentional or unintentional mutations. Mutant progeny having the same function or biological activity as the original transformed cell was screened for are encompassed by these terms. Where explicit designations are intended, this will be clear from the context.

As used herein, the term germline sequence refers to the sequence of non-rearranged immunoglobulin DNA sequences. Any suitable source of non-rearranged immunoglobulin sequences may be used.

Germline sequences of human proteins can be obtained, for example, from the JOINSOLVER germline sequence databases on the National Institute of Arthritis and Musculoskeletal and Skin Diseases website of the National Institutes of Health. Mouse protein germline sequences can be obtained, for example, as described in Giudicelli et al., 2005, Nucleic Acids Res. 33:D256-D261.

Physical and functional properties of illustrative antibodies to the RSV F protein.

The present invention relates to antibodies to the hRSV F protein and antigen-binding fragments thereof having the indicated structural and functional properties, as well as methods of using the antibodies or antigen-binding fragments thereof in the treatment or prevention of diseases/conditions associated with RSV infection.

An anti-RSV F protein antibody or antigen-binding fragment thereof of the present invention includes any antibody or antigen-binding fragment thereof described herein (eg, RB1) or a variant thereof (eg, with a variant sequence or functional variant); any antibody or antigennegative fragment containing any one or more of the CDR regions shown in table. 7; any antibody or antigen-binding fragment that binds to the same epitope on the human RSV F protein as the antibodies described herein (eg, RB1); and any antibody or antigen-binding fragment that cross-blocks (partially or completely) an antibody or cross-blocks (partially or completely) an antibody described herein (eg, RB1) when bound to RSV.

Cross-blocking antibodies and their antigen-binding fragments can be identified based on their ability to cross-compete with an antibody of the invention in standard binding assays (such as Biacore, ELISA, flow cytometry). For example, standard ELISA assays can be used in which a recombinant RSV F protein is immobilized on a plate, one of the antibodies is fluorescently labeled, and the ability of the unlabeled antibody to compete for binding with the labeled antibody is assessed. Additionally or alternatively, a Biacore assay can be used to evaluate the ability of an antibody to cross-compete. The ability of a test antibody to inhibit the binding of another antibody (eg, antibody D25) to the RSV F protein indicates that the test antibody can compete with another antibody (eg, D25) for binding to the RSV F protein, and thus, in some cases, may bind the same epitope on the RSV F protein as the D25 antibody, or an overlapping epitope.

As indicated above, antibodies and fragments thereof that bind to the same epitope as any of the antibodies or antigen-binding fragments thereof to the RSV F protein of the present invention are also part of the present invention. In addition, in specific embodiments, antibodies that bind to an epitope overlapping with an epitope bound by any of the RSV F protein antibodies of the invention are also part of the present invention. Several methods are known for mapping antibody epitopes to target antigens, including VDO mass spectrometry, X-ray crystallography, pepscan analysis, and site-directed mutagenesis. For example, VDO (hydrogen

- 14 039222 deuterium exchange) in combination with proteolysis and mass spectrometry can be used to determine antibody epitopes on a specific Y antigen. antibodies to it at different time intervals. Deuterium exchanges with hydrogen on the amide backbone of proteins in exposed regions, while antigen-antibody binding regions will be protected and show less or no exchange after LC-MS/MS analysis of proteolytic fragments.

Examples of immunoglobulin chains of the RSV F-protein antibodies of the invention, as well as their CDR regions, include, but are not limited to, those shown in Table 1. 7 (SEQ ID NOs: 1-8, 23 and 25). The present invention includes any polypeptide comprising, consisting essentially of, or consisting of the amino acid sequences of SEQ ID NOs: 1-8, 23, and 25, as well as recombinant nucleotides encoding such polypeptides.

The scope of the present invention includes isolated antibodies to hRSV F-protein and antigen-binding fragments containing a variant of the immunoglobulin chain described in the present description, for example, any of SEQ ID NO: 7, 8; wherein the variant exhibits one or more of the following properties: (i) binds to a pre-fusion shape of the human RSV F protein with a Kd value of from about 1 x 10 ^-9 to about 1 x 10' ¹² M as determined by surface plasmon resonance (eg, Biacore) or by a similar method (eg, KinExa or OCTET) or (ii) binds to the post-fusion form of the human RSV F protein with a Kd value of about 1 x 10 ^-9 to about 1 x 10 ^-1 M as determined by surface plasmon resonance (eg, Biacore) or equivalent method (for example, KinExa or OCTET). In specific embodiments, the heavy chain or heavy chain variable region does not contain the amino acid sequence of SEQ ID NO: 9.

In specific embodiments, the invention relates to an antibody or antigen-binding fragment thereof that binds human RSV F protein and contains VL domains and VH domains having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity. with SEQ ID NO: 8 (VL) and 7 (VH); wherein the variant exhibits the desired binding and properties, e.g. (i) binds to the pre-fusion form of the human RSV F protein with a Kd value of about 1 x 10 ^-9 to about 1 x 10 ^-12 M as determined by surface plasmon resonance (e.g., Biacore) or by a similar method (eg, KinExa or OCTET) or (ii) binds to the post-fusion form of the human RSV F protein with a Kd value of about 1 x 10 ^-9 to about 1 x 10' ^pM as determined by surface plasmon resonance (eg, Biacore) or equivalent method (for example, KinExa or OCTET). In specific embodiments, the heavy chain or heavy chain variable region does not contain the amino acid sequence of SEQ ID NO: 9.

The terms conservatively modified variants or conservative substitution refer to substitutions of amino acids in a protein with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.) such that changes can often be made without changing the biological activity of the protein. Those skilled in the art will appreciate that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not result in significant changes in biological activity (see, e.g., Watson et al. (1987), Molecular Biology of the Gene, The Benjamin/Cummings Pub Co., ^p.224 (4th Ed.)). In addition, substitutions for structurally or functionally similar amino acids are less likely to result in impaired biological activity. Illustrative conservative substitutions are given in table. one.

- 15 039222

Table 1

Illustrative conservative amino acid substitutions

original balance conservative replacement Ala (A) Gly; Ser Arg(R) Lys; His Asn(N) gin; His Asp(D) Glu; Asn Cys(C) Ser; Ala Gin (Q) Asn Glu(E) asp; gin Gly (G) Ala His(H) asn; gin He(I) Leu; Vai Leu(L) He; Vai Lys (K) Arg; His Met(M) Leu; He; Tyr Phe(F) Tyr; met; Leu Pro (P) Ala Ser(S) Thr Thr(T) Ser TRP(W) Tyr; Phe Tyr (Y) trp; Phe Wai (V) He; Leu

Functionally conserved variants of the antibodies of the invention are also contemplated by the present invention. As used herein, the term functionally conserved variants refers to antibodies or fragments thereof in which one or more amino acid residues have been changed without altering the desired property, such as affinity and/or specificity for an antigen. Such options include, but are not limited to, options with an amino acid substitution for an amino acid having similar properties, for example, options with conservative amino acid substitutions shown in table. 1. Also provided are isolated polypeptides containing the VL domains of antibodies to the hRSV F protein of the invention (e.g., SEQ ID NO: 8) and isolated polypeptides containing the VH domains (e.g., SEQ ID NO: 7) of antibodies to the hRSV F protein. according to the invention, having up to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid substitutions that may occur exclusively in the framework region or of which one or more may be in one or more of the CDR regions. In specific embodiments, the heavy chain or heavy chain variable region does not contain the amino acid sequence of SEQ ID NO: 9.

In another embodiment, an antibody or antigen-binding fragment thereof is provided that binds hRSV F protein and contains VL domains and VH domains having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, or 75% sequence identity with one or more of the VL domains or VH domains described herein and exhibits specific binding to hRSV F protein. In another embodiment, the binding antibody, or antigen-binding fragment thereof, of the present invention comprises VL and VH domains (with or without a signal sequence) having up to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid substitutions that may have site exclusively in the framework region, or of which one or more may be in one or more of the CDR regions and exhibits specific binding to the hRSV F protein. In specific embodiments, the heavy chain or heavy chain variable region does not contain the amino acid sequence of SEQ ID NO: 9.

polynucleotides and polypeptides.

The present invention also relates to polynucleotides encoding any of the polypeptides or immunoglobulin chains of antibodies and their antigen-binding fragments to the hRSV F protein of the invention. In one embodiment, the isolated polynucleotide encodes an antibody, or an antigen-binding fragment thereof, comprising at least one mature immunoglobulin (VL) light chain variable domain of the invention and/or at least one mature variable domain

- 16 039222 heavy chain immunoglobulin (VH) according to the invention. In some embodiments, an isolated polynucleotide encodes both a light chain and a heavy chain on a single polynucleotide molecule, and in other embodiments, the light and heavy chains are encoded on separate polynucleotide molecules. In another embodiment, the polynucleotide also encodes a signal sequence. For example, the present invention includes polynucleotides encoding the amino acid sequences of SEQ ID NOs: 1-8, 23, and 25, as well as polynucleotides that hybridize to them, and, in addition, any polypeptide encoded by such a hybridizing polynucleotide. In one embodiment, the invention relates to a nucleotide sequence comprising, consisting essentially of, or consisting of SEQ ID NO: 15 (heavy chain variable region) or SEQ ID NO: 16 (light chain variable region). In specific embodiments, codon optimization can be used to enhance the properties of a nucleic acid, such as expression in a particular host. In one embodiment, the invention relates to a nucleotide sequence comprising, consisting essentially of, or consisting of SEQ ID NO: 17 (codon-optimized heavy chain variable region) or SEQ ID NO: 18 (codon-optimized light chain variable region). In specific embodiments, a leader sequence may be used. In one embodiment, the invention relates to a nucleotide sequence comprising, consisting essentially of, or consisting of SEQ ID NO: 19 (leader sequence and heavy chain) or SEQ ID NO: 20 (leader sequence and light chain) linked to a heavy chain or light chain, with the formation of SEQ ID NO: 21 or SEQ ID NO: 22, respectively.

Typically, polynucleotides hybridize under conditions of low, medium, or high stringency and encode antibodies or antigen-binding fragments thereof that retain the ability to bind the hRSV F protein. A first polynucleotide molecule is hybridizable to a second polynucleotide molecule if the single stranded form of the first polynucleotide molecule can anneal to the second polynucleotide molecule under appropriate temperature and solution ionic strength conditions (see Sambrook, et al., supra). Temperature and ionic strength determine the stringency of the hybridization conditions. Typical low stringency hybridization conditions include 55°C, 5X SSC, 0.1% SDS and no formamide or 30% formamide with 5X SSC, 0.5% SDS at 42°C. Typical medium stringency hybridization conditions include 40% formamide with 5X or 6X SSC and 0.1% SDS at 42°C. High stringency hybridization conditions include 50% formamide with 5X or 6X SSC at 42°C or optionally higher temperature (eg 57, 59, 60, 62, 63, 65 or 68°C). Typically, SSC is a solution of 0.15 M NaCl and 0.015 M Na-citrate. Hybridization requires that the two polynucleotides contain complementary sequences, although base mismatches are possible depending on the stringency of the hybridization conditions. The appropriate stringency for hybridizing polynucleotides depends on the length of the polynucleotides and the degree of complementarity, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the stringency of the conditions under which nucleic acids can hybridize. For hybrids longer than 100 nucleotides, formulas have been developed to calculate the melting point (see Sambrook et al., supra, 9.50-9.51). In the case of hybridization with shorter polynucleotides, such as oligonucleotides, the number of mismatches becomes more important and the length of the oligonucleotide determines its specificity (see Sambrook, et al., supra, 11.7-11.8).

In one embodiment, the invention provides an isolated polynucleotide encoding the heavy chain variable domain (VH) of an antibody or antigen-binding fragment thereof, comprising CDR-H1 (SEQ ID NO: 1), CDR-H2 (SEQ ID NO: 2) and CDR-H3 (SEQ ID NO: 3).

In one embodiment, the invention provides an isolated polynucleotide encoding a light chain variable domain (VL) of an antibody or antigen-binding fragment thereof, comprising CDR-L1 (SEQ ID NO: 4), CDR-L2 (SEQ ID NO: 5), and CDR-L3 (SEQ ID NO: 6).

In one embodiment, the invention relates to an isolated polynucleotide encoding the heavy chain variable domain (VH) of an antibody of SEQ ID NO: 7 or the heavy chain of SEQ ID NO: 23.

In one embodiment, the invention relates to an isolated polynucleotide encoding a light chain variable domain (VL) of an antibody of SEQ ID NO: 8 or a light chain of SEQ ID NO: 25.

The present invention also relates to vectors, eg expression vectors such as plasmids, containing the isolated polynucleotides of the invention, wherein the polynucleotide is operably linked to control sequences that are recognized by the host cell when the host cell is transfected with the vector. Also provided are host cells containing the vector of the present invention, and methods for producing an antibody or antigen-binding fragment or polypeptide disclosed herein, including culturing a host cell containing an expression vector or a nucleic acid encoding immunoglobulin chains of the antibody or antigen-binding fragment thereof in culture medium and isolation of the antibody or its

- 17 039222 antigen-binding fragment from the host cell or culture medium.

The present invention also includes polypeptides, such as immunoglobulin polypeptides, containing amino acid sequences that are at least about 75% identical, 80% identical, more preferably at least about 90% identical, and most preferably at least about 95% identical. (e.g., 95, 96, 97, 98, 99, 100%) with the amino acid sequences of the antibodies described herein when compared using the BLAST algorithm when the algorithm parameters are chosen to achieve the best match between the corresponding sequences along the entire length of the corresponding reference sequences (e.g. wait threshold: 10; word length: 3; maximum hits in query range: 0; BLOSUM matrix 62; deletion penalties: insertion 11, continuation 1; conditional compositional adjustment of substitution matrix).

Sequence identity refers to the degree to which the amino acids of two polypeptides are the same at equivalent positions when the two sequences are optimally aligned.

The following literature sources describe BLAST algorithms commonly used for sequence analysis:

BLAST ALGORITHMS: Altschul et al. (2005), FEBS J. 272(20):5101-5109; Altschul, S.F., et al., (1990), J. Mol. Biol. 215:403-410;

Gish, W., et al., (1993), Nature, Genet. 3:266-272; Madden, T.L., et al., (1996), Meth. Enzymol. 266:131141;

Altschul, S.F., et al., (1997), Nucleic Acids Res. 25:3389-3402; Zhang, J., et al., (1997), Genome Res. 7:649-656;

Wootton, J.C., et al., (1993), Comput. Chem. 17:149-163; Hancock, J.M. et al., (1994), Comput. Appl. biosci. 10:67-70;

ALIGNMENT SCORING SYSTEMS: Dayhoff, M.O., et al., A model of evolutionary change in proteins, in Atlas of Protein Sequence and Structure, (1978), vol. 5 suppl. 3.M.O. Dayhoff (ed.), p. 345-352, Natl. Biomed. Res. Found., Washington, DC;

Schwartz, R.M., et al., Matrices for detecting distant relationships, in Atlas of Protein Sequence and Structure, (1978), vol. 5 suppl. 3.M.O. Dayhoff (ed.), p. 353-358, Natl. Biomed. Res. Found., Washington, DC;

Altschul, S.F., (1991), J. Mol. Biol. 219:555-565; States, D.J., et al., (1991), Methods. 3:66-70;

Henikoff, S., et al., (1992), Proc. Natl. Acad. sci. USA 89:10915-10919; Altschul, S.F., et al., (1993), J. Mol. Evol. 36:290-300;

ALIGNMENT STATISTICS: Karlin, S., et al., (1990), Proc. Natl. Acad. sci. USA 87:2264-2268;

Karlin, S., et al., (1993), Proc. Natl. Acad. sci. USA 90:5873-5877;

Dembo, A., et al., (1994), Ann. Prob. 22:2022-2039 and

Altschul, S.F. Evaluating the statistical significance of multiple distinct local alignments, in Theoretical and Computational Methods in Genome Research (S. Suhai, ed.), (1997), p. 1-14, Plenum, New York.

Binding affinity.

By way of example, and not limitation, the antibodies and antigen-binding fragments disclosed herein can bind the pre-fusion form of the F protein or the post-fusion form of the human RSV F protein with a KD value of at least about 1 x 10' ⁹ M (i.e. KD value of 1 x 10' ⁹ M or less) when determined by surface plasmon resonance (eg Biacore) or a similar method (eg KinExa or OCTET). In one embodiment, the antibodies and antigen-binding fragments disclosed herein can bind the pre-fusion form of the F protein or the post-fusion form of the human RSV F protein with a KD value of at least about 1 x 10 ^-9 to about 1 x 10 ^-12 M when defined by surface plasmon resonance (eg Biacore) or a similar method (eg KinExa or OCTET). In one embodiment, the antibodies and antigen-binding fragments disclosed herein can bind the pre-fusion form of the F protein or the post-fusion form of the human RSV F protein with a KD value of from about 1 x 10 ^-9 to about 1 x 10 ^-12 M as determined by surface plasmon resonance (eg Biacore) or a similar method (eg KinExa or OCTET). In one embodiment, the antibodies and antigen-binding fragments disclosed herein can bind the pre-fusion form of the F protein or the post-fusion form of the human RSV F protein with a K _D value of at least about 100 pM (i.e., a K _D value of about 100 pM or less) when determined by the Biacore method or a similar method. In one embodiment, the antibodies and antigen-binding fragments disclosed herein can bind the pre-fusion form of the F protein or the post-fusion form of the human RSV F protein with a K _D value of at least about 10 pM (i.e., a K _D value of about 10 pM or less) when determined by the Biacore method or a similar method. In one embodiment, the antibodies and antigen-binding fragments disclosed herein can bind the pre-fusion form of the F protein or the post-fusion form of the human RSV F protein with a KD value of from about 1 pM to about 100 pM as determined by the Biacore method or the like.

- 18 039222 method.

Methods for producing antibodies and their antigen-binding fragments.

The present invention relates to methods for producing an antibody or antigen-binding fragment to the hRSV F protein of the present invention, comprising culturing a cell line expressing the antibody or fragment under conditions favorable for such expression, and optionally isolating the antibody or fragment from the cells and/ or growth media (eg cell culture media).

Anti-hRSV F protein antibodies disclosed herein can also be generated by recombinant methods (eg, in an E. coli/T7 expression system, a mammalian cell expression system, or a lower eukaryotic expression system). In this embodiment, nucleic acids encoding antibody molecules of the invention (eg, VH or VL) can be inserted into a pET-based plasmid and expressed in the E. coli/T7 system. For example, the present invention includes methods for expressing an antibody, or an antigen-binding fragment thereof, or an immunoglobulin chain thereof in a host cell (e.g., a bacterial host cell such as E. coli, e.g., BL21 or BL21DE3), comprising expressing T7 RNA polymerase in a cell that also contains a polynucleotide encoding an immunoglobulin chain that is operably linked to the T7 promoter. For example, in one embodiment, a bacterial host cell, such as E. coli, contains a polynucleotide encoding a T7 RNA polymerase operably linked to a lac promoter, and polymerase and strand expression is induced by incubating the host cell with IPTG (isopropyl beta III-thiogalactopyranoside ).

Several methods for producing recombinant antibodies are known in the art. One example of a method for producing recombinant antibodies is given in US Pat. No. 4,816,567.

Transformation can be performed by any known method for introducing polynucleotides into the host cell. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotide(s) into liposomes, biolistic injection, and direct microinjection of DNA into the nucleus. In addition, nucleic acid molecules can be introduced into mammalian cells using viral vectors. Methods for transforming cells are well known in the art, see, for example, US Pat. Nos. 4,399,216; 4,912,040;

Thus, the present invention includes recombinant methods for producing an anti-hRSV antibody or antigen-binding fragment or immunoglobulin chain of the present invention, comprising introducing a polynucleotide encoding one or more immunoglobulin chains of the antibody or fragment (for example, heavy and/or light immunoglobulin chain); culturing the host cell (eg CHO or Pichia or Pichia pastoris) under conditions favorable for such expression; and optionally isolating the antibody or fragment or chain from the host cell and/or the medium in which the host cell is growing.

Antibodies to the hRSV F protein can also be synthesized by any of the methods described in US Pat. No. 6,331,415.

Eukaryotic and prokaryotic host cells, including mammalian cells, as hosts for expressing the antibodies or fragments or immunoglobulin chains disclosed herein are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, but are not limited to, Chinese Hamster Ovary (CHO) cells, NS0 cells, SP2 cells, HeLa cells, newborn hamster kidney (BHK) cells, monkey kidney (COS) cells, human hepatocellular carcinoma cells (e.g. HepG2) , A549 cells, 3T3 cells, HEK-293 cells, and a variety of other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, porcine, goat, bovine, horse and hamster cells. Particularly preferred cell lines are selected by identifying cell lines with high levels of expression. Other cell lines that can be used include insect cell lines such as Sf9 cells, amphibian cells, bacterial cells, plant cells, and fungal cells. Fungal cells include yeast cells and cells of filamentous fungi, including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum Pichia pijperi Pichia stiptis Pichia methanolica Pichia sp. Saccharomyces cerevisiae Saccharomyces sp. Hansenula polymorpha Kluyveromyces sp. Kluyveromyces lactis Candida albicans , Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens and Neurospora crassa, Pichia sp., any Saccharomyces sp., Hansenula polymorpha, any Kluyveromyces sp., Candida albicans, any Aspergillus sp., Trichoderma reesei, Chrysosporium lucknowense, any Fusarium sp., Yarrowia lipolytica and Neurospora crassa. When recombinant expression vectors encoding a heavy chain or an antigen-binding portion or fragment thereof, a light chain and/or an antigen-binding portion thereof, are introduced into mammalian host cells, antibodies are produced by culturing the host cells for a period of time sufficient to express the antibody or fragment or chain in the host cells, or their secretion into the culture medium in which the host cells grow.

Antibodies and their antigen-binding fragments and immunoglobulin chains can be recovered from the culture medium using standard protein purification methods. In addition, the expression of antibodies and their antigen-binding fragments and immunoglobulin chains according to the invention (or other fragments thereof) in a producer cell line can be increased using a number of known methods. For example, the use of a glutamine synthetase gene expression system (GS system) is a common approach to increase expression under certain conditions. The GS system is described in whole or in part in European Patent Nos. 0216846, 0256055 and 0323997 and European Patent Application No. 89303964.4. Thus, in one embodiment of the invention, mammalian host cells (e.g., CHO) lack the glutamine synthetase gene and grow in the absence of glutamine in the medium, however, the polynucleotide encoding the immunoglobulin chain contains the glutamine synthetase gene, which makes up for the absence of the gene in the host cell.

The present invention includes methods for purifying an anti-hRSV antibody or antigen-binding fragment thereof of the present invention, comprising applying a sample containing the antibody or fragment to a purification medium (e.g., cation exchange purification medium, anion exchange purification medium, hydrophobic purification medium, affinity purification (eg, protein-A, protein-G, protein-A/G, protein-L)) and either collecting the purified antibody or fragment in a flow-through fraction of said sample that does not bind to the medium; or discarding the running fraction and eluting the bound antibody or fragment from the medium and collecting the eluate. In one of the embodiments of the invention, the environment is in the column, which is applied to the sample. In one embodiment, the purification method is applied after the antibody or fragment has been recombinantly expressed in a host cell, for example, the host cell is first lysed and optionally the lysate is purified of insoluble materials prior to media purification.

Typically, glycoproteins produced in a particular cell line or transgenic animal will have a glycosylation pattern characteristic of glycoproteins produced in the cell line or transgenic animal. Thus, the particular glycosylation pattern of an antibody will depend on the particular cell line or transgenic animal that is used to produce the antibody. However, all antibodies encoded by the nucleic acid molecules provided herein or containing the amino acid sequences provided herein are included in the present invention, regardless of the glycosylation pattern the antibodies may have. Similarly, in specific embodiments, antibodies with a glycosylation pattern including only non-fucosylated N-glycans may be preferred because such antibodies have been shown to generally be more potent than their fucosylated counterparts, both in vitro and in vivo. (See, for example, Shinkawa et al., J. Biol. Chem. 278:3466-3473 (2003); US Pat. Nos. 6,946,292 and 7,214,775). These non-fucosylated N-glycan antibodies are unlikely to be immunogenic because their carbohydrate structures are a common component of the population found in human serum IgG.

The present invention includes bispecific and bifunctional antibodies and antigen-binding fragments having binding specificity for the hRSV F protein and another antigen such as, for example, the hRSV G protein, as well as methods of using them. A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be generated in a variety of ways, including hybridoma fusion or Fab' binding. See, for example, Songsivilai, et al., (1990), Clin. Exp. Immunol. 79:315-321, Kostelny, et al., (1992), J Immunol. 148:1547-1553. In addition, bispecific antibodies can be prepared as diabodies (Holliger, et al., (1993), PNAS USA, 90:6444-6448) or as janusins (Traunecker, et al., (1991). EMBO J. 10 :3655-3659 and Traunecker, et al., (1992), Int. J. Cancer Suppl. 7:51-52).

The present invention also includes antigen-binding fragments to the F protein of hRSV anti-hRSV antibodies disclosed herein. Antibody fragments include F(ab) ₂ fragments, which can be obtained by enzymatic cleavage of IgG, for example, with pepsin. Fab fragments can be obtained, for example, by reduction of F(ab) ₂ dithiothreitol or mercaptoethylamine.

Immunoglobulins can be assigned to different classes depending on the amino acid sequences of the constant domain of their heavy chains. There are at least five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, and IgG4; IgA1 and IgA2. The invention includes antibodies and antigen-binding fragments of any of these classes or subclasses of antibodies.

In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain constant region, eg, a human constant region, such as a γ1, γ2, γ3, or γ4 human heavy chain constant region, or a variant thereof. In another embodiment, the antibody or antigen binding fragment comprises a light chain constant region, eg, a human light chain constant region, such as a human lambda or kappa light chain constant region, or a variant thereof. By way of example, and not limitation, the human heavy chain constant region may be γ4 and the human light chain constant region may be kappa. In an alternative embodiment, the Fc region of the antibody is γ4 with a Ser228Pro mutation (Schuurman, J. et. al., 2001, Mol. Immunol. 38:1-8).

In one embodiment, the antibody or antigen-binding fragment has a heavy chain constant region of the IgG1 subtype.

In some embodiments, different constant domains may be added to VL and VH regions formed with the CDR regions provided herein. For example, if a particular intended use of an antibody (or fragment) of the present invention requires alteration of effector functions, then a heavy chain constant domain other than human IgG1 may be used, or an IgG1/IgG4 hybrid may be used.

Although human IgG1 antibodies are distinguished by their long half-life and the presence of effector functions such as complement activation and antibody-dependent cellular cytotoxicity, such activities may be undesirable for some uses of the antibody. In such cases, for example, a human IgG4 constant domain can be used. The present invention includes anti-hRSV F-protein antibodies and antigen-binding fragments thereof that contain an IgG4 constant domain, such as antagonistic humanized hRSV F-protein antibodies and fragments, as well as methods of using the same. In one embodiment, the IgG4 constant domain may differ from the native human IgG4 constant domain (Swiss-Prot Accession No. P01861.1) at a position corresponding to EU position 228 and CAVAT position 241, where the natural residue Ser108 is replaced by Pro to prevent potential formation of an interchain disulfide bond between Cys106 and Cys109 (corresponding to positions Cys 226 and Cys 229 in the EU system and positions Cys 239 and Cys 242 in the Kabat system), which can prevent the formation of the correct interchain disulfide bond. See Angal et al. (1993), Mol. Imunol. 30:105. In other cases, a modified IgG1 constant domain that has been modified to increase half-life or reduce effector function may be used.

Antibody construction.

The antibodies of the invention can be subjected to mutations in the framework region to improve the properties of the antibodies. One such modification of the framework region includes the mutation of one or more residues in the framework region, or even one or more regions of the CDR to remove T-cell epitopes in order to reduce the potential immunogenicity of the antibody. This approach is also referred to as immunogenicity deprivation and is described in more detail in US Pat. No. 7,125,689.

In specific embodiments, it will be desirable to replace some of the amino acids containing exposed side chains with a different amino acid residue to provide greater chemical stability to the final antibody to avoid deamidation or isomerization. Deamidation of an asparagine residue can occur in the sequences NG, DG, NG, NS, NA, NT, QG or QS and lead to the creation of an isoaspartic acid residue, which introduces a bend in the polypeptide chain and reduces its stability (the effect of isoaspartic acid). Isomerization can occur in the sequences DG, DS, DA or DT. In specific embodiments, the antibodies of the present invention do not contain deamidation or aspartic isomerization sites.

For example, an asparagine (Asn) residue can be replaced with Gln or Ala to reduce the likelihood of isoaspartate formation in any Asn-Gly sequences, especially in CDRs. A similar problem may occur in the Asp-Gly sequence. Reissner and Aswad (2003), Cell. Mol. life sci. 60:1281. The formation of isoaspartate can lead to a weakening or complete disappearance of the binding of an antibody to its target antigen. See, Presta (2005), J. Allergy Clin. Immunol. 116:731-734. In one embodiment, the asparagine residue is replaced with a glutamine (Gln) residue. It may also be desirable to change the amino acid adjacent to an asparagine (Asn) or glutamine (Gln) residue to reduce the likelihood of deamidation, which occurs more frequently when small amino acids are adjacent to an asparagine or glutamine residue. See, Bischoff & Kolbe (1994), J. Chromatog. 662:261. In addition, any methionine residues (typically solvent-exposed Met residues) in the CDR regions can be replaced with Lys, Leu, Ala, or Phe or other amino acids to reduce the chance that the methionine sulfur will be oxidized, which can lead to a decrease in binding affinity. antigen and furthermore contribute to molecular heterogeneity in the final antibody preparation, id. In addition, to prevent or minimize potentially cleavable Asn-Pro peptide bonds, it may be desirable to change any Asn-Pro combinations found in CDRs to Gin-Pro, Ala-Pro, or Asn-Ala. Antibodies with these substitutions are subsequently screened to confirm that the substitutions did not reduce the affinity or specificity of the antibody for the hRSV F protein or other desired biological activity to unacceptable levels.

- 21 039222

table 2

Exemplary Stabilizing CDR Variants

Remaining CDR Stabilizing Variant Sequence Asn-Gly (N-G) Gln-Gly, Ala-Gly or Asn-Ala (Q-G), (A-G) or (N-A) Asp-Gly (D-G) Glu-Gly, Ala-Gly or Asp-Ala (E-G), (A-G) or (D-A) Met (usually exhibited for solvent) (M) Lys, Leu, Ala or Phe (K) , (L) , (A) or (F) Asn (N) Gln or Ala (Q) or (A) Asn-Pro (N-P) Gin-Pro, Ala-Pro or Asn-Ala (Q-P), (A-P) or (N-A)

Modification of the Fc region of an antibody.

The antibodies and antigen-binding fragments thereof disclosed herein (e.g., RB1) can also be genetically modified to introduce changes in the Fc region, typically to change one or more properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding and/or effector function (eg, antigen-dependent cellular cytotoxicity). In addition, antibodies and their antigen-binding fragments as disclosed herein (e.g., RB1) may be chemically modified (e.g., one or more chemical moieties may be attached to the antibody) or be modified to change the glycosylation pattern, again to changing one or more properties of an antibody or fragment. Each of such embodiments is described in more detail below. The numbering of residues in the Fc region corresponds to the Kabat EU index.

The antibodies and antigen-binding fragments thereof disclosed herein (eg, RB1) also include antibodies and fragments with Fc regions modified (or blocked) to alter effector functions. See, for example, US patent No. 5624821, WO 2003/086310, WO 2005/120571, WO 2006/0057702. Such modifications can be used to enhance or suppress various immune system responses, with possible diagnostic and therapeutic benefits. Fc region changes include amino acid changes (substitutions, deletions and insertions), glycosylation or deglycosylation, and the addition of multiple Fc regions. Changes in Fc can also lead to a change in the half-life of antibodies in the case of therapeutic antibodies, which allows to reduce the frequency of dosing and, thus, increase convenience and reduce the amount of material used. See Presta, 2005, J. Allergy Clin. Immunol. 116:731, 734-35.

In one embodiment of the invention, the CH1 hinge region is modified such that the number of cysteine residues in the hinge region is increased or decreased. This approach is described in detail in US Pat. No. 5,677,425. The number of cysteine residues in the CH1 hinge region is changed, for example, to facilitate assembly of the light and heavy chains, or to increase or decrease the stability of the antibody.

In another embodiment, an antibody or antigen-binding fragment of the invention (eg, RB1) is modified to increase its biological half-life. Various approaches are possible. For example, one of the following mutations can be introduced: T252L, T254S, T256F, as described in US Pat. No. 6,277,375. CH2 loops of the IgG Fc region domain as described in US Pat. See, for example, Oganesyan et al., Mol. Immunol. 2009, 46:1750.

In other embodiments, the Fc region is altered by replacing at least one amino acid residue with another amino acid residue to alter the effector function(s) of the antibody or antigen-binding fragment. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, and 322 can be replaced with a different amino acid residue such that the affinity of the antibody for the effector ligand is altered and the antigen-binding ability of the parent antibody is maintained. The effector ligand for which the affinity is changed may be, for example, the Fc receptor or the C1 component of complement. This approach is described in more detail in US Pat. Nos. 5,624,821 and 5,648,260.

- 22 039222

In another example, one or more amino acids selected from amino acid residues 329, 331 and

322 can be replaced with a different amino acid residue such that the binding of the antibody to C1q is altered and/or complement dependent cytotoxicity (CDC) is reduced or eliminated. This approach is described in more detail in US patent No. 6194551.

In another example, one or more amino acid residues at amino acid positions 231 and 239 are changed to thereby alter the ability of the antibody to fix complement. This approach is described in more detail in International Patent Application Publication No. WO 94/29351.

In another example, the Fc region is modified to reduce the ability of an antibody or antigen-binding fragment of the invention (e.g., RB1) to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or to reduce the affinity of the antibody or fragment for the Fcγ receptor by changing one or more of the following amino acids: provisions: 238, 239, 243, 248, 249, 252, 254, 255, 256, 258, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290 292 293 294 295 296 298 301 303 305 307 309 312 315 320 322 324 326 327 329 330 331 333 334 335 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, or 439. This approach is described in more detail in the international patent application publication No. WO 00/42072. In addition, binding sites on human IgG1 for FcyR1, FcyRII, FcyRIII, and FcRn have been mapped and stronger binding variants have been described (see Shields et al. (2001), J. Biol. Chem. 276:6591-6604 ).

In one embodiment, the Fc region is modified to reduce the ability of an antibody of the invention (e.g., RB1) to mediate effector function and/or to enhance anti-inflammatory properties by modifying residues 243 and 264. In one embodiment, the Fc region of an antibody or fragment is modified by changes in residues at positions 243 and 264 to alanine. In one embodiment, the Fc region is modified to reduce the ability of the antibody or fragment to mediate effector function and/or to enhance anti-inflammatory properties by modifying residues 243, 264, 267, and 328.

Strengthening the effector function.

In some embodiments, the Fc region of an anti-hRSV antibody is modified to increase the ability of the antibody or antigen-binding fragment to mediate effector function and/or to enhance its binding to Fcy receptors (FcyRs).

As used herein, the term effector function refers to one or more of antibody-dependent cell-mediated cytotoxic activity (ADCC), complement-mediated cytotoxic activity (CDC), Fc-mediated phagocytosis or antibody-dependent cellular phagocytosis (ADCP), as well as antibody recycling through FcRn -receptor.

Interaction between the antigen-binding protein constant region and various Fc receptors (FcRs), including FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD16), is believed to mediate effector functions such as ADCC and CDC of the antigen-binding protein. The Fc receptor is also important for antibody crosslinking, which may be important for antitumor immunity.

Effector function can be measured in a variety of ways including, for example, FcyRIII binding to natural killer cells or FcyRI binding to monocytes/macrophages to measure ADCC effector function. For example, an antigen binding protein of the present invention can be evaluated for ADCC effector function in a natural killer cell assay. Examples of such assays can be found in Shields et al., 2001, J. Biol. Chem., Vol. 276, p. 6591-6604; Chappel et al., 1993, J. Biol. Chem. 268:25124-25131; Lazar et al., 2006, Proc Natl Acad Sci USA 103:4005-4010.

The ADCC or CDC properties of the antibodies of the present invention or their cross-linking ability can be enhanced in various ways.

It has been shown that human IgG1 constant regions with certain mutations or altered glycosylation at the Asn297 residue are more strongly bound to Fc receptors. It has also been shown that in some cases these mutations lead to an increase in ADCC and CDC (Lazar et al., Proc. Natl. Acad. Sci. USA, 2006, 103:4005-4010; Shields et al., J. Biol. Chem. 2001, 276:6591-6604; Nechansky et al., Mol. Immunol. 2007, 44:1815-1817).

In one embodiment of the present invention, such mutations occur at one or more of positions selected from 239, 332, and 330 (IgG1), or equivalent positions in IgGs of other isotypes. Examples of relevant mutations are S239D, I332E and A330L. In one embodiment, the antigen binding protein of the invention described herein has mutations at positions 239 and 332, such as S239D and I332E, or in the following embodiment, has mutations at three or more positions selected from 239, 332, and 330, such as S239D , I332E and A330L (EU numbering).

In an alternative embodiment, the present invention provides an antibody comprising a heavy chain constant region with an altered glycosylation profile such that the antigen binding protein has enhanced effector function. For example, when an antibody

- 23 039222 has enhanced ADCC or enhanced CDC, or it has both enhanced effector functions, both ADCC and CDC. Examples of suitable methodologies for obtaining antigen-binding proteins with an altered glycosylation profile are described in International Patent Application Publication Nos. WO 2003011878 and WO 2006014679 and European Patent Application No. EP 1229125.

In a further aspect, the present invention relates to non-fucosylated or afucosylated antibodies. Non-fucosylated antibodies contain in the Fc region the structure of the trimannose core of N-glycans of a complex type without fucose residues. Such glycosylation pattern-modified antibodies lacking a fucose residue in the core of the Fc region N-glycans may exhibit stronger ADCC than fucosylated counterparts due to increased FcyRIIIa binding capacity.

The present invention also relates to a method for producing an antibody of the invention, comprising the steps of a) culturing a recombinant host cell containing an expression vector containing an isolated nucleic acid described herein, while the recombinant host cell does not contain alpha-1,6-fucosyltransferase ; and b) recovering the antigen binding protein. The recombinant host cell may not originally contain a gene encoding alpha 1,6 fucosyl transferase (eg, yeast host cells such as Pichia sp.), or may be genetically modified to inactivate the alpha 1,6 fucosyl transferase gene. Recombinant host cells genetically modified to inactivate the FUT8 gene encoding alpha-1,6 fucosyltransferase are available. See, for example, the POTELLIGENT™ process system available from BioWa, Inc. (Princeton, N.J.), in which CHOK1SV cells lacking a functional copy of the FUT8 gene produce monoclonal antibodies having increased antibody-dependent cell-mediated cytotoxic (ADCC) activity that is increased relative to that of an identical monoclonal antibody produced in a cell with a functional FUT8 gene. Aspects of the POTELLIGENT™ process system are described in US Pat. Nos. 7,214,775; 6,946,292; Other suitable systems are known to those skilled in the art.

Those skilled in the art will appreciate that such modifications may not only be used alone, but may be used in combination with any other modifications to further enhance effector function.

Obtaining antibodies with modified glycosylation.

In another embodiment, the antibodies or antigen-binding fragments of the invention (eg, RB1) have a particular glycosylation pattern. For example, an afucosylated or aglycosylated antibody or fragment (ie, an antibody devoid of fucose or glycosylation, respectively) can be produced. The glycosylation pattern of an antibody or fragment can be altered, for example, to increase the affinity or avidity of the antibody or fragment for the hRSV F protein antigen. Such modifications can be made, for example, by changing one or more glycosylation sites in the sequence of the antibody or fragment. For example, one or more amino acid substitutions can be made that result in the removal of one or more variable region framework glycosylation sites to eliminate glycosylation at that site. Such aglycosylation may result in an increase in the affinity or avidity of the antibody or fragment for the antigen. See, for example, US Pat. Nos. 5,714,350 and 6,350,861.

Antibodies and antigen-binding fragments disclosed herein (e.g., RB1) may also include those generated in lower eukaryotic host cells, in particular fungal host cells such as yeast and filamentous fungi, that have been genetically modified to produce glycoproteins having glycosylation patterns characteristic of mammals or humans (see, for example, Choi et al., (2003), Proc. Natl. Acad. Sci. USA, 100:5022-5027; Hamilton et al., (2003), Science, 301:1244-1246; Hamilton et al., (2006), Science, 313:1441-1443; Nett et al., (2011), Yeast 28(3):237-52; Hamilton et al., (2007) , Curr Opin Biotechnol Oct 18(5):387-92). These genetically modified host cells have the ability to control the glycosylation profile of the glycoproteins that are produced in the cells, resulting in glycoprotein compositions dominated by a particular N-glycan structure (see, for example, US Pat. Nos. 7,029,872 and 7,449,308). Such genetically modified host cells have been used to produce antibodies that are dominated by specific N-glycan structures (see, for example, Li et al., (2006), Nat. Biotechnol. 24:210-215).

In specific embodiments, the antibodies and antigen-binding fragments thereof disclosed herein (e.g., RB1) further include those derived from lower eukaryotic host cells and which contain fucosylated and non-fucosylated hybrid and complex N-glycans, including bisected and polyantennary species including, but not limited to, N-glycans such as GlcNAc(1.4)Man ₃ GlcNAc2; Gal( ₁ .4)GlcNAc( _{1 .4} )Man ₃ GlcNAc ₂ ; NANA(1.4)Gal(1.4)GlcNAc(1.4)Man3GlcNAc ₂ .

In specific embodiments, the antibodies and antigen-binding fragments provided herein (e.g., RB1) may include antibodies or fragments having

- 24 039222 at least one hybrid N-glycan selected from the group consisting of GlcNAcMan ₅ GlcNAc ₂ ;

GalGlcNAcMan ₅ GlcNAc ₂ and NANAGalGlcNAcMan ₅ GlcNAc ₂ . In specific aspects, the hybrid N-glycan is the predominant type of N-glycans in the composition.

In specific embodiments, the antibodies and antigen-binding fragments provided herein (eg, RB1) include antibodies and fragments having at least one complex N-glycan selected from the group consisting of GlcNAcMan ₃ GlcNAc ₂ ; GalGlcNAcMan ₃ GlcNAc ₂ ; NANAGalGlcNAcMan ₃ GlcNAc2; GlcNAc ₂ Man ₃ GlcNAc ₂ ; GalGlcNAc ₂ Man ₃ GlcNAc ₂ ; Gal ₂ GlcNAc ₂ Man ₃ GlcNAc ₂ ; NANAGal ₂ GlcNAc ₂ Man ₃ GlcNAc ₂ and NANA ₂ Gal ₂ GlcNAc ₂ Man ₃ GlcNAc ₂ . In specific aspects, the complex N-glycan is the predominant type of N-glycans in the composition. In the following aspects, a complex N-glycan is a specific type of N-glycan that is about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% of the complex N-glycans in the composition. In one embodiment, the antibodies and antigen-binding fragments provided herein comprise complex N-glycans, wherein at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% complex N-glycans contain the structure NANA ₂ Gal ₂ GlcNAc ₂ Man ₃ GlcNAc ₂ and this structure is afucosylated. Such structures can be obtained, for example, in recombinant Pichia pastoris host cells.

In specific embodiments, the N-glycan is fucosylated. As a rule, fucose is in α1,3-bond with GlcNAc at the reducing end of the N-glycan, α1,6-bond with GlcNAc at the reducing end of the N-glycan, α1,2-bond with Gal at the non-reducing end of the N-glycan, a1, 3-links to GlcNac at the non-reducing end of the N-glycan or α1,4-links to GlcNAc at the non-reducing end of the N-glycan.

Thus, in specific aspects of the above glycoprotein compositions, the glycoform comprises α1,3-linked or α1,6-linked fucose to form a glycoform selected from the group consisting of

Man ₅ GlcNAc ₂ (Fuc), GlcNAcMan ₅ GlcNAc ₂ (Fuc), Man ₃ GlcNAc ₂ (Fuc), GlcNAcMan ₃ GlcNAc ₂ (Fuc), GlcNAc2Man3GlcNAc2(Fuc), GalGlcNAc2Man3GlcNAc2(Fuc), Gal2GlcNAc2Man3GlcNAc2(Fuc), NANAGal2GlcNAc2Man3Glc ) and NANA2Gal2GlcNAc2Man3GlcNAc2(Fuc);

a1,3-linked or a1,4-linked fucose to form a glycoform selected from the group consisting of

GlcNAc(Fuc)Man ₅ GlcNAc ₂ , GlcNAc(Fuc)Man ₃ GlcNAc ₂ , GlcNAc ₂ (Fuc1_ ₂ )Man ₃ GlcNAc ₂ ,

GalGlcNAc ₂ (Fuc1_ ₂ ) Man ₃ GlcNAc ₂ , Gal ₂ GlcNAc ₂ (Fuc1_ ₂ )Man ₃ GlcNAc ₂ ,

NANAGal ₂ GlcNAc ₂ (Fuc1_ ₂ )Man ₃ GlcNAc ₂ and NANA ₂ Gal ₂ GlcNAc ₂ (Fuc1_ ₂ )Man ₃ GlcNAc ₂ ; or α1,2-linked fucose to form a glycoform selected from the group consisting of Gal(Fuc)GlcNAc ₂ Man ₃ GlcNAc ₂ , Gal ₂ (Fuc1_ ₂ )GlcNAc ₂ Man ₃ GlcNAc ₂ ,

NANAGal ₂ (Fuc1_ ₂ )GlcNAc ₂ Man ₃ GlcNAc ₂ and NANA ₂ Gal ₂ (Fuc ₁ _ ₂ )GlcNAc ₂ Man ₃ GlcNAc ₂ .

In the following aspects, the antibodies or antigen-binding fragments thereof contain high-mannose N-glycans, including, but not limited to, Man ₈ GlcNAc ₂ , Man ₇ GlcNAc ₂ , Man6GlcNAc ₂ , Man ₅ GlcNAc ₂ , Man4GlcNAc ₂ , or N-glycans consisting of N- glycan structure Man ₃ GlcNAc ₂ .

In further aspects, the complex N-glycans described above further include fucosylated and non-fucosylated bisected and polyantennary varieties.

As used herein, the terms N-glycan and glycoform are used interchangeably and refer to an N-linked oligosaccharide, for example, attached via an asparagine-N-acetylglucosamine bond to an asparagine residue of a polypeptide. N-linked glycoproteins contain an N-acetylglucosamine residue linked to the nitrogen atom of the amide group of an asparagine residue in the protein. The predominant sugars on glycoproteins are glucose, galactose, mannose, fucose, N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc) and sialic acid (eg N-acetylneuraminic acid (NANA)). Processing of sugar groups occurs co-translationally in the ER lumen and continues post-translationally in the Golgi apparatus in the case of N-linked glycoproteins.

N-glycans share a pentasaccharide core of Man ₃ GlcNAc ₂ (Man means mannose; Glc means glucose and NAc means N-acetyl; GlcNAc means N-acetylglucosamine). Typically, N-glycan structures are presented with a non-reducing end on the left and a reducing end on the right. The reducing end of an N-glycan is the end that is attached to an Asn residue included in a glycosylation site on a protein. N-glycans differ in the number of branches (antennae) containing peripheral sugars (e.g., GlcNAc, galactose, fucose, and sialic acid) that complete the core structure from Man ₃ GlcNAc ₂ (Man3), also called the trimannose core, pentasaccharide core, or paucimannous nucleus. N-glycans are classified according to their branched components (eg, high mannose, complex or hybrid). The high-mannose type N-glycan has five or more mannose residues. Complex type N-glycan typically has at least one GlcNAc attached to the 1,3-mannose arm and at least

- 25 039222 one GlcNAc attached to the 1,6-mannose arm of the tri-mannose core. Complex N-glycans may also contain galactose (Gal) or N-acetylgalactosamine (GalNAc) residues, which are optionally modified with sialic acid or derivatives (eg NANA or NeuAc, where Neu is neuraminic acid and Ac is acetyl). Complex N-glycans can also have intrachain substitutions, including bisected GlcNAc and nuclear fucose (Fuc). Complex N-glycans can also have multiple antennae on the trimannose core and are often referred to as polyantennary glycans. The hybrid N-glycan contains at least one GlcNAc at the end of the 1,3-mannose arm of the tri-mannose core and zero or more mannose residues on the 1,6-mannose arm of the tri-mannose core. Various N-glycans are also called glycoforms.

When applied to complex N-glycans, the terms G-2, G-1, G0, G1, G2, A1 and A2 mean the following:

the term G-2 means an N-glycan structure, which can be characterized as Man ₃ GlcNAc ₂ ;

the term G-1 means an N-glycan structure, which can be characterized as GlcN AcMan ₃ GlcNAc ₂ ;

the term G0 means an N-glycan structure, which can be characterized as GlcNAc ₂ Man ₃ GlcNAc ₂ ;

the term G1 means an N-glycan structure, which can be characterized as GalGlcNAc ₂ Man ₃ G 1 cNAc ₂ ;

the term G2 means an N-glycan structure, which can be characterized as Gal ₂ GlcNAc ₂ Man ₃ GlcNAc ₂ ;

the term A1 means N-glycan structure, which can be characterized as NANAGal ₂ GlcNAc ₂ Man ₃ GlcNAc ₂ ; and the term A2 means an N-glycan structure, which can be characterized as NANA ₂ Gal ₂ GlcNAc ₂ Man ₃ GlcNAc ₂ .

Unless otherwise indicated, the terms G-2, G-1, G0, G1, G2, A1 and A2 mean N-glycan species that lack fucose residues attached to the GlcNAc residue at the reducing end of the N-glycan. If the term includes F, then F indicates that the N-glycan species contains a fucose residue on the GlcNAc residue at the reducing end of the N-glycan. For example, the designations G0F, G1F, G2F, A1F, and A2F all indicate that the N-glycan additionally contains a fucose residue attached to a GlcNAc residue at the reducing end of the N-glycan. Lower eukaryotes, such as yeasts and filamentous fungi, do not normally produce fucose-containing N-glycans.

In the case of polyantennary N-glycans, the term polyantennary N-glycan means N-glycans that additionally contain a GlcNAc residue on a mannose residue included in the non-reducing end of the 1,6-arm or 1,3-arm of the N-glycan, or a GlcNAc residue on each of mannose residues included in the non-reducing end of the 1,6-arm or 1,3-arm of the N-glycan. Thus, polyantennary N-glycans can have the formulas GlcNAc( ₂ .4)Man ₃ GlcNAc ₂ , Gal( _{1 .4} )GlcNAc( _{2 .4} )Man ₃ GlcNAc ₂ or NANA( _{1 .4} )Gal( _{1 .4} ) GlcNAC( ₂ .4)Man ₃ GlcNAc ₂ . The term 1-4 means 1, 2, 3 or 4 residues.

In the case of bisected N-glycans, the term bisected N-glycan refers to N-glycans in which a GlcNAc residue is linked to a mannose residue at the reducing end of the N-glycan. A bisected N-glycan may have the formula GlcNAc ₃ Man ₃ GlcNAc ₂ where each mannose residue is linked at the non-reducing end to a GlcNAc residue. In contrast, if a polyantennary N-glycan has the formula GlcNAc3Man3GlcNAc2, the formula indicates that two GlcNAc residues are linked to a mannose residue at the non-reducing end of one of the two N-glycan arms and one GlcNAc residue is linked to a mannose residue at the non-reducing end of the other arm of the N-glycan .

Physical properties of antibodies.

The antibodies and antigen-binding fragments thereof disclosed herein (eg, RB1) may further comprise one or more glycosylation sites in the variable region of either the immunoglobulin light or heavy chain. Certain glycosylation sites can result in a decrease in the immunogenicity of an antibody or fragment, or a change in the pK of an antibody due to altered antigen binding (Marshall et al., 1972, Annu Rev. Biochem. 41:673-702; Gala and Morrison, 2004, J. Immunol. 172: 5489-94; Wallick et al., 1988, J. Exp. Med. 168:1099-109; Spiro, 2002, Glycobiology, 12:43R-56R; Parekh et al., 1985, Nature, 316:452-7; Mimura et al., 2000, Mol. Immunol. 37:697-706). Glycosylation is known to occur on fragments containing the N-X-S/T sequence.

Each antibody or antigen-binding fragment (eg, RB1) will have a unique isoelectric point (pI) that typically falls within the pH range of 6 to 9.5. The pI value for an IgG1 antibody typically falls in the pH range of 7-9.5, and the pI value for an IgG4 antibody typically falls in the pH range of 6-8.

Each antibody or antigen-binding fragment (eg, RB1) will have a characteristic melting point, with a higher melting point indicating greater overall in vivo stability (Krishnamurthy R. and Manning MC (2002), Curr Pharm. Biotechnol. 3:361- 71). As a rule, T _M1 (initial unfolding temperature) can be more than 60°C, more

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65°C or more than 70°C. The melting point of an antibody or fragment can be measured by differential scanning calorimetry (Chen et al., (2003), Pharm. Res. 20:1952-60; Ghirlando et al., (1999), Immunol. Lett 68:47-52) or circular dichroism (Murray et al., (2002), J. Chromatogr. Sci.

40:343-9).

In a further embodiment, antibodies and their antigen-binding fragments (eg, RB1) are selected that do not rapidly degrade. The degradation of an antibody or fragment can be measured by capillary electrophoresis (CE) and MALDI-MS (Alexander A.J. and Hughes D.E. (1995), Anal. Chem. 67:3626-32).

In a further embodiment, antibodies (eg, RB1) and antigen-binding fragments thereof are selected to have minimal aggregation effects that may lead to the initiation of an undesirable immune response and/or altered or adverse pharmacokinetic properties. Generally, antibodies and fragments with aggregation of 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less are acceptable. Aggregation can be determined by several methods, including size exclusion chromatography (SEC) on a column, high performance liquid chromatography (HPLC) and light scattering.

Antibody conjugates.

Antibodies and their antigen-binding fragments to the hRSV F protein disclosed herein (eg, RB1) can also be conjugated to a chemical moiety. The chemical fragment may be, in particular, a polymer, a radioactive isotope or a cytotoxic factor. In specific embodiments, the implementation of the chemical fragment is a polymer that increases the half-life of the antibody or fragment in the patient's body. Suitable polymers include, but are not limited to, hydrophilic polymers, which include, but are not limited to, polyethylene glycol (PEG) (e.g., 2, 5, 10, 12, 20, 30, or 40 kDa PEG), dextran, and monomethoxypolyethylene glycol (mPEG ). Lee, et al., (1999), (Bioconj. Chem. 10:973-981) describes PEG-conjugated single chain antibodies. Wen, et al., (2001), (Bioconj. Chem. 12:545-553) describes the conjugation of antibodies to PEG, which is coupled to a radioactive metal chelator (diethylenetriaminepentaacetic acid (DTPA)).

The antibodies and antigen-binding fragments thereof disclosed herein (e.g., RB1) can also be conjugated with labels such as ⁹⁹ Tc, ⁹⁰ Y, ¹ⁿ In, ³² P, ¹⁴ C, ¹²⁵ I, ³ H, ¹³¹ I, ⁿ C, ¹⁵ O, ¹³ N, ¹⁸ F, ³⁵ S, ⁵¹ Cr, ⁵⁷ To, ²²⁶ Ra, ⁶⁰ Co, ⁵⁹ Fe, ⁵⁷ Se, ¹⁵² Eu, ⁶⁷ Cu, ²¹⁷ Ci, ²¹¹ At, ²¹² Pb, ⁴⁷ Sc, ¹⁰⁹ Pd, ²³⁴ Th and ⁴⁰ K, ¹⁵⁷ Gd, ⁵⁵ Mn, ⁵² Tr and ⁵⁶ Fe.

Antibodies and antigen-binding fragments disclosed herein (eg, RB1) can also be pegylated, eg, to increase their biological half-life (eg, in serum). To pegylate an antibody or fragment, the antibody or fragment is typically reacted with a reactive form of polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG moieties are attached to the antibody or antibody fragment. In particular embodiments, the PEGylation is carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or similar reactive water-soluble polymer). As used herein, the term polyethylene glycol encompasses any form of PEG that is used to derivatize other proteins, such as mono(C ₁ -C ₁₀ )alkoxy or aryloxy polyethylene glycol or polyethylene glycol-maleimide. In specific embodiments, the antibody or fragment that is pegylated is an aglycosylated antibody or fragment. Methods for PEGylating proteins are known in the art and can be applied to the antibodies of the invention. See, for example, European Patent Applications EP 0154316 and EP 0401384.

The antibodies and antigen-binding fragments disclosed herein (e.g., RB1) may also be conjugated to fluorescent or chemiluminescent labels, including fluorophores, e.g. rare earth chelates, fluorescein and its derivatives, rhodamine and its derivatives, isothiocyanate, phycoerythrin, phycocyanin, allophycocyanin , o-phthalaldehyde, fluorescamine, ¹⁵² Eu, dansyl, umbelliferone, luciferin, luminal labels, isoluminal labels, acridinium aromatic ester labels, imidazole labels, acridinium salt labels, oxalate ester labels, aequorin labels, 2,3 -dihydrophthalazinediones, biotin/avidin, spin labels and stable free radicals.

Antibodies and their antigen-binding fragments of the invention (for example, RB1) can also be conjugated to a cytotoxic factor such as diphtheria toxin, Pseudomonas aeruginosa exotoxin A chain, ricin A chain, abrin A chain, modecin A chain, alpha sarcin , proteins and compounds (e.g. fatty acids) Aleurites fordii, dianthine proteins, PAPI, PAPII and PAP-S proteins Phytoiacca americana, inhibitor from Momordica charantia, curcin, crotin, inhibitor from Saponaria officinalis, mitogellin, restrictocin, fenomycin and enomycin.

Any method known in the art can be used to conjugate antibodies and antigen-binding fragments of the invention (eg, RB1) to various fragments, including methods described in Hunter et al., 1962, Nature, 144:945; David et al., 1974, Biochemistry,

- 27 039222

13:1014; Pain et al., 1981, J. Immunol. Meth. 40:219 and Nygren, 1982, Histochem. and Cytochem. 30:407. Methods for conjugating antibodies and fragments are common and very well known in the art.

Prophylactic and therapeutic use of anti-hRSV antibodies.

Also provided are methods for preventing, treating, or ameliorating a symptom of RSV infection in patients, including human patients in need of such prevention, treatment, or amelioration, using the isolated antibodies or antigen-binding fragments thereof disclosed herein (e.g., RB1). In one embodiment of the invention, such a patient is suffering from an RSV infection. In one embodiment of the invention, such a patient is at risk of RSV infection.

In a specific embodiment, a prophylactic, therapeutic, or pharmaceutical composition comprising one or more antibodies or fragments thereof of the present invention is administered to a mammal, preferably a human, to treat, prevent, or ameliorate one or more symptoms associated with RSV infection, in an amount effective to reduce titers of RSV. According to such an embodiment, an effective amount of antibodies or antibody fragments results in a decrease in RSV titers in the lungs, as determined, for example, by the concentration of RSV in sputum samples or swabs from the lungs of a mammal. In another embodiment, a mammal, preferably a human, is administered a prophylactic, therapeutic, or pharmaceutical composition comprising one or more antibodies or fragments thereof of the present invention for the treatment, prevention, or amelioration of symptoms associated with RSV infection, in an amount effective to neutralize RSV and/ or to block RSV infection in a mammal.

Monoclonal antibodies, or antigen-binding fragments thereof, can also be used as immunotherapy for RSV-induced diseases in both humans and other animals. The terms immunotherapeutically or immunotherapy, as used herein in connection with the monoclonal antibodies or antigen-binding fragments thereof of the invention, mean prophylactic and therapeutic administration, passive immunization with substantially purified polypeptide products, and gene therapy by transfer of polynucleotide sequences encoding the product or part thereof. . Passive immunization involves the transfer of active humoral immunity or the administration of antibodies to a patient in need thereof. Accordingly, in specific embodiments, the present invention relates to methods for transferring active humoral immunity and methods for administering anti-RSV antibodies, or antigen-binding fragments thereof, such as IgG antibodies, to a patient at risk for RSV infection. Thus, monoclonal antibodies, or antigen-binding fragments thereof, can be administered to high-risk patients to reduce the likelihood and/or severity of RSV-induced disease, or administered to patients already showing signs of active RSV infection.

The present invention also relates to a method for modulating or treating at least one RSV-associated disease in adults or children, in a cell, tissue, organ, animal or patient, including, but not limited to, lower respiratory tract infections, pneumonia, tracheobronchitis, bronchiolitis , bronchitis, as well as any associated infections or inflammatory diseases, for example, but without limitation, at least one of the diseases or at least one inflammation associated with diseases such as systemic inflammatory response syndrome, septic syndrome, gram-positive sepsis, gram-negative sepsis , culture-negative sepsis, fungal sepsis, neutropenic fever, urosepsis, meningococcemia, adult respiratory distress syndrome, allergic rhinitis, perennial rhinitis, asthma, systemic anaphylaxis, receptor hypersensitivity reactions, chronic obstructive pulmonary disease (COPD), hypersensitivity pneumonia, granulomas due to inside ricellular microorganisms, drug sensitivity, cachexia, cystic fibrosis, neonatal chronic lung disease; at least one infectious disease in a cell, tissue, organ, animal, or patient, including, but not limited to, at least one of acute or chronic bacterial infection, acute and chronic parasitic or infectious processes, including bacterial, viral, and fungal infections , HIV infection, HIV neuropathy, meningitis, hepatitis (A, B or C or the like), septic arthritis, peritonitis, pneumonia, epiglottitis, E. coli 0157:h7, hemolytic uremic syndrome, thrombolytic thrombocytopenic purpura, malaria, dengue hemorrhagic fever, leishmaniasis, leprosy, toxic shock syndrome, streptococcal myositis, gas gangrene, mycobacterial tuberculosis, MAI infection, pneumocystis pneumonia, pelvic inflammatory disease, orchitis, epididymitis, legionella pneumonia, lyme disease, influenza A, Epstein-Barr virus , life-threatening hemophagocytic syndrome, life-threatening encephalitis, aseptic meningitis, and the like bnogo. Such a method may optionally include administering an effective amount of a composition or pharmaceutical composition comprising at least one anti-RSV antibody or antigen-binding fragment thereof to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy.

- 28 039222

In one embodiment, prophylactic, therapeutic, or pharmaceutical compositions comprising antibodies or fragments thereof of the invention are administered to a mammal, preferably a human, for the treatment, prevention, or amelioration of one or more symptoms associated with an RSV infection. In another embodiment, prophylactic, therapeutic or pharmaceutical compositions containing antibodies or fragments thereof according to the invention are administered to a person suffering from cystic fibrosis, bronchopulmonary dysplasia, congenital heart disease, congenital immunodeficiency or acquired immunodeficiency, or a person who has received a transplant (for example, after transplantation bone marrow, lung, or hematopoietic stem cell transplantation (HSCT)), to treat, prevent, or relieve one or more of the symptoms associated with RSV infection.

In another embodiment, prophylactic, therapeutic, or pharmaceutical compositions comprising antibodies or fragments thereof of the invention are administered to a child, preferably a premature infant or a child at risk of hospitalization due to RSV infection, to treat, prevent, or ameliorate one or more symptoms associated with RSV infection. . In another embodiment, prophylactic, therapeutic or pharmaceutical compositions containing antibodies or fragments thereof according to the invention are administered to elderly people or people in common residences (for example, nursing homes or rehabilitation centers), or people with immune deficiency.

It is preferred to use high affinity and/or effective in vivo inhibitory antibodies and/or neutralizing antibodies or antigen-binding fragments thereof that specifically bind to the RSV antigen, both to prevent RSV infection and to treat RSV infection. It is also preferred to use polynucleotides encoding high affinity and/or effective in vivo inhibitory antibodies and/or neutralizing antibodies or antigen-binding fragments thereof that specifically bind to the RSV antigen, both to prevent RSV infection and to treat RSV infection. Such antibodies or fragments thereof will preferably have an affinity for F-glycoprotein and/or F-glycoprotein fragments of RSV.

The antibodies and functional equivalents (such as their antigen-binding fragments) of the present invention recognize an epitope within the RSV F protein. Antibodies or their functional equivalents that specifically recognize said epitope can be combined with already known RSV-specific antibodies that bind to another epitope, such as palivizumab, D25, AM14, AM16 and AM23. When an antibody or functional equivalent of the invention specifically recognizing said epitope is combined with a known RSV-specific antibody, two or more different RSV epitopes are recognized during the same therapy. In this case, a stronger immune response to RSV and/or higher antibody specificity for RSV can be achieved. Due to a stronger immune response and higher specificity for RSV, this combination may lead to more effective treatment and/or prevention of RSV-associated disease.

One or more antibodies of the present invention, or fragments thereof that specifically bind to one or more RSV antigens, can be administered locally or systemically to the body as a prophylaxis. The antibodies of the invention, or fragments thereof, may also be advantageously used in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as, for example, IL-2, IL-3, IL-7, and IL-15), which , for example, serve to increase the number or activity of effector cells that interact with antibodies. The antibodies of the invention, or fragments thereof, can also be advantageously used in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as, for example, IL-2, IL-3, and IL-7), which, for example, serve to enhance the immune response. The antibodies of the invention, or fragments thereof, can also be advantageously used in combination with one or more drugs used to treat RSV infection, such as, for example, antiviral agents.

Antibodies of the invention or fragments can be used in combination with one or more of the following drugs: NIH-351 (Gemini Technologies), recombinant RSV vaccine (Aviron), PCBf-2 (Intracel), F-50042 (Pierre Fabre), T -786 (Trimeris), VP-36676 (ViroPharma), RFI-641 (American Home Products), VP-14637 (ViroPharma), PFP-1 and PFP-2 (American Home Products), RSV vaccine (Avant Immunotherapeutics), and F-50077 (Pierre Fabre).

The antibodies or antigen-binding fragments thereof of the invention may be administered alone or in combination with other therapies (eg hormone therapy, immunotherapy and anti-inflammatory therapy). In general, it is preferred to administer products derived from the same species or reactive (in the case of antibodies) with the same species as that of the patient. Thus, in a preferred embodiment, human or humanized antibodies, fragments, derivatives, analogs, or nucleic acids are administered to a human patient for therapy or prophylaxis.

The patient may be a mammal such as a human, dog, cat, horse, cow, mouse,

- 29 039222 rat, monkey (eg cynomolgus macaque, eg Masaca fascicularis) or rabbit. In preferred embodiments, the patient is a human.

In specific embodiments, the antibodies or antigen-binding fragments thereof disclosed herein (eg, RB1) may be used alone or in combination with antiviral therapy.

In specific embodiments, the antibodies or antigen-binding fragments thereof disclosed herein (eg, RB1) may be used alone or in combination with other anti-RSV monoclonal antibodies.

In specific embodiments, the antibodies or antigen-binding fragments thereof disclosed herein (eg, RB1) may be used alone or in combination with another anti-RSV vaccine.

The expression in combination with indicates that the components administered in the method of the present invention (e.g., an anti-hRSV antibody or antigen-binding fragment (e.g., RB1) together with, for example, palivizumab) can be formulated in one composition for simultaneous delivery or formulated separately in two or more compositions (eg, in a kit). Each component can be administered to the patient at a different time than the time of administration of the other component; for example, each administration may be performed non-simultaneously (eg, separately or sequentially) at some intervals over a specific period of time. In addition, the individual components may be administered to the patient by the same or different routes of administration.

Experimental and diagnostic applications.

Anti-hRSV F-protein antibodies and antigen-binding fragments thereof disclosed herein (eg, RB1) can be used as affinity purification agents. In this method, hRSV F protein antibodies and antigen-binding fragments thereof are immobilized on a solid phase such as Sephadex®, glass, agarose resin or filter paper by methods well known in the art. The immobilized antibody or fragment is contacted with a sample containing the hRSV F protein (or fragment) to be purified, then the substrate is washed with an appropriate solvent that will remove substantially all of the material in the sample except for the hRSV F protein bound to the immobilized antibody or fragment. Finally, the support is washed with a solvent that elutes the bound hRSV F-protein (eg, Protein A). Such immobilized antibodies and fragments are part of the present invention.

Antigens are also provided for the production of secondary antibodies useful, for example, for Western blot and other immunoassays described herein. In particular, disclosed are polypeptides containing sequences of the variable regions and/or CDR regions of a therapeutic antibody described herein (e.g., RB1) that can be used to generate anti-idiotypic antibodies used to specifically detect the presence of an antibody, for example, in a therapeutic context.

Anti-hRSV F-protein antibodies and antigen-binding fragments thereof may also be useful in diagnostic assays for hRSV F-protein, for example, to detect its expression in certain cells, tissues or serum. Such diagnostic methods can be useful in diagnosing various diseases.

The present invention includes an ELISA assay (enzyme-linked immunosorbent assay) using an anti-hRSV F protein antibody or antigen-binding fragment thereof as disclosed herein (eg, RB1).

For example, such a method includes the following steps:

(a) coating a substrate (eg, the well surface of a microtiter plate, eg, a plastic plate) with an anti-hRSV F protein antibody or an antigen-binding fragment thereof;

(b) applying a sample to be tested for the presence of RSV F protein to a substrate;

(c) washing the plate, which removes unbound material in the sample;

(d) applying detectably labeled antibodies (eg enzyme-linked antibodies) that are also specific for the RSV F protein antigen;

(e) washing the substrate, while unbound labeled antibodies are removed;

(f) if the labeled antibodies are bound to the enzyme, a chemical reagent is applied which upon interaction with the enzyme generates a fluorescent signal; and (g) detecting the presence of the labeled antibody.

Detection of a substrate-bound label indicates the presence of the hRSV F protein.

In a further embodiment, the labeled antibody, or antigen-binding fragment thereof, contains as a label a peroxidase that interacts with ABTS (e.g., 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)) or 3,3',5,5 '-tetramethylbenzidine, a color change occurs that is detectable. Alternatively, the labeled antibody or fragment contains as a label a detectable radioisotope (eg, ³ H) that can be detected in a scintillation counter in the presence of a scintillator.

An anti-hRSV F protein antibody or antigen-binding fragment thereof according to the invention (e.g., RB1)

- 30 039222 can be used in the method of Western blot or immune detection of the protein on the blot. Such a method is part of the present invention and includes, for example, the steps:

(1) optionally, transferring the proteins from the sample being tested for the presence of the hRSV F protein (e.g., after electrophoretic separation of the sample proteins in PAAG or SDS-PAGE) onto a membrane or other solid substrate by a method known in the art (e.g., semi-dry blotting or tank blotting); contacting a membrane or other solid substrate tested for the presence of bound hRSV F-protein or fragment thereof with an anti-hRSV antibody or antigen-binding fragment thereof according to the invention.

Such a membrane can be a nitrocellulose or vinyl (e.g., polyvinylidene fluoride (PVDF)) membrane onto which the proteins tested for the presence of hRSV have been transferred after non-denaturing PAAG (polyacrylamide gel electrophoresis) or SDS-PAGE (electrophoresis). in polyacrylamide gel with sodium dodecyl sulfate) (for example, after electrophoretic separation in the gel). Before contacting the membrane with the anti-hRSV antibody or fragment, the membrane is optionally blocked, for example, with a solution of non-fat dry milk or the like. to block non-specific protein binding sites on the membrane.

(2) washing the membrane one or more times to remove unbound hRSV F protein antibody or fragment, as well as other unbound substances; and (3) detecting a bound antibody or fragment to the hRSV F protein.

The detection of a bound antibody or fragment indicates that the hRSV protein is present on the membrane or substrate and in the sample. Bound antibody or fragment can be detected by binding the antibody or fragment to a secondary antibody (anti-immunoglobulin antibody) labeled with a detectable label, and then detecting the presence of the secondary antibody.

Antibodies and their antigen-binding fragments to the hRSV F protein disclosed herein (eg, RB1) can also be used for immunohistochemical analysis. Such a method is part of the present invention and includes, for example, (1) contacting a cell to be tested for the presence of hRSV F protein with an anti-hRSV F protein antibody or antigen-binding fragment thereof of the invention; and (2) detecting the antibody or fragment on or in the cell.

If an antibody or fragment is itself labeled with a detectable label, it can be detected directly. Alternatively, the antibody or fragment may be linked to a detectably labeled secondary antibody that is detected.

Pharmaceutical compositions and administration.

To prepare pharmaceutical or sterile compositions of antibodies and antigen-binding fragments to the hRSV F protein of the invention (eg, RB1), the antibody or antigen-binding fragment thereof is mixed with a pharmaceutically acceptable carrier or excipient. See, for example, Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984).

Therapeutic and diagnostic agents may be formulated by mixing with suitable carriers, excipients or stabilizers, for example in the form of lyophilized powders, thick suspensions, aqueous solutions or suspensions (see, for example, Hardman, et al. (2001), Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000), Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.) ( 1993), Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990), Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990 ) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000), Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY). In one embodiment, the antibodies or antigen-binding fragments of the present invention are diluted to an appropriate concentration in 1-20 mM histidine buffer, pH 5-7, and optionally NaCl or sucrose (e.g., 2-15% (w/v) is added). )) for tonicity. Additional agents such as polysorbate 20 or polysorbate 80 can be added to improve stability at a concentration of 0.01-0.10% (w/v). A representative preparation contains 10 mM L-histidine, 7% (w/v) sucrose, and 0.02% (w/v) polysorbate-80, pH 6.0.

The toxicity and therapeutic efficacy of antibodies or antigen-binding fragments of the invention, administered alone or in combination with another therapeutic agent, can be determined by standard pharmaceutical methods using cell cultures or experimental animals, for example, to determine the LD ₅₀ (dose lethal for 50% of the population) and ED ₅₀ (therapeutically effective dose in 50% of the population). The ratio of doses for toxic and therapeutic effects is called the therapeutic index (LD ₅₀ /ED ₅₀ ). The data obtained from these cell culture assays and animal studies can be used to determine the range of doses usable in humans. The dose of such compounds preferably lies in a range of circulating concentrations that include the ED ₅₀ with little or no toxicity. Within this range, doses may vary depending on the dosage form used and the route of administration.

The route of administration may vary. Routes of administration include oral, rectal, transmucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, dermal, percutaneous, or intra-arterial. Preferred routes of administration are intramuscular, intravenous and intranasal.

In specific embodiments, antibodies or antigen-binding fragments thereof to the hRSV F protein of the invention (eg, RB1) can be administered invasively, eg, by injection. In further embodiments, the hRSV F protein antibody, or antigen-binding fragment thereof, or pharmaceutical composition thereof is administered intravenously, subcutaneously, intramuscularly, intra-arterially, intratumorally, or by inhalation, aerosol delivery. Administration by non-invasive routes (eg, orally; eg, as a pill, capsule or tablet) is also within the scope of the present invention.

The present invention relates to a vessel (e.g. a plastic or glass vial, e.g. with a cap or a chromatographic column, a cannula or a syringe barrel) containing any of the antibodies or antigen-binding fragments of the invention (e.g. RB1), or a pharmaceutical composition thereof. The present invention also relates to an injectable device containing any of the antibodies or antigen-binding fragments of the invention (eg RB1) or a pharmaceutical composition thereof. An injection device is a device that administers a substance into a patient's body by parenteral route, such as intramuscular, subcutaneous or intravenous. For example, the injection device may be a syringe (for example, pre-filled with a pharmaceutical composition, such as an automatic medical syringe), which, for example, includes a barrel or housing for containing an injectable liquid (for example, an antibody, or a fragment, or a pharmaceutical composition thereof), a needle for piercing the skin and/or blood vessels for injecting fluid; and a piston for pushing fluid out of the barrel through the opening of the needle. In one of the embodiments of the invention, the injection device containing the antibody, or its antigen-binding fragment of the present invention, or its pharmaceutical composition, is an injection device for intravenous (in/in) administration. Such a device contains the antibody, or fragment, or pharmaceutical composition thereof in a cannula or trocar/needle, which can be connected to a tube, which can be connected to a bag or reservoir to contain fluid (for example, saline or lactate Ringer's solution containing NaCl, sodium lactate, KCl, CaCl ₂ and optionally containing glucose) introduced into the patient's body through a cannula or trocar/needle. The antibody, or fragment, or pharmaceutical composition thereof, in one embodiment of the invention, can be introduced into the device after the trocar and cannula have been inserted into the patient's vein and the trocar has been removed from the inserted cannula. The IV device may, for example, be inserted into a peripheral vein (eg, in the forearm or upper arm); superior vena cava or inferior vena cava, or into the right atrium of the heart (e.g. central vein), or into the subclavian, internal jugular, or femoral vein and, for example, advance towards the heart until it reaches the superior vena cava or right atrium ( e.g. central venous line). In one embodiment, the injection device is an automatic medical syringe, a needleless syringe, or an external infusion pump. The needleless syringe uses a narrow, high-pressure liquid jet that penetrates the epidermis to deliver the antibody or fragment or pharmaceutical composition thereof into the patient's body. External infusion pumps are medical devices that deliver an antibody or fragment or pharmaceutical composition thereof to a patient's body in controlled amounts. External infusion pumps may be electrical or mechanical. Different pumps operate in different ways, for example, a syringe pump holds fluid in the syringe reservoir and a movable piston element controls fluid delivery, an elastomeric pump holds fluid in an expandable ball reservoir, and pressure from the elastic walls of the ball delivers fluid. In a peristaltic pump, rollers roll along a flexible tube, squeezing it, pushing the fluid forward. In a multichannel pump, fluids can be delivered from multiple reservoirs at different rates.

The pharmaceutical compositions disclosed herein may also be administered by a hypodermic needleless injection device, such as those described in US Pat. pharmaceutical composition are also part of the present invention. The pharmaceutical compositions disclosed herein may also be administered by infusion. Examples of well-known implants and modules for administering pharmaceutical compositions include those described in US Pat. No. 4,487,603, which describes an implantable microinfusion pump for delivering drugs at a controlled rate; U.S. Patent No. 4,447,233, which describes an infusion pump for delivering drugs at a precise infusion rate.

- 32 039222 zii; US Pat. No. 4,447,224, which describes an implantable variable flow infusion device for sustained drug delivery; US patent No. 4439196, which describes an osmotic drug delivery system having a multi-chamber structure. Many other such implants, delivery systems and modules are well known to those skilled in the art and those containing the pharmaceutical compositions of the present invention are within the scope of the present invention.

The mode of administration depends on several factors, including the turnover rate of the therapeutic antibody or antigen-binding fragment in serum or tissue, the level of symptoms, the immunogenicity of the prophylactic/therapeutic antibody, and the availability of target cells in the biological matrix. Preferably, the mode of administration provides for the delivery of a sufficient amount of the therapeutic antibody or fragment to alleviate the disease being treated, while minimizing unwanted side effects. Accordingly, the amount of biological agent delivered depends in part on the specific prophylactic/therapeutic antibody and the severity of the condition being treated. Guidelines for selecting appropriate doses of therapeutic antibodies or fragments are available (see, for example, Wawrzynczak, (1996), Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991), Monoclonal Antibodies, Cytokines and Arthritis , Marcel Dekker, New York, NY; Bach (ed.) (1993), Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert, et al., 2003, New Engl. J. Med. 348:601-608; Milgrom et al., 1999, New Engl. J. Med. 341:1966-1973; Slamon et al., 2001, New Engl. J. Med. 344:783-792; Beniaminovitz et al. , 2000, New Engl. J. Med. 342:613-619; Ghosh et al., 2003, New Engl. J. Med. 348:24-32; Lipsky et al., 2000, New Engl. J. Med. 343:1594-1602).

The appropriate dose is determined by the clinician, for example, using parameters or factors known or suspected in the art for the purposes of preventing or treating disease. Typically, dosing is started with an amount slightly less than the optimal dose, and then the dose is increased in small increments until the desired or optimal effect is achieved with respect to any negative side effects. Important diagnostic measures include the determination of symptoms, such as inflammation or the level of production of inflammatory cytokines. It is generally desirable that the biological agent used be of the same species as the animal to be treated, in which case any immune response to the reagent is minimized. In the case of human patients, for example, it may be desirable to use humanized and fully human antibodies.

The antibodies or antigen-binding fragments thereof disclosed herein (e.g., RB1) may be administered by continuous infusion or doses, e.g. month, every two months, once a quarter, once every six months, once a year, etc. Doses can be administered, for example, intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracerebral, intraspinally, or by inhalation. The total weekly dose is usually at least 0.05 mcg/kg body weight, more often at least 0.2, 0.5, 1, 10, 100 mcg/kg, 0.25, 1.0, 2 .0, 5.0, 10, 25, 50 mg/kg or more (see e.g. Yang, et al., 2003, New Engl. J. Med. 349:427-434; Herold, et al., 2002, New Engl. J. Med. 346:1692-1698; Liu, et al., 1999, J. Neurol. Neurosurg. Psych. 67:451-456; Portielji, et al., 2003, Cancer Immunol. Immunother. 52:151-144). Doses may also be administered to achieve a predetermined target concentration of anti-hRSV antibody in the patient's serum, such as 0.1, 0.3, 1, 3, 10, 30, 100, 300 μg/ml or more. In other embodiments, the anti-hRSV antibody of the present invention is administered, for example, subcutaneously or intravenously, once a week, every other week, every 4 weeks, once a month, every other month, or quarterly at a dose of 10, 20 , 50, 80, 1θ0, 200, 500, 1000 or 2500 mg/patient.

As used herein, the term "effective amount" means the amount of an anti-RSV antibody or antigen-binding fragment of the invention (e.g., RB1) that, when administered alone or in combination with an additional therapeutic/prophylactic agent in a cell, tissue, or body of a patient, is effective to neutralize RSV and/or to prevent or cause a measurable improvement in one or more symptoms of a disease or condition associated with RSV infection. An effective dose also means an amount of antibody or fragment sufficient to at least partially prevent or alleviate symptoms. Where an individual active ingredient is administered separately, the term effective dose refers to that individual ingredient. When the ingredients are used in combination, the term effective dose refers to the total amount of active ingredients that results in a prophylactic or therapeutic effect when administered in combination, sequentially or simultaneously. In specific embodiments, an effective amount is an amount that provides a clinically targeted serum concentration of 10-30 μg/ml for 5 months. In one embodiment, the effective amount is a human dose that provides an approximate target concentration of 10-30 μg/ml for efficacy, as determined using a standard preclinical cotton rat model.

- 33 039222

Sets.

Also provided are kits containing one or more components, which include, but are not limited to, an antibody or antigen-binding fragment thereof to the hRSV F protein described herein (e.g., RB1), in combination with one or more additional components, including including, but not limited to, a pharmaceutically acceptable carrier and/or prophylactic/therapeutic agent described herein. The antibody or fragment and/or prophylactic/therapeutic agent can be formulated as a pure composition or in combination with a pharmaceutically acceptable carrier in a pharmaceutical composition.

In one embodiment, the kit comprises an antibody, or antigen-binding fragment thereof, to the hRSV F protein of the invention (e.g., RB1), or a pharmaceutical composition thereof in a single container (e.g., a sterile glass or plastic vial) and a pharmaceutical composition and/or prophylactic/ therapeutic agent in another container (for example, in a sterile glass or plastic vial).

In another embodiment, the kit comprises a combination of the invention comprising an antibody or antigen-binding fragment thereof to hRSV F protein of the invention (e.g., RB1) together with a pharmaceutically acceptable carrier, optionally in combination with one or more prophylactic/therapeutic agents formulated together, optionally, in a pharmaceutical composition in one common container.

If the kit includes a pharmaceutical composition for parenteral administration to a patient, the kit may include a device for performing such administration. For example, the kit may include one or more hypodermic needles or other injection devices described above.

The kit may include a package insert containing information about the pharmaceutical compositions and dosage forms in the kit. As a rule, such information helps patients and doctors to use the pharmaceutical compositions and dosage forms included in the kit effectively and safely. For example, the insert may contain the following information about the combination of the invention: pharmacokinetics and pharmacodynamics, results of clinical studies, efficacy parameters, indications for use, contraindications, warnings, precautions, adverse reactions, overdose information, proper doses and route of administration, description of the supplied forms, proper storage conditions, references, manufacturer/distributor information, and patent information.

Detection Kits and Prevention/Therapy Kits.

For convenience, an anti-hRSV antibody or antigen-binding fragment of the invention (eg, RB1) may be provided in a kit, i. as a packaged combination of reagents in predetermined quantities, with instructions for performing a diagnostic or detection assay. If the antibody or fragment contains an enzyme as a label, the kit will include the substrates and cofactors required by the enzyme (eg, a substrate precursor that creates a detectable chromophore or fluorophore). In addition, other additives such as stabilizers, buffers (eg blocking buffer or lysis buffer) and the like may be included. The relative amounts of the various reagents can vary widely to create solution concentrations of the reagents that greatly optimize the sensitivity of the assay. In particular, the reagents may be provided as dry powders, typically lyophilized, including excipients which, when dissolved, form a reagent solution having an appropriate concentration.

Also provided are diagnostic or detection reagents and kits containing one or more of such reagents for use in a variety of detection assays, including, for example, immunoassays such as ELISA (sandwich or competitive assay format). The components of the kit may be pre-bonded to the solid support or may be applied to the surface of the solid support when using the kit. In some embodiments, the signal generating means may be provided pre-coupled to an antibody or fragment of the invention, or may need to be combined with one or more components, such as buffers, antibody-enzyme conjugates, enzyme substrates, or the like, prior to use. Kits may also include additional reagents, such as blocking reagents to reduce non-specific binding to the surface of the solid phase, washing reagents, enzyme substrates, and the like. The surface of the solid phase may be in the form of a tube, bead, microtiter plate, microsphere, or other materials suitable for immobilizing proteins, peptides, or polypeptides. In specific aspects, an enzyme that catalyzes the formation of a chemiluminescent or chromogenic product or the reduction of a chemiluminescent or chromogenic substrate is a component of the signal generating means. Such enzymes are well known in the art. The kits may include any of the capture agents and detection reagents described herein. Optionally, the kit may also include instructions for carrying out the methods of the invention.

A kit is also proposed that includes an antibody or its antigen-binding fragment to the F-protein

- 34 039222 hRSV packaged in a container, such as a vial or bottle, and further including a label attached to or packaged with the container, the label stating the contents of the container and giving prescriptions and/or instructions relating to the use of the contents of the container to prevent/ treatment of one or more of the disease states described herein.

In one aspect, the kit is for the prevention or treatment of diseases/conditions associated with RSV infection and includes an antibody or antigen-binding fragment to hRSV F-protein and an additional prophylactic/therapeutic agent or vaccine. The kit may also optionally include a syringe for parenteral, eg intravenous, administration. In another aspect, the kit includes an antibody or antigen-binding fragment to the hRSV F protein and a label affixed to or packaged with the container containing information about the use of the antibody, or fragment, along with a vaccine or additional prophylactic/therapeutic agent. In another aspect, the kit includes a vaccine or additional prophylactic/therapeutic agent and a label attached to or packaged with the container containing information about the use of the vaccine or additional prophylactic/therapeutic agent together with an antibody or fragment thereof to hRSV F protein. In specific embodiments, the hRSV F protein antibody and the vaccine or additional prophylactic/therapeutic agent are in different vials or combined in the same pharmaceutical composition.

In the case of combined prophylaxis or therapy for the simultaneous administration of two prophylactic/therapeutic agents, it is not necessary to administer the agents at the same time or by the same route of administration, provided that there is an overlap of time periods during which the agents exert their prophylactic/therapeutic effect . Simultaneous or sequential administration is envisaged, as well as administration on different days or weeks.

The prophylaxis/therapy and detection kits disclosed herein can also be made, comprising at least one antibody, peptide, antigen-binding fragment or polynucleotide disclosed herein, and instructions for using the composition as a detection reagent or prophylactic/therapeutic agent. . Containers used in such kits can typically be at least one vial, test tube, flask, bottle, syringe, or other suitable container into which one or more of the detective and/or prophylactic/therapeutic compositions can be placed and, preferably divided into appropriate aliquots. When additionally providing a second prophylactic/therapeutic agent, the kit may also include a second container in which the second detection and/or prophylactic/therapeutic composition can be placed. Alternatively, multiple compounds may be formulated into a single pharmaceutical composition and may be packaged in a single container such as a vial, vial, syringe, bottle, or other suitable single container. The kits disclosed herein will generally also include means for containing the vial(s) in a tight protective sheath for commercial sale, such as, for example, injection molded or blow molded plastic containers that contain the desired vials. . If a radioactive, chromogenic, fluorogenic or other detectable label or detection agent is included in the kit, the labeling agent may either be provided in the same container as the detection or prophylactic/therapeutic composition itself, or alternatively may be placed in a second separate container into which this second composition can be placed and divided into appropriate aliquots. Alternatively, the detection reagent and the label can be prepared in the same container, and in most cases the kit will also typically include means to contain the commercially sealed vial(s) and/or convenient packaging and delivery system.

Also provided is a device, or apparatus, for implementing the detection or monitoring methods described herein. Such apparatus may include a chamber or tube into which the sample may be placed, a fluid supply system optionally including valves or pumps to direct sample flow through the device, optional filters to separate plasma or serum from blood cells, mixing chambers to add trapping reagents or detection reagents; and optionally, a detection device for determining the amount of a detectable label associated with the capture reagent's immune complex. Sample flow can be passive (eg, by capillary, hydrostatic, or other forces that do not require further manipulation of the device after sample application) or active (eg, by force generated by mechanical pumps, electroosmotic pumps, centrifugal force, or pressurization). air) or can be created by a combination of active and passive forces.

The following embodiments also provide a processor, a computer readable memory device, and a routine stored on the computer readable memory device and adapted to execute on the processor for implementing any of the methods described herein. Examples

- 35 039222 suitable computer systems, equipment and / or configurations include personal computers, server computers, portable and portable devices, multiprocessor systems, microprocessor systems, set-top boxes, programmable consumer electronic devices, network PCs, mini-computers, mainframe computers, distributed computing equipment, which includes any of the above systems or devices, or any other systems known in the art.

General Methods

Standard molecular biology techniques are described in Sambrook, Fritsch and Maniatis (1982, 1989, 2nd ^Ed ., and 2001, 3rd ^Ed .) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001), Molecular ^Cloning , 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993), Recombinant DNA, Vol. 217, Academic Press, San Diego, CA). Standard methods are also described in Ausbel et al. (2001), Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, NY, which describes cloning in bacterial cells and DNA mutagenesis (volume 1), cloning in mammalian and yeast cells (volume 2), glycoconjugates and protein expression (volume 3), and bioinformatics (volume 4).

Protein purification methods, including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization, are described (Coligan, et al. (2000), Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York).

Methods for chemical analysis, chemical modification, post-translational modification, fusion protein production, protein glycosylation are described (see, e.g., Coligan, et al. (2000), Current Protocols in Protein Science, Vol. 2, John Wiley and Sons, Inc., New York, Ausubel, et al (2001) Current Protocols in Molecular Biology, Vol 3, John Wiley and Sons, Inc., NY, NY, p. (2001), Products for Life Science Research, St. Louis, MO, pp. 45-89 Amersham Pharmacia Biotech (2001), BioDirectory, Piscataway, NJ, pp 384-391).

Methods for the preparation, purification, and fragmentation of polyclonal and monoclonal antibodies have been described (Coligan, et al. (2001), Current Protocols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999), Using Antibodies , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra).

Standard methods for characterizing ligand/receptor interactions are described (see, for example, Coligan, et al. (2001), Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York).

Single chain antibodies and diabodies have been described (see, for example, Malecki et al., 2002, Proc. Natl. Acad. Sci. USA 99:213-218; Conrath et al., 2001, J. Biol. Chem. 276:7346- 7350; Desmyter et al., 2001, J. Biol. Chem. 276:26285-26290; Hudson and Kortt, 1999, J. Immunol. Methods 231:177-189; and U.S. Patent No. 4,946,778).

Bifunctional antibodies have been described (see, for example, Mack, et al. (1995), Proc. Natl. Acad. Sci. USA, 92:7021-7025; Carter (2001), J. Immunol. Methods, 248:7-15 ; Volkel, et al. (2001), Protein Engineering, 14:815-823; Segal, et al. (2001), J. Immunol. Methods, 248:1-6; Brennan, et al. (1985), Science , 229:81-83; Raso, et al. (1997), J. Biol. Chem. 272:27623; Morrison (1985), Science, 229:202-1207; Traunecker, et al. (1991), EMBO J 10:3655-3659 and US Pat. Nos. 5,932,448, 5,532,210 and 6,129,914).

Bispecific antibodies have also been described (see, for example, Azzoni et al. (1998), J. Immunol. 161:3493; Kita et al. (1999), J. Immunol. 162:6901; Merchant et al. (2000), J Biol Chem 74:9115 Pandey et al (2000) J Biol Chem 275:38633 Zheng et al (2001) J Biol Chem 276:12999 Propst et al ( 2000), J. Immunol. 165:2214; Long (1999), Ann. Rev. Immunol. 17:875).

Antibodies can be conjugated to, for example, small molecule drugs, enzymes, liposomes, polyethylene glycol (PEG). Antibodies are useful for therapy, diagnostics, kitting, or other purposes, and include antibodies associated with, for example, dyes, radioisotopes, enzymes, or metals, such as colloidal gold (see, for example, Le Doussal et al. (1991), J. Immunol 146:169-175 Gibellini et al (1998) J Immunol 160:3891-3898 Hsing and Bishop (1999) J Immunol 162:2804-2811 Everts et al (2002) , J. Immunol. 168:883-889).

Flow cytometry techniques, including fluorescence-activated cell sorting (FACS), have been described (see, e.g., Owens, et al. (1994), Flow Cytometry Principles for Clinical Laboratory Practice, John Wiley and Sons, Hoboken, NJ; Givan (2001) Flow Cytometry, 2nd ^Ed .; Wiley-Liss, Hoboken, NJ; Shapiro (2003), Practical Flow Cytometry, John Wiley and Sons, Hoboken, NJ). Fluorescent reagents suitable for the modification of nucleic acids, including nucleotide primers and probes, polypeptides and antibodies, for use, for example, as diagnostic reagents, are available (Molecular Probes (2003), catalogue, Molecular Probes, Inc., Eugene, OR; Sigma -Aldrich (2003), catalogue, St. Louis, MO).

Standard techniques for immune system histology are described (see, for example, Muller-Harmelink (ed.) (1986), Human Thymus: Histopathology and Pathology, Springer Verlag, New York, NY; Hiatt, et al. (2000), Color Atlas of Histology, Lippincott, Williams, and Wilkins, Phila, PA Louis, et al (2002), Basic Histology: Text and Atlas, McGraw-Hill, New York, NY).

Software packages and databases for determining, for example, antigenic fragments, leader sequences, protein folding, functional domains, glycosylation sites, and sequence alignments are available (see, for example, GenBank, Vector NTI® Suite (Informax, Inc.,

Bethesda, M.D.); GCG Wisconsin Package (Accelrys, Inc., San Diego, CA); DeCypher® (TimeLogic Corp.,

Crystal Bay, Nevada); Menne, et al. (2000), Bioinformatics, 16:741-742; Menne, et al. (2000), Bioinformatics

Applications Note, 16:741-742; Wren, et al. (2002), Comput. Methods Programs Biomed. 68:177-181; von Heijne (1983), Eur. J Biochem. 133:17-21; von Heijne (1986), Nucleic Acids Res. 14:4683-4690).

Examples

Example 1 Identification of a fully human RSV neutralizing antibody.

To identify effective hRSV-neutralizing antibodies, sera were collected from informed consent donors and analyzed for their ability to neutralize hRSV virus in vitro. For neutralization assays, serum samples were first serially diluted and then incubated with 600 pfu of hRSV A strain expressing enhanced green fluorescent protein (RSV-GFP). PCB-GFP was mixed 1:1 with diluted serum in a total volume of 200 µl/well in 96-well round bottom plates and incubated at 37°C for 1 hour. -2 (15,000 cells/well). Plates were scanned on an acumen® Cellista instrument (TTP LabTech, Cambridge, MA) and data exported as number of GFP events and total fluorescence intensity per well. NT ₅₀ values were calculated using GraphPad Prism 6 (GraphPad Software, Inc., La Jolla, CA) by fitting a four parameter curve. Determination of titers in the binding reaction with forms before the fusion and after the fusion of the RSV F-protein by ELISA was performed as follows. Nunc C96 Maxisorp® Nunc-Immuno™ plates (Thermo Scientific, Inc.) were loaded with 50 µl per well of hRSV F-protein pre-fusion (see McLellan et al., 2013, Science, 342:592) or post-fusion (the post-fusion form of the LZF21 F protein consists of wild-type F ectodomain without fusion peptide (see McLellen et al., 2011, J. Virol. 85:7788)) at 1 μg/ml in PBS and incubated at 4°C during the night. The plates were washed with PBS/Tween 20 buffer and then blocked with 3% non-fat milk in PBS. Then 50 µl of serially diluted serum samples were added to the wells and incubated at room temperature for 90 minutes. The plates were washed and HRP-conjugated goat anti-human IgG (SouthernBiotech, Birmingham, AL) at a 1:2000 dilution was added. After 1 hour, the plates were washed and SuperBlu Turbo TMB solution (ViroLabs, Inc., Sterling, VA) was added to develop color. Absorbance (0D) was determined at 450 nm on a Wallac 1420 VICTOR2™ Multilabel Counter (Perkin Elmer, Waltham, MA). EC ₅₀ values were calculated using GraphPad Prism 6 by fitting a four parameter curve. A group of donors who were found to have high antibody titers when binding and neutralizing HRCV were re-invited to donate large volumes of blood to obtain PBMCs. PBMC preparations were obtained from a commercial supplier and purchased PBMCs were stored in liquid nitrogen until further use.

PBMCs from one patient showed good neutralization titers and had the highest titers in the binding ELISA to the post-F-fusion form, while also showing some of the best binding to the pre-F-fusion form (data not shown). Therefore, PBMCs from this donor were selected for the isolation of shape-specific post-fusion F-protein memory B cells by FACS.

Biotinylated trimeric F protein in post-fusion form (LZF21) was generated by biotinylation of LZF21 (McLellen et al., 2011, J. Virol. 85:7788) using the EZ Link™ Sulfo-NHS-LC biotinylation kit (Life Technologies, Grand Island, NY) according to the manufacturer's instructions. The LZF21 protein consists of the wild type F protein ectodomain without the fusion peptide (McLellen et al., 2011, J. Virol. 85:7788). F-protein-specific post-fusion murine hybridoma 4D7 is a murine hybridoma obtained by immunizing Balb/c mice with RSV A2. Balb/c mice were immunized twice ip with RSV A2 virus and boosted intravenously with 20 μg of purified RSV A2 (Advanced Biotechnologies, Inc., Columbia, MD) three days prior to cell fusion. Mouse spleens were harvested and splenocytes were fused with SP2/0 myeloma cells using polyethylene glycol. Cells were plated in D medium (StemCell Technologies Inc., Vancouver, BC), plated in 245x245 mm square Petri dishes, and incubated at 37°C, 5% CO2 for 2 weeks. Individual colonies were selected using ClonePix (Genetix), transferred to 96-well plates and incubated as above for 1 week. The supernatants were screened for anti-RSV activity by ELISA using purified RSV A2. Positive clones, including 4D7, were grown and then subcloned by serial dilution. Subclones were screened as described above and clone 4D7-8 was identified and used to optimize LZF21-6uotuh conjugate staining of memory B cells using FACS (data not shown). During these optimization experiments, it was determined that a concentration of 1.5 μg/ml of the LZF21-6uotuh conjugate was the best concentration for staining post-F-fusion form-specific memory B cells. The specificity of the staining reaction was demonstrated using an extraneous mouse hybridoma as a negative control and competing for binding with a 100-1000X excess of unlabeled

- 37 039222

LZF21.

Antigen-specific memory B cells were designated CD3-CD19 ⁺ IgG ⁺ LZF21 ⁺ . These cells were plated in 96-well plates (single cell/well) containing CD40 ligand-expressing HEK293 cells (prepared by standard molecular biology techniques) and IL-21 (Sino Biological Inc., North Wales, PA). After testing 30 samples, binding of the 6-well supernatant to the F-protein fusion mold was shown in an ELISA assay (performed as described above). These samples also differed in binding to the pre-fusion form of the F-protein (data not shown). These six samples were then tested in the neutralization assay as described above without dilution. The presence of neutralizing activity was demonstrated based on the reduction in the number of GFP events. Of the six samples, two samples (designated RB1 and RB11) showed complete neutralization of the hRSV A strain (see Table 3).

Table 3

ELISA, 450 nm Neutralization Hole IgG F-protein "after fusion" F-protein "before fusion" GFP events % neutralization A8 0.641 1.743 1.222 783 A9 1.743 1.915 1.754 689 AN 1.81 1.905 1.555 500 IN 1 1.82 1.925 1.910 0 one hundred% AT 11 1.851 1.748 1,900 one one hundred% AT 12 1,801 1.838 1.679 548 Control 0.037 0.038 0.044 596

RNA extraction and RT-PCR for a culture of sorted single memory B cells.

Part 1.

RNA from the lysate of the best RB1 clone in a 96-well plate was extracted using the RNeasy® Micro Kit (Qiagen, Inc., Valencia, CA) according to the manufacturer's instructions. RNA concentration was determined using NanoDrop™ 200°C (Thermo Fisher Scientific Inc., Wilmington, DE) at 260 nm UV light. The RNA extracted from the RB1 well was used as a template for RT-PCR amplification of the antibody heavy and light chain genes using primer sequences designed based on the leader (forward) and C'-terminus of IgG JH, kappa constant region, or lambda scopes (reverse).

The OneStep RT-PCR kit (Qiagen Inc, Valencia, CA) was used according to the manufacturer's instructions to amplify the antibody sequences. PCR conditions were as follows: 50°C for 30 min, 95°C for 15 min, [94°C for 30 s, 55°C for 30 s, 72°C for 1 min] x 40.72 °C for 10 min and storage at 4°C.

The RT-PCR product was directly used as a nested PCR template.

Part 2. Nested PCR.

RT-PCR products were used as templates in nested PCR to amplify the variable regions of an antibody with pfx50 DNA polymerase (Invitrogen, Cat. No. 12355-012).

Nested PCR primers were designed based on the human IgG heavy and light chain variable region framework 1 germline sequences.

µl RT-PCR product was mixed with 2.5 µl 10X PCR buffer, 2.5 µl 10X PCRX Enhancer solution (Invitrogen), 0.5 µl dNTP mix, 0.5 µl pooled forward primers (10 µM each), 0, 5 µl reverse primer (10 µM), 0.5 µl pfx50 DNA polymerase and 17 µl water. Nested PCR conditions were as follows: 2 min at 94°C, 10 cycles at 94°C for 30 s, 50°C for 30 s, 68°C for 1 min, followed by 30 cycles at 94°C for 30 s, 60°C for 30 s, 68°C for 1 min, followed by 7 min elongation at 68°C, followed by short term storage at 4°C.

Nested PCR products obtained for RB1 from PCR amplification of VH and VK or VH and VL were used as PCR templates with overlapping primers with specific linkers to co-anneal the antibody light and heavy chain genes to facilitate the next step of in-fusion cloning.

Part 3. PCR with overlapping primers and in-fusion cloning.

pfx50 DNA polymerase (Invitrogen) was used in this reaction. Forward and reverse primers have been developed to facilitate in-fusion cloning of PCR products with overlapping primers into a cloning vector. 1 µl nested heavy chain PCR product, 1 µl nested light chain PCR product and 1 µl linker were mixed with 5 µl 10X PCR buffer, 5 µl 10X PCRX enhancer, 1 µl dNTP mix, 1 µl forward primer (10 µM), 1 µl reverse primer (10 µM), 1 µl pfx50 DNA polymerase and 33 µl water.

PCR conditions were as follows: 94°C for 2 min, [94°C for 30 s, 60°C for 30 s, 68°C for 2 min] x 10, [94°C for 30 s, 65°C for 30 s, 68°C for 2 min] x 30, 68°C in

- 38 039222 for 7 minutes, storage at 4°C.

PCR products with overlapping primers were purified on an agarose gel for in-fusion cloning (a PCR product with VH+VK RB1 overlapping primers of approximately 1.2 kb was obtained). PCR products with VH+VK RB1 overlapping primers were cloned into pMab11Exp2 vector (with OmpA leader for light chain expression, with PelB leader for heavy chain expression) by in-fusion cloning. The In-Fusion HD® Cloning Kit (Clontech Laboratories, Inc., Mountain View, CA) was used according to the manufacturer's instructions. Transformants were selected and shipped to GeneWiz, Inc. (South Plainfield, NJ) for sequencing.

Sequencing results were analyzed using Sequencher (Gene Codes Corporation, Ann Arbor, MI).

Nucleotide and amino acid sequences of RB1 are presented in table. 7 (the amino acid sequences of the heavy chain variable region and the light chain variable region of RB1 isolated from a patient sample are shown in SEQ ID NO: 9 and SEQ ID NO: 8, respectively). Due to the design of Jh reverse primers having a nucleotide change, the isoleucine residue present in the natural sequence at position 125 was changed to a threonine residue in the expressed protein (the resulting RB1 heavy chain variable region is shown in SEQ ID NO: 7). The amino acid sequences for the RB1 antibody heavy and light chain variable domain genes were sent to GenScript USA, Inc. (Piscataway, NJ) for codon optimization and conversion of human IgG1, and for transient expression and production in CHO cells. The synthesized DNA molecules were subcloned into the pTT5 vector for expression in CHO-3E7 cells. Recombinant plasmids encoding the heavy and light chains of each antibody were transiently co-transfected into CHO-3B7 cell cultures. Cell culture supernatants harvested on day 6 were used for protein A column purification. Purified human IgG1 RB1 was used in the neutralization assay and other characterization experiments as described in Example 2.

Example 2 Characterization of anti-hRSV antibodies.

The RB1 antibody bound to both the pre-F-fusion form and the post-F-protein fusion form in an ELISA assay as described in Example 1 with an EC50 value in the range of 1-10 ng/mL, while the D25 antibody (see Kwakkenbos et al., 2010, Nature Medicine, 16:123-128) binds preferentially to the pre-F protein fusion form, see FIG. 1A, 1B.__________________________________________

mat Pre-fusion F protein (EU ₅₀ ng/mL) F-protein "after fusion" (EC ₅₀ ng/ml) D25 8.939 >10000 palivizumab 17.37 10.5 RB1 7.053 14.08

The neutralizing activity of RB1, RB11 and some control antibodies described in the literature (D25, palivizumab, full-length [D25 antibody was obtained in-house based on the published sequence and synagis® (palivizumab) was purchased from Myoderm, Norristown, PA]) was compared in against the RSV A Long strain (ATCC number VR-26™) and the RSV B strain Washington 18537 (ATCC number VR-1580™). The test samples were serially diluted three times in EMEM with the addition of 2% heat-inactivated EBS to obtain 11 concentration points. Then serially diluted samples were mixed with equal volumes of EMEM medium supplemented with 2% heat-inactivated EBS containing 100 pfu/well of RSV A or B strains. After incubation at 37°C for 1 h, 100 µl of HEp-2 cells at a cells/ml were transferred to 96-well plates containing virus/antibody mixture. 3 days after infection, the cells were washed once with PBS and then fixed in 80% acetone for 10 min at room temperature. A mixture of RSV F-protein (mAb143-F3-B138) and RSV N-protein (34C9) specific mouse mAbs (in-house) were added to the plates and incubated for 1 hour at room temperature. The plates were washed with PBS/0.05% Tween 20 buffer, biotinylated horse anti-mouse IgG was added to the plates and incubated for 1 hour at room temperature. The plates were washed with PBS/0.05% Tween buffer. A streptavidin IR absorbing dye conjugate was used to detect the RSV-specific signal and two cell standardization dyes were added to 96-well plates and incubated for 1 hour in the dark. After a 1-hour incubation, the plates were washed, air-dried for 20 min in the dark, and counted on the Licor Aerius® automated imaging system using a 700-channel laser for cell normalization and an 800-channel laser for RSV-specific signal detection. 800/700 ratios were calculated and percent neutralization and IC ₅₀ values were determined by plotting a four parameter curve in GraphPad.

- 39 039222

The RB1 antibody was able to neutralize both RSV A and RSV B strains with equal efficiency (IC ₅₀ 1-5 ng/ml). RB1 also showed more effective neutralization of RSV than control antibodies, see FIG. 2A, 2B and tab. 4.

Table 4

RB1 Binding and Neutralizing Capacity Compared to Control Antibodies

Binding F-squirrel "BEFORE Mergers" Binding F-protein "after fusion" Neutralizing activity, 1С ₅₀ (ng/ml) RSV A/Long RSV in/washington RB1 + + 3 1.7 D2 5 + - 3, 6 25, 9 AM22 + - fifty 172.8 131-2A, mouse (Millipore) +/- + 1046 >10000 4D7 (Merck), mouse +/- + 2408 >10000 palivizumab + + 211.5 166 MPE8 + - 106.6 46 101F mouse + + 67 43.6 AM14 + - 3.2 1.9

Determination of RB1 binding affinity for the pre-fusion form and the post-fusion form of the F protein: The binding kinetic activity of the RB1 antibody to the human RSV F protein (prepared as described in Example 1) was measured by surface plasmon resonance using the Biacore T200 system (Biacore, GE Healthcare, Piscataway, NJ). Approximately 5,000 EO of anti-mouse IgG, GE Healthcare, catalog number BR-1008-38, or approximately 13,000 EO of goat anti-rat IgG Fcy fragment-specific, Jackson ImmunoResearch, Cat. no. 112-006-071, was immobilized by amino binding on a CM5 S series sensor chip, Cat. no. BR-1005-30.

Sensograms of binding minus background were used to analyze the rate constants of association (k _a ) and dissociation (kd), as well as the equilibrium dissociation constant KD. The resulting data sets were fitted to the Langmuir model for 1:1 binding using the Biacore T200 evaluation program (version 2.0). In table. 5 shows the affinity values of the antibody for human RSV F protein when bound to the pre-fusion form and the post-fusion form of the RSV F protein.

Table 5

Measurement of the affinity of RB1 for the pre-fusion form of the F-protein and the form after the fusion of the RSV F-protein using Biacore

Protein K _op (M^sL K _off (with E K _D (nM) Form "before merger" F 4.4x10 ⁶ 1.4x10~ ⁴ 0.031 Post-merged form F 2.2x10 ⁶ 9x10~ ⁴ 0.41

RBI binds very strongly the pre-fusion form of the F-protein with a Kd of ~31 pM. The Kd value for form binding after fusion was an order of magnitude lower, 0.41 nM. The Kd value for D25 given in International Patent Application Publication No. WO 2014/121021 A1 was 57 pM. In addition, the RB1 antibody remains bound longer to the pre-fusion form than to the post-fusion form of the F protein, as evidenced by lower dissociation rates of 1.4 x 10 ^-4 compared to the post-fusion form of the F protein.

Example 3 Mapping of RB1 antibody epitopes.

The RB1 binding epitope on the F-protein after fusion was mapped by alanine-scanning mutagenesis. Epitope mapping was performed using shotgun mutagenesis at Integral Molecular as described. (Davidson and Doranz, 2014, Immunology, 143(1):13-20). To construct the library by shotgun mutagenesis, the RSV F-protein expression vector was mutated to create a library of clones, each representing a single point mutant, cumulatively covering all residues in the protein. Libraries were constructed using alanine-scanning mutagenesis, which is a more controlled method for determining the contribution of the side chains of each residue. Using semi-automated robotic operations, each mutant plasmid was individually cloned, sequenced, mini-prepared, and plated in a 384-well microplate format for multiple transfection, expression, and antigen binding assays in human cells. The alanine-scanning mutagenesis library was screened against RB1 for loss of antibody binding. Two residues, arginine-429 and isoleucine-432, have been identified as critical for RB1 binding. See below and FIG. 3A. RB1 appears to be an anti-site IV mAb (101F-like) and antibodies binding site IV have been reported in the literature to bind both the pre-fusion and post-fusion form of the F protein.

Binding capacity (% DT) Mutation mAt RB1 Fab RB1 mat D25 R429A 33, 1 (27) 5.1(4) 385.0 (13) I432A 36, 7 (39) 3.6 (7) 327.8 (161)

The inventors also co-crystallized Fab RBI with the pre-fusion form of the F protein to better understand epitope binding. Diffraction data were obtained from crystals with a resolution of 3.4-3.5 A. The RB1 antibody binds to the pre-fusion form of the F protein through interactions with both heavy and light chain CDR loops. The CDR3 loop of the light chain interacts with the Arg 429 side chain by forming two hydrogen bonds between the oxygen atoms of the Phe 91 and Leu 92 carbonyl groups and the nitrogen atoms of the Arg 429 guanidine groups. Also on the light chain, Asp 50 and Glu 55 in the CDR2 loop are located so that form hydrogen bonds with Asn 426 and Lys 445 in RSV. Extensive interactions occur through the CDR3 loop of the RB1 heavy chain, which, through Tyr 104 and Tyr 110, forms a surface for van der Waals interactions with Ile 432 in RSV. Lys 433 in RSV forms a hydrogen bond with Asn 107 in the CDR3 loop. Judging from the crystal structure, the RB1 light chain also folds opposite Glu 161 and Ser 182 or the adjacent trimer monomer prior to RSV fusion.

The binding epitope identified for RB1 is highly conserved among 944 out of 946 F protein sequences described in the literature. This indicates that very low resistance to antibodies directed to this region is expected.

Example 4 Anti-RSV Activity of Anti-RSV Antibodies in an Animal Model.

The RB1 antibody was compared with D25 and palivizumab for possible protection in a cotton rat challenge model. The study used antibodies palivizumab, D25 and RB1, administered at a dose of 2.5 mg/kg and serially diluted 10-fold to 0.25 mg/kg. In this model of passive immunotherapy, cotton hamsters were injected with RB1, D25, or palivizumab at different concentrations on day d0 and challenged with 105 PFU of RSV one day later. Titers of infecting RSV virus in the nose and lungs were determined four days after infection and used to determine virus spread in a plaque assay.

Cotton rats: At least five cotton rats (Sigmodon hispidus) aged 3-7 weeks with an average body weight of about 100 g were obtained from SAGE Labs (Boyertown, PA). Regular rodent chow and water were provided to the animals ad libitum.

Antibodies: 100 mg of lyophilized palivizumab (Myoderm, Norristown, PA) was dissolved in water to a concentration of 100 mg/mL. Other antibodies were expressed and purified in-house.

Antibody preparations: Formulation buffers were selected for each antibody in order to stabilize the proteins and prevent precipitation. The preparations were as follows: RB1 and D25 were diluted in 1x phosphate-buffered saline, pH 7.2. Palivizumab was formulated according to the manufacturer's recommendations by dissolving in distilled H ₂ O, leaving the protein in a buffer containing 25 mM histidine and 1.3 mM glycine at pH 6.0.

Preparation of solutions for dosing, administration and analysis.

Five animals were randomly weighed to determine the average body weight in the experimental groups. The preparations were prepared approximately 1 hour before administration to the animals. Frozen stock solutions of antibodies were thawed in an ice bath only once. Each antibody was diluted to the appropriate dose concentration to be administered to the animals in each group. On day 0 (start of study), animals randomly assigned to each group were lightly anesthetized with 1-4% isoflurane and injected 0.1 ml intramuscularly into the right quadriceps muscle with a 26G syringe and needle. Animals recovered after sedation within 2 min. 24 h later (±2 h), cotton hamsters were anesthetized with 1-4% isoflurane, blood was collected via the retroorbital sinus, and then immediately injected via the nostrils with 0.1 ml of 1x10 ^5.5 PFU wild-type RSV A2 or RSV B Washington in E Williams medium. Four days after inoculation, animals were euthanized by inhalation of CO2, lungs (left lobes) and turbinates were removed and homogenized in 10 volumes of Hank's balanced salt solution (Lonza) buffer SPG (gluta-41 039222 sucrose mat-phosphate) in an ice bath. Samples were clarified by centrifugation at 2000 rpm for 10 min, aliquoted, flash frozen and immediately stored at

-70°C before testing in plaque assay.

As shown in FIG. 4A-4D and 5A-5D, the RB1 antibody was able to induce a 2-3 log reduction in both RSV A and RSV B virus titers in the lungs and nose of animals at 2.5 mg/kg, which was similar to D25 but better. than palivizumab, which was unable to influence viral titers in the nose.

Example 5 Construction of Fc RB1.

The action of the neonatal Fc receptor for IgG (FcRn) in the process of transfer of humoral immunity from mother to fetus is well studied. In addition, FcRn protects IgG from degradation throughout life, which explains the long serum half-life of antibodies of this class. See, for example, Israel et al., 1996, Immunology, 89:573-8. The FcRn binds to the IgG Fc fragment at a site different from the classical FcyR binding sites or the C1q component of complement. The crystal structure of the FcRn-Fc complex showed that FcRn binds to the CH2-CH3 hinge region of an IgG antibody. A distinctive feature of the IgG-FcRn interaction is a strong dependence on pH. Binding of IgG-FcRn occurs at an acidic pH (6.0) in the lysosome, while dissociation occurs at a neutral pH (7.4) of the extracellular environment. The acidic environment (pH 6.0-6.5) in lysosomes allows FcRn to bind to the Fc region of IgG with affinity values in the lower micromolar range and protect it from catabolism. The protected FcRn-bound IgG is subsequently transferred to the cell surface and released into the extracellular environment. This process protects antibodies by reducing their susceptibility to extracellular degradation.

The Fc of the RB1 antibody has been genetically modified in an attempt to increase the half-life. RB1+YTE is a derivative of RB1 with a triple mutation (M252Y/S254T/T256E (YTE)) introduced into the Fc fragment of RB1. This set of YTE mutations results in increased binding of the antibody to neonatal Fc receptors, resulting in an increased half-life in humans. See, for example, Dall'Acqua et al., 2006, J. Biol. Chem. 281:23514-24. Mutations in three amino acids (YTE: M252Y/S254T/T256E) in the Fc region of motavizumab resulted in a 10-fold increase in FcRn binding in vitro at pH 6.0 in both human and monkey samples and subsequently shown to be 4 -fold increase in serum half-life in vivo monkeys. See Robbie et al., 2013, Antimicrob Agents Chemother. 57:6147-53.

The amino acid sequences of the heavy and light chain variable domains of the RB1+YTE antibody were sent to GenScript USA, Inc. (Piscataway, NJ) for codon optimization and conversion of human IgG1, and for transient expression and production in CHO cells. Nucleotide and amino acid sequences of RB1+YTE are given in table. 7.

The synthesized DNA molecules were subcloned into the pTT5 vector for expression in CHO-3E7 cells. Recombinant plasmids encoding the heavy and light chains of each antibody were transiently co-transfected into CHO-3E7 cell cultures. Cell culture supernatants harvested on day 6 were used for protein A column purification. Purified human IgG1 RB1+YTE was used in the neutralization assay and other characterization experiments.

Example 6 Characterization of RB1+YTE.

The RB1+YTE antibody bound to the F protein in an ELISA assay performed as described in Example 1 with an EC ₅₀ value ranging from 1.97 to 2.457 ng/mL, see FIG. 6. Neutralizing activity of RB1+YTE and a reference antibody described in the literature (motavizumab, MedImmune, Gaithersburg, MD; see U.S. Patent Application Publication No. US 20110158985) produced in-house based on a published sequence was compared against the RSV A Long strain (ATCC number VR-26™) and the RSV strain B Washington 18537 (ATCC number VR-1580™). The test samples were serially diluted three times in EMEM with the addition of 2% heat-inactivated EBS to obtain 11 concentration points. Then serially diluted samples were mixed with equal volumes of EMEM medium supplemented with 2% heat-inactivated EBS containing 100 pfu/well of RSV A or B strains. After incubation at 37°C for 1 h, 100 µl of HEp-2 cells at a cells/ml were transferred to 96-well plates containing virus/antibody mixture. 3 days after infection, the cells were washed once with PBS and then fixed in 80% acetone for 10 min at room temperature. A mixture of RSV F-protein (mAb143-F3-B138) and RSV N-protein (34C9) specific mouse mAbs (mAb143-F3-B138 and 34C9 antibodies) was produced in-house by immunizing mice with the appropriate antigens and immortalizing B cells using hybridoma technology) were added to the plates and incubated for 1 h at room temperature. The plates were washed with PBS/0.05% Tween 20 buffer, biotinylated horse anti-mouse IgG was added to the plates and incubated for 1 hour at room temperature. The plates were washed with PBS/0.05% Tween buffer. A streptavidin IR absorbing dye conjugate was used to detect the RSV-specific signal, and two cell-based dyes for normalization of the assay were added to 96-well plates and incubated for 1 hour in the dark. After a 1 hour incubation, the plates were washed, air dried for 20 min in the dark, and counted on the Licor Aerius® automated imaging system using

700-channel laser for cell normalization and 800-channel laser for RSV-specific signal detection. 800/700 ratios were calculated and percent neutralization and IC50 values were determined by plotting a four parameter curve in GraphPad.

Antibody RB-1+YTE was able to neutralize strains of RSV A and RSV B with equal efficiency (IC50 5-10 ng/ml), see table. 6.

Table 6

Neutralization and affinity measurement of RB1+YTE for the pre-F-protein fusion and post-F-protein fusion form of RSV

mat 1C ₅₀ in vitro neutralization (ng/ml) Kinetic constants (pre-fusion form F) Kinetic constants (form "after fusion" F) RSV A RSV in K _op (RSHchs- ¹ ) Koff (c- ¹ ) K _D (nM) Cop (RShchs- ¹ ) Koff (c- ¹ ) K _D (nM) RB1 3 1.7 4.4x10 ⁶ 1.4x10~ ⁴ 0.031 2.2x10 ⁶ 9x10~ ⁴ 0.41 RB1+YTE 3.6 3.49 3.2x10 ⁶ 2, Zx10~ ⁴ 0.071 1.4x10 ⁶ 7x10~ ⁴ 0.48

The introduction of a YTE mutation in the RB1 Fc fragment did not change the ability of the antibody to neutralize RSV A and B strains in vitro. The in vitro neutralization efficiency of RSV A was 3 and 3.6 ng/ml for RB1 and RB1+YTE, respectively. The in vitro neutralization efficiency of RSV B was 1.7 and 3.49 ng/ml for RB1 and RB1+YTE, respectively. The kinetic constants measured using Biacore were similar for RB1 and RB1+YTE, indicating that the introduction of YTE mutations in the Fc region of the antibody did not change its antigen-binding ability.

A non-GLP (Good Laboratory Practice) pharmacokinetic study was conducted at the New Iberia Research Center (UL Lafayette, LA). Eight biologic-naive male rhesus monkeys were randomly assigned to one of two study groups (n=4 per group). Each animal received a single intravenous (IV) dose of 10 mg/kg RB1+YTE or motavizumab-YTE. Blood samples were collected prior to dosing on day 0, 0.5, 1, 3, 8, and 24 hours post-dosing, and 1, 2, 3, 5, 7, and 10 days post-dosing. ECL immunoassays were used to quantify RB1+YTE (human x [RSV] mAb (RB1-YTE) IgG1/kappa (CE)) and motavizumab-YTE (humanized x [RSV] mAb IgG1/kappa) in rhesus macaque sera.

The assay used biotinylated mouse anti-human IgG kappa chain (BD Biosciences, San Jose, CA) as capture reagent and mouse anti-human IgG Fc region sulfoTAG as detection reagent (SouthernBiotech Birmingham, AL). The specified lower limit of quantification (LOQL) of the assay was 1.37 ng/mL with a minimum required dilution (MNR) of 20.

The pharmacokinetics of RB1+YTE and motavizumab-YTE were evaluated for up to 10 days in rhesus monkeys following an intravenous dose of 10 mg/kg using the same assay to quantify RB1+YTE and motavizumab-YTE. For each animal, a non-compartmental model was used to plot serum concentrations versus time curves using Phoenix Winnonlin 6.3 (Certara, NJ) to estimate area under the curve (AUC _{0-10 days} ). AUC values _{0-10 days were} averaged for 4 animals in each treatment group and presented as mean ± standard deviation. For RB1+YTE AUC _0-10gnee = 1159 ± 116 µg/ml x day and for motavizumab-YTE AUC _0-10gnee = 1381 ± 63.0 µg/ml x day.

RB1+YTE (designated RB1-YTE in the figure caption) and motavizumab-YTE showed similar serum concentration and pharmacokinetic profiles in rhesus monkeys, see FIG. 7. In addition, RB1+YTE PK in lower primates was found to be comparable to motavizumab YTE PK in cynomolgus monkeys described in the literature. See Dall'Acqua et al., 2006, J. Biol. Chem., 281:23514-24.

All references cited in this specification are incorporated by reference to the same extent as if it were specifically and individually stated that each individual publication, database information (e.g., Genbank sequences or GeneID information), patent application, or patent incorporated by reference. This statement of incorporation by reference is for the intent of the inventors and in accordance with 37 C.F.R. 1.57(b)(1), must refer to any and every single publication, database information (e.g., Genbank sequences or GeneID information), patent application, or patent, all of which are clearly identified in accordance with the requirements of 37 C.F.R. 1.57(b)(2), even if the citation of a literary source does not immediately include a statement to include it by reference. The presence of specific inclusion by reference statements, if any, in the body of the specification does not in any way detract from this general inclusion by reference statement. The citation of literature sources in the present description is not an admission that the literature source is the relevant prior art, nor is it an admission of the content or date of these publications or documents.

The present invention is not limited in scope to the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the above description and accompanying figures. Such modifications are to be included within the scope of the appended claims.

Table 7

Sequence Information

SEQID NO: Description SEQUENCES one RBI H - CDR1 DSAMS 2 RBI H - CDR2 FIKSKTYGGTKEYAASVKG 3 RBI H - CDR3 GAPYGGNSDYYYGLDV 4 RBI L-CDR1 RTSQDVRGALA five RBI L-CDR2 DASSLET 6 RBI L-CDR3 QQFLDFPFT 7 RBI VH EVQLVESGGGLVRPGRSLRLSCTVSGFSFDDSAMSWVRQAPG KGLEWISFIKSKTYGGTKEYAASVKGRFTISRDDSKNIAYLQ MNSLKTOflTAVYYCTRGAPYGGNSDYYYGLDVWGQGTTVTVS S 8 RBI VL (isolated from patient sample) DIQMTQSPSSLSASVGDRVTITCRTSQDVRGALAWYQQKPGK APKLLIFDASSLETGVPSRFSGSGSGTVFTLTISSLQPEDFA AYYCQQFLDFPFTFGQGTRLEIKRT nine RBI VH (isolated from patient sample) EVQLVESGGGLVRPGRSLRLSCTVSGFSFDDSAMSWVRQAPG KGLEWISFIKSKTYGGTKEYAASVKGRFTISRDDSKNIAYLQ MNSLKTOflTAVYYCTRGAPYGGNSDYYYGLDVWGQGTTVIVS S 10 Leader subsequence MGWS С11L FLVATAT GVH S

- 44 039222

eleven RBI VH+leader MGWSCIILFLVATATGVHSEVQLVESGGGLVRPGRSLRLSCT VSGFSFDDSAMSWVRQAPGKGLEWISFIKSKTYGGTKEYAAS VKGRFTISRDDSKNIAYLQMNSLKTGflTAVYYCTRGAPYGGN SDYYYGLDVWGQGTTVTVSS 12 RBI VL+leader MGWSCIILFLVATATGVHSDIQMTQSPSSLSASVGDRVTITC RTSQDVRGALAWYQQKPGKAPKLLIFDASSLETGVPSRFSGS GSGTVFTLTISSLQPEDFAAYYCQQFLDFPFTFGQGTRLEIK RT 13 IgGl heavy chain constant domain ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVT C VWD VS HEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK fourteen Kappa light chain constant domain VAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVfl HKLQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC 15 Nucleic acid encoding RBI VH GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACGGCCA GGGCGGTCCCTGAGACTCTCCTGCACAGTTTCTGGATTCAGC TTTGACGACTCTGCTATGAGCTGGGTCCGCCAGGCTCCAGGG AAGGGGCTGGAATGGATAAGTTTCATTAAAAGTAAAACTTAT GGTGGGACAAAAGAATACGCCGCGTCTGTGAAAGGCAGGTTC ACCATCTCAAGAGATGATTCCAAAAACATCGCCTATCTGCAA AT GAACAG C C T GAAAAC C GAG GAGACAG C C GT GTAT TAT T GT ACTAGAGGGGCGCCTTACGGCGGTAACTCCGATTACTACTAC GGTTTGGACGTCTGGGGCCAAGGGACCACGGTCACTGTCTCC TCA 16 Nucleic acid encoding RBI VL GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCT GTAGGAGACAGAGTCACCATCACTTGCCGGACAAGTCAGGAC GTTAGAGGTGCTTTAGCCTGGTATCAACAGAAACCAGGGAAA GCTCCTAAACTCCTGATCTTTGATGCCTCCAGTTTGGAGACT GGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGTT TTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCA GCTTATTACTGTCAGCAGTTTCTTGATTTCCCTTTCACCTTC GGCCAGGGGACACGACTGGAAATCAAACGTACG

- 45 039222

17 Nucleic acid encoding RBI VH (codon- optimized) GAGGTGCAGCTGGTCGAGAGCGGGGGGGGGCTGGTGCGGCCT GGCAGGTCTCTGAGACTGAGCTGCACCGTGAGCGGCTTCTCC TTTGACGATTCTGCCATGAGCTGGGTGCGGCAGGCTCCAGGC AAGGGACTGGAGTGGATCTCCTTCATCAAGTCTAAGACCTAC GGCGGCACAAAGGAGTACGCCGCTTCCGTGAAGGGCCGGTTT ACCATCAGCAGGGACGATTCCAAGAACATCGCCTATCTGCAG ATGAACAGCCTGAAGACCGAGGACACAGCCGTGTACTATTGC ACAAGAGGAGCTCCTTACGGAGGCAACAGCGACTACTATTAC GGACTGGACGTGTGGGGACAGGGAACCACAGTGACCGTGAGC TCC eighteen Nucleic acid encoding RBI VL (codon- optimized) GACATTCAGATGACTCAGTCCCCTTCAAGTCTGAGCGCCTCC GTGGGCGACAGAGTGACCATCACATGCCGGACCAGCCAGGAT GTGCGGGGCGCCCTGGCTTGGTACCAGCAGAAGCCAGGCAAG GCCCCCAAGCTGCTGATCTTTGACGCTAGCTCCCTGGAGACC GGCGTGCCCTCCAGGTTTTCTGGCAGCGGCTCCGGCACAGTG TTCACCCTGACAATCTCTAGCCTGCAGCCTGAGGACTTTGCC GCTTACTATTGCCAGCAGTTCCTGGATTTCCCCTTCACCTTC GGCCAAGGCACACGGCTGGAGATCAAGAGGACC 19 Nucleic acid encoding the heavy chain leader sequence ATGGGTTGGTCCTGTATTATCCTGTTCCTGGTCGCCACTGCT ACTGGGGTCCACTCA twenty Nucleic acid encoding light chain leader sequence ATGGGCTGGTCCTGTATTATCCTGTTCCTGGTGGCAACCGCA ACTGGTGTGCATAGC

- 46 039222

21 Nucleic acid encoding heavy chain constant domain - IgGl GCCTCTACAAAGGGCCCTAGCGTGTTCCCACTGGCTCCCTCT TCCAAGTCTACCAGCGGAGGAACAGCCGCTCTGGGATGTCTG GTGAAGGATTACTTCCCAGAGCCCGTGACCGTGTCCTGGAAC TCTGGCGCCCTGACCAGCGGAGTGCACACATTTCCAGCTGTG CTGCAGTCCTCTGGCCTGTATTCCCTGAGCTCCGTGGTGACC GTGCCCTCTAGCTCCCTGGGCACCCAGACATACATCTGTAAC GT GAAT CACAAGCCAAGCAATACAAAGGT GGACAAGAAGGT C GAGCCCAAGTCCTGTGATAAGACCCACACATGCCCCCCTTGT CCTGCTCCAGAGCTGCTGGGAGGACCTAGCGTGTTCCTGTTT CCACCCAAGCCTAAGGACACCCTGATGATCTCTAGGACCCCC GAGGTGACATGCGTGGTGGTGGACGTGAGCCACGAGGATCCT GAGGTGAAGTTTAACTGGTACGTCGATGGCGTGGAGGTGCAC AATGCCAAGACAAAGCCCAGAGAGGAGCAGTATAACTCCACC TACCGGGTGGTGTCTGTGCTGACAGTGCTGCACCAGGACTGG STGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAATAAGGCC CTGCCCGCTCCTATCGAGAAGACCATCTCTAAGGCCAAGGGC CAGCCTAGGGAGCCACAGGTGTATACACTGCCTCCATCCAGA GACGAGCTGACCAAGAACCAGGTGTSTSTGACATGTSTGGTG AAGGGCTTCTACCCTTCTGATATCGCCGTGGAGTGGGAGAGC AAT G G C C SAG SAGAGAACAAT TATAAGACCACACCCCCTGTG CTGGACAGCGATGGCTCCTTCTTTCTGTACAGCAAGCTGACC GTGGATAAGTCCCGGTGGCAGCAGGGCAACGTGTTCAGCTGT TSTGTGATGCACGAAGCCCTGCACAATSASTASASTCAGAAG AGCCTGTCCCTGTCACCTGGTAAA 22 Nucleic acid encoding kappa light chain constant domain GTGGCCGCTCCCTCCGTGTTTATCTTCCCCCCTTCTGACGAG CAGCTGAAGTCTGGCACAGCTAGCGTGGTGTGCCTGCTGAAC AATTTCTACCCTCGGGAGGCCAAGGTGCAGTGGAAGGTGGAT AACGCTCTGCAGTCTGGCAATAGCCAGGAGTCCGTGACCGAG CAGGACTCTAAGGATAGCACATATTCCCTGTCCTCTACCCTG ACACTGTCTAAGGCCGATTACGAGAAGCACAAGGTGTATGCT TGTGAAGTCACCCACCAGGGGCTGAGTTCACCAGTCACCAAG TCATTCAATCGGGGCGAGTGC 23 heavy chain RB1+YTE EVQLVESGGGLVRPGRSLRLSCTVSGFSFDDSAMSWVRQAPG KGLEWISFIKSKTYGGTKEYAASVKGRFTISRDDSKNIAYLQ MNSLKTCflTAVYYCTRGAPYGGNSDYYYGLDVWGQGTTVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLYITREPEVTCVWDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK

- 47 039222

24 Heavy chain nucleic acid RB1+YTE (codon- optimized) GAGGTGCAGCTGGTGGAATCCGGCGGCGGACTGGTCAGACCT GGCAGATCCCTGAGGCTCAGCTGTACCGTGAGCGGCTTCAGC TTCGACGACTCCGCCATGAGCTGGGTGAGACAGGCCCCTGGC AAGGGCCTGGAGTGGATCAGCTTCATCAAGAGCAAAACCTAT GGCGGAACCAAGGAATACGCCGCCTCCGTGAAGGGCAGGTTC ACCATTTCCAGGGACGACAGCAAGAACATCGCTTACCTCCAG ATGAACTCCCTCAAGACCGAGGATACCGCCGTGTATTATTGC ACCAGAGGCGCCCCCTACGGCGGCAATTCCGACTATTACTAC GGCCTGGATGTCTGGGGCCAAGGCACAACAGTGACCGTGAGC TCCGCTAGCACCAAGGGACCCAGCGTGTTCCCCCTGGCCCCC AGCAGCAAGAGCACAAGCGGAGGAACAGCCGCCCTCGGCTGT CTGGTGAAAGACTACTTCCCCGAGCCTGTGACAGTCAGCTGG AATAGCGGCGCTCTGACCAGCGGCGTCCACACCTTTCCCGCT GTCCTGCAGAGCTCCGGCCTGTACAGCCTGTCCTCCGTGGTC ACAGTGCCCTCCTCCAGCCTGGGCACACAAACCTACATCTGT AAC GT GAAC CACAAG CC CAG CAACAC CAAG GT G GACAAGAAG GTCGAACSSAAATSSTGTGACAAGACSCACACATGSSSSSSC TGCCCCGCCCCTGAGCTGCTGGGCGGCCCTTCCGTGTTCCTG TTCCCTCC CAAG CAAG GATAC C C C C T GTATAT CAC CAGAGAA CCCGAGGTGACCTGTGTGGTGGTCGACGTCAGCCACGAAGAT CCTGAGGTCAAGTTCAACTGGTATGTGGACGGCGTGGAGGTG CATAACGCCAAAACCAAGCCCAGGGAGGAACAGTATAACAGC ACCTACAGGGTGGTGTCCGTCCTGACCGTGCT GCACCAGGAC TGGCTGAACGGAAAGGAGTACAAATGTAAGGTCAGCAACAAA GCCCTGCCCGCTCCTATCGAAAAGACCATCTCCAAGGCCAAA GGCCAGCCCAGAGAACCCCAGGTGTACACCCTGCCCCCTAGC AGAGACGAGCTGACCAAAAACCAGGTCTCCCTGACCTGCCTG GTGAAAGGCTTCTACCCCAGCGATATCGCCGTGGAATGGGAA AGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCC GTGCTCGACAGCGATGGCAGCTTCTTTCTGTACAGCAAGCTG ACCGTGGACAAGAGCAGGTGGCAACAAGGCAACGTGTTCTCC TGCTCCGTGATGCACGAGGCTCTGCACAACCACTATACCCAG AAGTCCCTGAGCCTCAGCCCCGGAAAATGA 25 Easy 1 chain RB1+YTE DIQMTQSPSSLSASVGDRVTITCRTSQDVRGALAWYQQKPGK APKLLIFDASSLETGVPSRFSGSGSGTVFTLTISSLQPEDFA AYYCQQFLDFPFTFGQGTRLEIKRTVAAPSVFIFPPSDEQLK SGTASWCLLNNFYPREAKVQWKVflHKLQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYTKHKVYACE 26 Nucleic acid encoding light chain RB1+YTE GACATTCAGATGACTCAGTCCCCTTCAAGTCTGAGCGCCTCC GTGGGCGACAGAGTGACCATCACATGCCGGACCAGCCAGGAT GTGCGGGGCGCCCTGGCTTGGTACCAGCAGAAGCCAGGCAAG GCCCCCAAGCTGCTGATCTTTGACGCTAGCTCCCTGGAGACC GGCGTGCCCTCCAGGTTTTCTGGCAGCGGCTCCGGCACAGTG TTCACCCTGACAATCTCTAGCCTGCAGCCTGAGGACTTTGCC GCTTACTATTGCCAGCAGTTCCTGGATTTCCCCTTCACCTTC GGCCAAGGCACACGGCTGGAGATCAAGAGGACCGTGGCCGCT CCCTCCGTGTTTATCTTCCCCCCTTCTGACGAGCAGCTGAAG TCTGGCACAGCTAGCGTGGTGTGCCTGCTGAACAATTTCTAC CCTCGGGAGGCCAAGGTGCAGTGGAAGGTGGATAACGCTCTG CAGTCTGGCAATAGCCAGGAGTCCGTGACCGAGCAGGACTCT AAGGATAGCACATATTCCCTGTCCTCTACCCTGACACTGTCT AAGGCCGATTACGAGAAGCACAAGGTGTATGCTTGTGAAGTC ACCCACCAGGGGCTGAGTTCACCAGTCACCAAGTCATTCAAT CGGGGCGAGTGC

CLAIM

Claims (21)

1. An isolated antibody or antigen-binding fragment thereof that binds to human RSV F protein, wherein the antibody or antigen-binding fragment contains the variable region CDR1 - 48 039222 heavy chain, containing the amino acid sequence of SEQ ID NO: 1, CDR2 of the variable region of the heavy chain, containing the amino acid sequence of SEQ ID NO: 2, CDR3 of the variable region of the heavy chain, containing the amino acid sequence of SEQ ID NO: 3, CDR1 of the variable region of the light chain containing the amino acid sequence of SEQ ID NO: 4, a light chain variable region CDR2 containing the amino acid sequence of SEQ ID NO: 5, and a light chain variable region CDR3 containing the amino acid sequence of SEQ ID NO: 6. 2. An isolated antibody or an antigen-binding fragment thereof according to claim 1 that binds to the human RSV F protein, comprising an immunoglobulin light chain and an immunoglobulin heavy chain, selected from the group consisting of: a) an antibody or antigen-binding fragment thereof, comprising a heavy chain having at least 90, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 7 and a light chain having at least 90, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 8; and b) an antibody or antigen-binding fragment thereof, comprising a heavy chain variable region having at least 90, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 7 and a light chain variable region having at least 90, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 8, with any sequence variations occurring in the framework regions of the antibody. 3. An antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof binds to the pre-fusion form of the human RSV F protein with a Kd value of from about 1 x 10' ⁹ to about 1 x 10' ¹² M as determined by surface plasmon resonance (eg Biacore) or a similar method (eg KinExa or OCTET). 4. The isolated antibody or its antigen-binding fragment according to one of claims 1-3, containing the heavy chain variable region containing the amino acid sequence of SEQ ID NO: 7, and the light chain variable region containing the amino acid sequence of SEQ ID NO: 8. 5. The isolated antibody or antigen-binding fragment thereof according to claim 4, which is a full-length antibody having two light chains and two heavy chains, with each light chain containing a variable region containing SEQ ID NO: 8 and a human kappa light chain constant region (SEQ ID NO: 14); and each heavy chain contains a variable region containing SEQ ID NO: 7 and a human IgG1 constant region (SEQ ID NO: 13). 6. An isolated antibody according to claim 4, containing a heavy chain containing the amino acid sequence of SEQ ID NO: 23, and a light chain containing the amino acid sequence of SEQ ID NO: 25. 7. An isolated antibody according to claim 6, characterized in that the amino acid sequence of the heavy chain consists of the amino acid sequence of SEQ ID NO: 23 and the amino acid sequence of the light chain consists of the amino acid sequence of SEQ ID NO: 25. 8. An isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, which is an IgG antibody. 9. An isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, which is an antibody produced in a CHO cell. 10. An expression vector containing a nucleic acid encoding an antibody or an antigen-binding fragment thereof, containing a light chain variable region containing SEQ ID NO: 8, and a heavy chain variable region containing SEQ ID NO: 7. 11. A host cell containing an expression vector according to claim 10. 12. A host cell containing a nucleic acid encoding a light chain variable region comprising SEQ ID NO: 8 and a nucleic acid encoding a heavy chain variable region comprising SEQ ID NO: 7. 13. A composition for treating or preventing an RSV infection, comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier or diluent. 14. The composition according to claim 13, further comprising an antibody or antigen-binding fragment thereof, to a respiratory pathogen selected from influenza virus, human cytomegalovirus (hCMV), human metapneumovirus (hMPV), human parainfluenza virus (hPGV), human rhinovirus (hRV) , the causative agent of mycoplasmal pneumonia, the causative agent of streptococcal pneumonia, adenovirus, bocavirus, enterovirus, norovirus or VK virus. 15. The composition of claim 14, wherein the respiratory tract pathogen is an influenza virus, hCMV, hMPV, hPGV, norovirus, or VK virus. 16. A method for producing an antibody or its antigen-binding fragment, including: a) culturing a host cell containing a polynucleotide encoding a heavy chain and a light chain of any of the antibodies or antigen-binding fragments according to claims 1 to 9, under conditions favorable for the expression of the polynucleotide; and b) recovering the antibody or antigen-binding fragment from the host cell and/or culture - 49 039222 17. An immunogenic composition comprising an antibody or antigen-binding fragment according to any one of claims 1 to 9 and an antigen selected from RSV F protein (fusion protein) and RSV G protein (attachment protein) and fragments thereof. 18. A method for detecting the presence of a pre-fusion form of a human RSV F protein or fragment thereof in a sample, comprising contacting the sample with an antibody or fragment according to any one of paragraphs. 1-9 and detecting the presence of a complex between the antibody or fragment and the peptide; the detection of the complex indicates the presence of a pre-fusion form of the RSV F protein. 19. The use of an antibody or antigen-binding fragment according to any one of claims 1 to 9 for the preparation of a medicinal product for: a) the prevention or treatment of an infection or infectious disease, or b) prevention or treatment of respiratory diseases and post-transplant complications associated with infection. 20. The use of an antibody or antigen-binding fragment according to claims 1 to 9 for the preparation of a medicament for the treatment or prevention of RSV infection. 21. The use of a composition according to claims 13-15 for the preparation of a medicament for the treatment or prevention of RSV infection.